                 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
                            WASHINGTON, D.C.  20460
                                                              OFFICE OF

\* MERGEFORMAT

                                                         CHEMICAL SAFETY AND
                                                      POLLUTION PREVENTION

MEMORANDUM			

March 3, 2015						
SUBJECT:	Benalaxyl-M.   Human-Health Risk Assessment for Tolerances in/on Imported Grape and Tomato.  

PC Code:  113510
DP Barcode: D416044
Decision No.:  476296
Registration No.:  None
Petition No.: 3E8162
Regulatory Action:  Tolerance,  No U.S. Registration
Risk Assessment Type:  Single Chemical
Case No.:  NA
TXR No.:  NA
CAS No.: 688046-61-9
MRID No.:  NA 
40 CFR: 180.TBD

FROM:	William Donovan, Ph.D., Chemist/Risk Assessor
            Linda Taylor, Ph.D., Senior Toxicologist 
		Risk Assessment Branch V/VII
            Health Effects Division (HED) (7509P)

THROUGH:	Michael Metzger, Branch Chief
            Risk Assessment Branch V/VII, HED (7509P)
            
      Anna Lowit, Senior Toxicologist
		Immediate Office, HED (7509P)
      
      Christine Olinger, Branch Chief
      Risk Assessment Branch III, HED (7509P)
            
TO:		Bewanda Alexander, Risk Manager
		Rachel Holloman, Branch Chief
      Fungicide/Herbicide Branch 
		Registration Division (7505P)


The Health Effects Division (HED) of the Office of Pesticide Programs (OPP) is charged with estimating the risk to human health from exposure to pesticides.  The Registration Division (RD) of OPP has requested that HED evaluate hazard and exposure data and conduct assessments, as needed, to estimate the risk to human health that will result from the proposed uses of the new fungicide active ingredient benalaxyl-M.  

HED has evaluated the toxicity and exposure databases for the new active ingredient, benalaxyl-M, and has conducted a human health risk assessment in support of the proposed uses.  Based on this assessment, HED has determined that there are no potential risk estimates of concern for the proposed uses of benalaxyl-M.  HED recommends that the requested uses be granted.

A summary of the findings and an assessment of human health risk resulting from the proposed uses for benalaxyl-M are provided in this document.  The HED team members contributing to this risk assessment include Linda Taylor (hazard evaluation) and William Donovan (chemistry, dietary, and risk assessments).

                               Table of Contents
1.0 	Executive Summary	5
2.0	HED Recommendations	6
2.1	Data Deficiencies	6
2.1.1 	Toxicity Studies	6
2.2	Tolerance Considerations	7
2.2.1	Enforcement Analytical Method	7
2.2.2	International Harmonization	7
2.2.3	Recommended Tolerances	7
2.2.4	Revisions to Petitioned-For Tolerances	8
3.0	Ingredient Profile	8
3.1	Chemical Identity	8
3.2	Physical/Chemical Characteristics	8
3.3	Pesticide Use Pattern	8
3.4	Anticipated Exposure Pathways	10
3.5	Consideration of Environmental Justice	10
4.0	Hazard Characterization and Dose-Response Assessment	10
4.1	Toxicology Studies Available for Analysis	10
4.2  	Absorption, Distribution, Metabolism and Elimination (ADME)	11
4.2.1	Dermal Absorption	11
4.3      Toxicological Effects	12
4.4.1      Completeness of the Toxicology Database	13
4.4.2      Evidence of Neurotoxicity	13
4.4.3      Evidence of Sensitivity/Susceptibility in the Developing or Young Animal	13
4.4.4      Residual Uncertainty in the Exposure Database	13
4.5     Toxicity Endpoints and Points of Departure Selections	14
4.5.1     Dose-Response Assessment	14
4.5.2     Recommendation for Combining Routes of Exposure for Risk Assessment	14
4.5.3     Cancer Classification and Risk Assessment Recommendations	14
4.5.4     Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment	15
5.0	Dietary Exposure and Risk Assessment	16
5.1	Metabolite/Degradate Residue Profile	16
5.1.1	Summary of Plant and Animal Metabolism Studies	16
5.1.2	Comparison of Metabolic Pathways	16
5.1.3	Residues of Concern Summary and Rationale	16
5.2	Food Residue Profile	17
5.3	Water Residue Profile	18
5.4	Dietary Risk Assessment	18
5.4.1	Description of Residue Data Used in Dietary Assessment	18
5.4.2	Percent Crop Treated Used in Dietary Assessment	18
5.4.3	Acute Dietary Risk Assessment	18
5.4.4	Chronic Dietary Risk Assessment	18
5.4.5	Cancer Dietary Risk Assessment	18
5.4.6	Summary Table	19
6.0	Residential (Non-Occupational) Exposure/Risk Characterization	19
7.0	Aggregate Exposure/Risk Characterization	19
8.0	Cumulative Exposure/Risk Characterization	19
9.0	Occupational Exposure/Risk Characterization	20
10.0	References	20
Appendix A.  Toxicology Profile and Executive Summaries	21
A.1	Toxicology Data Requirements	21
A.2	Toxicity Profiles	22
A.3	Hazard Identification & Endpoint Selection	28
A.4	Executive Summaries	28
Appendix B.   Metabolism	47
B.1     Metabolism Summary Table	47
B.2     Metabolic Pathways	50
Appendix C.  Physical/Chemical Properties	52
Appendix D.  Review of Human Research	52



1.0 	Executive Summary

Benalaxyl-M is a new fungicide that is proposed for tolerances without U.S. registrations on grapes and tomatoes, with foliar application use proposed for these commodities.  The end-use products include water-dispersible granules and wettable powders, where benalaxyl-M is co-formulated with another active ingredient (mancozeb, folpet, or chlorothalonil).  These fungicide formulations are intended to control downy mildew (Plasmopara vitacola) in grapes and late blight (Phytophthora infestans) in tomato. 

The database of experimental toxicology studies available for benalaxyl-M provides a robust characterization of the hazard potential of this pesticide.  A comparative thyroid assay in rats is required to assess the potential impact of benalaxyl-M exposure on thyroid function in the young given the pivotal role of thyroid hormones in brain development.

The liver and thyroid are the primary target organs for benalaxyl-M.  In rats, increased liver weights, clinical chemistry changes indicative of liver toxicity, hepatocellular hypertrophy, and thyroid follicular cell hypertrophy were seen following subchronic and chronic exposure.  In mice, increased liver weight and microscopic lesions in the liver (hepatocellular hypertrophy, necrosis, eosinophilic foci) were observed following subchronic and chronic exposure. Additionally, chronic exposure in rats and mice led to increases in the incidence of liver (rat, mouse) and thyroid (rat) tumors.  In dogs, increased liver weight, changes in clinical chemistry indicative of liver toxicity, and hepatocellular hypertrophy were observed following subchronic exposure via the diet, whereas clinical chemistry changes indicative of liver toxicity, fat vacuolation in the liver, and thyroid follicular cell hypertrophy were observed following chronic exposure via capsules.  No evidence of increased quantitative or qualitative susceptibility was seen in the benalaxyl-M hazard database following in utero exposure with rats or rabbits in the prenatal developmental studies or in young rats in the 2 - generation reproduction study. No evidence of maternal toxicity or developmental effects was observed in the developmental toxicity studies in rabbits or rats.  There is no reproductive concern.  No neurotoxic effects were observed in the acute and subchronic neurotoxicity studies in rats, and no immunotoxic effects were observed in the immunotoxicity study in rats.  Benalaxyl-M was classified as "Likely to be Carcinogenic to Humans".  This determination was based on the treatment-related liver tumors observed in male mice, liver tumors observed in male and female rats; and thyroid follicular cell tumors observed in female rats.  No treatment-related tumors were observed in female mice.  A linear low-dose extrapolation model (Q*1) was used to estimate cancer risk, based on the male mouse liver tumor rates.  There is no mutagenicity concern from the in vivo or in vitro genetic toxicity assays.

Toxicological endpoints were selected for dietary exposure scenarios only.  An acute dietary endpoint was not selected because toxicity from a single exposure was not identified in the benalaxyl-M database.  The chronic population adjusted dose (cPAD) is derived from the chronic toxicity/carcinogenicity study in rats, which had a lowest observed adverse effect level (LOAEL) of 135 mg/kg/day based on an increase in gamma glutamyl transferase (GGT) in males, increased triglycerides in males, increased cholesterol in both sexes, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females and a no observed adverse effect level (NOAEL) of 20 mg/kg/day.  For the point of departure (POD) for the chronic dietary risk assessment, a total uncertainty factor of 1000X was applied (10X for interspecies variability, 10X for intraspecies variability, and 10X for database uncertainty to account for the lack of a comparative thyroid assay). ​ A full 10X factor was retained for interspecies variability since other effects not linked to thyroid perturbations were also the basis of the POD.  
 
The residue chemistry database is complete for the proposed uses, supporting a parent only residue definition for tolerance enforcement and risk assessment.  Based on the use of the Organization for Economic Cooperation and Development (OECD) Maximum Residue Limit (MRL) calculation procedures with residues reflecting critical Good Agricultural Practice (cGAP) use patterns, the recommended tolerance levels are 3.0 and 0.20 ppm for grape and tomato, respectively.  

A risk assessment was conducted for dietary exposure only based on the request for establishment of tolerances for import uses in/on grapes and tomatoes.  There are no drinking water, occupational or residential exposure assessments completed for non-domestic uses.   Chronic dietary analyses were performed using conservative assumptions (i.e., 100% crop treated and mean field trial residue levels).  There is no U.S. drinking water exposure associated with the establishment of an import tolerance; therefore, the chronic dietary exposure and risk assessments include food residues only.  The resulting chronic exposure estimates were less than HED's level of concern (<=100% chronic PAD) for general U.S. population (1.4% cPAD) and all population sub-groups. The most highly exposed population subgroup was Children 1-2 years old with an estimated risk of 7.1% cPAD.  The cancer risk estimate for the general U.S. population is 2 x 10[-6].
      
This risk assessment does not rely on data from studies in which human subjects were intentionally exposed to a pesticide or other chemical.  

2.0	HED Recommendations

Provided that benalaxyl-M reference standard is submitted to the National Pesticides Standards Repository, there are no data deficiencies that would preclude establishing the recommended tolerances for benalaxyl-M in/on grape and tomato.  The specific tolerance recommendations are provided in Section 2.2.3.  

2.1	Data Deficiencies

2.1.1 	Toxicity Studies

HED has determined that a comparative thyroid assay in rats is required to generate specific data on the thyroid hormone levels to protect the developing nervous system from thyroid hormone disrupting chemicals.  In the absence of these data, the 10X FQPA database uncertainty factor has been retained in this risk assessment for the uncertainty associated with the potential for enhanced sensitivity of the developing organism to prenatal or postnatal thyroid toxicity.  This uncertainty may be addressed by submission of a comparative thyroid assay or a proposal describing an alternative approach that adequately addresses the issue of disruption of thyroid hormone homeostasis during development.

2.2	Tolerance Considerations

2.2.1	Enforcement Analytical Method

An adequate analytical method is available to enforce the proposed tolerances for residues of benalaxyl-M in plant commodities.  Method RA.09.01, a high-performance liquid chromatography method with tandem mass spectrometry detection (HPLC/MS/MS), has been adequately validated and radiovalidated, and has undergone a successful ILV (independent laboratory validation).  The method has limits of detection and quantitation of 0.005 and 0.010 ppm, respectively.  

Benalaxyl-M has been tested using the Food and Drug Administration (FDA) multi-residue method (MRM) protocols.  Complete recovery of benalaxyl-M from tomato puree samples indicates that Protocol D of FDA Pesticide Analytical Methods (PAM) Vol. 1 may be applicable for determination of benalaxyl-M.

2.2.2	International Harmonization

Organization for Economic Cooperation and Development (OECD) Maximum Residue Limit (MRL) calculation procedures give rounded MRL estimates of 3 and 0.2 ppm for grape and tomato, respectively.  Codex has set benalaxyl MRLs of 0.3 and 0.2 mg/kg for grape and tomato, respectively.  Thus, the HED recommendations in this memo will result in harmonization of the U.S. tolerance with the Codex MRL for tomato, but not for grape since benalaxyl-M residues from the grape trials in Argentina were significantly higher than the Codex MRL.   

2.2.3	Recommended Tolerances

HED has reviewed the available residue data and determined the appropriate tolerance levels for residues of benalaxyl-M (Table 2.2.3).  The recommended tolerance expression is as follows:

      Tolerances are established for residues of the fungicide benalaxyl-M, including its metabolites and degradates, in or on the commodities in the table below.  Compliance with the tolerance levels specified below is to be determined by measuring only benalaxyl [methyl N-(2,6-dimethylphenyl)-N-(phenylacetyl)-DL-alaninate] in or on the commodity.

Table 2.2.3.  Tolerance Summary for Benalaxyl-M.
Commodity
                           Proposed Tolerance (ppm)
                        HED-Recommended Tolerance (ppm)
                                  Comments; 
                         Correct Commodity Definition
Grape
1.1
3.0
Grape
Grape, raisin
2.2
None
Covered by grape tolerance
Tomato
0.25
0.20
Tomato
                   
The tolerance regulation should include a footnote to the tolerance stating:
"There is no U.S. registration for use of benalaxyl-M on grape or tomato."

2.2.4	Revisions to Petitioned-For Tolerances

The proposed tolerance levels differ from those being recommended by HED.  The petitioner used the NAFTA calculator to determine proposed tolerance levels while HED used OECD MRL calculation procedures.  Further, for determination of the grape and tomato tolerance levels, the petitioner included the results from all trials.  In contrast, HED included only those data which matched the critical Good Agricultural Practice (cGAP).  
                                
3.0	Ingredient Profile

3.1	Chemical Identity

TABLE 3.1.	Test Compound Nomenclature.
Compound


Common name
Benalaxyl-M
Company experimental name
IR6141 and IR6141 M
IUPAC name
methyl N-(phenylacetyl)-N-(2,6-xylyl)-D-alaninate
CAS name
methyl N-(2,6-dimethylphenyl)-N-(phenylacetyl)-D-alaninate
CAS #
98243-83-5
End-use product/EP
Fantic, Sidecar

3.2	Physical/Chemical Characteristics

The physical and chemical properties of benalaxyl-M are provided in Appendix C.  Technical grade benalaxyl-M is a solid at room and biologically-relevant temperatures.  The compound has low volatility with little likelihood for exposure in the vapor phase.  The technical-grade material has low aqueous solubility and a log Kow of 3.5, indicative of potential for being a fat-soluble compound.  

3.3	Pesticide Use Pattern

The petitioner provided translations of the benalaxyl-M labels from Europe, Mexico, and Argentina.  Table 3.3 summarizes the key use parameters for benalaxyl-M application to grape and tomato.  
Table 3.	3.	Summary of Directions for Use of Benalaxyl-M.
Country
Formulation
                               Application Type
                               Application Rate 
                                   (kg ai/ha)
                          Max. No. Applic. per Season
                                    RTI[1]
                                    (days)
                          Max. Seasonal Applic. Rate
                                   (kg ai/ha)
                                    PHI[1]
                                    (days)
                                  Limitations
                                     Grape
Italy
Fantic M NC WG;
Fantic M blu; [WP]
Fantic F WG 
                                       
                                       
                                       
                                       
                                       
                                    Foliar
                                       
                                     0.10
                                     0.10
                                     0.075
                                       
                                       3
                                       3
                                       3
                                       
                                     10-14
                                     10-14
                                     10-14
                                       
                                     0.30
                                     0.30
                                     0.225
                                       
                                      42
                                      42
                                      42
Apply the first treatment at the beginning of flowering (BBCH 60).  Apply each subsequent treatment at an interval roughly coincident with the phenological stages of fully flowering (BBCH 65) and early development of the bunches (BBCH 73), when the berries reach the pea size.
France
Fantic F WG
Sidecar [WP]
                                       
                                       
                                     0.10
                                     0.10
                                       
                                       3
                                       3
                                       
                                     12-14
                                     10-14
                                       
                                     0.30
                                     0.30
                                       
                                      42
                                      42
For table grapes, do not treat
after flowering (Stage
BBCH69)
Spain
Fantic M [WP]
Fantic F [WG]
                                       
                                       
                                     0.10
                                     0.075
                                       
                                       2
                                       3
                                       
                                     10-14
                                     10-14
                                       
                                     0.20
                                     0.225
                                       
                                      40
                                      42
This product cannot be used in vineyards intended for multiplication or on wine trellises.
Argentina
Fantic M[2]  [WG]
                                       
                                       
                                     0.10
                                       
                                       4
                                       
                                     7-10

                                     0.40
                                       
                                      21
For sensitive varieties, it is 
advised  to  preventively
apply the product pre
and post-flowering until
the clusters close.
                                    Tomato
Italy
Fantic M NC WG;
Fantic M blu [WP]
                                       
                                       
                                       
                                       
                                    Foliar
                                       
                                     0.10
                                     0.10
                                       
                                       3
                                       3
                                       
                                     7-10
                                      10
                                       
                                     0.30
                                     0.30
                                       
                                      14
                                      14
Begin treatment when there are favorable conditions for the disease or when the main bud has developed (BBCH 61).
Spain
Fantic M [WP]
                                       
                                       
                                     0.10
                                       
                                       3
                                       
                                      10
                                       
                                     0.30
                                       
                                       3
Begin application when environmental conditions favor disease development.
Mexico
Sidecar M; [WG]
Fantic Star [WG]
                                       
                                       
                                     0.12
                                     0.12
                                       
                                       3
                                       3
                                       
                                       7
                                       7
                                       
                                     0.36
                                     0.36
                                       
                                      14
                                      14
Begin application when environmental conditions favor disease development (temperatures between 18 and 22[o]C and relative humidity above 90%).
Do not apply when temperatures are very high, when winds are stronger than 10 km/hour, or while it is raining.
   1 RTI = Retreatment interval.  PHI = Preharvest interval.
   2 Fantic M use in/on grape is pending in Argentina.

The use directions are adequate to allow evaluation of the residue data relative to the proposed uses.  The cGAP for grape and tomato were identified as the GAPs from Argentina and Spain, respectively. 

3.4	Anticipated Exposure Pathways

The Registration Division requested that HED perform an assessment of human health risk to support the petitioner's request for benalaxyl-M tolerances on imported grapes and tomatoes.  No previous Agency risk assessments have been conducted for benalaxyl-M since there are currently no U.S. registrations for benalaxyl-M-based products.  Humans may be exposed to benalaxyl-M through ingestion of imported grapes, tomatoes, and their processed commodities (i.e., grape juice, raisins, wine, dried tomato, tomato paste, puree, etc.).  Dietary exposures are expected based on detectable residues observed.  There are no U.S. drinking water, occupational or residential exposures associated with the requested tolerance.  

3.5	Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Populations and Low-Income Populations,"  (http://epa.gov/compliance/ej/resources/policy/exec_order_12898.pdf).  As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups according to well-established procedures.  In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on patterns of that subgroup's food and water consumption, and activities in and around the home that involve pesticide use in a residential setting.  Extensive data on food consumption patterns are compiled by the USDA under the National Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are used in pesticide risk assessments for all registered food uses of a pesticide.  These data are analyzed and categorized by subgroups based on age and ethnic group.  Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant.  Whenever appropriate, non-dietary exposures based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas postapplication are evaluated.  Further considerations are currently in development as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0	Hazard Characterization and Dose-Response Assessment
      
4.1	Toxicology Studies Available for Analysis

The database of experimental toxicology studies available for benalaxyl-M provides a robust characterization of the hazard potential of this pesticide.  HED has determined that a comparative thyroid assay in rats is required to assess the potential impact of benalaxyl-M exposure on thyroid function in the young (Dellarco et al., 2005).  The agency is requiring these data given the pivotal role of thyroid hormones in brain development.  In the absence of these data, the 10X FQPA database uncertainty factor has been retained.

The data from the following studies were used to evaluate the hazard potential of benalaxyl-M:

         * subchronic oral toxicity studies in rats, mice, and dogs 
         * chronic oral toxicity studies in rats and dogs 
         * carcinogenicity studies in rats and mice 
         * developmental toxicity studies in rats and rabbits 
         * reproduction toxicity study in rats 
         * acute and subchronic neurotoxicity studies in rats
         * subchronic dermal toxicity study in rats 
         * immunotoxicity study in rats  
         * complete mutagenicity study battery 
         * metabolism study in rats 

4.2  	Absorption, Distribution, Metabolism and Elimination (ADME)

Benalaxyl-M was well absorbed and rapidly excreted.  Absorption was rapid and the peak plasma concentrations (Tmax) were reached within 2-6 hours and the terminal half-lives T(1/2) ranged from 59 to 102 hrs.  There were no major sex differences in the maximum concentration (Cmax) or in the time taken to reach Cmax (Tmax), and the estimated total absorption was similar between the sexes (~89% for males and ~82% for females).  Radiolabel was excreted mainly via the feces within 72 hours (84% of administered dose).  Only 7% of the administered dose (AD) was excreted via the urine, and tissue radioactivity levels were low.  The highest tissue concentrations (as percent of administered dose) 0.5 hours after treatment were found in the liver and intestinal tract, which is indicative of active absorption.  Total radioactive dose recovered in the tissues 0.5 hours after treatment was 23%.  Tissue concentrations as percent of administered dose were <1% for both sexes by 72 hours post dose.  Metabolism of benalaxyl-M was extensive as at least 18 compounds were found.  Benalaxyl-M was a minor component excreted in the feces, and it was not found in the urine.  Feces contained a higher number of degrada - tion products, but the main metabolites were identical both in urine and feces.  The metabolic profile was the same in male and female rats.  Benalaxyl-M was extensively metabolized principally by oxidation of the methyl group on the aniline ring to the hydroxymethyl and finally to the carboxylic acid. Another metabolic pathway involved hydroxylation of the phenyl ring and hydrolysis of the carboxymethyl group.  Following single low and high doses, the main route of elimination was fecal via the bile (62-81% AD), while urinary excretion (8-19% AD) played a minor role. There was evidence of extensive enterohepatic recirculation.  Bile was an important route of excretion; a substantial portion of the administered dose was re-absorbed; biliary excretion was substantial for both sexes (80.5% of dose male; 62% of dose females), indicating most of the radiolabeled test material was absorbed and then excreted via the bile into the feces.  Excretion in the expired air was negligible (<0.1%).  Residual radioactivity was < 1% AD in the carcasses 96 hours after treatment.  

4.2.1	Dermal Absorption

Dermal exposure is not expected in the U.S. with an import tolerance.  There is no dermal absorption study on benalaxyl-M, but there is a subchronic dermal toxicity study available, which shows no dermal hazard at dose levels up to the limit dose.
	
4.3     Toxicological Effects

Benalaxyl-M is a new fungicide and a member of the N-acylalanine fungicides, which act by inhibiting RNA polymerase. There are no data on whether this pesticidal mode of action is relevant to mammalian toxicology.

The liver and thyroid are the primary target organs for benalaxyl-M.  In rats, increased liver weights, clinical chemistry changes indicative of liver toxicity, hepatocellular hypertrophy, and thyroid follicular cell hypertrophy were seen following subchronic and chronic exposure.  In mice, increased liver weight and microscopic lesions in the liver (hepatocellular hypertrophy, necrosis, eosinophilic foci) were observed following subchronic and chronic exposure. Additionally, chronic exposure in rats and mice led to increases in the incidence of liver (rat, mouse) and thyroid (rat) tumors.  In dogs, increased liver weight, changes in clinical chemistry indicative of liver toxicity, and hepatocellular hypertrophy were observed following subchronic exposure via the diet, whereas clinical chemistry changes indicative of liver toxicity, fat vacuolation in the liver, and thyroid follicular cell hypertrophy were observed following chronic exposure via capsules.

Benalaxyl-M was classified as "Likely to be Carcinogenic to Humans".  This determination was based on the treatment-related liver tumors observed in male mice, liver tumors observed in male and female rats; and thyroid follicular cell tumors observed in female rats.  No treatment-related tumors were observed in female mice.  There is no mutagenicity concern from the in vivo or in vitro genetic toxicity assays.  The negative data rule out mutagenicity as a possible mode of action (MOA) for the induction of tumors in the rat and mouse carcinogenicity studies.  A plausible non-genotoxic mode of action involving mitogenesis was not established for the development of liver tumors in the male mouse bioassay with benalaxyl-M.  MOA data for the liver tumors in male and female rats and the thyroid tumors in female rats were not provided. 

No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to benalaxyl-M with rats or rabbits in the prenatal developmental studies or in young rats in the 2-generation reproduction study.  The 2-generation rat reproduction study resulted in no effects on fertility, although decreased primordial follicles/growing follicles/corpora lutea were observed at the high dose.  However, there is a clear NOAEL for these findings, and there is no reproductive concern.  The offspring effects (decreased F2 pup body weight and increased incidence of hepatocellular hypertrophy in both sexes of F2 pups) occurred at the same dose that caused parental effects (increased liver weight and liver hypertrophy).  No evidence of maternal toxicity and no developmental effects were observed in developmental toxicity studies in rabbits or rats.  The rabbit was tested at the limit dose (1000 mg/kg/day), but the rat was not tested up to the limit dose.  Since the point of departure is 10-fold lower than the NOAEL in the rat study, a repeat rat study at the limit dose would not impact the risk assessment and is not needed.  
	
No neurotoxic effects were seen in the acute or the subchronic neurotoxicity study.  Benalaxyl-M was not found to be immunotoxic in the male rat.
4.4     FQPA Safety Factor for Infants and Children 

No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to benalaxyl-M with rats or rabbits in the prenatal developmental studies or in young rats in the 2-generation reproduction study.  There is no evidence for neurotoxicity following oral exposures to benalaxyl-M.  Thyroid toxicity was seen following subchronic and chronic exposures to adult rats.  There are, however, no data regarding the potential effects of benalaxyl-M on thyroid homeostasis in the young animals. This lack of characterization creates uncertainty with regards to potential life stage sensitivities due to exposure to benalaxyl-M, and raises the Agency's level of concern.  Therefore, the Agency is requiring a comparative thyroid assay in rats with benalaxyl-M.  This study will better address the concern for potential thyroid toxicity in the young.  There are no residual uncertainties in the benalaxyl-M residue database with regards to dietary exposure.  The FQPA Safety Factor is retained at 10X in the form of a database uncertainty factor (UFDB).
4.4.1    Completeness of the Toxicology Database 

There are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes and tomatoes; however, HED has determined that a comparative thyroid assay in rats is required, as discussed above.  The uncertainty for the lack of this study is addressed by retaining the FQPA Safety Factor of 10X as the UFDB.  
                                       
4.4.2    Evidence of Neurotoxicity

There is no evidence for neurotoxicity following oral exposures to benalaxyl-M. Following a single exposure, no evidence for neurotoxicity was observed at the limit dose and twice the limit dose.  Following repeated (dietary) exposures, there were no treatment-related clinical signs of neurotoxicity, behavioral changes, or neuropathology.  

4.4.3    Evidence of Sensitivity/Susceptibility in the Developing or Young Animal
No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to benalaxyl-M with rats or rabbits in the prenatal developmental toxicity studies or in young rats in the 2-generation reproduction study.  The 2-generation reproduction study resulted in no effects on reproductive function or fertility.  The offspring effects occurred at the same dose that caused parental effects.  No evidence of developmental delay or developmental toxicity was observed in developmental toxicity studies in rabbits or in rats.  The rabbit was tested at the limit dose (1000 mg/kg/day), but the rat was not tested up to the limit dose.  Since the point of departure is 10-fold lower than the NOAEL in the rat study, a repeat rat study at the limit dose would not impact the risk assessment and is not needed.   
4.4.4   Residual Uncertainty in the Exposure Database

There are no residential uses of benalaxyl-M in the U.S. and adequate food residue data are available for human health risk assessment.  Residue values used in the dietary risk assessments are unlikely to underestimate risk. 

Dietary exposure assessments were conducted using anticipated residues and assumed 100% crop treated.  Therefore, the chronic dietary (food only) exposure is considered a conservative estimate.  Drinking water exposure in the U.S. is not expected since there are no domestic registrations for benalaxyl-M. 

4.5    Toxicity Endpoints and Points of Departure Selections

4.5.1   Dose-Response Assessment

The details for the toxicity endpoint selection are presented in Appendix A2.  In risk assessments for import commodities, endpoints are typically selected for dietary exposure only.  Endpoints for incidental oral, dermal and inhalation exposures are not selected for import tolerances due to lack of potential U.S. occupational or residential exposure.  

An acute dietary endpoint for the general population, including infants and children and females 13+ was not selected for benalaxyl-M, based on the lack of toxicity resulting from a single exposure in the database.  

For chronic dietary risk assessment, a NOAEL of 20 mg/kg/day was selected based on an increase in gamma glutamyl transferase (GGT) in males, increased cholesterol in both sexes, increased triglycerides in males, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females  at a LOAEL of 135 mg/kg/day in the chronic toxicity/carcinogenicity study in rats.  For the POD for the chronic dietary risk assessment, a total UF of 1000X was applied (10X for interspecies variability, 10X for intraspecies variability, and 10X for database uncertainty) to account for the lack of a comparative thyroid assay.  When the POD is based on thyroid effects, the interspecies extrapolation UF is typically reduced to 3X due to the pharmacodynamic differences between rats and humans pertaining to thyroid perturbations.  However, in the case of benalaxyl-M, the full 10X factor was retained for interspecies UF since effects not related to thyroid function disruption (i.e., liver, ovarian stromal cell hyperplasia) were also the basis of the POD. 

4.5.2   Recommendation for Combining Routes of Exposure for Risk Assessment 

Since only oral exposure is anticipated for benalaxyl-M, it is not relevant to this action to discuss combining routes of exposure at this time.  

4.5.3   Cancer Classification and Risk Assessment Recommendations

In accordance with the EPA's Final Guidelines for Carcinogenic Risk Assessment (March, 2005): the Cancer Assessment Review Committee (CARC) classified benalaxyl-M as "Likely to be Carcinogenic to Humans", based on the following weight of evidence considerations:

   (i) Treatment-related hepatocellular adenomas and hepatocellular adenomas and/or carcinomas combined in male rats; 
   (ii) Treatment-related hepatocellular adenomas in female rats; 
   (iii) Treatment-related thyroid follicular cell adenomas and adenomas and/or carcinomas in female rats;  
   (iv) Treatment-related hepatocellular adenomas and hepatocellular adenomas and/or carcinomas combined in male mice;
   (v) Lack of mutagenic concern. Benalaxyl-M does not act through a mutagenic mode of action; and  
   (vi) The overall weight-of-evidence was not sufficient to indicate that benalaxyl-M induces liver tumors via a mitogenic mode of action. 

The CARC recommended that a low dose extrapolation model (Q1*) be applied to the experimental animal tumor data for quantification of cancer risk in humans from exposure to benalaxyl-M.  The most potent unit risk, Q1[*], is that for male mouse liver tumor rates at 5.90 x 10[-3] (mg/kg/day)[-1] in human equivalents.  The available data are inadequate to establish a mode of action (MOA) for the male mouse liver tumors.

4.5.4    Summary of Points of Departure and Toxicity Endpoints Used in Human Risk Assessment

Table 4.5.4.  Summary of Toxicological Doses and Endpoints for Benalaxyl-M for Use in Dietary Human Health Risk Assessments
                              Exposure/ Scenario
                              Point of Departure
                                 Uncertainty/
                              FQPA Safety Factors
                RfD, PAD, Level of Concern for Risk Assessment
                        Study and Toxicological Effects
Acute Dietary (General Population, including Infants, Children, and females 13+)
No appropriate acute endpoint was identified.
Chronic Dietary
(All Populations)
NOAEL= 20 mg/kg/day
UFA= 10x
UFH= 10x
FQPA UFDB = 10x
Chronic RfD = 
cPAD = 0.02 mg/kg/day
Chronic Toxicity/Carcinogenicity Study -rat (49040634)
LOAEL = 135 mg/kg/day based on based on an increase in γ-glutamyl transferase (GGT) in males, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females.  
Cancer (oral)
Classification:  "Likely to be Carcinogenic to Humans".  Based on male mouse liver tumors, Q1*= 5.90 x 10-3 (mg/kg/day)[-1]
Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and  used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures.  NOAEL = no observed adverse effect level.  LOAEL = lowest observed adverse effect level.  UF = uncertainty factor.  UFA = extrapolation from animal to human (interspecies).  UFH = potential variation in sensitivity among members of the human population (intraspecies).    UFDB = to account for the absence of a comparative thyroid study.  FQPA SF = FQPA Safety Factor.  PAD = population adjusted dose (c = chronic).  RfD = reference dose.
 
5.0	Dietary Exposure and Risk Assessment 

5.1	Metabolite/Degradate Residue Profile

5.1.1	Summary of Plant and Animal Metabolism Studies

The nature of the residue in grape, lettuce, and tomato is adequately understood.  The parent compound comprised 26-44% of the total residues found in the submitted grape, tomato, and lettuce metabolism studies.  The only metabolites found at more than 10% of the total residues were benzyl alcohol glucoside and malonylglucoside conjugates of the parent molecule, present at summed levels comparable to the parent molecule.  

There are no significant livestock feedstuffs associated with grape or tomato.  Therefore, no livestock metabolism data are relevant to the present tolerance petition. 

5.1.2	Comparison of Metabolic Pathways

The proposed metabolic pathways for benalaxyl-M in grape and rats are provided in Appendix B.  Metabolism in lettuce, grape, and tomato shared a common metabolic pathway with parent benalaxyl-M being the common and dominant residue.  Other major metabolites (>10% TRR) were identified as glucoside or malonylglucoside conjugates of the parent compound after oxidation.  The primary difference noted in the plant metabolism studies was the extent of benalaxyl-M metabolism, which corresponded with the PHIs of the metabolism studies.  Thus, relatively more parent was found in the short-PHI studies (lettuce and tomato) and relatively less in the longer PHI study (grape).  

In the rat metabolism study, benalaxyl-M was extensively metabolized to at least 18 compounds with parent present at only low levels.  The primary metabolic pathway involved oxidation of the methyl group on the aniline ring to the hydroxymethyl and then to the carboxylic acid.  Another pathway involved hydroxylation of the phenyl ring and hydrolysis of the carboxymethyl group.  Low levels of glucuronic acid conjugates were identified.     

Metabolism of benalaxyl-M is more extensive in the rat than in plants.  The metabolic pathway observed in plants also occurs in the rat, along with other pathways not found in plants.   

5.1.3	Residues of Concern Summary and Rationale

Major residue in the plant metabolism studies were the parent compound and its glucoside and malonylglucoside conjugates.  These conjugates are expected to be stable in the gastrointestinal tract.  Conjugation of the parent molecule is likely to decrease absorption and increase elimination, thereby reducing the toxicological concern of these residues.  Therefore, the residue of concern in plants is parent benalaxyl-M for tolerance enforcement and risk assessment.

Given that the only pathway for exposure at this time is via imported grapes and tomatoes, residue definitions for rotational crops, livestock, and water are not needed.  Table 5.1.3 summarizes the residues of concern for benalaxyl-M.

Table 5.1.3. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression
Matrix
Residues Included In Risk Assessment
Residues Included In Tolerance Expression
Plants


Primary Crop
Benalaxyl-M
Benalaxyl-M

Rotational Crop


Not Applicable


Not Applicable
Livestock


Ruminant



Poultry


Drinking Water





5.2	Food Residue Profile
	
The submitted residue chemistry studies are adequate for supporting regulatory conclusions, establishing appropriate tolerances levels for enforcement, and for purposes of risk assessment.  Analysis of residues can be accomplished through standard analytical techniques, and residues do not dissipate during frozen storage.  Following foliar spray applications to grape and tomato, benalaxyl-M residues translocate through the plant. Processing studies demonstrate no residue concentration in any grape or tomato processed commodities except for raisin, where an average concentration factor of 2.3x was determined.  Multiplying the concentration factor of 2.3x by the grape highest average field trial (HAFT) value of 0.98 ppm gives 2.3 ppm, which is less than the recommended grape tolerance of 3.0 ppm.  Thus, there is no need for a separate raisin tolerance since residue concentration in this processed commodity will be covered by the grape raw agricultural commodity (RAC) tolerance.  No confined rotational crop, livestock metabolism, or livestock feeding studies were submitted or required.  

At this time there are no U.S. registrations for benalaxyl-M.  Establishment of U.S. tolerances for grape and tomato was requested to support the import of treated crops from Europe, Central America, and South America.  For grape and tomato the maximum application rates are similar across countries; thus, critical Good Agriculture Practice (cGAP) selection was based on the shortest PHI, since residue decline trials showed a gradual decrease in residues with increasing PHI.  The Argentina use pattern constitutes the cGAP for grape, and the recommended grape tolerance of 0.20 ppm was based on trials reflecting the cGAP.  Three trials from Argentina conducted in 2011 gave residues approximately ten times higher than residues reported in any of the other grape trials and prevent harmonization with the Codex MRL for benalaxyl.  For tomato, the Spain use pattern, with a preharvest interval (PHI) of 3 days, was identified as the cGAP and formed the basis for the recommended tomato tolerance, which is harmonized with the Codex MRL for benalaxyl.  

5.3	Water Residue Profile

An assessment of residues in drinking water is not required for this assessment because there is no drinking water exposure in the U.S. associated with the establishment of an import tolerance.

5.4	Dietary Risk Assessment

5.4.1	Description of Residue Data Used in Dietary Assessment

For the chronic and cancer dietary analyses, anticipated residues (i.e., mean field trial residues) were used.  The residue chemistry database for benalaxyl-M included processing studies for all relevant processed commodities from grape and tomato except for dried tomato.  Thus, the default processing factor of 14.3x was used for dried tomato.  The only processed commodity showing residue concentration was for raisin, with an average concentration factor of 2.3x.  All other processed commodities showed residue reduction upon processing; for these commodities a processing factor of 1.0 was used in the analysis.  The Agency considers these to be conservative assessments.  

5.4.2	Percent Crop Treated Used in Dietary Assessment

For the chronic and cancer dietary analyses, a 100% crop treated assumption was used.

5.4.3	Acute Dietary Risk Assessment

Because no acute endpoint was identified in the toxicity database, no acute dietary analysis was conducted.

5.4.4	Chronic Dietary Risk Assessment
 
The results of the chronic dietary exposure analyses are reported in Table 5.4.6.  The resulting chronic risk estimates were less than HED's level of concern ( - <=100% cPAD) for the general U.S. population (1.4% cPAD) and all population sub-groups. The most highly exposed population subgroup was Children 1-2 years old with an estimated risk of 7.1% cPAD.

5.4.5	Cancer Dietary Risk Assessment

The results of the cancer dietary exposure analyses are reported in Table 5.4.6.  The cancer dietary assessment made use of the same input assumptions as the chronic analysis.  Benalaxyl-M has been classified as "Likely to be Carcinogenic to Humans" (TXR No. 0057093, L. Brunsman, 04-DEC-2014).  A linear low-dose extrapolation model (Q1*) was used to estimate cancer risk, with a Q1* = 5.90 x 10[-][3] (mg/kg/day)[-1].  The cancer risk estimate to the U.S. population is 1.7 x 10[-][6].




5.4.6	Summary Table

 Table 5.4.6.  Summary of Dietary (Food Only) Exposure and Risk for Benalaxyl-M.[1]
                              Population Subgroup
                                 Acute Dietary
                                Chronic Dietary
                                     Cancer
                                        
                          Dietary Exposure (mg/kg/day)
                                     % aPAD
                                Dietary Exposure
                                  (mg/kg/day)
                                     % cPAD
                                Dietary Exposure
                                  (mg/kg/day)
                                      Risk
 General U.S. Population
                                       NA
                                       NA
                                    0.000287
                                      1.4
                                    0.000287
                                1.7 x 10[-][6]
 All Infants (< 1 year old)
                                        
                                        
                                    0.000477
                                      2.4
                                       NA
                                       NA
 Children 1-2 years old
                                        
                                        
                                    0.001416
                                      7.1
                                        
                                        
 Children 3-5 years old
                                        
                                        
                                    0.000901
                                      4.5
                                        
                                        
 Children 6-12 years old
                                        
                                        
                                    0.000381
                                      1.9
                                        
                                        
 Youth 13-19 years old
                                        
                                        
                                    0.000148
                                     < 1
                                        
                                        
 Adults 20-49 years old
                                        
                                        
                                    0.000201
                                      1.0
                                        
                                        
 Adults 50-99 years old
                                        
                                        
                                    0.000225
                                      1.1
                                        
                                        
 Females 13-49 years old
                                        
                                        
                                    0.000206
                                      1.0
                                        
                                        
 [1]	Highest exposure identified in bold.
 
6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are no registered residential uses for benalaxyl-M.

7.0 Aggregate Exposure/Risk Characterization

As stated previously, there are no existing or proposed US registrations of benalaxyl-M and the only route of exposure is via dietary ingestion from imported grape and tomato commodities.  Therefore, aggregate exposure and risk estimates are equivalent to the dietary exposures and risk estimates discussed in Section 5.
	
8.0 Cumulative Exposure/Risk Characterization
      
Unlike other pesticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to benalaxyl-M and any other substances and benalaxyl-M does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assumed that benalaxyl-M has a common mechanism of toxicity with other substances. For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statements released by EPA's Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA's website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

An occupational assessment is not relevant for uses on imported commodities.

10.0	References

Dellarco, V. et al. 11/01/2005. Interim Guidance: Thyroid Disrupting Pesticides: Use of Rat Thyroid Data and Application of Uncertainty Factors for RfD Derivation. Prepared and Reviewed by the Hazard Science Policy Council.

OPP Guidance. 10/24/2005. Guidance for Thyroid Assays in Pregnant Animals, Fetuses and Postnatal Animals, and Adult Animals. Office of Pesticide Programs, Health Effects Division, Washington, DC. Cited in Dellarco, V. et al 2005

Benalaxyl-M.  Report of the Residues of Concern Knowledgebase Subcommittee (ROCKS).   -  E. Holman, D421423, 09/09/14.

Benalaxyl-M.  Report of the Cancer Assessment Review Committee.  J. Rowland and K. Middleton, TXR 0057035, 12/02/14.

Benalaxyl-M.  Quantitative Risk Assessment Based on Rat and Mouse Dietary Studies.  L. Brunsman, TXR 0057093, 12/04/14.

Residue Chemistry Assessment  -  W. Donovan, D416583, 12/02/14.

Dietary Exposure Assessment  -  W. Donovan, D416584, 01/13/15.



Appendix A.  Toxicology Profile and Executive Summaries

A.1	Toxicology Data Requirements
The requirements (40 CFR 158.340) for food (import tolerance) for benalaxyl-M are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.  
 
                                     Study
                                   Technical

                                   Required
                                   Satisfied
870.1100    Acute Oral Toxicity	
870.1200    Acute Dermal Toxicity	
870.1300    Acute Inhalation Toxicity	
870.2400    Primary Eye Irritation	
870.2500    Primary Dermal Irritation	
870.2600    Dermal Sensitization	
                                      yes
                                      no
                                      no
                                      no
                                      no
                                      no
                                      yes
                                      ---
                                      ---
                                      ---
                                      ---
                                      ---
870.3100    Oral Subchronic (rodent)	
870.3150    Oral Subchronic (nonrodent)	
870.3200    21-Day Dermal	
870.3250    90-Day Dermal	
870.3465    90-Day Inhalation	
                                      yes
                                      no
                                      no
                                      no
                                      no
                                      yes
                                      yes
                                      ---
                                      ---
                                      ---
870.3700a  Developmental Toxicity (rodent)	
870.3700b  Developmental Toxicity (nonrodent)	
870.3800    Reproduction	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.4100a  Chronic Toxicity (rodent)	
870.4100b  Chronic Toxicity (nonrodent)	
870.4200a  Oncogenicity (rat)	
870.4200b  Oncogenicity (mouse)	
870.4300    Chronic/Oncogenicity	
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
                                      yes
870.5100    Mutagenicity -- Gene Mutation - bacterial	
870.5300    Mutagenicity -- Gene Mutation - mammalian	
870.5375    Mutagenicity -- Structural Chromosomal Aberrations	
870.5xxx    Mutagenicity -- Other Genotoxic Effects	
                                      yes
                                      yes
                                      yes
                                      no
                                      yes
                                      yes
                                      yes
                                      ---
870.6100a  Acute Delayed Neurotoxicity (hen)	
870.6100b  90-Day Neurotoxicity (hen)	
870.6200a  Acute Neurotoxicity Screening Battery (rat)	
870.6200b  90-Day Neurotoxicity Screening Battery (rat)	
870.6300    Develop. Neurotoxicity	
                                      no
                                      no
                                      yes
                                      yes
                                      no
                                      ---
                                      ---
                                      yes
                                      yes
                                      ---
870.7485    General Metabolism	
870.7600    Dermal Penetration	
870.7800    Immunotoxicity	
                                      yes
                                      no
                                      yes
                                      yes
                                      ---
                                      yes

A.2		Toxicity Profiles 

A.2.1 	Acute Toxicity


Table A.2.1	Acute Toxicity Profile  -  Benalaxyl-M (technical)
Guideline No.
Study Type
MRID(s)
                                    Results
                               Toxicity Category
870.1100
Acute oral [rat]
                                   49040619
LD50 = >2000 mg/kg
No deaths or clinical signs
                                      III
870.1200
Acute dermal [rat]
                                   49040620
LD50 =  >2000 mg/kg
                                      III
870.1300
Acute inhalation [rat]
                                   49040621
                                   49040622
LC50 =  mg/L
Aerosol could not be generated (waiver granted)
                                       -
870.2400
Acute eye irritation [rabbit]
                                   49040623
No corneal opacity or iritis; + score of 2 for conjunctival redness at 24 hrs; no + scores at 48 hrs; all eyes free of irritation at 72 hrs
                                      III
870.2500
Acute dermal irritation [rabbit]
                                   49040624
Not an irritant
                                      IV
870.2600
Skin sensitization [Guinea pig]
                                   49040625
Not a sensitizer
                                       -


A.2.2	Subchronic, Chronic, Other Toxicity


Table A.2.2	Subchronic, Chronic and Other Toxicity Profile  -  Benalaxyl-M
                           Guideline No./ Study Type
                    MRID No. (year)/ Classification /Doses
                                    Results
870.3100
90-Day Oral Toxicity (Crl:CD BR rat)
49040627  (2000)
Acceptable/guideline 
0, 80, 400, 2000, or 10000 ppm (diet)
Males: 0, 6.2, 30.1, 154, or 861 mg/kg/day 
Females: 0, 6.7, 32.8, 167, or 892 mg/kg/day
NOAEL = 2000 ppm (males 154 mg/kg/day; females 167 mg/kg/day). 
LOAEL = 10000 ppm (males 861/females 892 mg/kg/day), based on elevated GGT values in both sexes, increased cholesterol and phospholipids in both sexes,  increased liver weights in both sexes, increased thymus weight in females, increased incidence of hepatocellular hypertrophy in both sexes, and increased incidence of  thyroid follicular cell hypertrophy in both sexes.
Findings at 2000 ppm are considered to be minor [increased GGT (both sexes); increased phospholipids (males), adaptive hepatocellular hypertrophy (both sexes), and not biologically adverse due to lack of severity (thyroid follicular cell hypertrophy]
870.3100
90-Day Oral Toxicity  (CD-1 mouse)
49040628  (2005)
Acceptable/guideline 
0, 600, 2000, or 7000 ppm (diet)
Males: 0, 93.2, 296.6, or 1053.8 mg/kg/day 
Females: 0, 111.8, 348.1, or 1253.3 mg/kg/day
Systemic NOAEL = 2000 ppm (296.6 mg/kg/day in males and 348.1 mg/kg/day in females).
Systemic LOAEL =  7000 ppm (1054 mg/kg/day in males and 1253 mg/kg/day in females), based on increased liver weights, increased incidence and severity of periportal hepatocellular hypertrophy in females, increased glucose in the urine in both sexes, focal necrosis in the liver in males, and depleted glycogen in the liver in females.
870.3150
90-Day Oral Toxicity  (Beagle dog)
49040629 (2000)
Acceptable/guideline 
0, 150, 450, 900, or 3000 ppm (diet)
Males: 0, 5.6, 16, 32, or 112 mg/kg bw/day 
Females: 0, 5.85, 17, 35, or 110 mg/kg bw/day 
Systemic NOAEL = 900 ppm (32 mg/kg/day in males and 35 mg/kg/day in females). 
Systemic LOAEL = 3000 ppm (112 mg/kg/day in males and 110 mg/kg/day in females), based on elevated alkaline phosphatase activity in both sexes, elevated triglyceride levels in males, increased liver weight in both sexes, and an increased incidence/ severity of hepatocyte hypertrophy in the centrilobular, midzonal, and periportal areas in males and females.
870.3200	
28-Day Dermal Toxicity (Wistar rat)
49040631 (2013)
Acceptable/guideline
0, 62.5, 250, or 1000 mg/kg/day 
6 hours/day, 7 days/week for 4 weeks 
NOAEL = 1000 mg/kg/day
LOAEL = not identified
Endpoints of concern (liver, thyroid) were assessed; no developmental, neurotoxicity, immunotoxicity, or reproductive concern.
870.3465
28/90-Day Inhalation Toxicity
49066308
Waiver request
49040621/49040622
See MRID49040621-49040622 (acute inhalation)
870.1300 waived. Aerosol could not be generated.
870.3700a
Prenatal developmental  (Crl: CD (SD) BR rat)
49049637 (2001)
Acceptable/non-guideline
0, 10, 50, or 250 mg/kg/day (dose volume 10 mL/kg/day)
(0.5% methylcellulose aqueous solution)
GD 6-15
Maternal NOAEL = 250  mg/kg/day (HDT)
Maternal LOAEL = not identified

Developmental NOAEL =250 mg/kg/day
Developmental LOAEL = not identified
Non-guideline since it did not test to limit dose and no adverse effect was observed in the maternal animals.
A new study is not required since an endpoint more sensitive than the one used for risk assessment would not be identified by a study at dose levels >250 mg/kg/day 
870.3700b
Prenatal developmental in (Himalayan rabbit)
49040638 (2003)
Acceptable/guideline
0, 50, 250, or 1000 mg/kg/day (0.5% methylcellulose)
GD 6-27
(dose volume 4 mL/kg/day)
Maternal NOAEL = 1000 mg/kg/day (HDT)
Maternal LOAEL = not identified

Developmental NOAEL = 1000 mg/kg/day
Developmental LOAEL = not identified
870.3800
Reproduction and Fertility Effects (Han Wistar rats)
49040639 (2008)
Acceptable/guideline
0, 120/80, 360/240, 1080/725, or 3240/2170 ppm 
Males: 0, 7.9, 23.5, 71.1, or 221.3 mg/kg/day
Females 0, 8.9, 26.7, 77.5, or 241.8 mg/kg/day
70-day pre-mating
Parental NOAEL = 1080 ppm (males 71/females 78 mg/kg/day)
Parental LOAEL = 3240 ppm (males 221/females 242 mg/kg/day), based on decreased # primordial follicles and corpora lutea in F1 females, decreased uterus weight in F0 females, and a slight increase in hepatocellular hypertrophy in both sexes and generations (males P 16/24, F1 12/24; females P 21/24; F1 23/24),.
Offspring NOAEL = 1080 ppm (males 71/females 78 mg/kg/day)
Offspring LOAEL = 3240 ppm (males 221/females 242 mg/kg/day), based on decreased pup body weight in F2 pups of both sexes and increased incidence of hepatocellular hypertrophy in F2 pups of both sexes.  
Reproductive NOAEL = 1080 ppm (males 71/females 78 mg/kg/day)
Reproductive LOAEL = 3240 ppm (males 221/ females 242 mg/kg/day), based on a decrease in ovarian follicle counts (primordial follicles and corpora lutea) in F1 females.
outside historical control and concurrent control values were generally low compared to the historical control values.
870.4100a
Chronic Toxicity
(Sprague Dawley CD Crl:CD(SD)BR rat)
49040634 (2013)

 See under 870.4200a
870.4100b		
Chronic toxicity 
(Beagle dog)
49040630 (2006)
Acceptable/guideline
0,  10/300[A], 30, or 100 mg/kg/day (52 weeks)
capsule  
[A]low dose ↑to 300 mkg (week 27)
NOAEL = not selected.
LOAEL = not selected.

870.4200a
Carcinogenicity 
(Sprague Dawley CD Crl:CD(SD)BR rat)
49040634 (2013)
Acceptable/guideline
0, 60, 390, or 2535 ppm
Main study: Males 0, 2.4, 15.66, 104.27 mg/kg/day
Females 0, 3.13, 20.04, or 135.28 mg/kg/day
52-week chronic study: Males: 0, 2.92, 19, 126 mg/kg/day
Females: 0, 3.82, 24.6, or 167 mg/kg/day
NOAEL = 390 ppm (males 15.7 mg/kg/day/females 20 mg/kg/day.
LOAEL = 2535 ppm (males 104/females 135 mg/kg/day), based on increase in GGT in males, increased cholesterol in both sexes, increased triglycerides in males, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females. 

evidence of carcinogenicity: increase in liver tumors (adenomas) at the high dose in both sexes; increase in thyroid follicular cell adenomas and follicular cell adenomas/carcinomas combined in high dose females (CARC 8/13/2014; Likely/Q*1).
870.4200b
Carcinogenicity (78 weeks)
(CD-1 mouse)
49040635 (2013)
Acceptable/guideline
0, 200, 1200, or 7000 ppm
Males: 0, 24.86, 156.1, or 962 mg/kg/day
Females: 0, 31.36, 196.7, or 1180 mg/kg/day
Systemic NOAEL = 1200 ppm (156 mg/kg bw/day for males and 197 mg/kg/day for females). 
Systemic LOAEL = 7000 ppm (962 mg/kg bw/day for males and 1180 mg/kg bw/day for females), based on increased liver weight in both sexes and an increased incidence of liver lesions (diffuse hepatocellular hypertrophy in both sexes, single cell necrosis in both sexes, focal/ multifocal necrosis in males, and eosinophilic foci in males).  

evidence of carcinogenicity: increased incidence of liver adenomas in males and liver adenomas/ carcinomas in both sexes at HDT; (CARC 8/13/2014; Likely/Q*1).
870.4300
Combined Chronic Toxicity/Carcinogenicity
(Sprague Dawley CD Crl:CD(SD)BR rat) 
49040634 (2013)

See under 870.4200a
870.5100
Bacterial reverse gene mutation (Ames Assay)
49040640 (2002)
Acceptable/guideline
5- 5000 μg/plate +/  - S9 (plate incorporation)
5- 1500 μg/plate +/  - S9 (preincubation) 
Negative up to the limit dose; compound precipitation at >=1500 μg/plate
870.5300
In vitro mammalian forward cell gene mutation (mouse lymphoma cells)
49040642 (1999)
Acceptable/guideline
5- 75 μg/mL   - S9 
15- 100 μg/mL +S9  
Negative up to cytotoxic concentration 
870.5375
In vitro mammalian chromosome aberration test
[Chinese Hamster Ovary (CHO cells)]
49040641 (1999)
Acceptable/guideline
Assay 1 5- 3000 μg/mL+/  - S9 (3 hrs.)
Assay 2 5- 150 μg/mL  - S9 (18 hrs.)
15- 150 μg/mL +S9 (3 hrs.) 
Negative up to cytotoxic & insoluble concentration
870.5395
Mammalian erythrocyte micronucleus assay (mouse)
49040643 (2000)
Acceptable/guideline
0, 100, 200, 400 mg/kg M
0, 75, 150, 300 mg/kg F
Single i.p. injections
Negative up to doses (>=300 mg/kg) causing overt toxicity in preliminary study (deaths and/or piloerection)
870.6200a
Acute Neurotoxicity Screening Battery
(CRL Wistar rat)
49040626 (2013)
Acceptable/guideline
0, 500, 1000, or 2000 mg/kg
10 mL/kg dose volume
0.5% aqueous CMC 
(peak effect time 30 minutes)
NOAEL =2000  mg/kg
LOAEL = not identified.

870.6200b
Subchronic Neurotoxicity Screening Battery
(CRL Wistar rat)
49143501 (2013)
Acceptable/guideline
0, 1000, 3000, or 10000 ppm
Males: 0, 67, 206, or 784 mg/kg/day
Females: 0,  78, 325, or 1139 mg/kg/day
Systemic NOAEL = 3000 ppm (males 206/females 325 mg/kg/day.
Systemic LOAEL =  10000 ppm (males 784/females 1139 mg/kg/day, based on increased serum GGT activity in both sexes and increased absolute and relative liver weights.  

No neurobehavioral or neuropathological effects were observed in either sex.
870.6300
Developmental Neurotoxicity
No study available or required.
870.7485
Metabolism and Pharmacokinetics
(Sprague-Dawley rat)
49040644
Acceptable/guideline

Radiolabel readily absorbed and excreted mainly via the feces (≈85%) and to a smaller extent via urine (≈8%). Biliary excretion explains high elimination of absorbed radiolabel via fecal route. By 72 hours, all tissue concentration were <1%; highest tissue concentrations in intestinal wall (8%), liver (8%), and stomach (6%); PK profile characterized by a peak level between 10 minutes and 1 hour post dose; elimination half-life of ≈18 hours; IR6141 was extensively metabolized, principally by oxidation of methyl group on aniline ring to the hydroxymethyl and finally to the carboxylic acid; minor pathways were hydroxylation of phenyl ring and hydrolysis of carboxymethyl group. shows urinary metabolic pathway identical to fecal metabolic pathway.
870.7485
Metabolism and Pharmacokinetics
(Sprague-Dawley rat)
49040645 (1999)
Acceptable/guideline


870.7485
Metabolism and Pharmacokinetics
(Sprague-Dawley rat)
4904046 (2008)
Acceptable/guideline

Total absorption similar to that in studies above, although females excreted more in urine than males (Females 17% vs males 8%); however, biliary excretion was substantial in both sexes (males 81%; females 62%), indicating most of radiolabel was absorbed and then excreted via the bile into feces; estimated total absorption was similar (males 89%; females 82%); recovery 97%-99%. Highest tissue concentrations in liver and GI tract in both sexes; Tmax was 1.8 hours post dose; terminal half-life of 62.4 hours; data suggest that absorption and elimination were not saturated since there was not a 10-fold increase in Cmax and tmax between the 10 mkd and 100 mkd doses.
870.7485
Metabolism and Pharmacokinetics
(Sprague-Dawley rat)
49040647 (2008)
Acceptable/guideline


870.7600
Dermal penetration 
No study available
870.7800
Immunotoxicity
(Wistar rat, male)
49040648 (2013)
Acceptable/guideline
0, 1000, 3000, or 10000 ppm (28 days) 
0, 80.5, 250.4, or 924.6 mg/kg/day
Systemic NOAEL = 1000 ppm (80.5 mg/kg/day)
Systemic LOAEL = 3000 ppm (250.4 mg/kg/day), based on increased absolute and relative-to-body liver weights.

Immunotoxicity NOAEL = 10000 ppm (924.6 mg/kg/day)
Immunotoxicity LOAEL = not identified.
Non-guideline (mechanistic)
(Crl:CD-1(Icr) BR mouse)
49040636 (2013)
Acceptable/non-guideline
0, 200, 1200, or 7000 ppm (diet; 3 or 14 day)
0, 30.9, 184.4, or 1094.1 mg/kg/day (males)
Assessment of cell proliferation not adequate; microsomal liver enzymes EROD, MROD, and LA12OH activities: slightly increased at 1200 and 7000 ppm; PROD, 6β-OH, and LA11OH activities: moderately increased at 7000 ppm; PROD activity: moderately increased at 1200 ppm; and 6β-OH and LA11OH activities: slightly increased at 1200 ppm. Expression of the Cyp1A-1, Cyp1A-2, Cyp2B10, and Cyp3A11 genes were increased at 7000 ppm; expression of Cyp1A-1, CypB10, and Cyp3A11 were increased at 1200 ppm; expression of Cyp2B10 was increased 45.8- and 195.7-fold at 1200 and 7000 ppm, respectively; expression of Cyp3A11 was increased 15.58-fold. Examination of the parameters of oxidative stress, reduced glutathione (GSH) and oxidized glutathione (GSSH) in liver, and thiobarbituric acid reactive substance (TBARS) in liver and serum, showed no effect of treatment with IR6141 for 14 days.


A.3	Hazard Identification & Endpoint Selection 

A.3.1	Acute Reference Dose (aRfD)  -  All Populations

An endpoint for an aRfD for either females age 13-49 or the general population was not identified in the toxicity database, including acute neurotoxicity or developmental toxicity studies. 

A.3.2.  Chronic Reference Dose (cRfD)

Study Selected: 2-year carcinogenicity study in rats
MRID No.:  
Executive Summary:  See Appendix A.4.5, Guideline § 870.4200.a 
Dose and Endpoint for Risk Assessment: NOAEL = 20 mg/kg/day based 
Comments about Study/Endpoint/Uncertainty Factors:   This study was the most sensitive endpoint and is the appropriate duration and route of exposure.  The LOAEL of 135 mg/kg/day is based on an increase in GGT in males, increased triglycerides in males, increased cholesterol in both sexes, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females. A 10X interspecies uncertainty factor to extrapolate from animals to humans; a 10X uncertainty factor to account for potential variation in sensitivity among the human population; and a 10X database uncertainty factor to account for the lack of a critical study (comparative thyroid assay (CTA)) were used. For the POD for the chronic dietary risk assessment, a total uncertainty factor of 1000X was applied (10X for interspecies variability, 10X for intraspecies variability, and 10X for database uncertainty to account for the lack of a CTA study). ​When the basis of the POD is based on thyroid effects, the interspecies extrapolation UF is typically reduced to 3X due to the pharmacodynamic differences between rats and humans pertaining to thyroid perturbations.  However, in the case of benalaxyl-M, the full 10X factor was retained for interspecies UF since effects not related to thyroid function disruption ​are also the basis of the POD. 


A.3.3	Incidental Oral Exposure, Dermal Exposure, Inhalation Exposure 

Endpoints for these exposure routes are not needed to assess non-domestic uses.  

A.4	Executive Summaries 

A.4.1	Subchronic Toxicity

	870.3100	90-Day Oral Toxicity - Rat

In a subchronic oral toxicity study (MRID 49040627), five groups of Crl:CD BR rats (10/sex/group; ~5 weeks of age) were administered 0, 80, 400, 2000, or 10,000 ppm of benalaxyl-M  [97.27% (Batch No. FCT/T/156-99 (20398/15)] in the diet and observed for 90 days. The respective dose levels based on food consumption were 0, 6.2, 30.1, 154, and 861 mg/kg bw/day for males and 0, 6.7, 32.8, 167, and 892 mg/kg bw/day for females. The animals in each test group were subjected to neurobehavioral testing, clinical pathology, and gross and microscopic examinations.

There was no adverse effect on survival for either sex, and no clinical signs attributed to the test substance were observed at any dietary concentration. Both sexes showed body weight deficits of 12%-14% throughout the 13-week exposure period at 10000 ppm, which may be attributed to an initial aversion to the diet. Decreased food consumption was observed in both sexes during the first week of the study but was comparable to the control thereafter 

No statistically significant differences were observed between any treatment group and the controls during the functional operational battery (FOB) and locomotor testing. The minor changes in hematology parameters were not considered adverse. The most notable changes in the clinical chemistry data were dose-related elevations in GGT activity in both sexes at the two highest dietary concentrations, elevations in cholesterol at the highest dietary concentration in both sexes, and elevations in phospholipids at the mid-high and high concentration in males and at the high concentration in females. There were no treatment-related or toxicologically significant findings in the urinalysis or gross examination findings.

Males and females in the high dose group had statistically higher absolute and relative liver weights compared to controls. Females in the mid-high dose group had statistically higher relative liver weights. Males in the high dose group had statistically higher relative brain, thyroid, testes, and epididymides weights compared to controls, which were likely related to the lower body weight at this dietary concentration. Females in the high dose group had statistically higher absolute and relative thymus weights, and lower absolute and relative heart weights. 

Generalized hepatocyte hypertrophy was observed in all males and females in the high dose group and in 8/10 males and 2/10 females in the mid-high dose group. Increased thyroid follicular cell hypertrophy was observed in five males and three females at the high dose and in three males at the mid-high dose compared to none in the control, low- and low-mid dose groups. There was an increase in the incidence and prominence of vacuolated cells in the pituitary pars distalis in six males at the high dose. There was an increased incidence of kidney cortico-medullary mineralization in females at the high dose (10/10) compared to the control (6/10) and other dose groups (5/10).

The NOAEL was 2000 ppm (males 154 mg/kg/day; females 167 mg/kg/day), based on elevated GGT values in both sexes, increased cholesterol and phospholipids in both sexes, increased liver weight in both sexes, increased thymus weight in females, increased incidence of hepatocellular hypertrophy (both sexes), and increased thyroid follicular cell hypertrophy (in both sexes) at the LOAEL of 10000 ppm (males 861 mg/kg/day; females 892 mg/kg/day.

This subchronic neurotoxicity study is classified as Acceptable/Guideline and satisfies the guideline requirement for a 13-week toxicity study in rats (OCSPP 870.3100; OECD 408).  

	870.3100	90-Day Oral Toxicity - Mouse

In a subchronic oral toxicity study (MRID 49040628), four groups of CD-1 mice (10/sex/group; 6 weeks of age) were administered 0, 600, 2000, or 7,000 ppm of benalaxyl-M [96.33% (Batch No. G018/04)] in the diet and observed for 90 days. The respective dose levels based on food consumption were 0, 93.2, 296.6, and 1053.8 mg/kg bw/day for males and 0, 111.8, 348.1, and 1253.3 mg/kg bw/day for females. The animals in each test group were subjected to clinical pathology testing, and gross and microscopic examinations.

All animals survived to scheduled sacrifice. No treatment-related clinical signs or body weight effects were observed. The most notable statistically significant clinical chemistry finding was a test material-related elevation in total bilirubin (p<0.01) in females at the high dietary concentration. Urinalysis data showed a treatment-related elevation in glucose in males and females at the mid and high dose levels. Males and females in the high dose group had statistically higher absolute and relative liver weights compared to controls (p<=0.01). Females in the mid-high dose group had statistically higher relative liver weights (p<=0.01). Minimal to slight periportal hepatocellular hypertrophy (without signs of necrosis) was observed in both sexes in all treated dose groups (none in controls), but only the females showed a dose-response. The average grade of hepatocellular glycogen deposits decreased in both sexes in all treated dose groups, but only the females showed a dose-response. 

The LOAEL in mice was 7000 ppm (1054 mg/kg/day in males and 1253 mg/kg/day in females), based on increased liver weights, increased incidence and severity of periportal hepatocellular hypertrophy in females, increased glucose in the urine in both sexes, focal necrosis in the liver in males, and depleted glycogen in the liver in females. The NOAEL is 2000 ppm (296.6 mg/kg/day in males and 348.1 mg/kg/day in females).

This subchronic oral toxicity study is classified as Acceptable/Guideline and satisfies the guideline requirement for a 13-week toxicity study in a rodent (OCSPP 870.3100; OECD 408).

	870.3150	90-Day Oral Toxicity  -  Dog

In a 13-week oral toxicity study (MRID 49040629), five groups of beagle dogs (4/sex/group; 23-36 weeks of age) were administered 0, 150, 450, 900, or 3000 ppm of benalaxyl-M [97.27% (Batch No. FCT/T/156-99 (20398/15))] via the diet. The respective dose levels based on food consumption were 0, 5.58, 16.24, 32.02, and 112 mg/kg bw/day for males and 0, 5.85, 17.34, 34.97, and 110 mg/kg bw/day for females. The animals in each test group were subjected to clinical pathology, and gross and microscopic examinations.

All animals survived to the end of the study. No clinical signs attributed to the test substance were observed at any dietary concentration. Body weights were slightly lower at the 3000 ppm dose level in both sexes at termination (males ↓4%/females ↓8%) compared to the control groups. Food consumption was not adversely affected in either sex. The most notable changes in clinical chemistry data were statistically elevated alkaline phosphatase activity (ALK) in both sexes (males ↑353%-494%/females ↑287%-374%) at the high dietary concentration, with the magnitude of the effect increasing with time. ALK was also elevated at the mid-high concentration in males (↑110%-141%) at both the 6- and 13-week sampling intervals and in females (↑85%) at the mid-high concentration at the 13-week interval. The historical control range for ALK activity was exceeded in males and females at the high dietary concentration. Triglycerides were significantly elevated at the high dose level in males (↑70%-96%) at 6 and 13 weeks and elevated in high-dose females (↑40%-68%) at the same time points but statistical significance was not attained. There were no treatment-related or toxicologically significant findings in the hematology or urinalysis data. Enlarged liver was observed in 2/4 males and 1/4 females at the high dietary concentration during gross examinations. Males and females in the high dose group had statistically higher absolute (↑19%) and relative (↑23%-29%) liver weights compared to controls (p<=0.01). Hepatocyte hypertrophy in centrilobular and midzonal areas were observed in all males and females in the mid-high and high dose groups. The change extended to the periportal area in two males and two females at the high dose and in one female from the mid-high dose group. No signs of liver necrosis were observed.
 
The no-observed-adverse effect level (NOAEL) for benalaxyl-M in beagle dogs was 900 ppm (32 mg/kg/day in males and 35 mg/kg/day in females). A lowest-observed-adverse effect level (LOAEL) for IR 6141 in beagle dogs was 3000 ppm (112 mg/kg/day in males and 110 mg/kg/day in females), based on elevated alkaline phosphatase activity in both sexes, elevated triglyceride levels in males, increased liver weight in both sexes, and an increased incidence/severity of hepatocyte hypertrophy in the centrilobular, midzonal, and periportal areas in males and females.

This subchronic toxicity study in the dog is classified as Acceptable/Guideline and satisfies the guideline requirement for a 13-week toxicity study in non-rodents (OCSPP 870.3150; OECD 409).  

	870.3200	21/28-Day Dermal Toxicity  -  Rat

In a 28-day dermal toxicity study (MRID 49040631), benalaxyl-M technical (96.53% a.i., Lot No. P/10/106) was applied to the shaved skin of 10 Wistar rats/sex/group at concentrations of 0, 62.5, 250, or 1000 mg/kg bw/day, 6 hours/day for 28 consecutive days.  Additional groups of 10 rats/sex were treated for the same time period at concentrations of 0 or 1000 mg/kg bw/day, followed by a two-week recovery period.  

No dermal irritation was observed during treatment, and no treatment-related moribundity or mortality were found.  No neurological effects were observed.  There were no treatment-related effects on body weight, food consumption, ophthalmoscopic parameters, the estrus cycle, or effects on hematology, clinical chemistry, or urinalysis parameters.  There were no treatment-related effects noted at necropsy on absolute or relative to body or brain organ weights, or microscopic treatment-related effects.  
   
The dermal toxicity NOAEL for male and female Wistar rats was 1000 mg/kg bw/day.  A LOAEL was not determined.  

This 28-day dermal toxicity study in the rat is Acceptable/Guideline and satisfies the guideline requirement for a 28-day dermal toxicity study (OPPTS 870.3200; OECD 410) in rats.

	870.3465	90-Day Inhalation  -  Rat

An inhalation study is not required for an import tolerance. 

A.4.2	Prenatal Developmental Toxicity

	870.3700a Prenatal Developmental Toxicity Study - Rat
  
In a developmental toxicity study (MRID 49040637), benalaxyl-M [>99% a.i.; batch # FCF/T/173-00 (ex 20493/57/B lotto II)] was administered to 25 female Crl:CD (SD) BR rats/dose in 0.5% aqueous methylcellulose by oral gavage at dose levels of 0, 10, 50, or 250 mg/kg bw/day on gestation days (GD) 6 through 15 (dose volume of 10 mL/kg bw/day).  Dams were sacrificed and necropsied on GD 20.  Pregnant uterus weight, numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded.  Individual fetal and placental weights were recorded.  All fetuses were sexed and subjected to gross external evaluations.  Approximately half of the fetuses per litter underwent skeletal evaluations while the other half underwent visceral examinations.

There was no evidence of maternal toxicity.  No clinical signs, behavioral changes, body weight effects, or deaths were observed in dams during the study.  An initial reduction in food consumption (GD 8-10) was observed in the dams at 250 mg/kg bw/day, but no differences were noted for absolute body weight or corrected body weight gain. No gross pathological effects were noted at sacrifice, and no treatment-related effects on cesarean parameters were observed at any dose level.    

The maternal lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in rats was not determined.  The maternal NOAEL was greater than or equal to 250 mg/kg bw/day.  

There were no treatment-related effects on fetal sex ratio, fetal weight, post-implantation loss, resorptions per litter, or live litter size.  Although the 10 and 250 mg/kg bw/day groups had increases in early resorptions, there was no dose response.  Further, a slightly lower mean number of live fetuses per litter was noted in the 250 mg/kg bw/day group but was not considered treatment-related due to small magnitude (<1 fetus), a corresponding lower number of mean implantations per dam, and the absence of a correlated increase in post-implantation loss.  One to two malformed fetuses were present in all groups, including the control group.  Therefore, no developmental effects were observed at doses up to 250 mg/kg bw/day.  

The developmental LOAEL for benalaxyl-M in rats was not determined, and the developmental NOAEL was greater than or equal to 250 mg/kg bw/day.

This developmental toxicity study in the rat is classified Acceptable/Non-Guideline, but it does satisfy the guideline requirement for a developmental toxicity study (OSCPP 870.3700; OECD 414) in the rat. It is non-guideline because no significant developmental or maternal toxicity occurred at the highest dose level tested.  However, a new study is not required at this time since a study at higher dose levels would not identify a more sensitive endpoint for risk assessment than the endpoint selected for risk assessment. 

	870.3700b Prenatal Developmental Toxicity Study - Rabbit

In a developmental toxicity study (MRID 49040638), benalaxyl-M [97.14% a.i., batch # FCF/T/173-00 (ex 20493/57/B lotto II)] was administered to 24 mated female Himalayan rabbits/dose in 0.5% aqueous methylcellulose by oral gavage (dose volume of 4 mL/kg bw/day) at dose levels of 0, 50, 250, or 1000 mg/kg bw/day on gestation days (GD) 6 through 27, inclusive.  Dams were sacrificed on day 28 of gestation and underwent gross macroscopic examination.  All fetuses were weighed, killed, sexed, examined for gross external abnormalities, and examined for both soft tissue and skeletal alterations.  Heads from one-half of the fetuses were subjected to evaluation of the internal structures, including the eyes, brain, nasal passages, and tongue.

Treatment with benalaxyl-M did not cause any maternal deaths, clinical signs, or effects on food consumption or body weight/body weight gain at doses up to and including 1000 mg/kg bw/day.  

The maternal LOAEL for benalaxyl-M in rabbits was not determined.  The maternal NOAEL was greater than or equal to 1000 mg/kg bw/day, the highest dose tested.

No treatment-related fetal deaths or resorptions were noted, and fetal sex ratio was unaffected by treatment.  At 1000 mg/kg bw/day, there was a slight decrease (6%) in fetal body weight, correlated with a slight delay in the stage of development in this dose group as indicated by increased percentages of fetuses with incomplete chondrification of cartilaginous sternebra(e) 2, 3, and/or 4 compared to controls.  However, these findings were significant only when analyzed on an individual fetal basis, while the litter should be considered the statistical unit of measure.  Skeletal abnormalities were limited to common sternebral abnormalities (including fused and abnormally shaped sternebrae) and abnormalities of cartilaginous sternebrae and/or costal cartilages (including bipartite and/or abnormally shaped cartilaginous sternebrae, fused or bifurcated costal cartilages).  The combined incidence of these abnormalities was 2 (2), 3 (2), 3 (3), and 6 (6) fetuses (litters) in the 0, 50, 250, and 1000 mg/kg bw/day groups, respectively; the slight increase in the high dose group was not statistically significant or considered adverse.  External and visceral abnormalities were incidental and not related to treatment and included one fetus in the 50 mg/kg bw/day group with an encephalocele and an open skull and two fetuses in the 1000 mg/kg bw/day group: one with flexure of the right forelimb and one with minimal bilateral dilation of the lateral brain ventricles.

The fetal LOAEL for benalaxyl-M in rabbits was not determined; and the fetal NOAEL was greater than or equal to 1000 mg/kg bw/day.  

The developmental toxicity study in the rabbit is classified Acceptable/Guideline and satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in rabbits.  
  
A.4.3	Reproductive Toxicity

	870.3800 Reproduction and Fertility Effects - Rat
  
In a two-generation reproduction study (MRID 49040639), benalaxyl-M (Benalaxyl-M; 96.33% a.i.; Batch No. G 018/04) was administered to groups of 24 HanRcc:WIST (SPF) rats/sex/dose in the diet at concentrations of 0, 120 [80], 360 [240], 1080 [725], or 3240 [2170] ppm, with lowered concentrations [those indicated in brackets] administered to lactating females and weaned pups to maintain a consistent intake of the test material in terms of mg/kg bw/day.  Premating doses were respectively 0, 7.9, 23.5, 71.1, or 221.3 mg/kg bw/day for F0 males, 0, 8.9, 26.7, 77.5, or 241.8 mg/kg bw/day for F0 females, 0, 8.9, 27.1, 81.0, or 257.0 mg/kg bw/day for F1 males, and 0, 9.9, 29.5, 85.8, or 280.2 mg/kg bw/day for F1 females.  Except for one high-dose F0 "replacement" male, the F0 and F1 parental animals were administered the treated or control diets for at least 70 days (for F0) or 90 days (for F1) prior to mating, throughout mating, gestation, and lactation, and until necropsy after weaning of litters, and one litter was produced by each generation.  The replacement animal was treated for only 55 days prior to mating.  Additional groups of 24 F1 pups/sex/dose were selected for post-weaning functional evaluations, including a functional observational battery/modified Irwin screen (FOB/modified Irwin) and locomotor activity measurement.

No significant treatment-related effects on mortality, clinical signs, body weight, or food consumption were noted in either sex or generation.  Treatment-related increased incidences of hepatocellular hypertrophy (mainly centrilobular) were observed in both sexes and generations at the high and high-mid doses and in low-mid-dose F0 males, F1 males, and F1 females.  Other findings included increased liver weights in high-dose males of both generations (absolute and relative-to-brain weights 20% and 16-17% greater than controls for F0 and F1 high-dose males, respectively; p<0.01), and increased incidence of gross enlargement of the liver in high-dose F1 females (9/24 high-dose F1 females vs. 0 controls; p<0.01). The liver is a target organ following benalaxyl-M exposure, and these liver findings are consistent with other benalaxyl-M rat studies. Treatment-related decreased spleen weights were seen in high-dose females of both generations, with the absolute and relative (to brain) spleen weights 14% lower than controls for F1 and 16%-17% lower than controls for F2.  Since hematology parameters and spleen histopathology were not examined as part of the current study, the toxicological significance of the decreased spleen weights is not known. A similar decrease was not observed in the chronic rat study in either sex at comparable dose levels.  No treatment-related effects were observed on fertility or reproductive performance in male or female rats in either generation.  Estrous cycle length, sperm measures, reproductive indices, precoital interval, gestation length, and postimplantation loss were not affected by treatment.  Significantly decreased numbers of primordial follicles and corpora lutea was observed in high-dose F1 females (respectively -26% and -29%, relative to controls); it is unknown whether this effect extended into the lower dose groups because the ovarian follicles were only counted in high-dose and control F1 females. However, the magnitude of the deficit at the high dose suggests that any treatment-related effects at lower dose levels would not be toxicologically significant.

Under the conditions of this study, the parental systemic LOAEL for benalaxyl-M in female rats is 3240/2170 ppm (241.8 mg/kg bw/day), based on decreased numbers of primordial follicles and corpora lutea in the F1 females, decreased uterus weights in F0 females, and a slight increase in the incidence of hepatocellular hypertrophy in both generations. The parental systemic NOAEL in females is 1080/725 ppm (77.5 mg/kg bw/day).  The parental systemic LOAEL in males is 3240 ppm (221.3 mg/kg bw/day), based on increased liver weights and an increased incidence of hepatocellular hypertrophy in both generation. The parental systemic NOAEL in males is 1080 ppm (71.1 mg/kg bw/day).
   
During postnatal days (PNDs) 7-14 and 14-21, the body weight gain of the high-dose F2 offspring of both sexes was significantly decreased (-11% to -13%) with resultant significantly decreased absolute body weights on PND 14 (males: -8%, p<0.05; females: -10%, p<0.01) and PND 21 (males: -10%; females: -11%; p<0.01 for both).  High-dose F1 females had a treatment-related 1.5-day delay in vaginal opening (p<0.05), with the group's mean body weight at completion of vaginal opening comparable to that of the control group.  Decreased absolute and relative (to brain) spleen weights were noted in high-dose males and females of the F1 generation (-16% for both sexes and both parameters) and the F2 generation (males: -21% to -22%; females: -17% to -19%). Since hematology parameters and spleen histopathology were not examined as part of the current study, the toxicological significance of the decreased spleen weights is not known.  There were no effects on pup survival/mortality, litter size from birth to weaning, postimplantation loss, viability indices, sex ratio, or male sexual maturation.  There were no treatment-related clinical observations or gross findings in pups or weanlings. There was an increased incidence of hepatocellular hypertrophy in both sexes of F2 pups at the high dose. Under the conditions of this study, postweaning functional investigations (FOB and locomotor testing) did not reveal any treatment-related effects.  However, due to the absence of positive control data demonstrating competence of the testing laboratory, the lack of provided information concerning the test methods and conditions, and the use of 15-minute subsessions for the locomotor testing, the reviewer does not consider the apparent absence of neurobehavioral effects to be definitive.  Since these parameters are not typically assessed in a reproduction study, and there are acute and subchronic neurotoxicity studies available to assess these parameters, these deficiencies do not interfere with the interpretation of this study.
   
The offspring LOAEL for benalaxyl-M in rats is 221.3 mg/kg bw/day for males and 241.8 mg/kg bw/day for females, based on decreased pup body weight in F2 pups of both sexes and increased incidence of hepatocellular hypertrophy in F2 pups of both sexes.  The offspring NOAEL is 1080/725 ppm (71.1 mg/kg bw/day) for males and 77.5 mg/kg bw/day for females. 

Findings potentially indicative of reproductive toxicity included decreased ovarian follicle counts (primordial follicles and corpora lutea). The decrease in ovarian follicle counts in the high-dose F1 females is considered treatment-related and adverse. The results in the treated group were outside the historical control range, and the concurrent control values were generally low compared to the historical control values. Although ovarian follicle counts were not assessed for the lower dose groups, it was concluded that the magnitude of the findings at the high dose are such that the results at lower doses would not be considered adverse. 

The reproductive toxicity LOAEL for benalaxyl-M is 3240/2170 ppm (241.8 mg/kg bw/day), based on decreased numbers of primordial follicles and corpora lutea in the F1 females. The reproductive toxicity NOAEL is 1080/725 ppm (77.5 mg/kg bw/day).    

This study is Acceptable/Guideline and satisfies the guideline requirement for a two-generation reproductive study [OCSPP 870.3800; OECD 416] in the rat.  Despite deficiencies in the conduct and reporting of this study and the occurrence of only minimal parental toxicity, offspring toxicity and potential reproductive toxicity were seen.  

A.4.4	Chronic Toxicity

	870.4100a (870.4300) Chronic Toxicity  -  Rat
  
  See under 870.4200a

	870.4100b Chronic Toxicity - Dog

In a 52-week oral toxicity study (MRID 49040630), four groups of beagle dogs (4/sex/group; 5.5-7.5 months of age) were administered via capsule 0, 10 (26 weeks)/300 (26 weeks), 30, or 100 mg/kg/day (mg/kg) of benalaxyl-M [96.33% (Batch No. G018/04] for 52 weeks. The 10 mg/kg/day dose was increased to 300 mg/kg beginning week 27 to increase the chance of detecting toxic effects. The animals in each test group were subjected to clinical pathology, and gross and microscopic examinations.

All animals survived to the end of the study. No clinical signs attributed to the test substance were observed at any dose. There were no treatment-related or toxicologically significant findings observed in the body weight data. No treatment-related effects were observed on hematology values, clinical chemistry values, urinalysis values, organ weight data, or during gross examination of tissues. Minimal or slight periportal fat vacuolation in the liver was observed in 3/4 males and 2/4 females from the 10/300 mg/kg/day dose group. All females from the 10/300 mg/kg/day dose group had a minimal diffuse hypertrophy of the thyroid gland. 

The lowest observed-adverse effect level (LOAEL) for benalaxyl-M in beagle dogs was not identified.
   
This chronic toxicity study in the dog is classified as Acceptable/Guideline. 

  
A.4.5	Carcinogenicity

	870.4200a Carcinogenicity Study - rat

In a combined chronic toxicity/carcinogenicity study (MRID 49040634), benalaxyl-M (96.33% a.i., Lot/Batch # G018/04) was administered in the diet to 20 male and 20 female Sprague Dawley CD Crl:CD(SD)BR rats at concentrations of 0, 60, 390, or 2535 ppm (0, 2.92, 19.0, and 126 mg/kg bw/day, respectively, for male rats and 0, 3.82, 24.6, and 167 mg/kg bw/day, respectively, for female rats) for 52 weeks (chronic study) and  groups of 65 male and 65 female rats were administered the same dietary concentrations for 104 weeks (main carcinogenicity study).  The equivalent doses for the main study were 0, 2.40, 15.7, and 104 mg/kg bw/day, respectively, for male rats and 0, 3.13, 20.0, and 135 mg/kg bw/day, respectively, for female rats. 

No treatment-related, adverse effects were observed on mortality, clinical signs, detailed clinical observations, FOB parameters, body weight and body weight gain, hematology, nodules/masses, water consumption, food consumption, or eyes in either sex. Increased γ-glutamyl transferase (GGT) levels were observed in both sexes at 7000 ppm, with males displaying the greater effect. Both sexes showed slightly increased cholesterol values also. Absolute liver weight was not significantly increased (11%-12%) by treatment with the test substance, but the liver/body weight ratio was significantly increased by 10% to 18% in high-dose male and female rats after 52 and 104 weeks of treatment.  The liver/brain weight ratio was not significantly affected by treatment with the test substance.  

Gross and microscopic examination showed treatment-related changes primarily in the liver and thyroid gland.  Gross examination showed accentuated lobulated pattern in the liver of 4/20 high-dose male rats compared with none of the controls.  In the chronic study (52 weeks), treatment-related microscopic lesions in the liver included fatty change and hepatocellular hypertrophy (primarily centrilobular) at the mid- and high-dose level in male rats, hepatocellular hypertrophy at the mid- and high-dose level in female rats, and fatty change and infiltrating inflammatory cells at the high-dose level in female rats.  The incidences of bile duct hyperplasia and peribiliary fibrosis were decreased in high-dose male and female rats compared with that of controls.  The incidence of thyroid follicular cell hypertrophy was significantly increased in male rats in the mid- and high-dose groups and in females in the high-dose group compared with that of controls.  In the main study (104 weeks), the incidences of eosinophilic foci and spongiosis hepatis in the liver of male rats and biliary cysts in females showed statistically significant positive dose-related trends but did not reach statistical significance at any dose; the incidence of eosinophilic foci was significantly increased in females in the high-dose group compared with that of controls.  The incidence of lesions in the thyroid gland was increased only in female rats in the high-dose group.  These included follicular cell hypertrophy, follicular cell hyperplasia, and follicular ectasia. The incidence of ovarian stromal cell hyperplasia was increased in females at the high-dose level.

The lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in rats is 2535 ppm (males 104/females 135 mg/kg bw/day), based on an increase in GGT in males, increased triglycerides in males, increased cholesterol in both sexes, slight increases liver weight in both sexes, increased incidence of hepatocellular hypertrophy in both sexes, increased incidence of thyroid follicular cell hypertrophy in both sexes, increased incidence of thyroid cell hyperplasia in females, increased incidence of thyroid follicular ectasia in females, and an increased incidence of ovarian stromal cell hyperplasia in females.  The no-observed-adverse-effect level (NOAEL) is 390 ppm (males 15.7 mg/kg bw/day and females 20 mg/kg bw/day).

At the doses tested, the incidences of thyroid follicular cell adenomas and follicular cell adenomas/carcinomas combined were marginally increased in females at the high dose compared with that of controls.  Additionally, there was an increased incidence of hepatocellular adenomas in both sexes at the high dose. 

This chronic/carcinogenicity study in the rat is Acceptable/Guideline and satisfies the guideline requirement for a chronic/carcinogenicity study (OCSPP 870.4300; OECD 453) in rats.  

	870.4200b Carcinogenicity (feeding) - Mouse

In a carcinogenicity study (MRID 49040635), benalaxyl-M (96.33% a.i., Batch/Lot # G 018/04) was administered in the diet to 50 male and 50 female CD-1 (SPF) mice per dose at concentrations of 0, 200, 1200 or 7000 ppm for 78 weeks (equivalent to 0, 24.84, 156.05, and 962.23 mg/kg bw/day for males and 0, 31.36, 196.72, and 1180.41 mg/kg bw/day for females). 

No treatment-related effects were observed on the following parameters: mortality, clinical signs, body weight, food consumption, and hematology parameters.  The liver was identified as the target organ with treatment-related effects observed at 1200 ppm in male mice and at 7000 ppm in both sexes.  At 7000 ppm, absolute liver weight, liver to brain weight ratio, and liver to body weight ratio were significant increased by 52%-63% in males and 52%-61% in females compared with that of controls.  Macroscopic examination showed a significant increase in liver nodules in males (18/50 vs 8/50 in controls, p<0.01) and in females 4/50 vs 1/50, N.S.) at 7000 ppm.  At 1200 ppm, the only treatment-related finding in the liver was an increased incidence of diffuse hepatocellular hypertrophy in male mice (21/50 vs 8/50, p<0.01).  Treatment-related microscopic liver lesions were observed in male mice at 7000 ppm were diffuse hepatocellular hypertrophy (49/50 vs 8/50 in controls, p<0.01), single cell necrosis (30/50 vs 10/50 in controls, p<0.01), focal/multifocal necrosis (12/50 vs 5/50 in controls, p<0.05), and eosinophilic focus (6/50 vs 1/50 in controls, p<0.01).  Treatment-related liver lesions were observed in female mice at 7000 ppm were diffuse hepatocellular hypertrophy (44/50 vs 0/50 in controls, p<0.01) and single cell necrosis (21/50 vs 5/50 in controls, p<001).  In addition, the severity of the liver lesions also increased in both sexes at 7000 ppm, as evidenced by the average severity grade and the incidence of mice with moderate and/or severe forms of the lesion. At 7000 ppm, one male and one female displayed thyroid follicular cell hyperplasia.    

The lowest-observed-adverse-effect level (LOAEL) for benalaxyl-M in male and female mice is 7000 ppm (962.23 mg/kg bw/day for males and 1180 mg/kg bw/day for females) based on increased liver weight in both sexes and an increased incidence of liver lesions (diffuse hepatocellular hypertrophy in both sexes, single cell necrosis in both sexes, focal/multifocal necrosis in males, and eosinophilic foci in males).  The NOAEL is 1200 ppm (156 mg/kg bw/day for males and 197 mg/kg/day for females).

At the doses tested, there was a treatment related increase in the incidence of hepatocellular adenoma in male mice, and an increase in the incidence of hepatocellular adenoma/carcinoma combined in male mice and female mice.  The increased incidences exceeded those of historical controls for hepatocellular adenoma in male mice and hepatocellular adenoma/carcinoma combined in both sexes.  

This carcinogenicity study in mice is Acceptable/guideline and satisfies the guideline requirement for a carcinogenicity study [OPPTS 870.4200; OECD 451] in mice.

A.4.6	Mutagenicity

                                Guideline No./
                                  Study Type
                     MRID No. (year)/ Classification/Doses
                                    Results
                                  Benalaxyl-M
                                   870.5100
                 Bacterial reverse gene mutation (Ames Assay)
                                49040640 (2002)
                             Acceptable/guideline
               5- 5000 μg/plate +/  - S9 (plate incorporation)
                  5- 1500 μg/plate +/  - S9 (preincubation)
  Negative up to the limit dose; compound precipitation at >=1500 μg/plate
                                   870.5300
     In vitro mammalian forward cell gene mutation (mouse lymphoma cells)
                                49040642 (1999)
                             Acceptable/guideline
                             5- 75 μg/mL   - S9 
                             15- 100 μg/mL +S9  
                    Negative up to cytotoxic concentration 
                                   870.5375
                 In vitro mammalian chromosome aberration test
                      [Chinese Hamster Ovary (CHO cells)]
                                49040641 (1999)
                             Acceptable/guideline
                    Assay 1 5- 3000 μg/mL+/  - S9 (3 hrs.)
                     Assay 2 5- 150 μg/mL  - S9 (18 hrs.)
                         15- 150 μg/mL +S9 (3 hrs.) 
            Negative up to cytotoxic & insoluble concentration
                                   870.5395
               Mammalian erythrocyte micronucleus assay (mouse)
                                49040643 (2000)
                             Acceptable/guideline
                           0, 100, 200, 400 mg/kg M
                            0, 75, 150, 300 mg/kg F
                            Single i.p. injections
                                       
Negative up to doses (>=300 mg/kg) causing overt toxicity in preliminary study (deaths and/or piloerection)
                     N-malonyl-N-(2, 6-xylyl)-D, L-alanine
                                   870.5100
                 Bacterial reverse gene mutation (Ames Assay)
                                49040606 (2002)
               33- 5000 μg/plate +/  - S9 Acceptable/guideline
    Negative up to the limit dose; plate incorporation & preincubation
                                   870.5300
         In vitro mammalian cell gene mutation (mouse lymphoma cells)
                                49040608 (2002)
                     168.8  -  2700 μg/mL  - S9 (4 hrs.)
                      168.8  -  2565 μg/mL +S9 (4 hrs.)
                     168.8  -  2700 μg/mL  - S9 (24 hrs.)
                             Acceptable/guideline
                    Negative up to insoluble concentrations
                                   870.5375
                 In vitro mammalian chromosome aberration test
                      [Chinese Hamster Ovary (CHO cells)]
                                49040609 (2002)
                  Assay 1 60.3  -  1930 μg/mL-/+S9 (4 hrs.)
                       60.3  -  1930 μg/mL-S9 (24 hrs.)
                                       
                                   Assay 2 
                      241.3  -  1930 μg/mL-S9 (46 hrs.)
                       241.3  -  1930 μg/mL+S9 (4 hrs.)
                                       
                             Acceptable/guideline
     Negative up to a cytotoxic concentration (1930 μg/mL-S9, 24 & 46
                                     hrs.)
                Methyl-N-malonyl-N-(2, 6-xylyl)-D, L-alaninate
                                   870.5100
                 Bacterial reverse gene mutation (Ames Assay)
                                49040605 (2002)
               33- 5000 μg/plate +/  - S9 Acceptable/guideline
    Negative up to the limit dose; plate incorporation & preincubation
                                   870.5300
         In vitro mammalian cell gene mutation (mouse lymphoma cells)
                                49040607(2002)
                    156.3  -  2500 μg/mL +/  - S9 (4 hrs.)
                   156.3  -  2500 μg/mL +/  - S9 (24 hrs.)
                             Acceptable/guideline 
                    Negative up to insoluble concentrations
                                   870.5375
                 In vitro mammalian chromosome aberration test
                      [Chinese Hamster Ovary (CHO cells)]
                                49040610 (2002)
                  Assay 1 19.5  -  2500 μg/mL-/+S9 (4 hrs.)
                                       
               Assay 2 625  -  1875 μg/mL-S9 (24 & 46 hrs.)
                       625  -  2500 μg/mL +S9 (4 hrs.)
                                       
                Assay 3 1000- 1500 μg/mL-S9 (24& 46 hrs.)
                             Acceptable/guideline
                              Confirmed positive:
Assay 2 937.5, 1250 & 1875 μg/mL-S9 (24 hrs.) & 937.5 μg/mL-S9 (46 hrs.).
                                       
                       Assay 3 1500 μg/mL-S9 (24 hrs.)
                                       
                                   870.5375
                 In vitro mammalian chromosome aberration test
                              (Human lymphocytes)
                                49040611 (2002)
                     Assay 1 0.078  -  10mM -/+S9 (3 hrs.)
                     Assay 2 0.313  -  10 mM -S9 (20 hrs.)
                         0.313  -  10 mM +S9 (3 hrs.)
                             Acceptable/guideline
                                 Positive: at 
                              10 mM -S9 (20 hrs.)
                                  Limit dose
                                   870.5395
               Mammalian erythrocyte micronucleus assay (mouse)
                                49040612 (2002)
                      0, 500, 1000, 2000 mg/kg M & F
                  Single i.p. injections Acceptable/guideline
               Negative up to an overtly toxic dose (2000 mg/kg)



A.4.7	Neurotoxicity

	870.6100 Delayed Neurotoxicity Study - Hen

A delayed neurotoxicity is not required and is not available.  

	870.6200 Acute Neurotoxicity Screening Battery

In an acute neurotoxicity study (MRID 49040626), groups of unfasted, 6- to 9-week-old CRL: Wistar rats (12/sex/dose) were given a single oral (gavage) dose of benalaxyl-M (96.53% w/w; Batch/Lot #P/10/106) in 0.5% aqueous carboxymethylcellulose (CMC) solution (dose volume of 10 mL/kg) at doses of 0, 500, 1000, or 2000 mg/kg bw and observed over a 14-day postdose period.  In addition to observations of all animals for clinical signs of overt toxicity conducted daily from the day of dosing to study termination on Day 15, weekly body weight measurements and neurobehavioral assessments (functional observational battery, landing foot splay testing, grip strength measurements, and motor activity testing) were conducted on all animals on pre-dose Day -7, dose Day 0 (starting at approximately 30 minutes after dosing), and post-dose Days 7 and 14. At study termination on Day 15, at least 6 animals/sex/group were perfusion-fixed. Other surviving animals were euthanized under pentobarbital anesthesia by exsanguination. Gross necropsy was performed on each animal. Brain weights of non-perfused animals were obtained on Day 15; brains of perfused animals were weighed on Day 17, after a 48 hour period of post-perfusion whole body fixation. Central nervous system and peripheral nervous system tissues from the control (0 mg/kg) and high dose (2000 mg/kg) groups of perfused animals (6 animals/sex/group) were processed into slides (embedded in paraffin or plastic Epoxy resin and sectioned) for histopathological evaluation. 

There were no treatment related effects on mortality, clinical signs, body weight, brain weight or gross and histologic pathology or neuropathology.  FOB, landing foot splay, fore- and hindlimb grip strength, and motor activity testing revealed no treatment related effects.
 
Based on the absence of treatment related effects in this study, a LOAEL was not identified. The NOAEL for benalaxyl-M technical product was 2000 mg/kg bw administered orally in adult male and female Wistar rats. 

This neurotoxicity study is classified as Acceptable/Guideline and satisfies the guideline requirement for an acute neurotoxicity study in rats (OCSPP 870.6200a; OECD 424). 

	870.6200 Subchronic Neurotoxicity Screening Battery
  
In a subchronic neurotoxicity study (MRID 49143501), benalaxyl-M technical product (Benalaxyl-M; 96.53% a.i.; Batch/Lot #P/10/106) was administered in the diet to 12 CRL:Wistar rats/sex/group at dietary levels of 0, 1000, 3000, or 10,000 ppm (resulting in calculated daily dose levels of 0, 67, 206 and 784 mg/kg bw/day for males and 0, 78, 325, and 1139 mg/kg bw/day for females, respectively) for 90 days.  Treatment was initiated on Day 0, and the neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed in all animals on Days -7, 14, 42, and 77 (Weeks -1, 3, 7 and 12).  At study termination (Day 90), at least 6 animals/sex/group were euthanized via in situ perfusion fixation, and the central and peripheral nervous system tissues from 6 male and 6 female perfused control and high-dose animals were subjected to histopathological evaluation.  Additional measures included ophthalmoscopic examination (all animals on Day -6 and control and high-dose at term), serum GGT activity (in all animals at term), gross pathology (of all surviving animals), and brain and liver weights (all surviving animals).
      
There were no treatment-related effects on mortality, general or detailed clinical signs, brain weight, or macroscopic and neurohistologic evaluations. Neurobehavioral test measures including FOB, landing foot splay, grip strength and motor activity testing also revealed no treatment-related effects for either males or females at any dietary level. Absolute body weights and food consumption were not affected by treatment in either sex. Liver weights (absolute and relative) were significantly increased in non-perfused males at the low-, mid-, and high-dose treatment levels (absolute weights: +29%. +42%, and +51%, respectively; p<0.05 or p<0.01) and in non-perfused mid- and high-dose females (absolute weights: +20% and +45%; p<0.01 for high-dose, only).  In perfused animals, liver weights were increased only at the high-dose treatment level (both sexes), but it is possible that liver weights of these animals were influenced by the perfusion/fixation process. Increased serum GGT activity was seen in high-dose animals of both sexes (males: 14.0 U/L; females: 16.1 U/L; both vs. 5.0 U/L for controls and p<0.01), which correlated with the increased liver weights in the high-dose animals of both sexes. 

Based on increased serum GGT activity in both sexes and increased absolute and relative liver weights, the LOAEL for benalaxyl-M in rats is 10000 ppm (equivalent to 784 mg/kg bw/day for males and 1139 mg/kg bw/day for females).  The NOAEL is 3000 ppm (equivalent to 206 mg/kg bw/day for males and 325 mg/kg bw/day for females).

The study is classified as Acceptable/Guideline and satisfies the guideline requirement for a subchronic neurotoxicity study in rats [OCSPP 870.6200b; OECD 424].  
 
	870.6300 Developmental Neurotoxicity Study

A developmental neurotoxicity study is not required and is not available.  

A.4.8	Metabolism

	870.7485	Metabolism - Rat

In four metabolism studies (MRIDs 49040644, 49040645 and 49040646, 49040647), [[14]C-U-aniline ring]-benalaxyl-M (>99% a.i., Batch/lot No. - MRID 49040644: FCF/T/155-98, MRID 49040645: 20376/76, MRIDs 49040646 and 49040647: B01/L-4) was administered to groups of 5 male Sprague Dawley rats at doses of 0 or 10 mg/kg bw or to groups of 2  -  4 male and female Sprague Dawley rats at doses of 10 or 100 mg/kg bw by oral gavage.  

(MRIDs 49040644 and 49060445).  Following a single 10 mg of [14]C [U-aniline ring]  - benalaxyl-M/kg bw oral dose to male rats, the test material was readily absorbed and excreted mainly through the fecal route (~85%) and to a smaller extent via the urine (~8%) within 48 hours.  Biliary excretion, as demonstrated in the subsequent studies, explains the high elimination of absorbed radioactivity via the fecal route.   The highest tissue concen - tra - tions, as percent of administered dose 0.5 hours after treatment, were found in the intestinal wall (8.0%), liver (8.3%), and stomach wall (6.4%).  Total radiolabel recovered in the tissues 0.5 hours after treatment was 23%.  By 72 hours after treatment, all tissue concentrations were <1% of the administered dose.  The pharmacokinetic profile was characterized by a peak level between 10 minutes (first sampling time) and 1 hour after admin - istration.  After peaking, a gradual decline of radioactivity was observed leading to an elimina - tion half-life (t(1/2)) of ~18 hours.  Cmax was 452 +- 158 ng eq./mL while AUC0-infinity was 6593 +- 470 hour :: ng eq./mL.  The average MRT (mean residence time extrapolated to infinity) was 25 +- 4 hours.    Excretion in the expired air was negligible (<0.1%).  No residual radioactivity was found in the carcasses 168 hours after treatment.  The total excretion accounted for 93.4% of the administered dose.  Benalaxyl-M was extensively metabo - liz - ed, principally by oxidation of the methyl group on the aniline ring to the hydroxymethyl and finally to the carboxylic acid.  Minor metabolic pathways were the hydroxylation of the phenyl ring and the hydrolysis of the carboxymethyl group.

(MRIDs 49040646 and 49040647).  The pharmacokinetics of total radioactivity in blood, plasma, tissue, urine, feces, bile and expired air following single oral dose of 10 mg/kg of  [14]C-[U-aniline ring]-benalaxyl-M was investigated in male and female Sprague Dawley rats. Elimination of absorbed radioactivity was mostly through the feces.  Biliary excretion was substantial for both sexes (80.5% of dose male; 62% of dose females within 72 hours), indicating most of the radiolabeled was absorbed and then excreted via the bile into the feces. Female rats excreted approximately double the amount of radiolabel into the urine (7.61% of dose males; 17.23% of dose females).  The highest tissue concentrations were found in the liver and GI tract of both male and female rats.  The amount of radiolabel recovered in the tissues was approximately double in males (17.29% of dose) relative to females (7.96% of dose) 1 hour after treatment.  Thereafter, tissue concentra - tions were <1% for both male and female rats at 72 and 168 hours.  Total recovered radioactivity was high, 99% in the male and 97% in the female, being most of the radioactivity was eliminated within 48 hours post dosing.  Negligible levels of radioactivity (<0.03%) were recovered in the expired air.  After oral administration of 10 mg/kg bw [14]C-benalaxyl-M to male rats, the blood Cmax was 1.22 +- 0.45 ug eq./g at tmax 1.8 hours post-dose.  The radioactivity was detectable up to 120-144 hours, resulting in a t(1/2),z (apparent terminal half-life of total radioactivity) of 62.4 hours.  The AUC0-infinity (area under the plasma concentration versus time curve up to infinite time) to the total radioactivity was 17.8 +- 1.9 ug eq.∙hour/g.  After oral admin - istration of 10 mg/kg bw [14]C-benalaxyl-M to female rats, Cmax was 1.93 +- 0.49 ug eq./g at 1.8 hours post-dose.  The levels of radioactivity declined with a t(1/2),z of 80 hours.  The AUC0-infinity was 34.3 +- 5.8 ug eq.∙hour/g.  

After oral administration of [14]C-benalaxyl-M at a dose of 100 mg/kg bw to male rats, Cmax was 5.44 +- 2.25 ug eq./g at 3.3 hours post-dose.  Detectable levels of radioactivity were measured until 96-120 hours post-dose, with an average t(1/2),z of 102 hours.  The AUC0-infinity was 155.9 +-26 ug eq.∙hour/g.  After oral administration of 100 mg/kg bw [14]C-benalaxyl-M to female rats, total radioactivity in blood reached a Cmax of 8.5 +- 3.21 ug eq./g at 6 hours post-dose.  The levels of radioactivity declined with a t(1/2),z of 59 hours.  The AUC0-infinitywas 212.3 +- 71.2 ug eq.∙hour/g.  The data suggest that absorption and elimination were not saturated as there was not a 10-fold increase in Cmax or tmax.  

Parent [14]C-benalaxyl-M was excreted exclusively in the feces as a minor component and accounted for 1.49% in females and 2.94% in males.  No parent compound was found in the urine.  [14]C-benalaxyl-M was extensively metabolized by oxidation of the methyl group on the aniline ring to hydroxymethyl and finally to carboxylic acid.  Other metabolic pathways were hydroxylation and di-hydroxylation (followed by methylation) of the phenyl ring. Conjugation of hydroxyl group with glucuronic acid was also found.  Total identified radioactivity accounted for 82.11% and 87.69% of the administered dose in the excreta of male and female rats respectively.

The above studies are well conducted and complement each other.  

Together, these metabolism studies are classified Acceptable / Guideline and satisfy the guideline requirement for a metabolism study [OCSPP 870.7485, OECD 417] in the rat. 

	870.7600	Dermal Absorption - Rat

A dermal absorption study is not required and is not available.  

A.4.9	Immunotoxicity

	870.7800	Immunotoxicity

In an immunotoxicity study (MRID 49040648), benalaxyl-M technical product (purity 96.53%, Lot No. P/10/106), was administered in the diet for 28 consecutive days at concen - tra - tions of 0, 1000, 3000, or 10,000 ppm (equivalent to 0.0, 80.5, 250.4, and 924.6 mg/kg bw/  day, respectively) to groups of 12 male Crl:WI rats/dose.  A concurrent cyclophosphamide (CPS) positive control group of 12 male Wistar rats received 25 mg CPS in saline/kg bw/day by IP injection from days 22  -  27.  The rats were inspected for signs of morbidity and mortality twice daily.  General clinical observations were made at least once a day while detailed clinical obser - vations were made weekly.  Body weight and food consumption were determined twice weekly while water consumption was measured daily.  All rats received a single IV injection into the tail vein of sheep red blood cells (SRBCs, 2x10[8] cells/rat) on Day 22.  The rats were sacrificed following an overnight fast and blood collected for an enzyme-linked immunosorbent assay for anti-SRBC IgM antibody and gamma glutamyl transferase (GGT) activity.  All rats were necropsied and observed macroscopically.  Histopathology was not done.  

No treatment-related effects were found on body weight, food consumption, food efficiency, or water consumption.  No clinical signs of toxicity were observed, and all rats survived until scheduled sacrifice.  There was a slight increase in GGT activity (6.5+-1.7 U/L vs 5.0+-0.0) in rats receiving 10,000 ppm, however, the significance of this result is obscured by the low sensitivity of the assay at the low end of the linear range.  No treatment-related effects were noted at necropsy.  The absolute liver weights of mid- and high-dose rats were statistically increased 17% and 39%, respectively, while the liver weight relative to body weight of low-, mid-, and high-dose rats were statistically increased 10%, 23%, and 48%, respectively.  In addition, the relative to body kidney weight of high-dose rats was statistically increased 8%.  While the increased absolute and relative to body liver weights were observed, no histopathology was done. The liver effect was a common observation of this chemical and was considered treatment-related.
   
The systemic LOAEL for male Wistar rats following dietary treat - ment for 28 days with benalaxyl-M was 3000 ppm (250.4 mg/kg bw/day) based on the increases in absolute and relative to body liver weights.  The NOAEL was 1000 ppm (80.5 mg/kg bw/day).  

Dietary treatment of male rats with 1000, 3000, or 10,000 ppm benalaxyl-M technical product for 28 continuous days did not affect spleen, thymus, or mesenteric lymph node weights or decrease the anti-SRBC IgM immune response. There were no statistically significant differences in anti-SRBC IgM levels in treated groups when compare to the vehicle control group.  High inter-individual variability was noted in all the treatment groups as well as in the control group.  Evaluation of the individual animal data of this study did not show any trend or distribution that would demonstrate significant suppression of anti-SRBC antibody response. Positive control group had statistically significant decrease in the anti SRBC IgM levels. This confirmed the ability of the test system to detect immuno-suppressive effects and confirmed the validity of the study design.

The Natural Killer (NK) cells activity was not evaluated in this study.  The toxicology database for benalaxyl-M does not reveal any evidence of immunotoxicity.  The overall weight of evidence suggests that the chemical does not directly target the immune system.  Under HED guidance a NK cell activity assay is not required at this time.
   
The immunotoxicity NOAEL for male Wistar rats treated for 28 consecutive days with benalaxyl-M was 10,000 ppm (924.6 mg/kg bw/day).  A LOAEL was not established.  

This immunotoxicity study is classified Acceptable / Guideline and satisfies the guideline requirement for an immunotoxicity study (OCSPP 870.7800) in the rat.  

A.4.10	Other
	Non-guideline	Mechanistic

In a 14-day mechanistic study (MRID 49040636), benalaxyl-M (96.53% a.i.; Lot/Batch # P/10/106) was administered in feed to groups of 18 male Crl: CD-1(Icr) Br mice at dietary concentrations of 0, 200, 1200, or 7000 ppm (0, 30.9, 184.4, and 1094.1 mg/kg bw/day, respectively).  Another group of 18 male mice were administered phenobarbitone (PB) in the diet at 850 ppm (139.6 mg/kg bw/day).  Six mice/groups were sacrificed for evaluation of cell proliferation after treatment for 3 and 14 days (Allocation A and B animals, respectively).   Allocation A and B animals were implanted subcutaneously with osmotic mini-pumps containing bromodeoxyuridine (BrdU) on days 6 and 8 of acclimatization, respectively.  Six mice/group were sacrificed after treatment for 14 days for evaluation of liver microsomal enzyme activity, mRNA gene expression, and oxidative stress (Allocation C animals).  

No effects were observed on mortality, clinical signs, body weight, or body weight gain, in mice administered benalaxyl-M or PB.  Absolute liver weight, liver/body weight ratio, and liver/brain weight ratio were increased 16% to 17% and 15% to 20% in mice treated with benalaxyl-M for 3 days or 14 days, respectively, and absolute and relative liver weights in mice treated with PB were increased by 32% to 36% and 33% to 41% after 3 and 14 days, respectively.  No benalaxyl-M- or PB-related macroscopic lesions were observed in this study.  Centrilobular to diffuse hepatocellular hypertrophy were observed in 0/6, 1/6, 2/6, and 4/6 mice treated with 0, 200, 1200, or 7000 ppm of the test substance, respectively, for 3 days and 0/6, 2/6, 4/6, and 3/6 mice, respectively, after 14 days.  All six mice treated with PB for 3 or 14 days had centrilobular to diffuse hypertrophy.  There was a dose-related increased severity of the lesions in mice treated with benalaxyl-M, and the lesions in PB-treated  mice was more severe than in those treated with IR6141. 

Analysis of the number of BrdU-positive nuclei in hepatocytes as an indicator of cell proliferation showed that cell proliferation was increased at all doses of IR6141 after 14 days; the data was equivocal after treatment for 3 days.  PB treatment resulted in an increase in cell proliferation after 3 and 14 days.  The effect of benalaxyl-M and PB treatment was analyzed on following microsomal liver enzymes: 7-ethoxyresorufin O-dealkylase (EROD, Cyp1a1 isoenzyme), 7-methoxyresorufin O-dealkylase (MROD, Cyp1a2 isoenzyme), 7-pentoxy-resorufin O-dealkylase (PROD, Cyp2b1), testosterone 6β-hydroxylase (6β-TOH, Cyp3a), lauric acid 12-hydroxylase (LA12OH, Cyp4a1), and lauric acid 11-hydroxylase (LA11OH, Cyp2e1).  EROD, MROD, and LA12OH activities were slightly increased at 1200 and 7000 ppm; PROD, 6β-OH, and LA11OH activities were moderately increased at 7000 ppm; and PROD activity was moderately increased at 1200 ppm and 6β-OH and LA11OH activities were slightly increased at 1200 ppm.  Treatment with PB for 14 days caused moderate increases in EROD, MROD, 6-β-TOH, and LA11OH activities, a pronounced increased in PROD activity and a slight increase in LA12OH activity.  The expression of the Cyp1A-1, Cyp1A-2, Cyp2B10, and Cyp3A11 genes were increased at 7000 ppm and the expression of Cyp1A-1, CypB10, and Cyp3A11 were increased at 1200 ppm.  The expression of Cyp2B10 was increased 45.8- and 195.7-fold at 1200 and 7000 ppm, respectively, and the expression of Cyp3A11 was increased 15.58-fold.  Treatment with PB for 14 days caused increased expression of all the genes tested.  Examination of the parameters of oxidative stress, reduced glutathione (GSH) and oxidized glutathione (GSSH) in liver, and thiobarbituric acid reactive substance (TBARS) in liver and serum, showed no effect of treatment with benalaxyl-M or PB for 14 days.
This 14-day mechanistic study in the mouse is Acceptable/Non-guideline. The study provides an assessment of possible liver toxicity for use in the evaluation of the mode of action of hepatocellular tumor induction in mice by benalaxyl-M.
Appendix B. Metabolism

B.1 Metabolism Summary Table

Table B.1.  Tabular Summary of Metabolites and Degradates
           Chemical Name (other names in parenthesis) and Structure
                                    Matrix
                              Percent TRR (PPM) 


                     Matrices - Major Residue (>10%TRR)
                     Matrices - Minor Residue (<10%TRR)
Benalaxyl-M

Grape - Fruit
26.4 % (0.023)
NA

Tomato - Fruit
43.5 % (0.025)
NA

Lettuce
41.5 % (0.84)
NA

Rat
NA
3% feces ♂
2% feces ♀
R5
N-(hydroxyphenylacetyl)-N-(2-hydroxymethyl-6-methylphenyl)-alanine 

Grapes - Fruit
NA
NA

Tomato - Fruit
NA
NA

Lettuce
NA
NA

Rat
22% feces ♀
3% feces ♂
R6
methyl-N-(phenylacetyl)-N-(2-carboxy-6-hydroxymethylphenyl)-alaninate

Grapes - Fruit
NA
NA

Tomato - Fruit
NA
NA

Lettuce
NA
NA

Rat
11% feces ♂
4% feces ♀
R7
(methyl-N-(hydroxyphenylacetyl)-N-(2-carboxy-6-methylphenyl)-alaninate



Grapes - Fruit
NA
NA

Tomato - Fruit
NA
NA

Lettuce
NA
NA

Rat
24% feces ♀
8% urine ♀

38% feces ♂
1% urine ♂

G10, T4, L4

Grapes - Fruit
11% (0.0093)
NA

Tomato - Fruit
NA
3.2% (0.0018)

Lettuce
11% (0.23)
NA

Rat
NA
NA

G13, [T9, T11], [L3, L5-L9, L11]


Grapes - Fruit
14% (0.012)
NA

Tomato - Fruit
NA
5.3% (0.0030)

Lettuce
10% (0.21)
NA

Rat
NA
NA
G15, T10, L10

Grapes - Fruit
11% (0.0097)
NA

Tomato - Fruit
32% (0.018)
NA

Lettuce
30% (0.61)
NA

Rat
NA
NA


B.2  Metabolic Pathways 

             Proposed Metabolic Pathway of Benalaxyl-M for Grapes

The figure below was copied without alteration from MRID 49040680.
                                       
                                       

                    Proposed Metabolic Pathway for the Rat.

The figure below was copied without alteration from MRID 49040647.




Appendix C.  Physical/Chemical Properties


TABLE C.1.	Physicochemical Properties of the Technical Grade Test Compound: Benalaxyl-M.  
                                   Parameter
                                     Value
                                   Reference
Melting point/range
                                   74.86 °C
                             D421271, S. Mathur, 
                                  22-JUL-2014
Boiling point
                                    330 °C
                                       
pH
                                      7.1
                                       
Density
                         1.19 +- 0.022 g/ml (19.5 °C)
                                       
Water solubility (at 25°C)
                                   37.0 mg/L
                                       
Solvent solubility (g/L)
                            n-heptane - 17074 mg/L
                            xylene - >39 (%w/w)
                            acetone - >49 (%w/w)
                         ethyl acetate - >49 (%w/w)
                      1,2-dichloroethane - >50 (%w/w)
                           methanol - >50 (%w/w)
                                       
Vapor pressure at 25°C
                                6.7 x 10[-4] Pa
                                       
Dissociation constant (pKa)
                                Not applicable
                                       
Octanol/water partition coefficient Log(KOW)
                                       
                                3.54 at 20 °C
                                       
                                       
UV/visible absorption spectrum
                  202 nm - 3.4 x 10[4] ξ (dm[3]/(mol x cm))
                    254 nm - 4.6 x 10[2]               "
                    259 nm - 4.6 x 10[2]               "
                    265 nm - 6.1 x 10[2]               "
                    275 nm - 4.4 x 10[2]               "
                                       

Appendix D.  Review of Human Research

This risk assessment does not rely on data from studies in which human subjects were intentionally exposed to a pesticide or other chemicals. 
