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




						                   OFFICE OF CHEMICAL SAFETY 							                                                            AND POLLUTION PREVENTION						


July 29, 2014

MEMORANDUM


                                    SUBJECT: Petition IN-10631; C.I. Pigment Red 112; Human Health Risk Assessment and Ecological Effects Assessment to Support Proposed Exemption from the Requirement of a Tolerance When Used as Inert Ingredients in Pesticide Formulations.


All PC Code:   804201 
                          

DP Barcode:  009297298

Regulatory Action:  Inert Tolerance Exemption; 40 CFR 180.920

Petition No: IN-10631

All CAS No:
6535-46-2


		
FROM:	Deirdre Sunderland, Industrial Hygienist  
		Inert Ingredient Assessment Branch (IIAB)
		Registration Division (7505P)




TO:		PV Shah, Chief
		Inert Ingredient Assessment Branch (IIAB)
		Registration Division (7505P)







                               Table of Contents

Executive Summary...........................................................................................3

1.0 Background................................................................................................5
2.0 Inert Ingredient Profile....................................................................................5
   2.1 Summary of Uses.......................................................................................5
   2.2 Physical and Chemical Properties.....................................................................5
   2.3 Metabolism and Pharmacokinetics....................................................................6

3.0 Hazard Assessment.......................................................................................7
   3.1 Acute Toxicity...........................................................................................7
   3.2 Subchronic Toxicity.....................................................................................8
   3.3 Reproductive and Developmental Toxicity.........................................................9
   3.4 Neurotoxicity............................................................................................9
   3.5 Mutagenicity.............................................................................................9
   3.6 Carcinogenic Potential................................................................................11
   3.7 Endpoint Selection and Levels of Concern.........................................................11
   3.8 FQPA Safety Factor Considerations................................................................11

4.0 Exposure Assessment....................................................................................12
   4.1 Dietary Exposure.......................................................................................12
   4.2 Cancer Exposure........................................................................................12
   4.3 Residential Exposure..................................................................................12
   4.4 Occupational Exposure................................................................................12

5.0 Aggregate Risk...........................................................................................12
   5.1 Acute Dietary Risk....................................................................................13
   5.2 Short-term/Intermediate Aggregate Risk...........................................................13

6.0 Cumulative Risk...........................................................................................13

7.0 Environmental Fate.......................................................................................13

8.0 Ecological Effects........................................................................................14
   8.1 Invertebrate Toxicity...................................................................................14
   8.2 Algal Toxicity............................................................................................14









EXECUTIVE SUMMARY

On September 5, 2013, Exponent, on behalf of Clariant Corporation, submitted a petition to the Environmental Protection Agency, herein referred to as the agency or EPA, to amend 40 CFR 180.920 by establishing an exemption from the requirement of a tolerance for the use of the inert ingredient C.I. Pigment Red 112 (CAS Reg. No. 6535-46-2) as a seed treatment pigment in pesticide formulations applied pre-harvest not to exceed 10% weight/weight (w/w). These seeds will not be used for food, feed, or oil processing. 

C.I. Pigment Red 112, also known as 3-hydroxy-N-(2-methylphenyl)-4-[2-(2,4,5-trichlorophenyl) diazenyl]-naphthalene-2-carboxamide, is a synthetic azo compound primarily used in industrial applications, such as a coloring agent for paints, plastics and inks. C.I. Pigment Red 112 has no approved pesticidal uses and the Agency is unaware of any current residential. 
 
Based on the physical and chemical properties of C.I. Pigment Red 112 it is unlikely that C.I. Pigment Red 112 will be absorbed in the body and therefore, it is unlikely that C.I. Pigment Red 112 becomes systemically bioavailable after exposure. In addition, azo reduction is likely to occur at low levels following oral ingestion limiting the potential for absorption and the subsequent formation of metabolites. It is expected that C.I. Pigment Red 112 will pass though the body and be excreted in the feces. The solubility of C.I. Pigment Red 112 in octanol is 3.31 mg/L resulting in a calculated Log Kow of 2.5 which would indicated that the potential for bioaccumulation would be low. 

Acute studies revealed low oral and dermal toxicity (Toxicity Category IV). When tested on rabbits, the chemical was shown to be non-irritating to the skin and eyes. Skin sensitization studies in guinea pigs showed that C.I. Pigment Red 112 was not skin sensitizer.

In a 28-day oral toxicity study in rats no treatment-related changes were noted in neurological evaluations, body weights, food consumption, hematology or clinical chemistry analyses, or organ weights at the limit dose of 1000 mg/kg/day. Although there were no reproductive or developmental toxicity studies available for C.I. Pigment Red 112, the 28-day oral study in rats included endpoints specific to reproductive toxicity including organ weights, gross pathology and histopathology. No adverse effects were seen at doses up to 1000 mg/kg/day. In addition, DEREK modeling was conducted and did not indicate any structural alerts for reproductive toxicity or endocrine-related toxicity.

 No neuropathological changes or effects were reported in any of the studies. The agency does not believe C.I. Pigment Red 112 will be neurotoxic

For the purpose of this risk assessment three mutagenicity studies were reviewed: a reverse gene mutation study, an in vitro cell mutagenicity study, and a Chinese hamster cell in vitro study. There was no evidence of cytotoxicity or mutagenicity in any of the reviewed studies. Although no carcinogenicity studies are available for C.I. Pigment Red 112, C.I. Pigment Red 112 is not expected to be absorbed in the body based on the physical/chemical properties of the substance. Many azo dyes contain benzidine in their structure and this human bladder carcinogen may be released upon azo reduction of the dye. In the case of C.I. Pigment Red 112, azo reduction is likely to occur at low levels following oral ingestion limiting the potential for the formation of genotoxic and carcinogenetic metabolites. Similarly, all mutagenicity studies on C.I. Pigment Red 112 were negative which makes it unlikely that C.I. Pigment Red 112 would be carcinogenic. 

No endpoint of concern was identified for any of the acute studies conducted. In addition, no endpoint of concern was determined in the 28-day study up to the limit dose of 1000mg/kg/day (highest dose tested). Since no endpoint of concern was identified in acute and subchronic studies and because C.I. Pigment Red 112 is not expected to be absorbed by the body, a quantitative risk assessment for C.I. Pigment Red 112 was not performed. As part of its qualitative assessment, the Agency did not use safety factors for assessing risk, and no additional safety factor is needed for assessing risk to infants and children. Furthermore, C.I. Pigment Red 112 will be used at a maximum of 10% w/w in seed treatments only. It is expected to be tightly bound to soil so the likelihood of consumers being exposed to C.I. Pigment Red 112 as an inert ingredient through foliar ingestion or water consumption is nominal. Seeds that have been chemically treated may NOT be used for food, feed, or oil processing. 

The proposed use of C.I. Pigment Red 112 as a seed treatment/dye under 40 CFR 180.920 is not expected to result in residential exposure to this chemical. In addition, there are no toxicological effects of concern in available studies and therefore, it is not necessary to conduct a quantitative assessments of residential (non-occupational) exposures and risks. Similarly, a quantitative occupational risk assessment is not necessary. 

Based on ecological and fate data the agency concluded that C.I. Pigment Red 112 does not pose an ecological risk to terrestrial or aquatic species. C.I. Pigment Red 112 is a solid material with a low solubility in water and is not expected to volatilize. As a result of the low water solubility coupled with the projected partitioning of C.I. Pigment Red 112, it is expected to be bound to soil and therefore, not expected to leach into ground water, be found in runoff or be taken up by the plant. Although C.I. Pigment Red 112 may persist in soils, due to the small amount of pigment used compared to the weight of the seeds used per acre, deposition in soil is not expected to significantly impact soil organisms. Bioaccumulation is also expected to be low. 

Based on the evaluated toxicity studies coupled with the expected exposure from the use of these chemicals as inert ingredients in pesticide products, EPA concludes that there is a reasonable certainty that no harm will result to the general population, including infants and children, from aggregate exposure to residues of C.I. Pigment Red 112 when used as an inert ingredient in pesticide formulations for seed treatment at a maximum of 10% w/w under 40 CFR 180.920.
 









 1.0       BACKGROUND

On September 5, 2013, Exponent, on behalf of Clariant Corporation, submitted a petition to the EPA, to amend 40 CFR 180.920 by establishing an exemption from the requirement of a tolerance for the use of the inert ingredient C.I. Pigment Red 112, CAS Reg. No. 6535-46-2 for use as a seed treatment pigment in pesticide formulations, not to exceed 10% w/w, applied pre-harvest. By regulation, seeds treated with a pesticide, with the exception of those used "solely as at-planting or hopper box treatments" or "products which are gaseous in form or are used as fumigants",  must also be treated with a "colorant" to indicate that the seeds are, in fact, treated with a pesticide (40 CFR 153.155). According to the Federal Seed Act section 201.31a (d), seeds that have been chemically treated may not be used for food, feed, or oil processing.

C.I. Pigment Red 112, also known as 3-hydroxy-N-(2-methylphenyl)-4-[2-(2,4,5-trichlorophenyl) diazenyl]-naphthalene-2-carboxamide, has no currently approved food or nonfood pesticidal uses. This chemical was the subject of an IUCLID Chemical Safety report prepared by Clariant Inc. in 2010. 

To access the Office of Chemical Safety and Pollution Prevention (OCSPP) test guidelines referenced in this document electronically, please go to http://www.epa.gov/ocspp and select "Test Methods and Guidelines."

2.0       INERT INGREDIENT PROFILE

2.1    Summary of Uses

C.I. Pigment Red 112 is primarily used in industrial applications, such as a coloring agent for paints, plastics and inks. C.I. Pigment Red 112 has no approved pesticidal uses. According to the U.S. National Institutes of Health's Household Product Database C.I. Pigment Red 112 is not found in any household consumer goods. It has been used in other countries in cosmetics, children's finger and face paints, and permanent tattoo ink. (Environment Canada, 2013) There are, however, no Federal Drug Administration (FDA) approved cosmetic uses for C.I. Pigment Red 112 in the US. 
 2.2    Physical and Chemical Properties

Some of the physical and chemical characteristics of C.I. Pigment Red 112 along with the chemical structure and nomenclature, are found in the table below.  










      Table 1.  Physical and Chemical Properties of C.I. Pigment Red 112
                                   Parameter
                                     Value
                                   Reference
Structure


ChemIDPlus, 2013
CAS Reg. No. 
6535-46-2
ChemIDPlus, 2013
Molecular Formula
C24H16C13N3O2
ChemIDPlus, 2013
Molecular Weight 
484.76
Clariant, 2010
Other Names
3-hydroxy-N-(2-methylphenyl)-4-[2-(2,4,5-trichlorophenyl) diazenyl]-naphthalene-2-carboxamide; 
CI 12370
ChemIDPlus, 2013
Physical State
solid
Clariant, 2010
Melting Point
None, decomposition at >= 270°C
Clariant, 2010
Particle Size distribution
Medium:4.51 um
Clariant, 2010
Density 
1.48 g/ml 
Clariant, 2010
Vapor Pressure 
8.51 x10-11 mm Hg (calculated)
Clariant, 2010
Log Kow @ 25°C
2.5 (calculated) 
Clariant, 2005, (MRID 49336101)
Water Solubility
9.8ug/L
Clariant, 2005, (MRID 49336101)

 2.3    Metabolism and Pharmacokinetics 

C.I. Pigment Red 112 has very low water solubility (9.8 ug/L) and therefore, it is unlikely that C.I. Pigment Red 112 will be absorbed by the body and become systemically bioavailable. In addition, azo reduction is unlikely to occur following oral ingestion limiting the potential for absorption and the subsequent formation of metabolites. It is expected that C.I. Pigment Red 112 will pass though the body and be excreted in the feces. This is supported by the 28-day oral study which found reddish staining in the feces and gastrointestinal tract of rats treated with C.I. Pigment Red 112. The solubility of C.I. Pigment Red 112 in octanol is 3.31 mg/L resulting in a calculated Log Kow of 2.5 which would indicated that the potential for bioaccumulation would be low. Based on the physical and chemical properties of C.I. Pigment Red 112 it is unlikely that C.I. Pigment Red 112 will be absorbed in the body and therefore, it is unlikely that C.I. Pigment Red 112 becomes systemically bioavailable after exposure. 






3.0       HAZARD ASSESSMENT 
 3.1    Acute Toxicity 

Acute Oral Toxicity (OSCPP 870.1100)
Wistar rats (5/sex/dose) were administered 5000 mg/kg of C.I. Pigment Red 112 (97% C.I. Pigment Red 112) via gavage and observed for 14 days. No mortalities occurred at 5000 mg/kg. (MRID 49148201)
LD50 > 5000 mg/kg
 
According to the 2010 IUCLID report on C.I. Pigment Red 112, various other acute oral (gavage) rat studies were conducted with LD50s ranging from >10,000 to >15,000 mg/kg. These studies were not available to the Agency and were reported in the IUCLID document as "reliable with restrictions" and therefore, they are presented here as supporting studies only. 

Acute Dermal Toxicity (OSCPP 870.1200)
Albino Wistar rats (5 males) were administered a single dose of 5000 mg/kg of C.I. Pigment Red 112 applied directly to the skin for 24 hours and were observed for 14 days after removal of excess test substance. No mortality or signs of toxicity were observed. (MRID 49148202)
LD50 > 5000 mg/kg

Acute Inhalation Toxicity
No acute inhalation toxicity studies are available for C.I. Pigment Red 112. 

Primary Dermal Irritation (OSCPP 870.2500)
A test substance comprised 97% C.I. Pigment Red 112 was tested for acute skin irritation when a single dermal dose of 500 mg applied to a 2.5 x 2.5 cm surgical gauze patch and applied to an clipped area of the skin of three albino New Zealand White rabbits held in place with semi-occlusive for four hours (OSCPP 870.2500). Skin examinations were performed at 30 minutes, 24, 48, and 72 hours after removal of dressing. The skin areas treated were slightly stained light pink until 24 hours after removal of the patch; however, the evaluation of skin redness was still possible despite the slight staining. At 30 minutes post exposure one animal displayed very slight edema and erythema. No reactions were observed in any of the animals at 24 hours after the removal of the test substance. (MRID 49148203)
Based on the results of this study C.I. Pigment Red 112 is not considered a skin irritant. 

Primary Eye Irritation (OSCPP 870.2400)
A test for acute ocular irritation was conducted with C.I. Pigment Red 112 by applying a single dose of 100 mg (as a paste in 0.05 ml polyethylene glycol), into the conjunctival sac of three Albino New Zealand White rabbits (OSCPP 870.2400). One eye remained untreated and served as a control. Treated eyes were rinsed 24 hours after application. Ocular examinations were performed at 1, 24, 48, and 72 hours after instillation. 

No iritis or corneal opacity was observed throughout the observation period in any of the animals. Conjunctival redness was observed at 1 hour in all animals (score 1), and in one animal 24 hours after instillation (score 1). Chemosis (score 1) was observed in one animal at 1 hour after instillation. All effects cleared by 48 hours post instillation. Based on the results of this study C.I. Pigment Red 112 is considered nonirritating. (MRID 49148204)

                   Table 2.  Summary of Acute Toxicity Data
                                   Endpoint
                                      DMS
                               Toxicity Category
                                   Reference
Acute Oral LD50 (mg/kg)
                                   >5000
                                      IV
                                 MRID 49148201
Acute Dermal LD50 (mg/kg)
                                   >5000
                                      IV
                                 MRID 49148202
Acute Inhalation LD50 (mg/L)
                                    No Data
                                      N/A
                                      N/A
Skin Irritation (4 hour exposure)
                              Not a skin irritant
                                      IV
                                 MRID 49148203
Eye Irritation
                              Not an eye irritant
                                      IV
                                 MRID 49148204

Dermal Sensitization
Skin sensitization testing of C.I. Pigment Red 112 (containing 3% Naphtol AS-D) was performed on 20 female guinea pigs according to the Buehler method (OCSPP 870.2600). Due to the red color of the test substance a determination of erythema could not be made using a test substance of 100% C.I. Pigment Red 112; therefore, the study portion for erythma was performed with a 20% pigment composition in sesame oil with a control group (10 animals) receiving sesame oil only. 

After 24 hours, no irritating effects (erythema or edema) were observed following the application of concentrations of 100% or 20% C.I. Pigment Red 112. Based on the results of this study, the mixture was not considered to be a dermal sensitizer. (MRID 49148206)
	
Two Local Lymph Node Assays were conducted with C.I. Pigment Red 112; one study tested 0.5, 10, and 20% C.I. Pigment Red 112 with <0.01% Naphtol AS-D (MRID 49148205) and the other tested 5, 10, and 20% C.I. Pigment Red 112 with a suggested 4.1% Naphtol AS-D (MRID 49148214). The second test substance containing what the petitioner claimed was 4.1% Naphtol AS-D was positive for skin sensitization whereas the test with 0.01% Naphtol AS-D was not, indicating that skin sensitization is possible when Naphtol AS-D is present at sufficiently high concentrations. The percent of Napthol AS-D in the second study could not be confirmed with the study data available. However, the two negative skin sensitization studies did have documented proof of the Napthol AS-D content at up to 3%. The manufacturing process for C.I. Pigment Red 112 that will be used as a seed treatment inert ingredient is specifically manufactured to contain less than 1% Naphtol AS-D which, based on the study results, would not result in skin sensitization. Therefore, the C.I. Pigment Red 112 manufactured for use as a seed treatment inert ingredient is not considered a skin irritant. 

3.2    Subchronic Toxicity 

In a 28-day oral toxicity study (OCSPP 870.3050) C.I. Pigment Red 112 (99.15%) was administered in propylene glycol to 5 Crl:WI (Han) rats/sex/dose by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day daily for 28 days. Animals were observed daily for clinical signs; body weight and food consumption were recorded weekly; and selected neurological evaluations (i.e., hearing ability, pupillary reflex, static righting reflex, grip strength, and motor activity testing (recording period of 12 hours overnight using a computerized monitoring system)) were performed at the end of Week 1, 2, 3, and 4. Gross and microscopic examination (e.g., hematology and clinical chemistry, tissue and organ examination, and organ weights) was conducted at study termination after 4 weeks of treatment. 

Treatment-related effects were limited to red staining of feces in all treated males and females and staining of body surfaces, but this pigmentation was not apparent in the slides of tissues examined microscopically and was not considered adverse. No treatment-related changes were noted in neurological evaluations, body weights, food consumption, hematology or clinical chemistry analyses, or organ weights. Although a LOAEL was not established, the limit dose was tested. (MRID 49148210)
The NOAEL was 1000 mg/kg/day, and the LOAEL was not established.
 3.3    Reproductive and Developmental Toxicity

Although there were no reproductive or developmental toxicity studies available for C.I. Pigment Red 112, the 28-day oral study in rats included endpoints specific to reproductive toxicity including organ weights, gross pathology and histopathology. The study showed that there were no treatment-related effects on relative and absolute organ weights for the testes or epididymides. Gross pathology of the reproductive organs did not reveal any treatment-related effects. Furthermore, histopathology of epididymides, prostate gland, seminal vesicles, testes, ovaries, vagina, cervix, and uterus demonstrated no effect at doses up to 1000 mg/kg/day. In addition to this study DEREK modeling was conducted and did not indicate any structural alerts for reproductive toxicity or endocrine-related toxicity.
 3.4    Neurotoxicity
      
No adverse effects on selected neurological evaluations (i.e., hearing ability, pupillary reflex, static righting reflex, grip strength, and motor activity testing) were observed in the 28-day toxicity study in rats. 
 3.5    Mutagenicity/Cytotoxicity 

Reverse Gene Mutation (MRID 49148207)
In a reverse gene mutation assay (OCSPP 870.5100) in bacteria, S. typhimurium strains TA98, TA100, TA1535, and TA1537 were exposed to C.I. Pigment Red 112 dissolved in Dimethyl sulfoxide (DMSO) in two independent assays using triplicate plating. Experiment 1, which was both a mutagenesis and a range-finding assay, used the standard plate assay and tested concentrations of 0, 4, 20, 100, 500, 2500, and 5000 ug/plate, with and without S9-mix derived from livers of induced rats. In addition, it tested the same concentrations with metabolic activation in a preincubation assay that used S9-mix derived from livers of non-induced hamsters. The confirmatory mutagenicity assay (experiment 2) tested mutagenesis and cytotoxicity under exactly the same three sets of conditions as in experiment 1. For the tests with metabolic activation, the S9 fraction for the standard plate assay was obtained from Aroclor-1254-induced male Sprague-Dawley rat liver and that for the preincubation test was obtained from non-induced, male, Syrian golden hamster liver.

C.I. Pigment Red 112 was tested at concentrations up to the limit concentration of the assay in both experiments. There was no evidence that the assays were affected by cytotoxicity. Precipitate was observed on all test plates at concentrations of 500 ug/plate and above. While the precipitate did not interfere with the counting of the numbers of revertant colonies, it did make it impossible to evaluate bacterial lawns at the maximum concentration tested. The number of revertants per plate was not increased over the concurrent solvent control value at any test material concentration, with or without S9-mix, in any tester strain. The solvent and positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In vitro Cell Mutagenicity (MRID 49148209)
In an in vitro mammalian cell gene mutation assay (OSCPP 870.5300), Chinese hamster V79 cells cultured in vitro were exposed to C.I. Pigment Red 112 dissolved in DMSO and culture medium for 4 hours at concentrations of 0, 3.1, 6.3, 12.5, 25.0, 50.0, and 400 ug/mL in the absence or the presence of metabolic activation in the first experiment or for 24 hours at concentrations of 0, 3.1, 6.3, 12.5, 25.0, 50.0, and 400 ug/mL in the absence of metabolic activation in the second experiment. All doses except for 50.0 ug/mL were continued to determine mutant frequencies. The S9 fraction was obtained from phenobarbital and β-naphthoflavone-induced male Wistar Hanlbm rat liver.

C.I. Pigment Red 112 was tested up to the limit of solubility in DMSO and aqueous media. In both the presence and absence of metabolic activation, the test article was tested up to 16 times the lowest concentration at which precipitation was consistently observed with the naked eye in the culture medium. There was no convincing evidence of any cytotoxicity. No significant increase in mutant frequency over that of the solvent controls was observed for any test article concentration. The positive controls did induce the appropriate responses. There was no evidence of induced mutant colonies over background.

Chinese Hamster Cell in vitro (MRID 49148208)
In an in vitro test on the induction of micronuclei in cultured V79 Chinese hamster cells, V79 cell cultures were treated with Pigment Red 112 dissolved in DMSO, for 4 or 24 hr without activation, and for 4 hr with activation. The preparation interval was 24 h in all tests. In order to assess the toxicity of the test article to the cultured V7 cells, a cell Proliferative Index (PI) was calculated for all treated and control V79 cultures. Only those cultures which showed a PI > 1.3 were evaluated. Concentrations of the test article selected for micronucleus analysis were 7.8, 15.6 and 31.3P ug/mL (Test 1, without activation), 3.9, 7.8 and 15.6[P] ug/mL (Test 2, without activation), 2.0, 3.9 and 7.8 ug/mL (Test 1, with activation), and 2.0, 3.9 and 7.8 ug/mL (Test 2, with activation). With activation, the maximum concentrations tested for micronucleus induction were well below the limit of solubility (7.8 ug/mL was the highest concentration used in either Test with activation, while 31.3 ug/mL was the concentration at which precipitation was first observed). S9 came from male Wistar rat liver induced with phenobarbital and β-naphthoflavone.

The test article was found to cause no significant increases in micronucleus induction in cultured V79 cells without or with activation at the concentrations that were tested. However, the highest concentration tested with activation was well below a precipitating concentration, and no data were presented to indicate that higher concentrations were cytotoxic.

P=Precipitating Concentration

Supporting Data
Although the full study reports were not available, the IUCLID Chemical Safety Report for C.I. Pigment Red 112 illustrates two additional Bacterial Reverse Mutation Assays which support the studies presented above. In both studies no evidence of mutagenicity or cytotoxicity was seen with and without metabolic activation in S. typhimurium TA 1535, 1537. 98, or 100 or E.coli WP uvr A. 
  3.6    Carcinogenic Potential 

No carcinogenicity studies are available for C.I. Pigment Red 112. In the absence of quantitative data a qualitative structure activity relationship (SAR) database, DEREK Version 11, was used to determine if there were structural alerts suggestive of carcinogenicity. Although DEREK results indicate "plausible" alerts for carcinogenicity, this is unlikely to be the case because C.I. Pigment Red 112 is not expected to be absorbed in the body based on the large molecular weight and low water solubility. Many azo dyes contain benzidine in their structure and this human bladder carcinogen may be released upon azo reduction of the dye. (Levine, 1991) In the case of C.I. Pigment Red 112, azo reduction is likely to occur at low levels following oral ingestion limiting the potential for the formation of genotoxic and carcinogenetic metabolites. (Environment Canada, 2013) Similarly, all mutagenicity studies on C.I. Pigment Red 112 were negative which makes it unlikely that C.I. Pigment Red 112 would be carcinogenic.

3.7    Endpoint Selection and Levels of Concern

Available toxicity studies indicate that C.I. Pigment Red 112 has a very low acute and subchronic toxicity. No endpoint of concern was identified for any of the acute studies conducted. In addition, no endpoint of concern was determined in the 28-day study up to the limit dose of 1000mg/kg/day (highest dose tested). Since no endpoint of concern was identified in acute and subchronic studies and because C.I. Pigment Red 112 is not expected to be absorbed by the body based on the physical/chemical properties, a quantitative risk assessment for C.I. Pigment Red 112 is not necessary.
   
3.8    FQPA Safety Factor Considerations

FFDCA Section 408(b)(2)(c) provides that EPA shall apply an additional tenfold (10X) margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure unless EPA determines based on reliable data that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the FQPA safety factor (SF). In applying this provision, EPA either retains the default value of 10X, or uses a different additional safety factor when reliable data available to EPA support the choice of a different factor. 

The available acute and repeat dose toxicity studies indicate that C.I. Pigment Red 112 has low toxicity with no effects seen at the limit dose of 1000 mg/kg/day. DEREK modeling was conducted and did not indicate any structural alerts for reproductive toxicity or endocrine-related toxicity. There is no indication, based upon the available data, that C.I. Pigment Red 112 is neurotoxic or immunotoxic and therefore, would not result in increased susceptibility in infants or children. Although the toxicity database for C.I. Pigment Red 112 is limited to acute studies, a 28-day repeat dose study and mutagenicity studies the physical chemical properties suggest that it is unlikely that C.I. Pigment Red 112 will be absorbed by the body and become bioavailable and therefore, unlikely to cause adverse effects in infant and children. As part of its qualitative assessment, the Agency did not use safety factors for assessing risk, and no additional safety factor is needed for assessing risk to infants and children. Furthermore, C.I. Pigment Red 112 will be used at a maximum of 10% w/w in seed treatments only. It is expected to be tightly bound to soil so the likelihood of consumers being exposed to C.I. Pigment Red 112 as an inert ingredient through foliar ingestion or water consumption is nominal. Taking into consideration all available information, there is no concern, at this time, for increased sensitivity to infants and children to this chemical when used as inert ingredient in pesticides formulations.

4.0       EXPOSURE 

4.1    Dietary Exposure
Although dietary exposure to C.I. Pigment Red 112 may occur from eating foods grown from seeds treated with pesticide formulations containing this inert ingredient and drinking water containing runoff from soils containing the treated seeds, the amount of residue that a consumer would be exposed to would be relatively small because the C.I. Pigment Red 112 is expected to be tightly bound to soil and therefore, it is unlikely the pigment would be taken up by the plant and subsequently available for consumption through eating the plant. In addition, runoff is unlikely based on the low water solubility and soil binding of the pigment. The amount of C.I. Pigment Red 112 used in formulation is not to exceed 10% w/w. According to the Federal Seed Act section 201.31a (d), seeds that have been chemically treated may NOT be used for food, feed, or oil processing. There are no other known food uses for C.I. Pigment Red 112. Furthermore, no endpoint of concern was identified and therefore, a quantitative dietary exposure assessment for C.I. Pigment Red 112 was not conducted. 

4.2    Cancer Exposure 
Although there are no animal carcinogenicity studies available in the database, C.I. Pigment Red 112 tested negative in mutagenicity assays and no evidence of specific target organ toxicity was observed in the repeat dose studies. Based on the available toxicity data, C.I. Pigment Red 112 not expected to be carcinogenic. Therefore, a cancer dietary exposure assessment is not necessary to assess cancer risk. 

4.3    Residential (Non-Occupational) Exposure 
The term "residential exposure" is used in this document to refer to non-occupational, non-dietary exposure (e.g., for lawn and garden pest control, indoor pest control, termiticides, and flea and tick control on pets). The proposed use of C.I. Pigment Red 112 as a seed treatment/dye under 40 CFR 180.920 is not expected to result in residential exposure to this chemical. In addition, there are no toxicological effects of concern in available studies and therefore, it is not necessary to conduct a quantitative assessments of residential (non-occupational) exposures and risks. 

4.4    Occupational Exposure 
Typically, seeds used in the propagation of agricultural crops are treated with one or more pesticide products. By regulation, seeds treated with a pesticide, with the exception of those used "solely as at-planting or hopper box treatments" or "products which are gaseous in form or are used as fumigants",  must also be treated with a "colorant" to indicate that the seeds are, in fact, treated with a pesticide (40 CFR 153.155). This is generally done in a closed system with minimal worker exposure. According to the Federal Seed Act section 201.31a (d), seeds that have been chemically treated may NOT be used for food, feed, or oil processing. 

The Agency has reviewed the available toxicological information for C.I. Pigment Red 112 and there are no adverse toxicological effects observed in the studies submitted at the limit dose of 1000 mg/kg/day (highest dose tested), therefore a quantitative occupational risk assessment is not necessary. 

5.0       AGGREGATE RISK 

5.1    Acute Aggregate Risk
There was no hazard attributable to a single exposure seen the in the toxicity database for the C.I. Pigment Red 112; therefore an acute aggregate risk assessment is not required.
 
5.2    Short-term/Intermediate Aggregate Risk
Short-term aggregate exposure takes into account short-term residential exposure plus chronic exposure to food and water (considered to be a background exposure level). Since there are no oral, dermal or inhalation toxicological endpoints of concern, it is not necessary to aggregate dermal and inhalation residential exposures with estimated dietary exposures. 

6.0       CUMMULATIVE EXPOSURE 

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider "available information" concerning the cumulative effects of a particular pesticide's residues and "other substances that have a common mechanism of toxicity." EPA has not found C.I. Pigment Red 112 to share a common mechanism of toxicity with any other substances, and C.I. Pigment Red 112 does do not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has assumed that the C.I. Pigment Red 112 does not have 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 EPA's website at http://www.epa.gov/pesticides/cumulative. 
 7.0       ENVIRONMENTAL FATE 

In determining the environmental fate of C.I. Pigment Red 112 the Agency considered physical/chemical properties (Table 1) and modeled data from the EPISuite v4.11 the PBT Profiler (Persistent, Bioaccumulative, and Toxic Profiles Estimated for Organic Chemicals).

C.I. Pigment Red 112 is a solid material with a low solubility in water (9.8ug/L) and is not expected to volatilize (Vapor Pressure 8.51 x10-11 mm Hg; Henry's Law Constant estimated 1.082E -12 atm-m[3]/mole). Based on the results of the Level III Fugacity Model (Table 3) C.I. Pigment Red 112's highest affinity would be for sediment (52%) and soil (47%). As a result of the low water solubility coupled with the projected partitioning of C.I. Pigment Red 112, C.I. Pigment Red 112 is expected to be bound to soil and therefore, not expected to leach into ground water, be found in runoff or be taken up by the plant. Although C.I. Pigment Red 112 may persist in soil (EPISuite and PBT Profiler Estimate), due to the small amount of pigment used compared to the weight of the seeds used per acre, deposition in soil is not expected to significantly impact soil organisms. Bioaccumulation is also expected to be low (Log Kow 2.5). 





                       Table 3: Level III Fugacity Model
Media
Mass Amount (%)
Half-Life (days)
Air
0.026
0.41
Water
0.943
180
Soil
47
360
Sediment
52
1620

 

 
 
 8.0      ECOLOGICAL EFFECTS
 8.1    Invertebrate Toxicity 
Daphnia magna Reproduction Test
A 21-day Reproduction Test (OECD 211) was performed with Daphnia magna (ten/dose) under semi-static conditions. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was <1 %, which is seen to be not biologically significant. Therefore, a nominal loading of 1 mg/L showed no biologically or statistically significant effects including mortality, immobilization, and reproduction. The No Observed Effect Loading (NOEL) and the Lowest Observed Effect Loading (LOEL) were assessed at 1 mg/L and >1 mg/L, respectively. (MRID 49148212)

Earthworm Reproduction Test-Eisenia fetida
An 8-week Earthworm Reproduction Test (OECD 222) was carried out under static conditions to determine the lethal and sublethal effects (i.e., mortality, biomass and reproductive performance) of C.I. Pigment Red 112 on the earthworm species Eisenia fetida (10/replicates(8)/dose) treated with a limit test item concentration of 1000 mg/kg dry soil. Adult earthworms were placed on artificial soil treated with the limit test item concentration as well as on artificial soil serving as control and on control soil which was treated with the solvent. Application of the test item was carried out prior to introducing the earthworms into the test vessels. After the first 4 weeks of the test, mortality and biomass of the adult worms were determined. After a further 4 weeks the reproduction rate was determined by counting the hatched juveniles. Under the conditions of this study C.I. Pigment Red 112 did not induce mortality or pathological symptoms of adult earthworms after exposure to the test item at a concentration of 1000 mg CI/kg soil dry weight for 28 days. The biomass increase of the adult earthworms after 28-days was not statistically significantly reduced compared to the pooled control at the limit test item concentration of 1000 mg/kg soil dry weight. After 8-weeks of exposure earthworm reproduction was not reduced, but instead increased at the limit test concentration of 1000 mg CI/kg soil dry weight.

Thus, the No Observed Effect Concentration (NOEC) with respect to mortality, body weight and reproduction of Eisenia fetida after 8 weeks of exposure in artificial soil was determined  to be at 1000 mg/kg soil dry weight (highest dose tested) and the Lowest Observed Effect Concentration (LOEC) was >1000 mg/kg soil dry weight. (MRID 49148213)




8.2    Algal Toxicity 

Alga Growth Inhibition Test-Desmodesmus subspicatus
A 72-hour Alga Growth Inhibition Test (OECD 201) was conducted under static conditions with unicellular freshwater green alga species Desmodesmus subspicatus to determine the effect of C.I. Pigment Red 112 on growth rate and yield. Six replicated were tested with the limit concentration (1mg/L) and the control. The NOEC was 1 mg/L. The LOEC and EC50 were >1 mg/L. (MRID 49148211)


9.0       RISK CHARACTERIZATION   

The Agency is unaware of any current residential or pesticidal uses for C.I. Pigment Red 112. C.I. Pigment Red 112 is primarily used in industrial applications, such as a coloring agent for paints, plastics and inks. Under the proposed use pattern C.I. Pigment Red 112 will be uses in seed treatment at no more than 10% w/w to identify seeds that have been treated with a pesticide. These seeds will not be used for food, feed, or oil processing. 

Based on the physical and chemical properties of C.I. Pigment Red 112 it is unlikely that C.I. Pigment Red 112 will be absorbed in the body and therefore, it is unlikely that C.I. Pigment Red 112 becomes systemically bioavailable after exposure. In addition, azo reduction is likely to occur at low levels following oral ingestion limiting the potential for absorption and the subsequent formation of metabolites. It is expected that C.I. Pigment Red 112 will pass though the body and be excreted in the feces. The solubility of C.I. Pigment Red 112 in octanol is 3.31 mg/L resulting in a calculated Log Kow of 2.5 which would indicated that the potential for bioaccumulation would be low. 

Acute studies revealed low oral and dermal toxicity (Toxicity Category IV). When tested on rabbits, the chemical was shown to be non-irritating to the skin and eyes. Skin sensitization studies in guinea pigs showed that C.I. Pigment Red 112 was not skin sensitizer.

In a 28-day oral toxicity study Crl:WI(Han) rats were gavaged with 0, 100, 300, or 1000 mg/kg/day for 28 days. Animals were observed daily for clinical signs; body weight and food consumption were recorded weekly; and selected neurological evaluations (i.e., hearing ability, pupillary reflex, static righting reflex, grip strength, and motor activity testing) were performed at the end of Week 1, 2, 3, and 4. Gross and microscopic examinations were conducted at study termination. Treatment-related effects were limited to red staining of feces in all treated males and females and staining of body surfaces, but this pigmentation was not apparent in the slides of tissues examined microscopically and was not considered adverse. No treatment-related changes were noted in neurological evaluations, body weights, food consumption, hematology or clinical chemistry analyses, or organ weights. The NOAEL was 1000 mg/kg/day (highest dose tested), and a LOAEL was not established.

Although there were no reproductive or developmental toxicity studies available for C.I. Pigment Red 112, the 28-day oral study in rats included endpoints specific to reproductive toxicity including organ weights, gross pathology and histopathology. The study showed that there were no treatment-related effects on relative and absolute organ weights for the testes or epididymides. Gross pathology of the reproductive organs did not reveal any treatment-related effects. Furthermore, histopathology of epididymides, prostate gland, seminal vesicles, testes, ovaries, vagina, cervix, and uterus demonstrated no effect at doses up to 1000 mg/kg/day. In addition to this study DEREK modeling was conducted and did not indicate any structural alerts for reproductive toxicity or endocrine-related toxicity.

Three mutagenicity studies were reviewed in this risk assessment: a reverse gene mutation study, an in vitro cell mutagenicity study, and a Chinese hamster cell in vitro study. In the reverse gene mutation assay S. typhimurium strains TA98, TA100, TA1535, and TA1537 were exposed to C.I. Pigment Red 112 dissolved in Dimethyl sulfoxide (DMSO) in two independent assays using triplicate plating. Experiment 1 used the standard plate assay with and without S9 and a preincubation assay up using S9 up to 5000 ug/plate. The confirmatory mutagenicity assay (experiment 2) tested mutagenesis and cytotoxicity under exactly the same three sets of conditions as in experiment 1. C.I. Pigment Red 112 was tested at concentrations up to the limit concentration of the assay in both experiments. There was no evidence that the assays were affected by cytotoxicity and no evidence of induced mutant colonies over background.

In an in vitro mammalian cell gene mutation assay Chinese hamster V79 cells cultured in vitro were exposed to C.I. Pigment Red 112 dissolved in DMSO and culture medium for 4 hours at concentrations of 0, 3.1, 6.3, 12.5, 25.0, 50.0, and 400 ug/mL in the absence or the presence of metabolic activation in the first experiment or for 24 hours at concentrations of 0, 3.1, 6.3, 12.5, 25.0, 50.0, and 400 ug/mL in the absence of metabolic activation in the second experiment. There was no evidence of any cytotoxicity and no significant increase in mutant frequency over that of the solvent controls. 

In an in vitro test on the induction of micronuclei in cultured V79 Chinese hamster cells, V79 cell cultures were treated with Pigment Red 112 dissolved in DMSO, for 4 or 24 hrs without activation, and for 4 hrs with activation. The preparation interval was 24 hrs in all tests. In order to assess the toxicity a cell Proliferative Index (PI) was calculated. Only those cultures which showed a PI > 1.3 were evaluated. The test article was found to cause no significant increases in micronucleus induction in cultured V79 cells without or with activation at the concentrations that were tested. However, the highest concentration tested with activation was well below a precipitating concentration, and no data were presented to indicate that higher concentrations were cytotoxic.

Although the full study reports were not available, the IUCLID Chemical Safety Report for C.I. Pigment Red 112 illustrates two additional Bacterial Reverse Mutation Assays which support the studies presented above. In both studies no evidence of mutagenicity or cytotoxicity was seen with and without metabolic activation in S. typhimurium TA 1535, 1537. 98, or 100 or E.coli WP uvr A. 

No neuropathological changes or effects were reported in any of the studies. The agency does not believe C.I. Pigment Red 112 would be neurotoxic. No carcinogenicity studies are available for C.I. Pigment Red 112. Although DEREK modeling results indicate "plausible" alerts for carcinogenicity, this is unlikely to be the case because C.I. Pigment Red 112 is not expected to be absorbed in the body based on the physical/chemical properties of the chemical. Many azo dyes contain benzidine in their structure and this human bladder carcinogen may be released upon azo reduction of the dye. In the case of C.I. Pigment Red 112, azo reduction is likely to occur at low levels following oral ingestion limiting the potential for the formation of genotoxic and carcinogenetic metabolites. Similarly, all mutagenicity studies on C.I. Pigment Red 112 were negative which makes it unlikely that C.I. Pigment Red 112 would be carcinogenic.

Available toxicity studies indicate that C.I. Pigment Red 112 has a very low acute and subchronic toxicity. No endpoint of concern was identified for any of the acute studies conducted. In addition, no endpoint of concern was determined in the 28-day study up to the limit dose of 1000 mg/kg/day (highest dose tested). Since no endpoint of concern was identified in acute and subchronic studies and because C.I. Pigment Red 112 is not expected to be absorbed by the body based on the physical/chemical properties, a quantitative risk assessment for C.I. Pigment Red 112 was not performed. As part of its qualitative assessment, the Agency did not use safety factors for assessing risk, and no additional safety factor is needed for assessing risk to infants and children. Furthermore, C.I. Pigment Red 112 will be used at a maximum of 10% w/w in seed treatments only. It is expected to be tightly bound to soil so the likelihood of consumers being exposed to C.I. Pigment Red 112 as an inert ingredient through foliar ingestion or water consumption is nominal.

Although dietary exposure to C.I. Pigment Red 112 may occur from eating foods grown from seeds treated with pesticide formulations containing this inert ingredient and drinking water containing runoff from soils containing the treated seeds, the amount of residue that a consumer would be exposed to would be relatively small because C.I. Pigment Red 112 is expected to be tightly bound to soil and therefore, it is unlikely the pigment would be taken up by the plant and subsequently available for consumption through eating the plant. Seeds that have been chemically treated may NOT be used for food, feed, or oil processing. In addition, runoff is unlikely based on the low water solubility and soil binding of the pigment. There are no other known food uses for C.I. Pigment Red 112. 

The proposed use of C.I. Pigment Red 112 as a seed treatment/dye under 40 CFR 180.920 is not expected to result in residential exposure to this chemical. In addition, there are no toxicological effects of concern in available studies and therefore, it is not necessary to conduct a quantitative assessments of residential (non-occupational) exposures and risks. Similarly, a quantitative occupational risk assessment is not necessary. 

Based on ecological (i.e., earthworm, water flea, and algae) and fate data the agency concluded that C.I. Pigment Red 112 does not pose an ecological risk to terrestrial or aquatic species. C.I. Pigment Red 112 is a solid material with a low solubility in water and is not expected to volatilize. Based on the results of the Fugacity Model C.I. Pigment Red 112's highest affinity would be for sediment (52%) and soil (47%). As mentioned above, as a result of the low water solubility coupled with the projected partitioning of C.I. Pigment Red 112, it is expected to be bound to soil and therefore, not expected to leach into ground water or be found in runoff. Although C.I. Pigment Red 112 may persist in soils, due to the small amount of pigment used compared to the weight of the seeds used per acre, deposition in soil is not expected to significantly impact soil organisms. Bioaccumulation is also expected to be low. 

Based on the evaluated toxicity studies coupled with the expected exposure from the use of these chemicals as inert ingredients in pesticide products, EPA concludes that there is a reasonable certainty that no harm will result to the general population, including infants and children, from aggregate exposure to residues of C.I. Pigment Red 112 when used as an inert ingredient in pesticide formulations for seed treatment at a maximum of 10% w/w under 40 CFR 180.920.




                                 Bibliography
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ChemIDPlus. (2013) U.S. National Library of Medicine. National Institutes of Health, Department of Health & Human Services. Retrieved on January 2, 2014 from http://chem.sis.nlm.nih.gov/chemidplus

Clariant Corporation. (2010) IUCLID Chemical Safety Report for 3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl) azo]naphthalene-2-carboxamide; CAS RN 6535-46-2. July 14, 2010. (MRID 49242001)

Dynterova, A. (2007) (Inert Ingredient) Acute Dermal Toxicity (in Rats). Unpublished study prepared by Research Institute for Organic Syntheses. Project Number: 07122. June 15, 2007. (MRID 49148202)

Ehling, G. (2002) (Inert Ingredient) Guinea Pig Sensitization (Buehler Test). Unpublished study prepared by Aventis Pharma Deutschland GmbH. Project Number: PT02-0042. June 27, 2002. (MRID 49148206)

Environment Canada, Health Canada (2013) Draft Screening Assessment: Aromatic Azo and Benzidine-based Substance Grouping, Certain Monoazo Pigments. November 2013. Retrieved on February 12, 2014 from http://www.ec.gc.ca/ese-ees/default.asp?lang=En&n=9C4DA306-1

Hall, C. (2009) In vitro Micronucleus Test in Chinese Hamster V79 Cells with Pigment Red 112. Unpublished study prepared by Harlan Cytotest Cell Research Gmbh. Project Number: 1282200. September 30, 2009. (MRID 49148208)

Krome, K. (2010) Pigment Red 112 Earthworm (Eisenia fetida), Effects on Reproduction. Unpublished study prepared by Dr. U. Noack-Laboratorien. Study Number RBN13439. March 5, 2010. (MRID 49148213)

Levine, W. (1991) Metabolism of Azo Dyes: Implication for Detoxification and Activation. Drug Metabolism Reviews 23(3&4): 235-309. (MRID 49242002)

Meurer, K. (2008) Local Lymph Node Assay (LLNA) in Mice with Pigment Red 112. Unpublished study prepared by RCC Cytotest Cell Research Gmbh. Project Number: 1131902. January 25, 2008. (MRID 49148205)

Muller, W. (1996) (Inert Ingredient): Bacterial Reverse Mutation Test (Standard Plate Test) and Prival Modification (Preincubation Test). Unpublished study prepared by Hoechst Aktiengesellschaft. Project Number: 96.0210. April 10, 1996. (MRID 49148207)

Noack, M. (2010) Pigment Red 112 Daphnia magna Reproduction Test, Limit Test, Semi-Static, 21 d. Unpublished study prepared by Dr. U. Noack-Laboratorien. Study Number DRE13439. January 3, 2010. (MRID 49148212)

Rupprich, N.; Weigand, W. (1983) Permanent Red FRG Test of the Acute Oral Toxicity in the Male and Female Wistar-Rats. Unpublished study prepared by Hoechst Aktiengesellschaft. Project Number: 83.0401. July 27, 1983. (MRID 49148201)

Rupprich, N.; Weigand, W. (1983) (Inert Ingredient) Test for Acute Dermal Irritation/Corrosion on the Rabbit. Unpublished study prepared by Hoechst Aktiengesellschaft. Project Number: 83.0375. July 21, 1983. (MRID 49148203)

Rupprich, N.; Weigand, W. (1983) (Inert Ingredient) Test on Acute Eye Irritation/Corrosion (in Rabbits). Unpublished study prepared by Hoechst Aktiengesellschaft. Project Number: 83.0376. July 21, 1983. (MRID 49148204)

Scheerbaum, D. (2010) Pigment Red 112 Alga, Growth Inhibition Test with Desmodesmus subspicatus, 72 h. Unpublished study prepared by Dr. U. Noack-Laboratorien. Project Number: 090707CP. March 11, 2010. (MRID 49148211)

Ullmann, L. (2002) (Inert Ingredient): Local Lymph Node Assay (LLNA) in Mice (Identification of Contact Allergens). Unpublished study prepared by RCC Umweltchemie Ag. Study Number: 856583. January 8, 2002. (MRID 49148214)

van Otterdijk, F. (2008) Repeated Dose 28-Day Oral Toxicity Study with Pigment Red 112 by Daily Gavage in the Rat. Unpublished study prepared by Notox B.V. Project Number: 487395. June 2, 2008. (MRID 49148210)

Wollny, H. (2008) Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) with Pigment Red 112. Unpublished study prepared by RCC Cytotest Cell Research Gmbh. Project Number: 1154200. June 5, 2008. (MRID 49148209)

  
