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


							OFFICE OF CHEMICAL SAFETY 								AND POLLUTION PREVENTION



MEMORANDUM  

DATE:		June 3, 2021

SUBJECT:      IN-11384; Pyrrolo[3,4-C]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- 
            Human Health Risk and Ecological Effects Assessment of a Food Use Pesticide Inert Ingredient 
 
            CAS Reg. No.: 84632-65-5 
                  PC Code:  790437     
                  Decision:  558542

FROM:	Deirdre Sunderland, MHS 
      Industrial Hygienist
		Chemistry, Inerts & Toxicology Assessment Branch (CITAB)
		Registration Division (RD) 
	

TO:		Kerry B. Leifer 
      Branch Chief
		CITAB/RD 




 EXECUTIVE SUMMARY

In December 2019, BASF Corporation submitted a petition (IN-11384) to the Environmental Protection Agency (herein referred to as EPA or the Agency) requesting an exemption from the requirement of a tolerance for pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- (CAS No. 84632-65-5), hereby referred to as `Pigment Red 254' under 40 CFR § 180.910 when used as a pesticide inert ingredient pre- and post-harvest. Pigment Red 254 is currently approved as a nonfood inert ingredient when used as a pigment in antimicrobial paint formulations at a maximum end-use concentration of 225 ppm (0.0225%) w/w. In addition, Pigment Red 254 has various non-pesticidal uses in consumer and industrial products as a colorant and is also approved by the U.S. Food and Drug Administration (FDA) as an indirect food additive (colorant) in food contact polymers at levels not to exceed 1.0% by weight of the polymer (21 CFR §178.3297).

A metabolism study on Pigment Red 254 showed that by 168 hours, it was almost completely eliminated in the feces. The majority was excreted within 24 hours with very little found in the urine, blood or tissue, indicating that Pigment Red 254 is not likely to be bioavailable. This is further supported by the lack of adverse effects seen in the acute and repeat dose toxicity studies. 

Acute toxicity studies conducted with Pigment Red 254 indicate low toxicity via the oral (LD50 > 5000 mg/kg), dermal (LD50 > 2000 mg/kg), and inhalation (LC50 > 2.25 mg/l) routes of exposure. Pigment Red 254 was non-irritating to the eyes and skin of rabbits. It is also not a dermal sensitizer in guinea pigs. 

In a 28-day repeated dose oral toxicity study in rats treated with Pigment Red 254 up to 1000 mg/kg/day, the only effects observed as a result of treatment were red extremities and feces in the high-dose group due to the color of the test article. No other treatment related effects were observed. Therefore, the No-Observe-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day (the highest dose tested). 

A reproductive/developmental toxicity screening test on a surrogate chemical, Irgazin Orange EH 1287, showed no parental, reproductive, or developmental toxicity up to the highest dose tested (1000 mg/kg/day). Therefore, the parental, reproductive, and developmental NOAEL in this study was 1000 mg/kg/day. Pigment Red 254 was negative for genotoxicity and mutagenicity in various in vitro and in vivo studies. It is not expected to be a carcinogen nor is it expected to be neurotoxic or immunotoxic. 

Based on the current and proposed use pattern of Pigment Red 254, dietary and residential exposure from pesticidal uses are expected to be low. In addition, no endpoint of concern was identified in the database below the limit dose of 1000 mg/kg/day; therefore, a quantitative assessment of human exposure was not necessary. 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. 

Pigment Red 254 is not expected to readily biodegrade in the environment. However, the exposure from the use of Pigment Red 254 in antimicrobial paints at 225 ppm and as a pigment for seed treatment, as proposed, is expected to be negligible and therefore, does not present a hazard for the environment or add significantly to the existing exposure from baseline consumer and industrial used of Pigment Red 254. Pigment Red 254 was non-toxic to aquatic systems and earthworms. 

Overall, the low toxicity of Pigment Red 254, coupled with the negligible exposure from the current and proposed use as inert ingredients in pesticide products, indicate that residues of this compound do not pose a safety risk at aggregate exposure levels, including exposure expected to result from the proposed uses as an inert ingredient in pesticide formulations. Based upon the factors summarized above, the Agency is establishing an exemption from the requirement of a tolerance for pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- (CAS No. 84632-65-5) (i.e., Pigment Red 254) under 40 CFR § 180.910 as an inert ingredient in food use pesticide formulations pre- and post- harvest. 
 BACKGROUND

In December 2019, BASF Corporation submitted petition IN-11384 to the EPA requesting an exemption from the requirement of a tolerance for pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- (CAS No. 84632-65-5), also referred to as `Pigment Red 254', under 40 CFR § 180.910 when used as a pesticide inert ingredient pre- and post-harvest. Pigment Red 254 is currently approved as an inert ingredient for nonfood use as a pigment in antimicrobial paint formulations at a maximum end-use concentration of 225 ppm (0.0225%) w/w. Pigment Red 254 is approved by the FDA for use as an indirect food additive (colorant) in food contact polymers at levels not to exceed 1.0% by weight of the polymer (21 CFR §178.3297). It is intended for use in finished articles that contact food under FDA conditions of use B through H described in 21 CFR § 176.170(c).

Pigment Red 254 also has widespread use in consumer products, by professional workers, in formulation or re-packing, at industrial sites and in manufacturing end use products such as: coating products, inks and toners, and polymers. Due to its resistance to degradation by light, heat, solvents, and weather, it is popular in the manufacture of paints for automobiles and other outdoor uses. 

 PHYSICAL/CHEMICAL PROPERTIES 

Table 1 lists the available physical and chemical properties for Pigment Red 254. 

           Table 1: Physical/Chemical Properties of Pigment Red 254
Characteristic
Value
Source 
Synonyms 
1) 1,4-bis(4-chlorophenyl)-3-hydroxy-2H-pyrrolo[3,4-c]pyrrol-6-one
2) Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-
3) Pigment Red 254
ChemID*
Structure


Multiple
Structural Formula
C18H10Cl2N2O2 
ChemID*
Molecular Weight
357.195
ChemID*
Appearance
Red solid, powder
Various Studies
Relative Density 
 1.58
MRID 51595401
Melting point
>300 °C
MRID 51595402
Boiling point 
597.9°C

USEPA EPISuite v.4.10
Vapor Pressure
2.36 x 10[-13] mmHg @ 25°C 
USEPA EPISuite v.4.10
Water Solubility 
0.1 mg/L at 20°C
ECHA
Log Kow
5 at 20°C
ECHA
*ChemID: https://chem.nlm.nih.gov/chemidplus/rn/startswith/84632-65-5
                                                                               
 METABOLISM

In an OECD 417 toxicokinetics study (MRID 51027015), the absorption, distribution, and excretion of Pigment Red 254 were evaluated when administered orally at two target dose levels (i.e., 100 mg/kg and 1000 mg/kg) to male rats. Radioactivity in the urine, feces, blood/plasma, and organs/tissues was followed for 168 hours. Due to lack of solubility of the test article, the amount of Pigment Red 254 in the stock solutions was indirectly determined by means of weighing. The primary route of excretion for Pigment Red 254 is via the feces. 

The amount present in the feces after 168 hours, on average, was 119.7 % and 96.0 % at the low and high dose level, respectively. After 24 hours, fecal excretion already accounted for 101.4 % (low dose level) and 79.7 % (high dose level) of the radioactivity administered. Together with the cage wash (1.8 % and 7.7 %), total excreted radioactivity amounted to 121.5 % and 104.3 % at the low and high dose level, respectively. 

At 168 hours post-administration, 0.3 % and 0.6 % of radioactivity was found in the urine at the low and high dose level, respectively. After 24 hours, radioactivity in the urine had already reached 0.3 % and 0.4 %, respectively, and according to the study author "may for the most part originate from a contamination via the feces". At both dose levels, negligible amounts of radioactivity (<0.05 %) were found in the blood, organs/tissues, intestinal tract, and residual body.  

At the low and high dose level, maximal levels of radioactivity (Cmax) in blood (0.139 μg/g and 1.152 μg/g, respectively) and plasma (0.140 μg/g and 1.048 μg/g, respectively) were reached one or two hours after the administration. These Cmax-values were below two times the limit of quantitation. Already 4 to 12 hours after the administration, radioactivity levels in blood and plasma were below the limit of quantitation. Because of the low radioactivity levels found in blood and plasma, no elimination kinetics and no area under the curves could be calculated. 

Based on the resultant amount of Pigment Red 254 in the feces, urine, and tissues of test animals, it appears that oral absorption of Pigment Red 254 is negligible and therefore, it is not expected to be bioavailable. As similarly noted in EPA's 2012 nonfood use risk assessment for Pigment Red 254, due to its low solubility in water and low solubility in fat (<0.01 g/100 g fat simulant were soluble at 37° C (MRID 49390901)), Pigment Red 254 "does not seem likely to be significantly biologically available in mammalian systems". 

  -  - TOXICOLOGY
                                                                               
This section contains data on acute and repeat dose toxicity studies as well as information on genotoxicity and reproductive/developmental toxicity. A summary of the acute, repeat dose, and reproductive/developmental toxicity data can be found in Section 5.4, Table 2. No reproductive/developmental studies were available for Pigment Red 254, however, data on a surrogate chemical, Irgazin Orange EH 1287, was used to assess for the reproductive/developmental toxicity potential of Pigment Red 254. Irgazin Orange EH 1287 was determined to be an acceptable surrogate for Pigment Red 254 because of its comparable chemical structure (see Figure 1 below) and physical/chemical properties. 

                                  Figure 1-Irgazin Orange EH 1287

When appropriate, the OCSPP Test Guideline number and/or the OECD Guideline for the Testing of Chemicals study numbers are provided. The OCSPP Test Guidelines can be found at https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/final-test-guidelines-pesticides-and-toxic and the OECD guidelines can be accessed through https://www.oecd.org/chemicalsafety/testing/oecdguidelinesforthetestingofchemicals.htm. Many of the studies were conducted in the mid- to late-1980s so testing standards may have been different.

 Acute Toxicity

Data on acute toxicity, as well as data on eye and skin irritation and skin sensitization, are discussed in this section. 

 Acute Oral Toxicity 
                                                                               
An OECD 401 guideline study was conducted on ten KFM-Han. Wistar rats (5/sex) dosed with a single oral gavage dose of 5000 mg/kg of Pigment Red 254 and observed for 15 days. (MRID 51027001) No deaths related to treatment were observed during the study. Clinical signs included sedation, rales (males), dyspnea, curved body position, and ruffled fur. All animals recovered within 3 days of treatment. No macroscopic organ changes were observed. The acute median lethal dose (LD50) was found to be >5000 mg/kg.
 Acute Dermal Toxicity
An acute dermal toxicity study in rats was conducted according to OECD 402. (MRID 51027002) A single topical application of 2000 mg/kg of Pigment Red 254 was applied to ten KFM-Han. Wistar rats (5/sex). The treatment area (approximately 10% of the total body surface) was covered for 24 hours then rinsed, dried, and observed for 15 days. 
        
No deaths occurred during the study. All rats showed a reddish discoloration on the treated skin during the observation period. No other signs of local or systemic toxicity was observed. The acute median lethal dose (LD50) was >2000 mg/kg.

 Acute Inhalation Toxicity

In an OECD 403 guideline study albino Tif: RAI f (SPF)) (Hybrids of RII/1 x RII/2) rats (5/sex) were exposed to nose only Pigment Red 254 at a mean exposure concentration of 2252 mg/m[3] for a four-hour single exposure. Animals were observed for 14 days post administration. Due to the properties of the test material, it was not possible to generate higher concentrations. The mass median aerodynamic diameter (MMAD) was reported as 0.4 um with a geometric standard deviation (GSD) of 3.6-5.2. 
        
Symptoms included piloerection, hunched posture, and dyspnea but these symptoms recovered within 5 days. No mortality occurred. No remarkable findings were noted during the necropsy. Histopathological examinations of the lungs revealed minimal congestion. The minimal emphysema and the minimal and multifocal bronchiolar dilatation seen in all animals is a common response in rats treated by inhalation and it was regarded not to be treatment related. The LC50 was determined to be > 2250 mg/m[3] (2.25 mg/L). (MRID 51027003) 
 Skin Irritation 
A primary skin irritation study (OECD 404; MRID 51027007) was performed with 3 New Zealand White rabbits. The test substance (0.5 g) was moistened with polyethylene glycol (PEG 400) and applied under an occlusive patch to a 10 cm x 10 cm area of intact skin for 4 hours. The site was then washed, and observations were made 1, 24, 48, and 72 hours after removal of the test article.  

Except for the severe red discoloration, most likely caused by the test substance, there was no treatment related effects (i.e., erythema, edema, or corrosion) seen throughout the study. Pigment Red 254 was considered non-irritating to the skin. 
 Eye Irritation 
An OECD 405 guideline study was performed using New Zealand White rabbits to assess the eye irritation potential of Pigment Red 254. (MRID 51027006) Three rabbits were given a single ocular dose of 0.1 g and observed for three days. 

At 1-hour post installation some of the test article remained in the eye but after 24 hours there was no discoloration of the cornea and conjunctivae of the treated eyes. No corrosion was observed. Under the conditions of this study Pigment Red 254 was non-irritating to the rabbit's eye
 Skin Sensitization 
Pigment Red 254 was evaluated for skin sensitization potential according to OECD 406. (MRID 51027008) Guinea pigs (Pirbright White Strain (Tif: DHP)) (10/sex/group) were treated first with intradermal injections into the neck region and then a closed patch exposure (0.4 g paste, 2 x 4 cm) over the injection sites one week later which remained occluded for 48 hours. The intradermal injections were done in 3 pairs (0.1 ml per injections). 
None of the animals in the test group showed skin reactions 24 or 48 hours after removing the dressings. Under the conditions of this study, Pigment Red 254 was not a skin sensitizer in guinea pigs.
 Repeat Dose Toxicity

In an OECD 407: 28-day oral toxicity study (MRID 51027009), Pigment Red 254 was administered via gavage to four groups of SPF-bred Wistar rats (5/sex/dose) at concentrations of 0, 100, 300, or 1000 mg/kg/day. Two females, one in the control group and one in the 300 mg/kg/day group, died spontaneously on days 17 and 20, respectively. These deaths are mostly likely due to an intubation error. There were no test substance-related deaths. 

There were no changes in clinical observations, neurological parameters, food consumption, body weight gain, or gross pathology. In addition, there were no changes in hematological or clinical chemistry parameters, urinalysis, organ weights, or macroscopic pathology. "All pathology findings recorded, were of a spontaneous nature common in rats of this age and strain." There was no evidence of abnormal histopathological finding resulting from treatment with Pigment Red 254. 

The No Observed Adverse Effect Level (NOAEL) for Pigment Red 254 in this study is 1000 mg/kg/day, the highest dose tested. 

 Reproductive and Developmental Toxicity
In an OCSPP 870.3550/OECD 421 Reproduction/Developmental Toxicity Screening Test (MRID 51336201), Irgazin Orange EH 1287 (a surrogate of Pigment Red 254) was administered to four groups of Wistar Han rats (10/sex/dose) via gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day. Males were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 40-45 days (2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation). 

Evaluated parameters included mortality/viability, daily clinical observations, body weight and food consumption, organ weights, and histopathology on a selection of tissues. Hematology and clotting tests (Week 4, males only) and macroscopy at termination were evaluated. Reproduction/developmental parameters consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio, and early postnatal pup development (mortality, clinical signs, body weights and macroscopy) were also evaluated. 

No parental, reproductive, or developmental toxicity was observed up to the highest dose tested. Based on these results, a parental, reproduction, and developmental NOAEL of 1000 mg/kg/day was derived. 






 Toxicology Summary Profile
               Table 2:  Toxicology Profile for Pigment Red 254
                                  Study Type
                                     Doses
                                    Results
                                     Acute
Acute Oral Toxicity (rat)
0 or 5000 mg/kg
LD50 >5000 mg/kg
Acute Dermal Toxicity (rat)
0 or 2000 mg/day 
LD50 >2000 mg/kg
Acute Inhalation Toxicity (rat)
2252 mg/m[3]
LD50 > 2.25 mg/L 
Skin Irritation (rabbit)
0.5 g
No signs of irritation were observed
Eye Irritation (rabbit)
0.1 g
No signs of irritation were observed
Skin Sensitization (guinea pig)
Patch 0.4 g

No signs of sensitization were observed
                                  Repeat Dose
28-Day -Oral Toxicity: Gavage (rat)
0, 100, 300, or 1000 mg/kg/day 
NOAEL = 1000 mg/kg/day  
LOAEL = not determined
                          Reproductive/Developmental
Reproduction/ Developmental Toxicity Screening Test: Gavage (rat)
0, 100, 300, or 1000 mg/kg/day
Maternal NOAEL = 1000 mg/kg/day  
Maternal LOAEL = not determined
Developmental/Offspring NOAEL = 1000 mg/kg/day
Developmental/Offspring LOAEL = not determined 
Reproductive NOAEL = 1000 mg/kg/day
Reproductive LOAEL = not determined
 Genotoxicity and Mutagenicity
Various in vitro and in vivo studies have shown that Pigment Red 254 is not genotoxic or mutagenic. These studies are detailed below. 
 In vitro
  Reverse Gene Mutation
In a reverse gene mutation assay in bacteria (OECD 471; MRID 51027012), strains TA98, TA100, TA102, TA1535, and TA1537 of S. typhimurium were exposed to Pigment Red 254 at concentrations of 20, 78, 313, 1250, and 5000 ug/0.1 ml per plate in the presence and absence of microsomal activation. 

Pigment Red 254 was tested up to a limit concentration of 5000 ug/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
  Reverse Gene Mutation
In a second bacterial reverse gene mutation assay (OECD 471; MRID 51027013), strains TA1535, TA1537, TA98, and TA100 of S. typhimurium and E. coli WP2 uvrA were exposed to Pigment Red 254 diluted in dimethyl sulphoxide (DMSO) which were applied to plates in concentrations of 15, 50, 150, 500, 1500, or 5000 ug/plate in the presence and absence of metabolic activation. Pigment Red 254 was tested up to a limit concentration of 5000 ug/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
  Mammalian Cell Gene Mutation Assay
In a mammalian cell gene mutation assay (OCSPP 870.5300/OECD 476; MRID 51027014) in thymidine kinase locus (TK[+/-]), mouse lymphoma L5178Y TK[+/-] 3.7.2 C cells cultured in vitro were exposed to Pigment Red 254 at concentrations of 0, 3.2, 10. 32, 100, 316, and 1000 ug/mL in the presence of mammalian metabolic activation for 3 hours and in the absence of mammalian metabolic activation (at the same concentrations) for 3 hours and for 24 hours. 

Noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. In the performed assays the obtained mutation frequencies (in absence and in the presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant global evaluation factor (GEF) thresholds for positive call, and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett's Test, α = 0.05) from that of the corresponding vehicle control at the 3-hour treatments (+-S9 Mix) and the obtained statistical significances at the 24-hour treatment at the concentration levels of 316 and 1000 μg/mL were not considered as biologically relevant for this study.
 
Under the conditions of this study, the test item Pigment Red 254 did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line). 
 In vivo
A Micronucleus test was conducted with Chinese Hamsters (Cricetulus Griseus). (MRID 51027011) Hamsters (8/sex/dose) were treated by gavage with three different doses, one receiving the maximum dose of 5000 mg/kg and the other two doses of 1/5 and 1/25 of that amount, respectively. A positive and negative control group were also included. This experiment treated the animals with the highest applicable dose of 5000 mg/kg and sacrificed them at 16, 24, and 48 hours. Bone marrow smears were prepared.

There was no significant increase in the number of micronucleated polychromatic erythrocytes in animals treated with 5000 mg/kg Pigment Red 254 compared to control animals. The positive control yielded a marked increase of the percentage of micronucleated cells. The mean percentage of polychromatic erythrocytes with micronuclei was 3.34. 

Under the study conditions utilized, no evidence of mutagenic activity was observed in Chinese Hamsters treated with Pigment Red 254.
 Carcinogenicity
Although no long-term studies are available to evaluate the carcinogenic potential of Pigment Red 254, no target organ toxicity was seen in the repeat dose study and no genotoxic or mutagenic effects were seen in any of the in vitro or in vitro tests (Section 5.5). Therefore, the Agency does not believe Pigment Red 254 is carcinogenic.

 Neurotoxicity

No neurotoxicity studies are available for Pigment Red 254; however, toxicity tests have not provided any evidence that this compound produces neurotoxic effects. 
                                                                               
 Immunotoxicity

No immunotoxicity studies are available for Pigment Red 254; however, no effects were seen in the current toxicity database that would indicate that Pigment Red 254 would be immunotoxic.

 TOXICITY ENDPOINT SELECTION

No toxicological endpoint of concern has been identified in the database below the limit dose of 1000 mg/kg/day; therefore, a quantitative assessment of human exposure is not necessary.

 SPECIAL CONSIDERATION for INFANTS and CHILDREN

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 applies 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 on Pigment Red 254 indicate that it has low toxicity. There is no indication, based upon the available data, that Pigment Red 254 is neurotoxic or immunotoxic and therefore, would not result in increased susceptibility in infants or 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. Taking into consideration all available information, there is no concern, at this time, for increased sensitivity to infants and children to Pigment Red 254 when used as inert ingredient in pesticides formulations.

 EXPOSURE
        
Pigment Red 254 is currently approved as a nonfood use pesticide inert ingredient for use as a pigment in antimicrobial paint formulations at a maximum end-use concentration of 225 ppm (0.0225%) w/w. BASF Corporation is requesting that EPA establish an exemption from the requirement of a tolerance for Pigment Red 254 when used as a dye or coloring agent in pesticide formulations under 40 CFR §180.910 for use pre- and post- harvest. 
Although dietary and residential exposure are possible through consumer and food uses, exposure data on the current pesticidal and non-pesticidal use of Pigment Red 254 is not available. Based on its current use as a pigment in antimicrobial paint formulations with a maximum end-use concentration of 225 ppm (0.0225%) w/w and the proposed use, dietary and residential exposures from use as an inert ingredient are expected to be low. In addition, no endpoint of concern was identified in the database below the limit dose of 1000 mg/kg/day; therefore, a quantitative risk assessment was not necessary.  
 Dietary Exposure
Although dietary exposure to Pigment Red 254 may occur from eating foods resulting from crops treated with pesticides containing the inert ingredient or foods containing Pigment Red 254 as a FDA food additive, no endpoint of concern was identified below the limit dose and therefore, a quantitative dietary exposure assessment for Pigment Red 254 was not conducted. 
 Residential 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 current and proposed use of Pigment Red 254, as an inert ingredient in pesticide formulations and use in consumer products, may result in residential exposures; however, there are no toxicological effects of concern below the limit dose in available studies and therefore, it is not necessary to conduct a quantitative assessment of residential (non-occupational) exposures and risks. 

 Occupational Exposure/Risk
The Agency has reviewed the available toxicological information for Pigment Red 254 found that there are no adverse toxicological effects observed below the limit dose in the studies submitted; therefore, a quantitative occupational risk assessment is not necessary. 

  AGGREGATE EXPOSURE

The Federal Food, Drug, and Cosmetic Act (FFDCA) section 408 directs EPA to consider available information concerning exposure from the pesticide residue in food and other non-occupational exposures to determine that there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information.

Because no adverse effect below the limit dose was seen in the toxicity database for Pigment Red 254, a qualitative risk assessment was conducted and subsequently, it is not necessary to aggregate dermal and inhalation residential exposures with estimated dietary exposures. 

    CUMULATIVE EXPOSURE

Cumulative effects from substances with a common mechanism of toxicity. 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 Pigment Red 254 to share a common mechanism of toxicity with any other substances, and Pigment Red 254 does do not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA assumed that Pigment Red 254 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.

    ENVIRONMENTAL FATE & EFFECT

Limited data on the environmental fate and effects of Pigment Red 254 are available. In order to predict how Pigment Red 254 might behave in the environment EPA's EPI Suite v4.1 was utilized. The EPI (Estimation Programs Interface) Suite(TM) is a Windows(R)-based suite of physical/chemical property and environmental fate estimation programs developed by EPA's and Syracuse Research Corp. (SRC) and can be found at https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-program-interface. The full EPI Suite report can be found in Appendix A. 

 Overview

EPI Suite's Fugacity model predicts that, once released to the environment, 84.1% of Pigment Red 254 would be found in the soil, 7.78% would be in the water, 8.11% would be in the sediment, and a very small percent (0.0002) would partition to air. If released to the atmosphere, Pigment Red 254 is expected to exist in mostly the solid phase and not volatilize (VP: 2.36 x x 10[-13] mmHg @ 25°C). Based on the Henry's Law Constant (2.89 x 10-15 atm-m3/mole at 25°C) Pigment Red 254 is expected to be nonvolatile from surface waters. 

The Koc indicates whether a compound is likely to partition to soil or sediments from water. The estimated log Koc values for Pigment Red 254 are 4.136 (Koc= 13660 L/kg) and 0.875 (Koc = 150 L/kg) based on EPI Suite's MCI and Kow models, respectively. These value vary greatly in their interpretation but based on the totality of information on Pigment Red 254, the Agency believes it is likely that Pigment Red 254 will bind to soils or sediments and will tend to have negligible to slow migration to groundwater. 

The water solubility (<0.1 mg/L at 20°C) and Octanol/Water Partition Coefficient (log Kow = 5 at 25°C) indicate that Pigment Red 254 it is not likely to be soluble in water. The low predicted bioconcentration factor (BCF), a ratio of a chemical's concentration in the tissue of an aquatic organism to the chemical's concentration in the ambient water, of 13.11 L/kg wet-wt indicates that there is a low potential for Pigment Red 254 to bioconcentrate in aquatic organisms. Pigment Red 254 was not readily biodegradable in two biodegradation tests (Section 11.2). 

        Table 3:  Environmental Fate Characteristics of Pigment Red 254
Parameter
Value
Source
Vapor Pressure (VP)
2.36 x x 10[-13] mmHg @ 25°C 

USEPA EPISuite v.4.10
Henry's Law Constant 

2.89 x 10[-]15 atm-m3/mole at 25°C (Bond method)
USEPA EPISuite v.4.10
Water Solubility 
<0.01 g/L at 20°C
<0.1 mg/L at 20°C
 MRID 51595403
 ECHA*
Log Kow 
 5 at 25°C
 ECHA*
Log Koc
MCI method 
Kow method

4.136
0.875

USEPA EPISuite v.4.10
Ready Biodegradability 
No
MRIDs 51027023, 51027025
BCF 
13.11 L/kg wet-wt
USEPA EPISuite v.4.10
Fugacity Model Compartment/Mass Amount (%)
Air 
Water 
Soil
Sediment
Mass Amt (%)        

0.000229
7.78
84.1
8.11
Half-Life (hr)

0.23
1440
2880
13000
USEPA EPISuite v.4.10
 *ECHA: https://echa.europa.eu/es/registration-dossier/-/registered-dossier/13744/1/1

 Biodegradation

The results of two biodegradability tests conducted with Pigment Red 254 are presented in Sections 11.2.1 and 11.2.2 below. Both tests show that Pigment Red 254 is not readily biodegradable.  

    Ready Biodegradability

The ready biodegradability of Pigment Red 254 was assessed in a Modified Sturm Test conducted under OECD 301B guidelines. (MRID 51027025) The biodegradation was calculated based on the theoretical carbon content and the cumulative quantities of carbon dioxide determined on the days that measurements were taken (i.e., day 6, 9, 13,16, 20, 23, 27, and 28). The biodegradation calculated for the 10 mg test substance/L was 10% and for the 20 mg test substance/L it was 4% in 28 days. Pigment Red 254 is not readily biodegradable in this test.

    Zahn-Wellens/Carbon Dioxide Evolution Test

The inherent biodegradability of Pigment Red 254 was assessed in a combined Zahn-Wellens/Carbon Dioxide Evolution Test. (MRID 51027023). The carbon dioxide evolution test used was a Modified Sturm Test. Biodegradation was calculated as percentage of the measured amount of carbon dioxide over the theory. Results showed 0% degradation in both the 11.5 mg and 20.1 mg test substance/L after 28 days. Therefore, Pigment Red 254 is not biodegradable in this test.

      ECOTOXICITY 

Pigment Red 254 had no effect on both lethal and non-lethal parameters in fish toxicity studies with Rainbow Trout and Zebra Fish. There were similarly no effects on aquatic invertebrates, algae, or earthworms at the highest dose tested in each study (i.e., 100 mg/l, 100 mg/l and 1000 mg, respectively). 

Table 4: Toxicity of Pigment Red 254 to Aquatic Species 
Species
Parameter
Dose (mg/L)
Source
 Fish
Brachydanio rerio
 96-hr LC50
 >100
 MRID 51027017
Rainbow Trout
 21-Day NOEC, LOEC
 >sat. solution
 
 Water Flea
Daphnia magna
 24-hr LC50
 >100
 MRID 51027018
Daphnia magna
 21-Day NOEC, LOEC
 >sat. solution
 MRID 51027019
Algae
Scenedesmus subspicatus
 72-hour EbC50
  >sat. solution
 MRID 51027020

    Fish
                                                                               
    Acute Toxicity 
                  
The acute toxicity of Pigment Red 254 to Zebra Fish (Brachydanio rerio) was determined according to OECD 203 guideline. (MRID 51027017) Although the English translation of the study is not clear, it appears that between 10-20 fish per test substance and control were used.  A concentration of 100 mg/l of Pigment Red 254 and 4 mg/l alkylbenzolesuphonate were premixed with water, then added to aquarium. No effects of toxicity were seen during the study. Therefore, the 96-hour LC50 was determined to be >100 mg/l. 

    Prolonged Toxicity

The toxicity of Pigment Red 254 to Rainbow Trout (10/dose) was determined according to OECD 204. (MRID 51027016) Twenty fish were exposed to Pigment Red 254 for 21 days. These fish were split into two groups and an additional 10 fish were used as controls. For the treated fish the loading dose was 0.79 g/l/day at the beginning of the study and 1.07 g/l/day at the end. There were no "observable and significant effect on fish for both lethal and non-lethal parameter used in this study". Therefore, the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were determined to be ">sat. sol."
   Aquatic Invertebrates: Water Flea
The acute toxicity from Pigment Red 254 to Daphnia magna was evaluated according to an OECD 202 guideline study. Pigment Red 254 (50 mg) and 2 mg alkylbenzenesulphonate were mixed with water and made into a solution. Daphnia magna (20/concentration and control) were added to nominal concentrations of 0, 10, 18, 32, 58 and 100 mg/l of the stock solution. (MRID 51027018). No immobilization was seen during the study. The 24-hour EC50 was >100 mg/l as was the EC100.

A second OECD 202* study evaluated the effects of Pigment Red 254 on Daphnia magna. (MRID 51027019) Pigment Red 254 was administer to 10 daphnia per concentration and controls for 21 days. The test substance (2.0 g) was added to test water for a total volume of 2000 ml of the test solution. There was no observable effect on daphnids for both mortality and reproduction. Therefore, the 21-day NOEC was "saturated solution mg/l" and the LOEC was ">saturated solution mg/l".  

* The original OECD Guideline 202 included two parts: Part I - the 24h EC50 acute immobilization test and Part II - the reproduction test. In 1998 the adoption of Test Guideline 211 "Daphnia magna Reproduction" Test led to a new version of Guideline 202, restricted to acute immobilization. This study was conducted before the change. 
   Algae
An OPPTS 850.5400/OECD 201 growth inhibition test was performed using Scenedesmus subspicatus, in the presence of Pigment Red 254. (MRID 51027020) The suspension of 100 mg of the test substance in 1000ml test water was continuously agitated for 48-hours. The stock solution and stock solution water mixture were 1:1. The blank contained water, and each test concentration was tested in 3 replicates, and the blank in 6. The 72-hour EbC50 calculated: >saturated solution (EbC50: the concentration of test substance which results in a 50 percent reduction in biomass growth) 
   Earthworm
An acute earthworm toxicity test (MRID 51027021) was carried out with Eisenia Foetida foetida earthworms (40/concentration and control) for 14 days. A stock mixture was prepared for each concentration (12.3, 37, 111, 333, and 1000 mg) by premixing the corresponding amount of Pigment Red 254 with 200 g fine quartz. The LC50 was determined to be >1000 mg/kg as was the LC100. 

    RISK CHARACTERIZATION

BASF Corporation is requesting that EPA establish an exemption from the requirement of a tolerance for pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- (CAS No. 84632-65-5) under 40 CFR § 180.910 when used as a pesticide inert ingredient pre- and post-harvest. Pigment Red 254 is currently approved as a nonfood inert ingredient for use only as a pigment in antimicrobial paint formulations at a maximum end-use concentration of 225 ppm (0.0225%) w/w. BASF intends to use Pigment Red 254 in seed treatment pesticide products. 

Pigment Red 254 has widespread use as a pigment in consumer and industrial products and has various uses in coatings, inks, and polymers. It is approved by the U.S. Food and Drug Administration (FDA) for use as an indirect food additive (colorant) in food contact polymers at levels not to exceed 1.0% by weight of the polymer (21 CFR §178.3297).

A metabolism study on Pigment Red 254 showed that it was almost completely eliminated in the feces with the majority excreted within 24 hours. With very little found elsewhere in the urine, tissue or blood is unlikely that Pigment Red 254 is bioavailable. This is further supported by the lack of adverse effects seen in the acute and repeat dose studies. Acute toxicity studies conducted with Pigment Red 254 indicate low toxicity and it was non-irritating to the eyes and skin of rabbits. It was also not a dermal sensitizer. 

In a 28-day repeated dose oral study in rats treated with Pigment Red 254 up to 1000 mg/kg/day, the only effects observed as a result of treatment were red extremities and feces in the high-dose group due to the color of the test article. No other treatment related effects were observed. Therefore, the NOAEL was 1000 mg/kg/day (the highest dose tested). 

A reproductive/developmental toxicity screening test on the surrogate compound Irgazin Orange EH 1287 showed no parental, reproduction and developmental toxicity up to the highest dose tested (1000 mg/kg/day). Therefore, the parental, reproductive, and developmental NOAEL was determined to be 1000 mg/kg/day. Pigment Red 254 was negative for genotoxicity and mutagenicity in various in vitro and in vivo studies. It is not expected to be a carcinogen nor is it expected to be neurotoxic or immunotoxic. 

Based on the current and proposed use pattern of Pigment Red 254, dietary and residential exposure from pesticidal uses are expected to be low. In addition, no endpoint of concern was identified in the database below the limit dose of 1000 mg/kg/day; therefore, a quantitative assessment of human exposure was not necessary. 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. 

Pigment Red 254 is not expected to biodegrade however, the increased exposure from use of Pigment Red 254 in antimicrobial paints at 225 ppm and as a pigment for seed treatment as proposed, is expected to be negligible and therefore, does not present a hazard for the environment. Pigment Red 254 was non-toxic to aquatic systems and earthworms. 

Overall, the low toxicity of Pigment Red 254, coupled with the negligible exposure from the current and proposed use as an inert ingredient in pesticide products, indicate that residues of this compound do not pose a safety risk at aggregate exposure levels, including exposure expected to result from the proposed uses as an inert ingredient in pesticide formulations. Based upon the factors summarized above, the Agency is establishing an exemption from the requirement of a tolerance for pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro- (CAS No. 84632-65-5) under 40 CFR § 180.910 as an inert ingredient in food use pesticide formulations pre- and post- harvest. 


                                 Bibliography

(MRID 51027001) Ullman, L. (1986) Acute Oral Toxicity Study with T K 11627 in Rats. Project Number: 062245, 851241. Unpublished study prepared by Research & Consulting Company, Limited. 28p.
(MRID 51027002) Ullman, L. (2019) Acute Dermal Toxicity Study with T K 11627 in Rats. Project Number: 062278, 851244. Unpublished study prepared by Research & Consulting Company, Limited. 28p.
(MRID 51027003) Hartmann, P. (1991) Acute Inhalation Toxicity in the Rat (TK 11627). Project Number: 904061. Unpublished study prepared by Ciba-Geigy Limited. 79p.
(MRID 51027006) Ullman, L. (1986) Primary Eye Irritation Study with TK 11627 in Rabbits. Project Number: 062256, 851242. Unpublished study prepared by Research & Consulting Company Limited. 29p.
(MRID 51027007) Ullman, L. (2019) Primary Skin Irritation Study with TK 11627 in Rabbits (4-hour Occlusive Application). Project Number: 062267. Unpublished study prepared by Research & Consulting Company, Limited. 29p.
(MRID 51027008) Maurer, P. (1988) Skin Sensitisation Test in the Guinea Pig with TK 11627 Maximisation Test. Project Number: 884357. Unpublished study prepared by CIBA-GEIGY KLYBECK. 26p.
(MRID 51027009) Ullman, L. (1986) Subacute 28-day Oral Toxicity (Gavage) Study with TK 11627 in the Rat. Project Number: 062280, 851245. Unpublished study prepared by Research & Consulting Company, Limited. 168p.
(MRID 51027011) Strasser, F. (1986) Micronucleus Test (Chinese Hamster). Project Number: 851247. Unpublished study prepared by CIBA-GEIGY Limited. 19p.
(MRID 51027012) Deparade, E. (1986) Salmonella/Mammalian-Microsome Mdtagenicity Test. Project Number: 851248. Unpublished study prepared by CIBA-GEIGY Limited. 21p.
(MRID 51027013) Taublova, E. (2011) Pigment Red 254 ¿ Bacterial Reverse Mutation Test: Final Report. Project Number: 97/11/12. Unpublished study prepared by VUOS- Research Institute for Organic Syntheses. 22p.
(MRID 51027014) Vertesi, A. (2015) In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK Mouse Lymphoma Assay) with C.I. Pigment Red 254: Final Report. Project Number: 678/476/0792. Unpublished study prepared by Toxi-Coop ZRT. 69p.
(MRID 51027015) Dijk, A. (1992) Irgazin DPP Red Bo: Absorption, Distribution and Excretion after Single Oral Administration to Male Rats. Project Number: 272812, 904150. Unpublished study prepared by RCC Umweltchemie Ag. 69p.
(MRID 51027016) Vial, A. (1990) Report on the Prolonged Toxicity Test of TK 11627 to Rainbow Trout. Project Number: 904067. Unpublished study prepared by CIBA-GEIGY Limited. 27p.
(MRID 51027017) Rufli, H. (1986) Report on the Test for Acute Toxicity of TK 11627 to Zebrafish. Project Number: 851251. Unpublished study prepared by CIBA-GEIGY Limited. 14p.
(MRID 51027018) Rufli, H. (1986) Report on the Test for Acute Toxicity of TKH627 to Daphnia Magna. Project Number: 851251. Unpublished study prepared by CIBA-GEIGY Limited. 15p.
(MRID 51027019) Vial, A. (1990) Report on the Reproduction Test of TK 11627 to Daphnia (Daphnia magna straus 1820). Project Number: 904065. Unpublished study prepared by CIBA-GEIGY KLYBECK. 22p.
(MRID 51027020) Vial, A. (1990) Report on Growth Inhibition Test of TK 11627 to Green Algae. Project Number: 904064. Unpublished study prepared by CIBA-GEIGY LIMITED. 19p.
(MRID 51027021) Vial, A. (1990) Report on Acute Toxicity of TK 11627 on Earthworms (Eisenia Foetida foetida). Project Number: 904065. Unpublished study prepared by CIBA-GEIGY Limited. 19p.
(MRID 51027023) Baumann, W. (1993) Report on the Test for Inherent Biodegradability in a Combined Zahn-Wellens / Carbondioxide Evolution Test of IRGAZIN DPP Red BO. Project Number: 938034. Unpublished study prepared by CIBA-GEIGY KLYBECK. 18p.
(MRID 51027025) Baumann, W. (1986) Report on the Test for Ready Biodegradability of TK 11627 in the Modified Sturm Test. Project Number: 851249. Unpublished study prepared by CIBA-GEIGY KLYBECK. 13p.
(MRID 51336201) Beekhuijzen, M. (2012) Reproduction/Development Toxicity Screening Test of (Inert Ingredient) in Rats by Oral Gavage. Project Number: 499724. Unpublished study prepared by WIL Research Group. 253p.
(MRID 51595401) To be added
(MRID 51595402) To be added
(MRID 51595403) To be added
(MRID 51595404) To be added
U.S. Environmental Protection Agency. Estimation Programs Interface Suite(TM) for Microsoft(R) Windows, v 4.00. United States Environmental Protection Agency, Washington, DC, USA. 

European Chemicals Agency (ECHA). REACH Database https://echa.europa.eu/es/registration-dossier/-/registered-dossier/13744/1/1. Last accessed 6/1/2021.

Appendix A
Appendix A

