5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No indication of adverse effects on fertility were observed in a screening study (OECD 421, GLP) that was performed with the pigment in the nanoform (BASF 2013).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Aug 2012 - 27 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

yes

Remarks:

20 animals per sex were used.

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 1 - 11 wks
- Housing: individually in Makrolon type M III cages, except during mating and postnatal days 0-4
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single-housing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice.

The stability of the test substance in deionized water for a period of 5 days at room temperature was proven before the start of the study.

Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control.

Duration of treatment / exposure:

40 to 55 days
males: 2 weeks premating and during mating
females: 2 weeks premating, during mating, gestation and 4 days lactation

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: limit test with 1000 mg/kg bw was done based on unexpected total implantation loss at that dose in another OECD 421 study (80R0323/08X008)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during work days and once on weekends

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: twice a day during work days and once on weekends

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of dosing and weekly thereafter; females: on the first day of dosing and once weekly during the premating period, on days 0, 7, 14 and 20 of gestation and during lactation on the same day as litters (days 0 and 4 postnatal)

FOOD CONSUMPTION:
- Time schedule: weekly during premating, during gestation and lactation periods on days 0, 7, 14 and 20 (gestation) and 0 and 4 (lactation), no food consumption was determined for males during mating and postmating periods and for females without positive evidence of sperm and without litter

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after mating
- Maternal animals: All surviving animals on day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed: testes and epididymides. Furthermore, the following tissues were prepared for microscopic examination: all gross lesions, testes, epididymides, ovaries, uterus and oviducts, prostate, seminal vesicles and coagulating gland. Full histopathology of ovaries, testes and epididymides was performed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal cavities.

Statistics:

For the hypothesis of equal means student's t-test (two-sided) was performed. For the hypothesis of equal proportions Fisher's exact test (one-sided) was applied. For the hypothesis of equal medians one-sided Wilcoxon test was performed. Results were found to be significant at p<0.05.

Reproductive indices:

Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
(*defined by a female with vaginal sperm or with implants in utero)
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
(*defined by a female with implants in utero)
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
(*defined as the number of females with vaginal sperm or with implants in utero)
Female fertility index (%) = (number of females pregnant*/number of females mated**) x 100
(*defined as the number of females with implants in utero; **defined as the number of females with vaginal sperm or with implants in utero)
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
(*defined as the number of females with implants in utero)

Offspring viability indices:

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Post implanatation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities observed during the study period. All male and female animals of test group 1 (1000 mg/kg bw/d) showed orange discolored feces from study day 2 until the end of the study. Two sperm positive females of test group 1 (Nos. 128 and 133 – 1000 mg/kg bw/d) did not deliver F1 pups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the male and female F0 generation parental animals in test group 1 (1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation. Mean body weights and mean body weight gain of the F0 males and females in test group 1 (1000 mg/kg bw/d) were comparable to the concurrent control group throughout the entire study period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The male mating index was 85% in control group and 100% in test group 1 (1000 mg/kg bw/d). This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Three males of control group and two males of test group 1 did not generate F1 pups with paired females.
Thus, the male fertility index was 85% in the control group and 90% in test group 1. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The female mating index calculated after the mating period for F1 litter was 85% in the control group and 100% in test group 1.
The mean duration until sperm was detected (GD 0) varied between 3.4 in test group 0 and 3.7 days (in test group 1).
All sperm positive rats delivered pups or had implants in utero with the following exception:
• Control group female Nos. 101, 116 and 117 (mated with male No. 1, 16 and 17) did not become pregnant.
• high-dose females Nos. 128 and 133 (mated with males Nos. 28 and 33) did not become pregnant.
The female fertility index was 100% in the control group and 90% in test group 1. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The mean duration of gestation was similar in all test groups (22.0 days in test group 1 and 22.3 days in the control group).
The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (11.2 and 13.0 implants/dam in all test groups (0, and 1000 mg/kg body weight/day)). There were no statistically significant differences in post-implantation loss between the groups (6.5% / 5.1%), and the mean number of F1 pups delivered per dam remained unaffected (10.4 and 12.3 pups/dam at 0 and 1000 mg/kg bw/d).

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean absolute or relative weight parameters did not show significant differences when compared to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A treatment-related yellow discoloration of contents was observed in the glandular stomach, jejunum, cecum, and the colon.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
One male animal (No. 1, control group) showed a severe (grade 4) multifocal degeneration of the testes resulting in an aspermia of the epididymides, correlating with the gross finding size reduction. These findings explain why the female animal (No. 101) was not pregnant. All other females that were not pregnant as well as their male mating partners did not show relevant findings in the investigated organs. The non-pregnant female rats of the control group (Nos. 108 and 115) exhibited a cyst in the right oviduct as well as a dilated uterus and vagina. High-dose female No. 132 (1000 mg/kg bw/d) had a dilated uterus at gross necropsy. None of the other non-pregnant females (Nos. 115, 120, 125 and 134) had any relevant gross lesions.

Dose descriptor:

NOAEL

Remarks:

systemic/reproductive performance/fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the limit dose

Critical effects observed:

no

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index indicating pup mortality during lactation (PND 0 - 4) was 96.5% in the control group and 99.6% in test group 1. Due to the lack of dose response relationship this was assessed as being incidental. The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Body weight of female pups in test group 1 was slightly decreased on day 4, but the overall mean pup weight of both sexes was comparable to the control group. Thus, this finding was assessed as being incidental. One male runt was seen in test group 0. One male and five female runts were seen in test group 1 (1000 mg/kg bw/d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

GROSS PATHOLOGY (OFFSPRING)
Respectively one male F1 pup of control group showed an empty stomach. One female F1 pup of test group 1 (1000 mg/kg bw/d) showed post mortem autolysis. One female pup of test group 1 (1000 mg/kg bw/d) was cannibalized. This finding was assessed as being spontaneous in nature and without biological relevance.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed (as far as F1 can be assessed in a screening study)

Critical effects observed:

no

Reproductive effects observed:

no

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

other justification

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Two screening studies are available.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are two reproduction/developmental toxicity screening tests available performed with the test substance (CAS 36888-99-0, Pigment yellow 139).

 

The first study conducted is considered as supporting study and was done as a full screening study according to OECD 421 and under GLP (BIOAGRI, 2012).

The study was performed with the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day via gavage with 10 male and 10 female Wistar rats per dose group.

The following findings were seen:

Total post-implantation loss occurred in two dams at 1000 mg/kg bw./day.

Mild to severe nephropathy were observed in two male rats at 1000 mg/kg bw./day; these males also had slight, diffuse vacuolation in the liver.

Total post-implantation loss in principle gives rise of concern.

However, total post-implantation loss was also observed in 3 out of the 9 historical control groups at BIOAGRI. Therefore, the interpretation of the findings observed at the high dose Ievel is limited by the low number of animals and the comparably high incidence in the historical control of BIOAGRI, precluding a final assessment. Remaining reproductive parameters (pre-coital interval, fertility index, mating index, gestation length, mean litter size, litter weight) were comparable in all groups.

The experience with organic pigments has shown that they are inert substances that fail to cause indication of systemic uptake. The best examples are Pigment Red 112 (MW = 484.76), which did not show adverse effects at the limit dose in the subchronic oral toxicity study (OECD 408) and all diketopyrrolopyrrol-pigments (eg Pigment Red 254, MW = 357 g/mol – 14C toxicokinetic data showed absence of systemic uptake). (For note, the molecular weight of Pigment Yellow 139 is 367 g/mol).

It is therefore the intention to waive higher TIER studies for the REACH registration. A basic prerequisite is that there is certainty that no hazard is missed. Since the study performed at BIOAGRI showed findings which could not be properly evaluated, it was decided to repeat the study in a limit test design – only with the highest test group of 1000 mg/kg bw and a control group - and to use a higher number of animals (20 per sex and dose). This design was considered to be adequate to make a final assessment on whether the substance causes treatment related effects. The same batch of the test substance and the same vehicle were used.

 

The second study, applied as key study, was equally performed according to OECD 421 and under GLP, but as a limit test and with the modification of 20 animals per sex and group (BASF, 2013).

Vehicle and 1000 mg/kg bw/d were administered to wistar rats daily via gavage.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of the test group (1000 mg/kg bw/d) during the entire study period. All animals of the test group showed orange discolored feces. This finding was substance-related due to the yellow color of the dye stuff.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of the test group during the entire study. Regarding developmental toxicity, no signs of toxicity were observed in male or female pups. During pathology a yellow discoloration of contents occurred in the glandular stomach, jejunum, and in the cecum in all males and most females as well as in the colon in 4 out of 20 females of the test group. The yellow discoloration was related to the yellow color of the test substance. All other findings recorded were considered to be incidental in nature and not related to treatment.

Thus, under the conditions of this modified reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/d. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d. Compared to the control group, no adverse treatment-related findings were noted.

In summary, A NOAEL of 1000 mg/kg bw/d for systemic toxicity, reproductive performance and fertility was derived from the screening studies. Therefore, the test substance does not show any evidence of a fertility impairing effect.

Effects on developmental toxicity

Description of key information

OECD 421, GLP (limit test), BASF, 2013, NOAEL (systemic, development) = 1000 mg/kg bw/d
OECD 421, GLP (full), BIOAGRI, 2012, NOAEL (systemic) = 1000 mg(kg bw/d, NOAEL (development) = 300 mg/kg bw/d

OECD 414 ongoing.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Aug 2012 - 27 Feb 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

yes

Remarks:

20 animals per sex were used

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 1 - 11 wks
- Housing: individually in Makrolon type M III cages, except during mating and postnatal days 0 - 4
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Paliotol Gelb K 2142 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice.

The stability of the test substance in deionized water for a period of 5 days at room temperature was proven before the start of the study.

Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

40 to 55 days

Frequency of treatment:

daily, 7 days/week

Duration of test:

males: 2 weeks premating and during mating
females: 2 weeks premating, during mating, gestation and 4 days lactation

Remarks:

Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: limit test with 1000 mg/kg bw was done based on unexpected total implantation loss at that dose in another OECD 421 study (80R0323/08X008)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during work days and once on weekends

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: twice a day during work days and once on weekends

BODY WEIGHT: Yes
- Time schedule for examinations: males: on the first day of dosing and weekly thereafter; females: on the first day of dosing and once weekly during the premating period, on days 0, 7, 14 and 20 of gestation and during lactation on the same day as litters (days 0 and 4 postnatal)

FOOD CONSUMPTION:
- Time schedule: weekly during premating, during gestation and lactation periods on days 0, 7, 14 and 20 (gestation) and 0 and 4 (lactation), no food consumption was determined for males during mating and postmating periods and for females without positive evidence of sperm and without litter

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 4
- Organs examined: all gross lesions, ovaries, uterus and oviducts

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: all per litte
- Soft tissue examinations: Yes: all per litter

Statistics:

For the hypothesis of equal means student's t-test (two-sided) was performed. For the hypothesis of equal proportions Fisher's exact test (one-sided) was applied. For the hypothesis of equal medians one-sided Wilcoxon test was performed. Results were found to be significant at p<0.05.

Indices:

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Post implanatation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Details on maternal toxic effects:

Details on maternal toxic effects:
Neither parental mortality nor clinical signs were observed during the study.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No substance related embryotoxic/teratogenic effects were observed until lactation day 4. Slight effects on viability index (96.5% in control and 99.6% in 1000 mg/kg bw/day group) were deemed to be incidental and the rates of liveborn and stillborn pups were evenly distributed about both groups.

Remarks on result:

not measured/tested

Abnormalities:

not specified

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are two reproduction/developmental toxicity screening tests available performed with the test substance (CAS 36888-99-0). Studies according to OECD 414 are ongoing.

 

The first study conducted is considered as supporting study and was done as a full screening study according to OECD 421 and under GLP (BIOAGRI, 2012).

The study was performed with the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day via gavage with 10 male and 10 female Wistar rats per dose group.

The following findings were seen:

Total post-implantation loss occurred in two dams at 1000 mg/kg bw./day.

Mild to severe nephropathy were observed in two male rats at 1000 mg/kg bw./day; these males also had slight, diffuse vacuolation in the liver.

 

Total post-implantation loss in principle gives rise of concern.

 

However, total post-implantation loss was also observed in 3 out of the 9 historical control groups at BIOAGRI. Therefore, the interpretation of the findings observed at the high dose Ievel is limited by the low number of animals and the comparably high incidence in the historical control of BIOAGRI, precluding a final assessment. Remaining reproductive parameters (pre-coital interval, fertility index, mating index, gestation length, mean litter size, litter weight) were comparable in all groups.

The experience with organic pigments has shown that they are inert substances that fail to cause indication of systemic uptake. The best examples are Pigment Red 112 (MW = 484.76), which did not show adverse effects at the limit dose in the subchronic oral toxicity study (OECD 408) and all diketopyrrolopyrrol-pigments (eg Pigment Red 254, MW = 357 g/mol – 14Ctoxicokinetic data showed absence of systemic uptake). (For note, the molecular weight of Pigment Yellow 139 is 367 g/mol).

It is therefore the intention to waive higher TIER studies for the REACH registration. A basic prerequisite is that there is certainty that no hazard is missed. Since the study performed at BIOAGRI showed findings which could not be properly evaluated, it was decided to repeat the study in a limit test design – only with the highest test group of 1000 mg/kg bw and a control group - and to use a higher number of animals (20 per sex and dose). This design was considered to be adequate to make a final assessment on whether the substance causes treatment related effects. The same batch of the test substance and the same vehicle were used.

 

The second study, applied as key study, was equally performed according to OECD 421 and under GLP, but as a limit test and with the modification of 20 animals per sex and group (BASF, 2013).

 

Vehicle and 1000 mg/kg bw/d were administered to Wistar rats daily via gavage.

 

Regarding developmental toxicity, no signs of toxicity were observed in male or female pups.

Thus, under the conditions of this modified reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for developmental toxicity in the F1 progeny was found to be1000 mg/kg bw/d. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d.

Compared to the control group, no adverse treatment-related findings were noted.

 

In summary, a NOAEL of 1000 mg/kg bw/d for developmental toxicity was derived from the screening studies. Therefore, the test substance does not show any evidence of a teratogenic/developmental toxic effect.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects were observed in a screening study regarding fertility and developmental toxicity (OECD 422/421). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.

During the four days covered in the screening study, no effects via lactation were observed.