Perylene-3,4:9,10-tetracarboxydiimide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive/developmental toxicity screening study: NOAEL (systemic toxicity and reproductve toxicity) >= 1000 mg/kg bw/d, according to OECD 421, GLP compliant, 2013, K1

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, (Jul 2000)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical state: Solid violet
- Analytical purity: >90%, dose calculation adjusted to purity
- Storage condition of test material: Room temperature

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) 11-12 wks
- Weight at study initiation: (P) Males: about 330 g; Females: about 200 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions: during overnight matings, male and female mating partners were housed together in Makrolon type M III cages; pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): of 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 material was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking 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. The test substance preparations were produced at least once a week.

Details on mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

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 drinking water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed. The samples were homogenous within the different levels of concentration. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90-110% of the nominal concentrations. These results demonstrate the correctness of the concentrations of the test article.

Duration of treatment / exposure:

The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

MORTALITY
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CLINICAL OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without litter, waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore these values are only documented in the Individual Tables (PART II).
• Females between PND 4 and sacrifice were weighed once a week.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Epididymides weights, testes weights, staging of spermatogenesis

Litter observations:

Viability/mortality:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

Clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

Necropsy:
All animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights:
Weight assessment was carried out on all animals. The following weights were determined:
1. Anesthetized animals
2. Epididymides
3. Testes

Histopathology:
The following organs / tissues of all parental animals were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
All gross lesions, Cervix, Coagulating gland, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed. The following tissues were examined: testes, epididymides, ovaries from all control and high dose animals.

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

The following statistical methods were used:
- Comparison of the dose group with the control group was performed using the Student’s t-test (two-sided) for the hypothesis of equal means (Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x)
- Comparison of the dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions (Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups)
- Comparison of the dose group with the control group was performed using the WILCOXON test (one-sided+) for the hypothesis of equal medians (Mating days until day 0 pc)
- Comparison of the dose group with the control group was performed using the WILCOXON test (one-sided-) for the hypothesis of equal medians (Viability Index)
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians (Weight parameters)

Reproductive indices:

male and female mating index, male and female fertility index, gestation index, postimplantation loss

Offspring viability indices:

Live birth index, pup number and status at delivery, viability index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed black discolored feces from study day 1 until the end of the study. During gestation period on study days 41-43 one female animal (No. 122) of test group 2 (300 mg/kg bw/d) showed a palpable mass in the abdominal region. Because of a single occurrence and no other clinical observation in this animal this finding was assessed as being incidental. Two sperm positive females of the control group (Nos. 106 and 107) and one sperm positive female of test group 1, 2 and 3 (Nos. 111, 122 and 139 – 100, 300, 1000 mg/kg bw/d) did not deliver F1 pups.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the test groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of the F0 males and females in all test groups were comparable to the concurrent control group throughout the entire study period. The mean body weight gain of the F0 males in test group 2 in the entire premating phase was decreased (-37.4%). The mean body weight gain of the F0 females in test group 1 in the gestation period from study day 7 to 14 was increased (+24.2%). Because of single incidences and no dose response relationship these findings were assessed as being incidental.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of the male animals in test group 1 (100 mg/kg bw/d) was slightly decreased in the premating phase from study days 7-13 (-5.9%) and in the entire premating phase (-5.9%). Because of a single incidence this finding was assessed as being incidental.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of violet color. Therefore, the gross findings in the remaining animals were not investigated in addition.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The male mating index was 100% in all test groups.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Two males of the control group (Nos. 6 and 7 mated with females Nos. 106 and 107), one male of test group 1 (No. 11 mated with female No. 111 – 100 mg/kg bw/d), one male of test group 2 (No. 22 mated with female No. 122 – 300 mg/kg bw/d) and one male of test group 3 (No 39 mated with female No. 139 – 1000 mg/kg bw/d) did not generate F1 pups. Thus, the male fertility index was 80% in the control group and 90% in all test groups. 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 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.7, 2.9, 3.1 and 3.2 days in test groups 0 - 3. 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.
All sperm positive rats delivered pups or had implants in utero with the following exception:
• Control group females Nos. 106 and 107 (mated with male No. 6 and 7) did not become pregnant.
• Low-dose female No. 111 (mated with male No. 11) did not become pregnant.
• Mid-dose female No. 122 (mated with male No. 22) did not become pregnant.
• High-dose female No. 139 (mated with male No. 39) did not become pregnant.
The female fertility index was 80% in the control group and 90% in all test groups. 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 (22.1 days in the control group, 22 days in test group 1, 22.2 days in test group 2 and 22 days in test group 3). The gestation index was 100% in all test groups.
The postimplantation loss was 8.86% in test group 0, 7.90% in test group 1, 1.78% in test group 2 and 4.28% in test group 3. 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.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of delivered F1 pups was 10.6 in test group 0, 12.7 in test group 1, 13.1 in test group 2 and 12.7 in test group 3. The liveborn birth index was 98.8% in the control group, 96.5 in test group 1, 99.2 in test group 2 and 99.1% in test group 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% in all test groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
Two male runts were seen in test group 0. One female runt was seen in test group 1 and 2 (100 and 300 mg/kg bw/d). 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.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male and one female F1 pup of test group 1 showed post mortem autolysis. One female F1 pup of test group 2 and 3 (300 and 1000 mg/kg bw/d) showed an empty stomach. This finding was assessed as being spontaneous in nature and without biological relevance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this modified reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility and for general, systemic toxicity was 1000 mg/kg bw/day.

Executive summary:

In a GLP-compliant Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421, the test article was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. No signs of general systemic toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study period. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed black discolored feces from study day 1 until the end of the study. Regarding pathology, macroscopically black discoloration of the content of the digestive tract in numerous animals was observed. Beside the discoloration no signs of toxicity in the respective tissues were noted macroscopically. This finding is regarded to be a consequence to the oral intake of the violet test substance and therefore treatment related but not adverse in nature. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. 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/day. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/Developmental Toxicity Screening Study:

In a GLP-compliant reproduction/developmental toxicity screening study according to OECD guideline 421 the first read across candidate was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 100, 300 and 1000 mg/kg bw/day (BASF, 2013). Drinking water served as vehicle. The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females. Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period. All animals of test group 2-3 showed black discolored feces. This finding was substance-related due to the black color of the dye stuff. Regarding pathology, there was a black discoloration of the gastro-intestinal tract of animals in test group 2 (300 mg/kg bw/day) and 3 (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices). Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000mg/kg bw/d for the F0 parental rats. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/day.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the reproductive toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

For other category members additional screening studies according to OECD TG 422 or 421 are available. In none of the studies a evidence for reproductive toxicity was found, supporting the results of this screening study.

Effects on developmental toxicity

Description of key information

Prenatal development: Read across, NOAEL (systemic toxicity/developmental toxicity) >= 1000 mg/kg, according OECD 414, GLP compliance, 2022, K1

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Remarks:

sytsemic toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Aug 2021 - 22 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories CRL, Rhone, 327 Impasse du Domaine Les Oncines, 69210 Saint Germain Nuelles (France)
- Age at order: Males: at least 11 weeks; Females: 9 weeks
- Weight at order: Males: 325-350 g; Females: 200-225 g
- Housing: no more than 5 of one sex to a cage (before and after mating); 1 male and 1 female per cage (during mating)
- Diet: ad libitum, 4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 2 °C
- Humidity: 55 % +/- 15 %
- Air changes (per hr): approximately 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 2021 To: 22 Nov 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Suspensions of the test item, in 0.5 % CMC, were prepared using the following procedure:
1. the required amount of test item were weighed.
2. the required amount of vehicle was added.
3. the mixture was treated with a Silverson for 3 minutes.
4. the resulting suspension was left under magnetic stirring for at least 16 hour, at room temperature, prior to dosing and during dosing
The formulation was prepared daily at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw
- Concentration in vehicle: 0, 10, 40, 120 mg/ml
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4346 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%). A 28 hour stability at room temperature and a 9 day stability at 2-8°C (followed by one day at room temperature under magnetic stirring) were verified in the range from 10 to 100 mg/mL.
Samples of the preparations prepared on Week 1 and Last Week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: not specified
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

from Day 6 through Day 19 post coitum (14 Days)

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid

Dose / conc.:

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

Remarks:

High

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: given by Sponsor
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for rat dam blood sampling: in the morning
- Other: on Day 20 post coitum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twiche daily and once daily at weekends an public holidays
- Cage side observations: Check for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: all animals, on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION: Yes
- measured on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: thyroid and brain

THYROID HORMONE DETERMINATION (T3, T4, TSH): Yes
- Blood sampled in the morning on day 20 post coitum (day of necropsy)
- slight isoflurane anaesthesia

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of intra-uterine deaths: Early resorptions (only placental remnants visible), Late resorptions (placental and foetal remnants visible)
- Number, sex and weight of all live foetuses
- Number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing) and in each foetuses allocated to the skeletal examination
- Gross evaluation of placentae

- The uteri from females without visible implantations (one in group 1, 3, 4; five in group 2) were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation

- The uteri from dams showing unilateral implantations on the right horn (one in group 1 and 3), were also immersed in a 20% solution of ammonium sulphide, revealing the non-pregnant left horn

Blood sampling:

- Serum: Yes
- Volume: 1 mL
- in the morning of Day 20 post coitum

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: Yes

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:

> Pre impl. Loss [%] = (no. of corpora lutea − no. of implantations) x 100/ no. of corpora lutea

Post-implantation loss was calculated as a percentage from the formula:

> Post impl. Loss [%] = (no. of implantations − no. of live foetuses) x 100/ no. of implantations

Total implantation loss was calculated as a percentage from the formula:

> Total impl. Loss [%] = (no. of corpora lutea − no. of live foetuses) x 100/ no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males.

All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Historical control data:

Historical control data from 2014 to 2022 was provided. Data included: foetal external abnormalities, skeletal examination, fixed visceral examination, fate of females, macroscopic observation of females at final cesarean section, litter data and sex ratio of dams with live foetuses at necropsy. Tables were extracted from reproductive toxicology historical control data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss was observed in one control, two mid-dose and one high dose females. Considering the low incidence of hairloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the treatment period a presence of couloured faeces (red) in all treated group. Considering that the test item is a red solid the colour indicated the presence of the substance in the faeces and not an abnormalities.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and weight gain were unaffected in females treated up to 1000 mg/kg/day over the entire administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The statistically significant increase in food consumption (29.7 g of high dose group versus 27.4 g of controls) was considered incidental and unrelated to treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were observed in the determination of T3, T4 and TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus weight, corrected maternal body weight and weight gain were not affected by treatment.
There were no treatment-related organ weight changes (brain and thyroid gland) at the end of the treatment period. Any variations were considered to be within the range of expected spontaneous changes in rats of the same age and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations in the thyroid gland at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Litter data of treated females were comparable to controls.
Presence of foetuses with a weight less that 2.7 g and classified as small were noted in control, mid and high dose groups. Considering that the incidence of the high dose group is lower than controls and in the absence of the dose relation trend the presence is consider incidental and unrelated to treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus with malformation was observed in the mid- dose group. The foetus showed unilateral fusion of thoracic arches no. 10th and 11th in combination with the fusion of the 10th and 11th ribs. Considering that no other observations were noted related to the thoracic arches or ribs and in the absence of the relationship the changes were considered spontaneous in origin.
The other changes noted were comparable between the control and the treated groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

The variations observed occurred with similar incidence across all groups including controls.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

 

 

Table 1: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE
Interval: 0 - 20 Days Group	1		2		3		4
Observation	(25)		(25)		(25)		(25)
APPEARANCE	a	b	a	b	a	b	a	b
Hairloss	1	4.0	0	0.0	2	8.0	1	4.0
() = Number of animals alive at start of interval a = Number of animals affected b = Percent of animals with observation during interval

 

 

Table 2: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA
Organ: Brain	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.853	1.848	1.860	1.851
Standard deviation	0.073	0.066	0.091	0.070
Group diff. at p < 0.05		0.051	0.051	0.051
Group diff. at p < 0.01		0.064	0.064	0.064
Analysis of variance: F ratio = 0.12; Df = 3/ 96; F probability = 0.942 Note: a * indicates group mean is significantly different from control at level of significance shown.
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	0.0218	0.0236	0.0219	0.0232
Standard deviation	0.0048	0.0037	0.0045	0.0044
Group diff. at p < 0.05		0.0030	0.0030	0.0030
Group diff. at p < 0.01		0.0037	0.0037	0.0037
Analysis of variance: F ratio = 1.09; Df = 3/ 96; F probability = 0.358 Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

Table 3: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.181	1.280	1.181	1.256
Standard deviation	0.268	0.198	0.254	0.240
Group diff. at p < 0.05		0.163	0.163	0.163
Group diff. at p < 0.01		0.204	0.204	0.204
Analysis of variance: F ratio = 1.11; Df = 3/ 96; F probability = 0.349 Note: a * indicates group mean is significantly different from control at level of significance shown. ° = expressed as % organ to brain weight ratio

 

 

Table 4: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE
Group	1	2	3	4
Number in group	25	25	25	25
Caecum
-        Abnormal contents	0	0	0	1
Forelimbs
-        Hairloss	1	0	1	1
Uterus
-        Unilateral implantation	1	0	1	0
-        Not pregnant	1	4	1	1
-        Total resorption	0	1	0	0
Whole animal
-        No abnormalities detected	22	20	22	23

 

 

Table 5: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Group	Organ	Cat	Observation(s)	No. Observed	Foetuses Affected	%	No.             Observed	Litters Affected	%
1	Whole foetus		No abnormalities detected	285	280	98.25	24	-	-
	Whole foetus	AN	Small	285	5	1.75	24	4	16.67
2	Whole foetus		No abnormalities detected	228	228	100.00	20	20	100.00
3	Whole foetus		No abnormalities detected	272	265	97.43	24	-	-
	Whole foetus	AN	Small	272	7	2.57	24	5	20.83
4	Whole foetus		No abnormalities detected	290	289	99.66	24	-	-
	Whole foetus	AN	Small	290	1	0.34	24	1	4.17

 

 

 

Table 6: SKELETAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
				No. Foetuses			No. Litters
Group	Organ	Cat	Observation(s)	Obs	Aff	%	Obs	Aff	%
1	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	70	46.67	24	23	95.83
	Lumbar vertebrae	VA	Centrum incomplete ossification	150	1	0.67	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification	150	1	0.67	24	1	4.17
	Ribs	AN	Wavy	150	10	6.67	24	8	33.33
	Ribs	VA	Short 14th	150	65	43.33	24	22	91.67
	Ribs	VA	Rudimentary 14th	150	11	7.33	24	9	37.50
	Ribs	VA	14 ribs	150	15	10.00	24	9	37.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	8	5.33	24	3	12.50
	Skull	AN	Hyoid no ossification	150	4	2.67	24	3	12.50
	Skull	AN	Temporal incomplete ossification	150	16	10.67	24	10	41.67
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	General incomplete ossification	150	5	3.33	24	4	16.67
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	20	83.33
	Skull	VA	Interparietal incomplete ossification	150	41	27.33	24	17	70.83
	Skull	VA	Parietal incomplete ossification	150	28	18.67	24	15	62.50
	Sternebrae	AN	Fused	150	1	0.67	24	1	4.17
	Sternebrae	AN	No ossification	150	2	1.33	24	2	8.33
	Sternebrae	VA	No ossification 5th	150	7	4.67	24	6	25.00
	Sternebrae	VA	Incomplete ossification 5th	150	22	14.67	24	11	45.83
	Sternebrae	VA	Incomplete ossification 6th	150	43	28.67	24	17	70.83
	Thoracic vertebrae	AN	Centrum bipartite and asymmetical	150	2	1.33	24	2	8.33
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	6	4.00	24	3	12.50
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	150	1	0.67	24	1	4.17
2	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	119	1	0.84	20	1	5.00
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	119	37	31.09	20	15	75.00
	Ribs	AN	Wavy	119	9	7.56	20	6	30.00
	Ribs	VA	14 ribs	119	10	8.40	20	6	30.00
	Ribs	VA	Short 14th	119	52	43.70	20	18	90.00
	Ribs	VA	Rudimentary 14th	119	12	10.08	20	11	55.00
	Sacral vertebrae	AN	Arch(es) incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Frontal incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Temporal incomplete ossification	119	6	5.04	20	4	20.00
	Skull	AN	Hyoid no ossification	119	1	0.84	20	1	5.00
	Skull	VA	Interparietal incomplete ossification	119	26	21.85	20	11	55.00
	Skull	VA	Parietal incomplete ossification	119	11	9.24	20	8	40.00
	Skull	VA	Supraoccipital incomplete ossification	119	42	35.29	20	19	95.00
	Sternebrae	VA	Incomplete ossification	119	1	0.84	20	1	5.00
	Sternebrae	VA	Incomplete ossification 6th	119	21	17.65	20	10	50.00
	Sternebrae	VA	No ossification 5th	119	3	2.52	20	3	15.00
	Sternebrae	VA	Incomplete ossification 5th	119	20	16.81	20	11	55.00
	Thoracic vertebrae	AN	Centrum bipartite	119	1	0.84	20	1	5.00
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	119	4	3.36	20	2	10.00
3	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	144	4	2.78	24	1	4.17
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	144	65	45.14	24	20	83.33
	Ribs	AN	Wavy	144	7	4.86	24	5	20.83
	Ribs	MA	Fused	144	1	0.69	24	1	4.17
	Ribs	VA	14 ribs	144	16	11.11	24	10	41.67
	Ribs	VA	Rudimentary 14th	144	11	7.64	24	9	37.50
	Ribs	VA	Short 14th	144	78	54.17	24	22	91.67
	Sacral vertebrae	AN	Arch(es) incomplete ossification	144	5	3.47	24	3	12.50
	Skull	AN	Frontal incomplete ossification	144	1	0.69	24	1	4.17
	Skull	AN	Hyoid no ossification	144	4	2.78	24	1	4.17
	Skull	AN	General incomplete ossification	144	2	1.39	24	2	8.33
	Skull	AN	Temporal incomplete ossification	144	14	9.72	24	6	25.00
	Skull	VA	Parietal incomplete ossification	144	28	19.44	24	12	50.00
	Skull	VA	Interparietal incomplete ossification	144	36	25.00	24	14	58.33
	Skull	VA	Supraoccipital incomplete ossification	144	58	40.28	24	21	87.50
	Sternebrae	AN	Bipartite 5th	144	1	0.69	24	1	4.17
	Sternebrae	AN	No ossification	144	1	0.69	24	1	4.17
	Sternebrae	VA	Incomplete ossification	144	5	3.47	24	3	12.50
	Sternebrae	VA	Incomplete ossification 6th	144	36	25.00	24	13	54.17
	Sternebrae	VA	No ossification 5th	144	10	6.94	24	5	20.83
	Sternebrae	VA	Incomplete ossification 5th	144	22	15.28	24	16	66.67
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	144	2	1.39	24	2	8.33
	Thoracic vertebrae	AN	Centrum asymmetrical ossification	144	1	0.69	24	1	4.17
	Thoracic vertebrae	AN	Centrum bipartite	144	1	0.69	24	1	4.17
	Thoracic vertebrae	MA	Arch(es) fused	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	144	1	0.69	24	1	4.17
4	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	52	34.67	24	19	79.17
	Ribs	AN	Wavy	150	12	8.00	24	6	25.00
	Ribs	VA	Rudimentary 14th	150	6	4.00	24	6	25.00
	Ribs	VA	14 ribs	150	11	7.33	24	8	33.33
	Ribs	VA	Short 14th	150	71	47.33	24	21	87.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	5	3.33	24	4	16.67
	Skull	AN	General incomplete ossification	150	2	1.33	24	2	8.33
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	Hyoid no ossification	150	4	2.67	24	4	16.67
	Skull	AN	Temporal incomplete ossification	150	17	11.33	24	9	37.50
	Skull	VA	Interparietal incomplete ossification	150	46	30.67	24	18	75.00
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	19	79.17
	Skull	VA	Parietal incomplete ossification	150	30	20.00	24	14	58.33
	Sternebrae	AN	Asymmetrical ossification 5th	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 5th	150	32	21.33	24	17	70.83
	Sternebrae	VA	No ossification 5th	150	4	2.67	24	3	12.50
	Sternebrae	VA	Incomplete ossification	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 6th	150	33	22.00	24	13	54.17
	Thoracic vertebrae	AN	Centrum bipartite	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	4	2.67	24	4	16.67

Conclusions:

On the basis of the results, the dosage of >=1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.

Executive summary:

The effects of the test material were investigated, in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19 in a GLP compliant study according to OECD TG 414. 

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The plan for this study was to investigate doses of 100, 300 and 1000 mg/kg bw/day of the test item with a dose volume of 10 mL/kg body weight, during the gestation period from Day 6 through Day 19 post coitum. Control females received the vehicle (0.5 % carboxymethyl cellulose (CMC)) at the same dose volume during the same treatment period.
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms from the treatment with the test item were observed. No macroscopic findings were noted during necropsy of the females. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of foetuses per dam were not affected by treatment with the test item at all dose levels. No test item-related effects on foetal body weights were noted. No test item-related effects on foetal sex ratios or anogenital distance were noted in any dose group.
During the external examination of the foetuses, no test item-related abnormal findings were noted. Hormone levels of dams were comparable between groups. Females did not show any macroscopic or microscopic (thyroid) changes related to treatment. Skeletal and visceral examinations were comparable between groups.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Prenatal Developmental Toxicity:

As no data for the test substance is available regarding prenatal developmental toxicity a read-across to the category member CAS 5521-31-3 was performed.

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The plan for this study was to investigate doses of 100, 300 and 1000 mg/kg bw/day of the test item with a dose volume of 10 mL/kg body weight, during the gestation period from Day 6 through Day 19 post coitum. Control females received the vehicle (0.5 % carboxymethyl cellulose (CMC)) at the same dose volume during the same treatment period.
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms from the treatment with the test item were observed. No macroscopic findings were noted during necropsy of the females. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of foetuses per dam were not affected by treatment with the test item at all dose levels. No test item-related effects on foetal body weights were noted. No test item-related effects on foetal sex ratios or anogenital distance were noted in any dose group.
During the external examination of the foetuses, no test item-related abnormal findings were noted. Hormone levels of dams were comparable between groups. Females did not show any macroscopic or microscopic (thyroid) changes related to treatment. Skeletal and visceral examinations were comparable between groups.

 

In a GLP-compliant supporting reproduction/developmental toxicity screening study according to OECD guideline 421 the test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 100, 300 and 1000 mg/kg bw/d (BASF, 2013). Drinking water served as vehicle. The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and the entire gestation period as well as 4 days of lactation in females. No signs of general systemic toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study period. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed black discolored feces from study day 1 until the end of the study. This finding was substance-related due to the dark violet color of the dye stuff. Regarding pathology, macroscopically black discoloration of the content of the digestive tract in numerous animals was observed. Beside the discoloration no signs of toxicity in the respective tissues were noted macroscopically. This finding is regarded to be a consequence to the oral intake of the violet test substance and therefore treatment related but not adverse in nature. Regarding developmental toxicity, no signs of toxicity were observed in male or female pups of all test groups. Thus, under the conditions of this modified reproduction/developmental toxicity screening test The NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/day. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/day.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the developmental toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

For other category members additional screening studies according to OECD TG 422 or 421 are available. In none of the studies a evidence for reproductive or developmental toxicity was found, supporting the results of this read-across study.

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 on fertility or development were observed in a screening study in rats (OECD 421) and no adverse effects were observed in a teratogenicity/developmental toxicity study in rats (OECD 414). 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 thirteen days covered in the screening study, no effects via lactation were observed.