Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects were observed in the screening study at the limit dose (OECD 422) for Pigment Yellow 155 (BASF 2013).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Specific details on test material used for the study:

- Lot/batch No.: DEB2 146870
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- 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) 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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:
Pigment Yellow 155 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.

VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100mL, respectively

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation)

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:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, littering and lactation behavior of the dams, parturition behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, except during mating period, during gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: Functional observational battery was performed in the first five parental males and females (with litter) per group at the end of the administration period. The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving animals: Males approximately one week post-mating and females after 4 days of lactation and two weeks thereafter.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Oviducts, Peyer’s patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Organn weights: Epididymides, Testes, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: 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.

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter where analysed by simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy where analysed by pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
Proportions of affected pups per litter with necropsy observations where analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity where analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Reproductive indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, post implantation loss

Offspring viability indices:

Viability index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

yellow discolored feces, no mortality

Body weight and weight changes:

no effects observed

Description (incidence and severity):

except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No animal died prematurely in the present study. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. Except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two males of test group 1 (Nos. 12, 20 mated with females Nos. 112, 120) did not generate F1 pups.
The male fertility index ranged between 80% and 100% (see Tab. 1).
The apparently infertile male animals Nos. 12 and 20 (test group 1; 100 mg/kg bw/d) were not examined histopathologically.
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.
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 3.2, 3.8, 2.9 and 4.2 days in test groups 0-3. 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. All sperm positive rats delivered pups with the exception of females Nos. 112 and 120 (test group 1), which were mated with male Nos. 12 and 20 did not become pregnant. The female fertility index varied between 80% and 100% (Table 1). Female animals Nos. 112 and 120, which delivered no pups, showed no implantation sites. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.3 days). The gestation index was 100% in all test groups.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.0% in test group 1, 99.1% in test group 3 and 99.2% in control and test group 2. Moreover, the number of stillborn pups was comparable between the groups. The postimplantation loss was 2.3% in test group 0, 0.98% in test group 1, 5.6% in test group 2 and 6.5% 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.

ORGAN WEIGHTS (PARENTAL ANIMALS): The weight decrease in absolute epididymis and liver weight in males of test group 2 (300 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw). The decrease in thymus weights of females in test group 3 (1000 mg/kg bw/day) was also regarded to be incidental as no histopathologic correlate could explain the weight decrease.
All other mean weight parameters did not show significant differences when compared to the control groups.

GROSS PATHOLOGY (PARENTAL ANIMALS):Several animals of test group 3 (1000 mg/kg bw) revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related.
Each one male and female of test groups 1 and 2 (100 and 300 mg/kg bw/day) (animals No. 12, 21, 111, 125) revealed either some or all of the following findings: deposition or foci on the lung, deposition on the diaphragm, discoloration and enlargement of the mediastinal lymph nodes, deposition on the thymus, and deposition on the sternum. All these findings were regarded to be treatment related by gavage errors.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS): 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 yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
The discoloration and depositions described for animals No. 12, 21, 111, 125 in several organs of the thoracic cavity were multifocal granulomas with intrahistiocytically located yellow particles, interpreted as test substance. The granulomas were foreign body reactions interpreted as a consequence to a gavage error. The discoloration of the mediastinal lymph nodes was regarded to be the physiologic clearing route of the lung. Particles that were located intraalveolar were transported via macrophages to the regional lymph nodes and caused there the activation of the lymph nodes and the discoloration. Therefore these depositions and discolorations were caused by the test substance due to a gavage error and were not regarded to be an adverse finding.
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.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING): The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% in test group 0, 98.2% in test group 1 and 100% in test groups 2 and 3. Due to the lack of dose response relationship this was assessed as being incidental.

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): Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
One female runt was seen in test group 0. One male and six female runts were seen in test group 1 (100 mg/kg bw/d); 1 female runt was seen in test group 3 (1000 mg/kg bw/d).

GROSS PATHOLOGY (OFFSPRING): Respectively one male F1 pup of test group 2 (300 mg/kg bw/d) and test group 3 (1000 mg/kg bw/d) showed post mortem autolysis at gross necropsy. Two female pups of test group 1 (100 mg/kg bw/d) were cannibalized. This finding was assessed as being spontaneous in nature and without biological relevance.

OTHER FINDINGS (OFFSPRING): 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 (for details see table 2).

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Table 10: Fertility index

	Test group (mg/kg bw/day)
Test groups (mg/kg bw/day	0 (0)	1 (100)	2 (300)	3 (1000)
Male fertility index [%]	100	80	100	100
Female fertility index [%]	100	80	100	100

Table 11: Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	40.3	50.0	50.4	57.4
Live females [%]	59.7	50.0	49.6	42.6
PND 4
Live males [%]	40.3	51.0	50.4	57.4
Live females [%]	59.7	49.0	49.6	42.6

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

A GLP-compliant investigation of the toxicological effects resulting from repeated oral-gavage administration to rats was performed following OECD 422 without deviations (BASF, 2013). Pigment Yellow 155 (CAS 68516-73-4) was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Pigment Yellow 155 was administered to male rats for 41 days and to female rats for 52 days including time prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Treatment with the test item up and including 1000 mg/kg bw/day did not reveal any clinical signs or histological findings and did not affect reproduction.

All dose-treated males and females had dose-related yellow discolored feces during the treatment period. This finding is considered to be a typical effect resulting from oral administration of a yellow dyestuff and not adverse.

Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day.

Effects on developmental toxicity

Description of key information

OECD 414, GLP (rat): NOAEL = 1000 mg/kg bw (BASF 2022)

OECD 422, GLP (rat) NOAEL = 1000 mg/kg bw (BASF 2013)

Pigment Yellow 155 is not teratogenic in rats. In addition, no developmental toxicity was observed in offspring raised until postnatal day 4.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Content: 95.7%
Storage stability: Expiry date: 08 Nov 2028
Physical state / appearance: solid / yellow
Storage conditions: Room temperature

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: 10-12 weeks
- Weight at study initiation: ca 190g
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination 6 am to 6 pm)

IN-LIFE DATES: From: 19/20/21 Oct (Cohort 1/2/3, resp.) 2021 To: 8/9/10 Nov (Cohort 1/2/3, resp.) 2021

Route of administration:

oral: gavage

Vehicle:

other: Tween 80

Remarks:

0.5% in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Beginning of administration and thereafter at regular intervals
- Volume applied: 10 ml/kg bw/day
- Concentration: 1.00, 3.00, 10.00 g/100 ml for test groups 100, 300, 1000 mg/kg bw/day, respectively

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate
study in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water at room temperature over a period of 7
days had been verified prior to the start of the study in a similar batch.
Samples of the test substance preparations were sent thrice to the analytical laboratory for
verification of the concentrations. Due to difficulties in the manufacture of the test substance preparations, the attained results of homogeneity and
concentration control analyses of samples taken during the in-life phase were inconclusive. Therefore, a third set
of homogeneity/concentration control samples were generated after the end of the in-life phase, using the same
procedures and under the same conditions as the previous samples.
The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). For the latter purpose three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C).

Details on mating procedure:

Time-mated
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

daily

Duration of test:

from implantation to one day prior to the expected day of parturition

Dose / conc.:

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

Remarks:

nominal 100 mg/kg bw

Dose / conc.:

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

Remarks:

nominal 300 mg/kg bw

Dose / conc.:

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

Remarks:

nominal 1000 mg/kg bw

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Result of OECD 422 study
- Rationale for route: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Due to technical reasons, the study was carried out in 3 cohorts.
- Reason for species selection: The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked: morbidity, pertinent behavioral changes and/or signs of overt toxicity

CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
- Clinical observations checked: mortality

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD20
- Blood samples obtained: Yes, from all animals
- Anesthesia: Yes (Isoflurane)
- Organs examined: Thyroid glands (with parathyroid glands), All paired organs were weighed together (left and right).
- Histopathology: Yes, Thyroid glands

THYROID HORMONES
- Parameters checked: Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Blood sampling:

On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance: Yes
- Fetuses were sexed, weighed, condition of placenta checked and umbilical cords, fetal membranes, and fluids were examined.

Statistics:

DUNNETT-test, FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test.
* for p < 0.05
** for p < 0.01

Indices:

The conception rate, The preimplantation loss, The postimplantation loss, anogenital index

Clinical signs:

no effects observed

Description (incidence and severity):

Yellowish discolored feces were recorded for each eight mid- and high-dose females (300 and 1000 mg/kg bw/d) from GD 8, furthermore, for all mid- and high-dose females from GD 10
onwards. Whereas up to 15 (out of 25) low-dose females (100 mg/kg bw/d) where affected from GD 12 onwards until terminal sacrifice (GD 20). This feces discoloration mirrors the presence of the yellowish test substance in the gastrointestinal tract. An adverse, toxic effect is not assumed.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. No weight changes were noted in in the thyroid glands.

Gross pathological findings:

no effects observed

Description (incidence and severity):

A yellowish discolored content of the duodenum was recorded in 9 (out of 25) high-dose females, while a discolored content of the rectum as well as of the caecum was seen in 3 highdose females, respectively. These discolorations mirror the presence of the test substance in the gastrointestinal tract. They are considered as non-adverse, causing no toxic effects by themselves.
Additionally, two spontaneous findings were noted in individual females of test groups 2 and 3 (300 and 1000 mg/kg bw/d). A diaphragmatic hernia was seen in mid-dose female No. 61, while a dilated renal pelvis was recorded for mid-dose female No. 66 and high-dose female No. 94.
Since these findings occurred in single females without a dose response relationship, they were assessed as incidental and not treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

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:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Remarks:

maternal developmental

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

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:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Dose descriptor:

NOAEL

Remarks:

prenatal development

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

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 295	25 287	25 292	25 300
Fetal incidence	N (%)	0.0	1 (0.3)	0.0	0.0
Litter incidence	N (%)	0.0	1 (4.0)	0.0	0.0
Affected fetuses/litter	Mean%	0.0	0.3	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One female fetus in test group 1 showed micromelia.

Table 2: Total external variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 295	25 287	25 292	25 300
Fetal incidence	N (%)	0.0	1 (0.3)	0.0	0.0
Litter incidence	N (%)	0.0	1 (4.0)	0.0	0.0
Affected fetuses/litter	Mean%	0.0	0.3	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One external variation in one single fetus (No. 46-09) was recorded in test group 1 (100 mg/kg bw/d), i.e. limb hyperextension. The incidence of this single finding was not statistically significantly different from control and there is no dose response relationship visible. This finding can also be found in the historical control data at comparable incidences.

Table 3: Total external unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 295	25 287	25 292	25 300
Fetal incidence	N (%)	0.0	0.0	2 (0.7)	1 (0.3)
Litter incidence	N (%)	0.0	0.0	2 (8.0)	1 (4.0)
Affected fetuses/litter	Mean%	0.0	0.0	0.6	0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One external unclassified observation (placentae fused) was recorded in two mid-dose and one high-dose fetuses without relation to dose. This is a common finding which can be found in the historical control data, thus, it is considered as not treatment-related.

Table 4: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 140	25 137	25 140	25 142
Fetal incidence	N (%)	4 (2.9)	5 (3.6)	4 (2.9)	4 (2.8)
Litter incidence	N (%)	4 (16)	5 (20)	4 (16)	4 (16)
Affected fetuses/litter	Mean%	2.4	3.3	2.9	2.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Three soft tissue variations were detected, i.e. short innominate in one control fetus, dilated renal pelvis and dilated ureter in several fetuses of all test groups including the control. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences.

Table 5: Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	30-12 Fa) 50-09 F	multiple skeletal malformations cleft sternum
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	81-01 M	shortened scapula

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional external malformation

Table 6: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 155	25 150	25 152	25 158
Fetal incidence	N (%)	0.0	2 (1.3)	0.0	1 (0.6)
Litter incidence	N (%)	0.0	2 (8.0)	0.0	1 (4.0)
Affected fetuses/litter	Mean%	0.0	1.2	0.0	0.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 155	25 150	25 152	25 158
Fetal incidence	N (%)	152 (98)	143 (95)	151 (99)	151 (96)
Litter incidence	N (%)	25 (100)	25 (100)	25 (100)	25 (100)
Affected fetuses/litter	Mean%	98.2	95.4	99.2	95.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

Table 8: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
Litter Fetuses	N N	25 155	25 150	25 152	25 158
Fetal incidence	N (%)	113 (73)	117 (78)	104 (68)	130 (82)
Litter incidence	N (%)	25 (100)	24 (96)	24 (96)	25 (100)
Affected fetuses/litter	Mean%	72.6	77.1	70.1	81.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Table 9:Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of thoracic centrum; unchanged cartilage	0.0	0.7	0.0	2.0*	0.4 (0.0 - 1.6)
Incomplete ossification of sacral arch; cartilage present	0.0	1.8*	2.0	0.8	0.9 (0.0 - 3.8)
Misshapen sternebra; unchanged cartilage	19.5	28.4	16.0	29.2*	24.3 (11.4 - 32.4)
Supernumerary rib (14th); cartilage not present	48.6	63.0**	56.6	51.5	51.0 (37.5 - 64.9)
Incomplete ossification of tuberositas deltoidea; cartilage present	0.0	0.7	0.8	2.0*	0.3 (0.0 - 1.8)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

** = p <= 0.01 (Wilcoxon-test [one-sided])

The findings ‘incomplete ossification of sacral arch (cartilage present)’, ‘misshapen sternebra (unchanged cartilage)’ and ‘supernumerary rib (14th) (cartilage not present)’ were not related to dose and the mean values were clearly inside the historical control ranges. Therefore, these findings are assessed as not treatment-related. There was an increase in incidence at 1000 mg/kg bw/d of incomplete ossification of thoracic centrum (unchanged cartilage) and of tuberositas deltoidea (cartilage present), when

compared to concurrent control. However, the values were only slightly outside the historical control range. Incomplete ossification is a transient stage in pre – and postnatal development which is amended over time when the offspring gets more mature. As the underlying cartilages were entirely intact a complete recovery of the affected offspring is assumed. Therefore, those two findings might be regarded as treatment-related but not as adverse.

Conclusions:

Pigment Yellow 155 causes neithr teratogenicity nor developmental toxicity in rats.

Executive summary:

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the

expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the stability of the test substance in the vehicle over a period of 7 days. The results of the analyses of the test substance preparations of the high-dose group samples confirmed the correctness of the prepared concentrations or undercut the limits of the analytical method only marginally. Whereas the mean concentration values of the low- and mid-dose samples showed deviations below 80% of the nominal concentrations. The homogeneous distribution of the test substance in the vehicle (Tween 80 in drinking water (50 mg/100 mL)) was demonstrated in the high-dose group samples, but not in the samples of the low- and middose group. A comparison of the sample mass and volume (density) lead to the conclusion that an inclusion of air and hence a formation of foam took place while trying to extract a definitevolume of liquid sample with a syringe. However, the outcome of this study remained unaffected since the administered high-dose level was within or only marginally below the intended concentration.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 and 1000 mg/kg bw/d Pigment Yellow 155 and controls.

Concerning thyroid hormone measurement, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, no treatment related findings were noted in the thyroid glands. 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.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

There was an increase in incidence at 1000 mg/kg bw/d of incomplete ossification of thoraciccentrum (unchanged cartilage) and of tuberositas deltoidea (cartilage present), when compared to concurrent control. However, the values were only slightly outside the historical control range. Incomplete ossification is a transient stage in pre– and postnatal developmen which is amended over time when the offspring gets more mature. As the underlying cartilages were entirely intact a complete recovery of the affected offspring is assumed. Therefore, those two findings might be regarded as treatment-related but not as adverse.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Pigment Yellow 155 to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

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

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity (BASF 2022). Analyses confirmed the stability of the test substance in the vehicle over a period of 7 days. The results of the analyses of the test substance preparations of the high-dose group samples confirmed the correctness of the prepared concentrations or undercut the limits of the analytical method only marginally. Whereas the mean concentration values of the low- and mid-dose samples showed deviations below 80% of the nominal concentrations. The homogeneous distribution of the test substance in the vehicle (Tween 80 in drinking water (50 mg/100 mL)) was demonstrated in the high-dose group samples, but not in the samples of the low- and middose group. A comparison of the sample mass and volume (density) lead to the conclusion that an inclusion of air and hence a formation of foam took place while trying to extract a definite volume of liquid sample with a syringe. However, the outcome of this study remained unaffected since the administered high-dose level was within or only marginally below the intended concentration. Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 and 1000 mg/kg bw/d Pigment Yellow 155 and controls. Concerning thyroid hormone measurement, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no treatment related findings were noted in the thyroid glands. 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.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. There was an increase in incidence at 1000 mg/kg bw/d of incomplete ossification of thoracic centrum (unchanged cartilage) and of tuberositas deltoidea (cartilage present), when compared to concurrent control. However, the values were only slightly outside the historical control range. Incomplete ossification is a transient stage in pre– and postnatal development which is amended over time when the offspring gets more mature. As the underlying cartilages were entirely intact a complete recovery of the affected offspring is assumed. Therefore, those two findings might be regarded as treatment-related but not as adverse. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Pigment Yellow 155 to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d.

The test material used in this study has been analysed for particles below the size of 100 nm and was found to fulfill the critera of a nanomaterial in the European Union.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on toxicity to reproduction,(OECD 422 and OECD 414) the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).