4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

One key study (OECD 422, GLP) to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats to the reproductive/developmental system after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg bw/day is available.

The NOAEL for the test item under the condition of this study is considered to be > 1000 mg/kg bw/day for reproductive/developmental toxicity.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.02-23.08.2017

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

Version / remarks:

July, 2016

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Develop mental Toxicity Screening Test. EPA 712-C-00-368

Version / remarks:

July, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Wistar rat, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 327 - 487 g (mean: 397.89 g); females: 213 - 277 g (mean: 240.33 g)
- Fasting period before study: not reported
- Housing: Animals were housed in groups of 5 per sex in type IV polysulphone cages. During mating period, males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage.
- In each cage Altromin saw fibre was used as bedding
- Nesting material were provided latest on GD 18 for all mated females
- Diet (ad libitum): free access to Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours artificial light / 12 hours dark

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Prior to the start of the treatment: detailed clinical observation was made. None of the animal showed pathological signs.
- Before dosing: all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
Before the first administration: all animals were weighed and assigned to the experimental groups. Each animal was marked with its identification number by individual ear tattoo marking.

The animals were treated with the test item formulation or vehicle on 7 days per week for a max. period of 63 days, i.e. during 14 days of pre-mating and max. 14 days of mating in both males and females.
Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Details on mating procedure:

- Mating at a ratio of 1:1 (male to female) (if possible).
- Vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented.
- The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
- The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period:
- formulation analysis for stability and homogeneity of the test item in the selected vehicle.
- pre-start stability analysis for high and low dose group samples, at 0h, 6h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day (-15 to -35 °C) (10 samples).
- pre-start homogeneity investigation for samples from various dose levels (top, middle and bottom) of high dose and low dose groups (6 samples).
Result: the test item was homogenous (after 60 min without stirring) ==> during the study samples were collected for the investigation of homogeneity and samples for substance concentration were only collected in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and the last week of the study (gestation / lactation) from all dose groups (12 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

Treatment with test item formulation or vehicle: 7 days/week with max. of 63 days, i.e. during 14 days of pre-mating and max. 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to postnatal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days / week

Details on study schedule:

- General clinical observations: at least 1x/day (preferably at the same time)
- Observations for morbidity and mortality: 2x/day (weekends and public holidays: 1x/day)

Dose / conc.:

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

Remarks:

control group

Dose / conc.:

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

Remarks:

low dose group

Dose / conc.:

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

Remarks:

medium dose group

Dose / conc.:

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

Remarks:

high dose group

No. of animals per sex per dose:

10 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

100 animals (40 males and 60 females) were included in the study.

Prior to the start of the treatment:
- detailed clinical observation was made
- no pathological signs observed before the first administration

Before the first administration:
- all animals were weighed & assigned to the experimental groups (homogenous variation in body weight throughout the groups of males and females)(randomisation with IDBS Workbook 10.1.2 software).

Dose selection rationale:
- Based on DRF study 3 dose groups were determined (LD = low dose, MD = medium dose, HD = high dose).

Body weight and food consumption:
- weighed once before the assignment to the experimental groups
- weighed on the first day of dosing
- weighed weekly
- weighed at the end of the study
During pregnancy, females:
- were weighed on gestation days (GD) 0, 7, 14 and 20
- weighed within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups.
- all animals were weighed directly before termination.

Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Positive control:

not applicable

Parental animals: Observations and examinations:

Clinical observations:
- General clinical observations: 1x/day (same time each day) (recording of health condition)
- Observed for morbidity and mortality: 2x/day (weekends & public holidays: 1x/day)
- Detailed clinical observations: 1x before first exposure, at least 1x/week thereafter
- Clinical observations: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocal isation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional observations:
- Detailed behavioural observations: week before first treatment, during last week of treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females) of each group.
- Observations: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anteriorchamber of the eye and fundus of eye).

Oestrous cyclicity (parental animals):

- Estrous cycles: monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study.
- Vaginal smears: examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

- Testes: detailed qualitative examination, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

- duration of gestation was recorded & calculated from day 0 of the pregnancy.
- litter examination: as soon as possible after the delivery of the dam.
- recordings: number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Litters were weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing.
In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

- males were sacrificed any time after the completion of the mating period (after a minimum dosingperiod of 28 days)
- females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine).
- non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear or from the last day of mating period.

All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ weights at necropsy:
- wet weight of the organs of 5 randomly selected male and female animals (only lactating females) from each group was recorded.
- paired organs were weighed together.
- organ weights of animals found dead were not recorded.
- reproductive organs were weighed from all animals.
- thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

The following tissues/organs were examined: testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from all adult males and females), liver, kidneys (paired weight), heart.Further tissues/organs from the same selected animals were preserved.

All animals found dead were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination.

Histopathology:
- full histopathology: on the preserved organs and tissues of the selected animals of the control and HD groups which were sacrificed at the end of the treatment.
- evaluation of thyroid/parathyroid glands from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyro
id gland of selected animals and there was also no test item related effect observed on T4 hormonelevel in males sacrificed on PND 13.
- full histopathology was carried out on the preserved organs and tissues of all animals which diedduring the study.
- any gross lesion macroscopically identified was examined microscopically in all animals.

Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery

Postmortem examinations (offspring):

Clinical biochemistry:
- from 2 female pups/litter on day 4 after birth
- from 2 pups/litter (1 male and 1 female) at termination on day 13 blood samples were collected. Blood samples from the day 13 pups were assessed for serum levels for thyroid hormones (T4) & total T4 hormone levels.

Further assessment of T4 in blood samples from the day 4 pups was not deemed necessary. Assessment of TSH in day 4 pups and day 13 pups were not considered necessary based on the fact that no histopathological findings were observed in T4 hormone levels of day 13 pups. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated where litter size dropped below 8 pups. When there was only one pup available above a litter size of 8, only one pup was sacrificed on PND 4.

Pathology:
- Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
- All surviving pups were killed by cervical dislocation on PND 13.
- Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal- Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Reproductive indices:

- Reproductive indices: copulation, viability and delivery indices
- Fertility index: number of females pregnant/ No. of females copulated X 100

Offspring viability indices:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical signs salivation and moving the bedding on few days were observed immediately after the dose administration and for few minutes and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no adverse toxicological relevance. During the weekly detailed clinical observation, no relevant differences between the groups were found.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, two mortalities were observed:
- Female (Control) was found dead on post natal day (PND) 13. No specific clinical signs were observed in this animal before death or during entire study period
- Male (Low Dose) was found dead on premating day (PMD) 9. Clinical sign observed before death was severely increased salivation on PMD 8

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- males and females: no test item treatment related effect observed on bw & bw gain; no statistically significant differences for bw & bw gain except statistically significantly lower body weight gain in LD males during premating day 1-7 and statistically significantly higher body weight gain during gestation day 14-20 in HD group when compared with the controls.

These differences were either marginal, within the biological variation or due to lack of dose dependency, not considered to be adverse effects due to treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period except statistically significantly higher food consumption in HD group females during PND 0-4 when compared with the controls.This marginal increase in food consumption has no toxicological significance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- males sacrificed at the end of treatment period ==> no test item related adverse effects for haematological parameters
- females sacrificed at the end of treatment period ==> no test item related effect or statistically significant effect observed on any of the haematology parameters except statistically lower reticulocytes in MD group when compared with the controls. Due to lack of dose dependency, this effect on reticulocytes in MD group was considered as incidental.

All other group mean and most of the individual values for haematological parameters in male and females were comparable with the controls. No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- males sacrificed at the end of treatment period ==> no test item related or statistically significant effect on any clinical biochemistry parameter except statistically significantly lower ASAT group mean value in all treatment groups and ALAT in MD group. These lower liver enzyme values have no toxicological significance. In the light of no histopathological findings in liver and all individual values were within the historical data range (ASAT- 26-175.5 U/L, ALAT- 14.8-126.5 U/L), this statistically significant effect in males was considered as incidental and not related to test item.

- females sacrificed at the end of treatment period ==> statistically significantly higher ASAT in HD group and lower ASAT, ALAT, Creatinine, urea, cholesterol, glucose and sodium in LD group was observed when compared with the controls. As all individual values were within the historical data range (ASAT- 21.9- 219.8 U/L) and due to lack of dose dependency, this effect on few clinical chemistry parameters in females was considered as incidental and toxicologically irrelevant.

All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- selected male and female animals sacrificed at the end of treatment period ==> no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. This effect on urine parameters was not considered to be test item related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- males and females ==> no relevant effects in any of the parameters of the functional observation battery before and at the end of the treatment period. No biologically relevant differences observed in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One female from the control group (Animal No. 49) & one male from the LD group (Animal No. 85) were found dead on study day 51 and 9 respectively. For both
animals the cause of morbidity was not evident at histopathological evaluation. All findings observed at necropsy were considered not to be test item related and deemed to be incidental.

The histopathology evaluation did not reveal any test item related morphological evidence of human relevant toxicities in the organs and tissues examined. The accumulation of hyaline droplets in renal tubular epithelial cells form males belonging to the control and high dose group is a male rat specific phenomenon with no toxicological relevance in humans.

Further, the test item did not produce histological evidence of toxicity in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, and vagina. The sperm staging did not reveal any treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. The treatment with the test item did not induce histomorphological effects in the reproductive organs of non-pregnant females (no. 41, 113, 115, 125, 133 and 138) and their pairing partners (no. 1, 83, 85, 95, 103 and 108).

In conclusion, a histopathological NOAEL (No Observed Adverse Effect Level) could be established at 1000 mg//kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

- no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups compared to the controls.
- no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
- a statistically significant increase in number of normal cycles observed in LD group which was not considered to be toxicologically relevant.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.
- no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group.
- a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 80 % was observed in the LD and HD group as compared to 90 % in control group.
Although there was reduction in fertility index in LD and HD group, it was within the standard pregnancy rate of rat i.e. ≥ 80 %, this effect on fertility index was considered as biological variation and not related to treatment with the test item administration

- no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls. None of the females showed signs of abortion or premature delivery.

- no test item related effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group. However, statistically significantly higher precoital interval was observed in LD group when compared with the controls. Due to lack of dose dependency, this effect in the form of marginally higher precoital interval in LD group was not considered to be toxicologically relevant.

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: general reproductive toxicity

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- no test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group.
- a marginally higher percent mean mortality of pups between PND 0 and PND 4 was observed in the LD (0.83%), MD group (0.74%) and HD group (0.74%) compared to the control group (0.00%).
This outcome did not achieve statistical significance and was attributed to missing (possible cannibalism) one pup of each 1 dam from LD, MD and HD group on PND 4 (pup no. 1 of LD dam no. 117, pup no. 1 of MD dam no. 127 and pup no. 15 of HD dam no. 139). This pup mortality was considered as incidental or biological variation and not related to the treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- no test item related effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls.
- no statistically significant change observed in dose groups compared to controls except statistically significantly higher pup mean weights in MD group on PND 0.
Due to lack of dose dependency and consistency, this effect on pup mean weight on PND 0 in MD group was not considered to be test item related.

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:

no effects observed

Description (incidence and severity):

- no test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like dark head (pup no. 1 from dam 50 of control group on PND 0) and dark snout (pup no.1 from dam 122 of MD group on PND 0) were observed.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Precoital interval and duration of gestation:
- no test item related effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.
- statistically significantly higher precoital interval was observed in LD group when compared with the controls. Due to lack of dose dependency, this effect in the form of marginally higher precoital interval in LD group was not considered to be toxicologically relevant.

Anogenital distance and nipple retention:
*males:
-statistically significant lower cube root of pup weight, absolute and relative anogenital distance on the day of anogenital measurement was observed in male LD group when compared with the controls.
- statistically significantly higher male pup weight in MD group and statistically significantly lower cube root of male pup weight and relative anogenital distance in HD group observed when compared with the controls.
*females:
- statistically significantly lower absolute and relative anogenital distance was observed in LD, MD and HD groups when compared to the controls.
- statistically significantly higher female pup weight and cube root of female pup weight observed in MD group when compared with the controls.

Anogenital distance (AGD) is always correlated with cube root of pup body weight, litter size and sex ratio (for data normalization to simulate linear measurement). In male and females, parameters like pup body weight, litter size and sex ratio were not affected in treatment groups and these parameters are correlated with AGD. Although statistically significant effect was observed in males and females, In the light of absence of dose dependency and consistency, effect on pup weight and cube root of pup weight, effect on AGD in males and females cannot be considered as test item related.
No statistically significant effect of toxicological relevance was observed on nipple retention in the male pups of any of the groups when compared with the controls.

Thyroid hormone (T4) analysis:
- no test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity screening

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

The NOAEL for the test item Reactive Yellow 42 in this study is considered to be 1000 mg/kg bw/day for reproductive toxicity screening.

Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats to the reproductive/developmental system after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day. The treatment period was 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.

In conclusion:

No adverse effects of test item were found on oestrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data,

reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in pups sacrificed on PND 13. pup external findings.

There were no test item-related adverse effects to the reproductive/developmental system up to 1000 mg/kg bw/day. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity is considered to be at least 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

Quality of whole database:

No issues with the quality of the study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The aim of this study was to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats to the reproductive/developmental system after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day. The treatment period was 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.

No adverse effects of test item were found on oestrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in pups sacrificed on PND 13. pup external findings.

There were no test item-related adverse effects to the reproductive/developmental system up to 1000 mg/kg bw/day. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity is considered to be at least 1000 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Justification for classification or non-classification

28 -Day repeated dose toxicity with reproduction/developmental toxicity screening

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats to the reproductive/developmental system after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day.

There were no test item-related adverse effects to the reproductive/developmental system up to 1000 mg/kg bw/day.

Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity is considered to be at least 1000 mg/kg bw/day.

Based on the available data on toxicity to reproduction the substance does not meet the criteria for classification according to CLP Regulation No 1272/2008 in section 3.7. The substance is therefore considered not toxic to reproduction.