N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Justification for selection of Effect on fertility via oral route:
The test substance, Solvent Yellow 124, was tested for reproduction and sub-acute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996. The NOAEL from this study was 100mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10.9.2012-15.4.2013

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

Deviations:

yes

Remarks:

(see Overall remarks)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF Breeding VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 10 weeks – on arrival
- Weight at study initiation: (P) Males: cca 284 g; Females: cca 186g

- Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight
Identification of animals: the animals were identified by the colour marks on their fur, each cage were marked with the number of animals, sex, number of cage, name and dose level of the test substance

- Fasting period before study: no
- Housing: SPF conditions – 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
-Bedding: sterilized soft wood shavings

- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding (ST BERGMAN, manufacturer: Ing Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using and was analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis)
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Relative humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour

STUDY TIME SCHEDULE
Administration: 24. 10. – 18. 12. 2012 (details below Duration of treatment..)

- Photoperiod (hrs dark / hrs light): light: 12 hour light/12 hour dark

STUDY TIME SCHEDULE
Experimental part: 10.9.2012-15.4.2013
Administration: 24. 10. – 18. 12. 2012

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The test substance solution was administered to the stomach by gavage. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by olive oil in the same volume.

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighted into glass beaker and the beaker was replenished by olive oil. The solution was mixed by magnetic stirrer (650 rpm) for 60 minutes and then it was mixed continually during administration. The application form was prepared daily just before administration.

- Concentration in vehicle: The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
- Lot/batch no. (if required): 5211201(Dr. Kulich Pharma, s.r.o.)

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation:
Animals were mated from the 15th day of study (see below Duration of treatment).

- Proof of pregnancy: Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
- After successful mating each pregnant female was caged (how): SPF conditions – pregnant females – individually, offspring – with mother
- Any other deviations from standard protocol: see Overall remarks - with no impact on condition of animals and course of study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity were determined by evaluation of absorbance measured at maximum of the test substance absorbance spectra by spectrophotometric method. The method was developed at the test facility (Analytical Group I).

Duration of treatment / exposure:

Parental males:
1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study
Parental females:
1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum
Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)
Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study
Satellite males:
1st day → 42nd day (administration) → 56th day (observation)
Satellite females:
1st day → 42nd day (administration) → 56th day (observation)

Frequency of treatment:

7 days per week at the same time

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females and 12 males per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding study
(14 days, dose levels 0, 10,30 and 100 mg/kg bw/day)

- Animal assignment random

- Other:

Positive control:

None

Parental animals: Observations and examinations:

HEALTH CONDITION CONTROL
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before, during application and immediately after application.

MORTALITY
Twice daily

CLINICAL OBSERVATION
Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day
(11.00 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATION
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.
males - weekly
females - weekly in pre-mating and mating period, during pregnancy 0., 7th, 14th, 20th day, during lactation 0. or 1st, 3rd and 4th day
Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption x 100) was calculated for animals of repeated dose toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. (Numbers of females for repeated dose toxicity part of study were chosen at the end of study).

WATER CONSUMPTION
The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

Sperm parameters (parental animals):

In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.
Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension.
The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, and abnormal form of neck – were recorded.

Litter observations:

All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):

SACRIFICE
Parental males: 43th day of study
Parental females: 4th day of lactation
Non-pregnant females: 55th day of study or 26th day after confirmed mating

GROSS NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.

Reproductive indices:

Male mating index= (number of males with confirmed mating / number of males cohabited) x 100
Female mating index = (number of sperm-positive females / number of females cohabited) x 100
Male fertility index = (number of males impregnating a females / number of males cohabited) x 100
Female fertility index = (number of pregnant females / number of sperm-positive females) x 100
Gestation index = (number of females with live born pups / number of pregnant females) x 100
Survival index = (number of live pups on day 4 post partum* / number of pups born alive+) x 100

* without still born pups (dead pups with anaerial lungs)
+ with dead pups with aerial lungs

Offspring viability indices:

Pre-implantation loss Number of corpora lutea – number of implantations
Post-implantation loss Number of implantations – number of live births
Post-natal loss Number of live births – number of alive at postnatal day 4

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Females consuption decreased

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Females consuption decreased

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

MORTALITY
There were no unscheduled deaths during the whole study.

HEALTH CONDITION CONTROL
Males
In treated males of the lowest dose level no significant changes of health condition before, during and immediately after application of the test substance were observed. In all males of the middle dose level the anaemia of ear skin (in the 5th week of application) and yellowish colour of ear skin (in the 6th week of application) were observed. At the highest dose level the following changes were observed: anaemia of ear skin from the 1st to the 2nd week of the study and yellow colour of ear skin from the 2nd to the 6th week of study.

Females
In treated females of the lowest dose level no significant changes of health condition before, during and immediately after application of the test substance were observed. In all females of the middle dose level the anaemia of ear skin (in the 5th week of application) and yellowish colour of ear skin (in the 6th week of application) were observed. At the highest dose level the following changes were observed: anaemia of ear skin from the 1st to the 2nd week of the study and yellow colour of ear skin from the 2nd to the 6th week of study.


CLINICAL SIGNS
Males
At the lowest dose level no clinical changes after application of the test substance were observed. In all males of the middle dose level the yellowish colour of ear skin (in the 5th and 6th week of application) were observed. In two males at this dose level salivation was recorded in the last week of application. In all males at the highest dose level the increased activity and yellowish colour of skin, fur and visible mucous membranes were observed from the 3rd to the 6th week of study. At the highest dose level the salivation was observed in part of males from the 4th to the 6th week of application.

Females
No clinical changes after application of the test substance were observed in females at the lowest dose level. In all females of the middle dose level the yellowish or yellow colour of ear skin (from the 5th to the 8th week of application) were observed. In all females at the highest dose level the increased activity and yellowish colour of skin, fur and visible mucous membranes were observed from the 2nd to the 8th week of study. At the highest dose level the salivation was observed in part of females from the 5th to the 8th week of application.


BODY WEIGHT AND BODY WEIGHT INCREMENT

Males
The statistical analysis of the data revealed no significant intergroup differences.
The body weight of males of all dose levels was similar compared to control.

Females
Pre-mating mating period
The mean body weight increments of the control females and females at all dose levels were well balanced in the pre-mating period. Statistically significant differences were not detected.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy.
The mean body weight increment of treated mothers at all dose levels was analogous to control mothers. Statistically significant differences were not detected.
Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period.
The mean body weight increments of treated mothers at all dose levels and control mothers were similar. Statistically significant differences were not detected.

FOOD CONSUMPTION
Parental males
The food conversion males of all dose levels were variable in comparison with control without treatment related effect.


Females
Pre-mating period
The mean food consumption of females at all dose levels was balanced with control females in the 1st week of pre-mating period. In the 2nd week the food consumption of females of the middle and the highest dose levels was decreased.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy.
Dose-dependent effect (decrease) manifested till 0-7 days of pregnancy than the mean food consumption of mothers at all dose levels was analogous to control mothers.
Lactation
Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period.
The mean food consumptions of mothers of the all dose levels were slightly decreased than in control females.

OBSERVATION OF SPERM
Sperm motility was quite the same in control males and treated males. Presence of “non-motile sperms” was not detected.
Significantly increased presence of morphologically changed sperms was not detected in any dose level.

REPRODUCTIVE PERFORMANCE
Treated females of all dose levels were mated. Evidence of copulation was not found in one female at the control group, at the middle and highest dose levels and in two females at the lowest dose level.
The number of females achieving pregnancy in treated females was balanced with the control group. Abortion was not occurred. The duration of mating of females and duration of pregnancy of all dose level it was similar to the control.
The number of females bearing live pups and females with live pups at day 4 after parturition in females at all dose levels was similar to the control.
The numbers of corpora lutea in females of all dose levels were similar to the control. The number of implantation was slightly decreased at the middle and highest dose levels. The numbers of live pups at birth and at day 4 after parturition in females at the lowest and middle dose levels were conformable to the control group. At the highest dose level these numbers were slightly decreased against control.
No significant differences of mating and fertility indexes were observed. Survival and gestation indexes of treated groups were analogous to control.
Slightly increased pre-implantation losses were recorded at the highest dose level. Post-implantation losses and post-natal losses were similar to the control group.

BIOMETRY OF REPRODUCTIVE ORGANS
Males
The statistical analysis of the data revealed no significant intergroup differences in relative weight of reproductive organs and pituitary gland.
Absolute and relative weights of organs were similar in treated and control males.
Females
The statistical analysis of the data did not reveal significant intergroup differences in absolute and relative weights of ovaries, uterus and pituitary gland. Absolute and relative weights of examined organs were conformable to the control group.
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of biometry results.

GROSS PATHOLOGY
Males: No macroscopical findings were recorded in 12-0-0-0 males.
In 12-12-12-12 males no macroscopical changes were observed in reproductive organs and pituitary gland.
All treated males showed yellowish or yellow colouring of some tissue: yellowish or yellow colour of fatty tissue in 0-12-12-12 males, yellowish or yellow colour of ear skin in 0-0-12-12 males, yellow colour of hair in 0-0-0-12 males and yellow colour of mucous membrane in forestomach in 0-0-0-12 males.
Females:
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-10-30-100 mg/kg/day further in the text.
No macroscopical changes of reproduction organs and pituitary gland were recorded in 12-12-12-12 females.
No macroscopical findings were recorded in 12-0-8-5 females.
All treated females showed yellowish or yellow colouring of some tissue: yellowish or yellow colour of fatty tissue in 0-12-12-12 females, yellowish or yellow colour of ear skin in 0-0-4-12 females, yellow colour of hair in 0-0-0-12 females and yellow colour of mucous membrane in forestomach, stomach or yellow colour of chyme in 0-2-7-12 females.

HISTOPATHOLOGY
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-10-30-100 mg/kg/day further in the text.
In 12-12-12-12 males no histopathological changes were detected in testes, epididymis, coagulation glands and seminal vesicles.
Focal chronic inflammation was recorded in prostate gland of 1-1-1-1 males. Cysts in pituitary gland were recorded in 2-0-0-0 males.

In all females no histological changes were detected in ovaries, uterus, vagina and pituitary gland. In reproductive organs only the changes related to previous pregnancy were found.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly decreased

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Number and Sex Ratio of Pups
The statistical evaluation of the number of live born pups/per litter was performed. No statistically significant intergroup differences were recorded.
The total numbers of live pups and mean number of pups per litter at the dose level 30 and 100 mg/kg/day were slightly decreased in comparison with the control.
In sex ratio no significant differences were recorded in treated groups.

DEVELOPMENT (OFFSPRING)
Presence of stillborn pups was not recorded at any groups of females.
Mortality of pup (one female) in lactation period was detected at the lowest dose level.
No differences in development of pups were observed at the control and treated groups.

BODY WEIGHT (OFFSPRING)
The statistical evaluation of mean weight of pup on the 0./1st day and mean weight of pup on the 4th day was performed. No statistically significant intergroup differences were recorded.
The mean weight of litter was slightly decreased at all treated groups.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. In examined pups of control and at the lowest dose level no pathological findings were recorded. At the middle and highest dose levels all examined pups showed yellow colour of fatty tissue, subcutis and milk in stomach. Intensity of colouring was more marked at the highest dose level.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Reproductive effects observed:

not specified

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and DEVELOPMENT was established higher than 100 mg/kg body weight/day.

Executive summary:

The test substance, Solvent Yellow 124, was tested for reproduction and sub-acute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996.

Results of Reproduction Part of study

 

The oral administration of Solvent Yellow 124 to rats by gavage, the dose levels 10, 30 and 100 mg/kg/day, did not cause mortality.

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, spermiogenesis and sperm parameters,biometry of reproductive organs and pituitary gland, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals and sex ratio of pups were not adversely affected by the test substance treatment. The slight intergroup differences were considered to be of no toxicological significance.

Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy was not significantly changed – number of females achieving pregnancy was similar in control and treated groups. The total number of live pups and mean number of pups per litter were decreased in high-dose females. Pre-implantation losses were slightly increased in females of the dose level 100 mg/kg/day but the pos-implantation and post-natal development of pups were not influenced.

Evaluation of pup weight revealed no effect on intrauterine pup growth attributable to test substance. The mean pup weight at birth was similar at all dose levels and control group. The slightly decreased mean weight of litter in treated groups was related with the decreased number of pups per litters. The body weight increments of pups at the treated groups from the birth to the 4th day after parturition were similar to the weight increment of pups in control group.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

C.I. Solvent Yellow 124, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 100 mg/kg/day, was associated with maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity. The extent of the findings was sufficient to make a clear positive conclusion even with the group size of 5 litters. A potentially adverse test item related effects (discoloration of foetuses, indicating presence/accumulation of test item) was observed at all dose levels. Since the test item was positive, it is considered that because of this finding the LOAEL was 40 mg/kg.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 to 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The enclosed study is a Dose Range Finding Study. The multiple adverse developmental effects observed at 100 mg/kg bw/day indicates that the test item is teratogenic. The extent of the findings in the group size of 5 litters is sufficient to classify the test item as having adverse effects on development of rat foetuses. It is not considered to be justified for classification purposes, on animal experiment ethical grounds, to repeat this study with a larger group size as in a full OECD 414 study design. The results are considered suitable for classification and the full study was not conducted. The full report is appended below for ECHA's reference.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 414, Prenatal Developmental Toxicity Study, Organisation for Economic Co-Operation and Development, Paris, 22 January 2001

Deviations:

yes

Remarks:

Study was stopped at the Dose Range Finder stage; see below.

GLP compliance:

no

Remarks:

Study was a non-GLP DRF study. The intention was to progress to the full OECD 414 study; however the effects indicated that the substance was teratogenic.

Limit test:

no

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain: Hannover Wistar rats (CRL:WI(Han))
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
Justification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to the experience of the Test Facility with this strain in teratology studies.
Housing condition: Standard laboratory conditions; individual housing
Number of animals: 40 female animals, 6 mated female animals/group, 5 groups (one control and 4 test item-treated groups); 6, 6, 6, 6, 5 pregnant and evaluated female animals (with implantation sites at necropsy) per Control, Low, Mid 1, Mid 2, and High dose groups, respectively; 40 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from Citoxlab Hungary Ltd. spare colony were used.
Age of animals: Young adult female rats, nulliparous and non-pregnant, at least 15 weeks old at mating.
Starting body weight: 207-272 g (the variation did not exceed ± 20% of the mean weight)
Acclimation period: at least 33 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian.
Cage type: Type II polycarbonate cages were used during mating and gestation period and Type III polycarbonate cages were used during the acclimatisation period
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany were used in the study (Batch number: 03018170329, Expiry date: 29 March 2020 and 03018170529, Expiry date: 29 May 2020).
Nesting: Arbocel crinklets natural nesting material produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) was used in the study (Batch number: 05072170228, Expiry date: 28 February 2020).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1-22.6°C (target: 22 ± 3°C)
Relative humidity: 33-70% (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Successfully mated animals were housed individually. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
The temperature and relative humidity were monitored continuously and recorded twice on each day during the study. The bedding and nest building material were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Diet and water supply
The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Batch number: 262 21592, Expiry date: January 2018, Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum. The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis of the water is performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36, Hungary).

Animal Identification
Adult animals were identified by temporary numbers written with indelible ink on the tail during the entire study. During necropsy and Caesarean section procedures each evaluated dam was given an additional number (evaluation number indicating group number), and cross-referenced with the numbers used during the in-life phase of the study.
The cages were marked with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date. Cages were arranged to minimise any possible effects due to cage placement.
The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording data were identified only by the litter numbers until the end of foetal examinations (facilitating blind examination of foetuses).
The foetuses were identified during the Caesarean section with individual numbers according to their implantation sites. For visceral examination they were identified by digit-clipping; for skeletal examination the foetuses were identified by means of a water-proof plastic ribbon tied around their neck.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The test item was formulated in the vehicle (olive oil) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of Citoxlab Hungary Ltd.
Formulations were prepared prior to administration to the animals at the appropriate frequency (not more than 7 days before use).

Based on the available information, olive oil was selected as a suitable vehicle for this study in agreement with the Sponsor.
Relevant data of the of the vehicle are shown below:
Name: Olive oil
Supplier: Sigma-Aldrich Co.
Lot No.: BCBT7822
Expiry date: 28 February 2019
Storage conditions: Room temperature

Animals (one control and four test item treated groups) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle was assessed in the conditions employed on the study during the analytical method validation (Citoxlab study code: 17/122-316AN ). In this study, test item formulation samples in the 1-50 mg/mL concentration range (using olive oil as vehicle) were proven to be stable for at least 7 days when stored at room temperature (20±5°C).
Analysis of test item formulations for concentration and/or homogeneity was performed using a validated HPLC method. Top, middle and bottom samples were taken from the test item formulations once during the study. Samples were taken in duplicate (0.1 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) had to be less than 10%.

Details on mating procedure:

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 6 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Duration of treatment / exposure:

The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats from gestation day 6 (GD 6) to gestation day 19 (GD 19).

Frequency of treatment:

The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats daily by oral gavage on a 7 days/week basis, approximately at similar times.

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 mated females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-selection and route of administration
The dose levels were set by the Sponsor in agreement with the Study Director based on the available data, including the results of a repeated dose DRF toxicity study by oral gavage in rats performed at the Test Facility with the aim of inducing toxic effects but no death or suffering at the highest dose (100 mg/kg bw/day).
As in the previous DRF study even the lowest, 100 mg/kg bw/day dose group showed haematology changes and related tissue enlargement that might compromise a healthy pregnancy, four doses were selected for a more precise dose setting before the main study.
The oral route is a possible route of exposure to the test item in humans and mammals in the environment and it was considered suitable to provide the exposure required for this developmental toxicology study.

Randomisation
The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Provantis was used to verify homogeneity/variation among/within groups.

Maternal examinations:

IN-LIFE PROCEDURES
Clinical observations
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day). Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).
Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Body weight measurement
The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

Food consumption measurement
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

PATHOLOGY
Caesarean section and necropsy
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (RELEASE®, 300 mg/mL sodium pentobarbital solution; Supplier: Wirtshaftsgenossenschaft Deutscher Tierärzte (Address: Siemensstrasse 14., D-30827 Garbsen, Germany), Batch number: 106075, Expiry date: 31 July 2018) administered by intramuscular injection and followed by exsanguination was used for euthanasia.
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.

Histopathology
No histopathology evaluation was performed in the study (as it was not required for interpretation).

Ovaries and uterine content:

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the appearance of the anogenital distance.
Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination.
For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sannomiya mixture (a mixture of 920 mL concentrated isopropanol, 30 g sulfosalicylic acid, and 50 mL acetic acid), then, after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid – ethanol/isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.
All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.

Statistics:

The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.

Indices:

Maternal Data:
Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
Clinical signs (by gestation day)
Mortality (by gestation day)
Body weight and body weight gain: mean ± S.D.
Corrected body weight on GD 20 (body weight minus gravid uterine weight) and corrected body weight gain GD 0-20 (body weight gain (GD 0-20) minus gravid uterine weight): mean ± S.D.
Net body weight change (body weight gain during the treatment period (GD 6-20) minus gravid uterine weight): mean ± S.D.
Gravid uterine weight: mean ± S.D.
Food consumption: mean ± S.D.
Gross pathology findings, placenta findings

Caesarean Section and Necropsy Data:
Number of corpora lutea: mean ± S.D.
Number of implantations: mean ± S.D.
Number and percentage of live foetuses: mean ± S.D.
Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss, as well as foetal death
Preimplantation loss: %, group mean
[(Number of corpora lutea - Number of implantations) x100] / Number of corpora lutea
Postimplantation loss: %, group mean

[(Number of implantations-Number of live foetuses) x100] / Number of implantations

Foetal Data:
Sex distribution: %, group mean
(Number of male (female) foetuses x100) / Number of foetuses
Foetal body weight (accuracy 0.01 g): mean * S.D.
External abnormalities*/litter: %, group mean
(Number of foetuses with abnormality x100) / Number of foetuses
Visceral abnormalities*/litter: %, group mean
(Number of foetuses with abnormality x100) / Number of foetuses
Skeletal abnormalities*/litter: %, group mean
(Number of foetuses with abnormality x100) / Number of foetuses

Historical control data:

Data provided where considered useful.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red discharge was observed in 1 out of 6 animals in the Control group (animal number #1501) from Day 7. This change was considered to be incidental.
Abnormal skin colour was present in the Low (40 mg/kg bw/day, 5 out of 6 animals, from Day 19), Mid 1, Mid 2 and High dose animals (60, 80, and 100 mg/kg bw/day, 6/6, 6/6 and 5/5 animals, from Day 15-19, Day 15-18, Day 11-13, respectively). This was related to treatment.
Piloerection was present in the Low (40 mg/kg bw/day, 1 out of 6 animals from Day 17 and 2 out of 6 animals from Day 19), Mid 1 (60 mg/kg bw/day, 1 out of 6 animals from Day 18 and 3 out of 6 animals from Day 19), Mid 2 (80 mg/kg bw/day, 1 out of 6 animals from Day 17, 1 out of 6 animals from Day 18, and 3 out of 6 animals from Day 19), and High dose animals (100 mg/kg bw/day, 4 out of 5 animals from Day 10, and 1 out of 5 animals from Day 11). This change was considered to be related to treatment, but piloerection alone is not considered to be a clear indication of an adverse effect due to the minor/subjective nature of the observation.
Hunched back was present in the Mid 2 (80 mg/kg bw/day, 1 out of 6 animals from Day 17 and 3 out of 6 animals from Day 19), and High dose animals (100 mg/kg bw/day, 4 out of 5 animals from Day 11). This change was considered to be an adverse effect related to treatment.
The animal found dead on Day 16 in the High dose group (100 mg/kg bw/day) showed similar symptoms: piloerection from Day 10, abnormal skin colour and hunched back from Day 11. Based on the presence of the symptoms from Day 10, the death was considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal on Gestation Day 16 was found dead in the High (100 mg/kg bw/day) group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item related effect on body weight was observed in the Low and Mid 1 dose groups (40 and 60 mg/kg bw/day) when compared to control. Test item related effect on body weight was observed in the Mid 2 (10 % below control at termination but not statistically different) and High dose (16 % below control at termination, p>0.01) when compared to control.
Consequently, statistically significant differences were seen in the body weight gain, corrected body weight gain, and net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value in the in the Mid 2 and High dose groups (80 and 100 mg/kg bw/day) when compared to control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-dependent, reduced mean daily food consumption during the treatment period were observed in all treated groups when compared to the control value, which reached statistical significance between GD10-12 and GD18-20 in the High dose group (100 mg/kg bw/day, p < 0.05), and in the Mid dose 2 group (80 mg/kg bw/day, p < 0.01).
For these two groups, the difference in the daily mean food consumption (calculated for the entire period of the study) was larger than 20 % when compared to the control.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Diffuse yellow discoloration in the adipose tissue was present in all groups of Test Item treated animals. The fact that all dose levels showed a yellow discolouration demonstrates systemic exposure (and hence absorption) of the test item at all dose levels. Furthermore, similar discoloration was present in the non-glandular mucosal region of the Mid 2 and High dose groups (80 and 100 mg/kg bw and 3 out of 6, and 4 out of 5 animals, respectively).
In 1 out of 5 High dose animals, thickened non-glandular mucosal region was present in the stomach.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increase of the late embryonic loss and, consequently, in the total intrauterine mortality was observed in the High dose group.
The mean number of corpora lutea was comparable with the control in all test item treated groups (this is formed before treatment starts). No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.
The early embryonic loss values of the test item treated groups were comparable with control. Late embryonic loss was significantly higher in the High dose group. The number of dead foetuses were similar across the groups. There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
Total intrauterine mortality was significantly higher in the High dose group when compared to the controls.

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Thirty females (6 for each group) were mated in the study. The number of confirmed pregnant, evaluated dams was 6 in the Control, Low, Mid 1, Mid 2 dose group (0, 40, 60 and 80 mg/kg bw/day, respectively), and 4 in the High dose group (100 mg/kg bw/day).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of placentas
No abnormalities were observed on the placentas in the Control, Low Mid 1 and Mid 2 dose group (0, 40, 60. 80 mg/kg bw/day, respectively). In the High dose group, in one litter (animal number 5501) all placentas (13 out of 13) showed paleness and yellow discoloration, and in one animal (animal number 5502) 3 out of 8 placentas were observed as pale. In the other two litters in the High dose group, the placentas were normal. These records are in consistent with the colour changes of the foetuses, where paleness was considered to be a significant sign of the presence of Test Item in the foetuses, and indicate that the Test Item is able to pass through the placental barrier. It must be also noted that the naturally very dark red colour of the placentas would mask the visible signs of accumulation of the test item at lower concentrations; it could only be visible a relatively high levels of a strongly coloured substance.

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

placenta

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

With the exception of foetal body weight, no toxicologically relevant adverse effect of the test item was observed on the foetal parameters in the Low, Mid 1, Mid 2 and High dose groups (40, 60. 80 and 100 mg/kg bw/day, respectively).
The mean foetal weight per litter in the test item treated groups showed a dose-dependent decrease which reached significance (p <0.05) in the Mid 2 and High dose group when compared to the control mean value.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Although no similar observation is included in the Historical Control data, the variation localized oedema in one foetus in the Low dose group was considered to be an incidental finding.
The malformation of generalized oedema in all foetuses (13 out of 13) in one High dose litter were considered to be a Test Item related finding. This finding is rare in the historical control rats, a whole litter being affected has never been seen in controls.
Based on the external findings in the Low, Mid 1 and Mid 2 dose groups (40, 60 and 80 mg/kg bw/day, respectively) the number of malformed / variant / intact foetuses in these groups were comparable with the control.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Most of the skeletal findings correspond with the current historical control (HC) or the concurrent study control or were considered to be incidental findings without dose response. Reduced ossification in the High dose foetuses with retarded body weight, and the increased incidence of the bent radius and ulna in one High dose litter were considered to be a Test Item related effect.
Based on the skeletal findings the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid 1 and Mid 2 dose groups (40, 60 and 80 mg/kg bw/day, respectively), but showed a Test Item related effect in the High dose group (100 mg/kg bw/day).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

No visceral variations could be found in any of the animals in the study.
Malformations in the genital organs (Absent oviduct and malposition of the epididymis) were found in one female and one male foetus in the highly affected High dose litter (dam’s animal number: 5501). Neither of the two findings occurs in the Historical Control, and while the occurrence is low for both changes, in relations with the other findings in this litter it is considered a Test Item related effect

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Discolouration:
While it cannot be classified in classical teratology terms (that is, we could not regard discolouration as variations or malformations), the discolouration indicates a dose-dependent Test Item related effect, that would potentially affect normal development.
From the findings, it is clear that the Test Item can pass through the placental barrier (hence the discoloured placentas in the higher doses and the presence of the yellow colour in the foetal tissues). We could also find evidence that it can pass through the developing blood-brain barrier (yellowish, discoloured cerebrum was present in the Mid 2 and High dose foetuses even after the fixation in 80% 2-propanol for the visceral examination – that is, after the fixation method could have extracted test item from the tissues and masked any subtle effects.)
A clear dose-dependency could be seen in the external evaluations, where paleness indicating the start of accumulation of the Test Item, reaching a tipping point in higher doses (Mid 2 to High doses), where the concentrations are high enough to be described as yellowish discoloration.
It must be also noted that in parallel studies with the same Test Item in adult Wistar rats, yellow discoloration of the brain tissue at 150 mg/kg bw/day (an evidence that the Test Item could pass through the fully developed blood-brain barrier), and hyperactivity in the 100 mg/kg bw/day female dose group, as a Test Item related effect was present.
Based on these results, we would expect a potential functional neuroteratology effect, as the Test Item appears to be “neuroactive” in adult animals, and with the developing blood-brain barrier (which is known to be more open, or less restrictive, than the adult one), we would expect a potentially higher concentration in the foetal brain, thus potentially a more severe, permanent effect on the developing brain than in the adult.

Details on embryotoxic / teratogenic effects:

Malformations such as Malformed ribs (Rib 10 intercostal, left; Rib 11-12, branched proximally, right) and Vertebrae, misshapen in 1 out of 31 foetuses in the Mid 1 dose group were observed. Based on the low incidence and the lack of any dose response, these findings were considered to be incidental findings.
The malformation Radius, Ulna, bent in the High dose group (3 out of 20 foetuses in 1 out of 4 litters) showed an elevated incidence compared to the Historical Control data, and is in line with other (external, visceral) malformations observed in the same litter, hence it is considered to be a test item related effect.
In the case of most skeletal variations the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.
However, the increased occurrence of variations in the High dose litters where malformed foetuses were present indicates a test item related effect. It must be noted that retarded ossification is a common finding in litters where there is maternal toxicity or lower maternal body weight; hence the retarded ossification is interpreted as secondary to maternal toxicity rather than a foetotoxic effect.

Summary of examination of the foetuses:
In conclusion, foetal findings in the High dose litters indicates an observable test item related effect. Malformations ascribed to test item were observed in the High dose at External, Visceral and Skeletal examination. A dose-dependent pale colour or yellow discoloration was observed, indicating the presence of the Test Item in the foetal tissues, suggesting potential neuro- and developmental toxicity effects in the offspring’s generation. Classical foetal findings in the rest of the treated groups, in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment.

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: A dose-dependent pale colour or yellow discoloration was observed, indicating the presence of the Test Item in the foetal tissues, suggesting potential neuro- and developmental toxicity effects in the offspring’s generation.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Selected body weight parameters

Parameters	Dose (mg/kg bw/day)
	0	40	60	80	100
Number of evaluated dams	6	6	6	6	5
Body weight on GD20 (g)	328.3	307.0	317.3	295.8	276.2**	DN
Body weight gain GD6-20 (g)	86.2	59.7	63.8	46.8**	12.8**	D
Body weight gain GD0-20 (g)	98.5	73.7	82.2	60.7**	35.2**	D
Corrected body weight gain on GD20 (g)	268.50	259.33	258.83	238.67	242.00	NS
Corrected body weight gain GD0-20 (g)	38.67	26.00	23.67	3.50**	1.00*	D
Net body weight gain GD6-20 (g)	26.33	12.00	5.33	-10.33**	-21.40**	D

Notes: Body weight data were rounded to one decimal place. Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

NS: Statistically not significant when compared to the vehicle control.

DN: Dunnett’s test

D: Dunn test

** = p <0.01

 

Summary of pregnancy data

Parameter	Dose (mg/kg bw/day)
	0	40	60	80	100
Number of mated females	6	6	6	6	6
Pre-terminal death or euthanasia	0	0	0	0	1
Number of non-pregnant females	0	0	0	0	0
Number of females with ≤ 5 implantation sites	0	1	0	1	1
Number of evaluated females on GD20 (Caesarean section)	6	6	6	6	5

 

Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	40	60	80	100
Number of evaluated dams	6	6	6	6	5
Mean number of corpora lutea	12.2	10.8	12.0	13.8	12.4	NS
Preimplantation loss, mean	1.0	2.0	1.5	1.8	2.4	NS
Preimplantation loss (%), mean	7.78	24.21	12.09	12.43	22.95	NS
Mean number of implantations	11.2	8.8	10.5	12.0	10.0	NS
Early embryonic loss, mean	0.8	0.7	0.0	0.3	0.8	NS
Early embryonic loss (%), mean	7.49	5.90	0.00	3.24	12.48	NS
Late embryonic loss, mean	0.0	0.2	0.2	0.2	1.4**	D
Late embryonic loss (%), mean	0.00	1.28	1.52	1.37	21.47**	D
Dead foetuses, mean	0.2	0.0	0.2	0.3	0.2	NS
Dead foetuses (%), mean	1.52	0.00	1.52	2.78	1.25	NS
Postimplantation loss, mean	1.0	0.8	0.2	0.8	2.4	NS
Postimplantation loss (%), mean	9.01	7.18	3.03	7.41	35.20	NS
Total intrauterine mortality, mean	2.0	2.8	1.8	2.7	4.8*	DN
Total intrauterine mortality (%), mean	16.09	30.83	15.00	19.50	43.11	NS
Viable foetuses, mean	10.2	8.0	10.2	11.2	7.6	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to vehicle control.

DN: Dunnett’s test

D: Dunn test

*= p <0.05

**= p<0.01

 

Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	40	60	80	100
Number of examined litters	6	6	6	6	4(5†)
Viable foetuses, mean	10.2	8.0	10.2	11.2	7.6†	NS
Male foetuses, mean	6.0	4.7	6.0	5.2	3.8	NS
Female foetuses, mean	4.2	3.3	4.2	6.0	5.8	NS
Total number of foetuses	61	48	61	67	38	NS
Total number of male foetuses	36	28	36	31	15	NS
Total number of female foetuses	25	20	25	36	23	NS
Sex distribution (% of males / females)	59 / 41	58 / 42	59 / 41	46 / 54	39 / 61	NS
Mean foetal weight / litter (g)	3.541	3.553	3.356	2.958*	2.714*	D
Number of foetuses with retarded body weight	1	1	4	31	19
Number of affected litters (with runts)	1	1	3	5	3

Notes: Most important parameters are shown in bold.

†= animal number 5505 in the High dose group showed total intrauterine mortality. This litter was included in the number of examined letters and in the calculation of the mean viable foetuses, but with no viable foetuses, it is excluded for further calculations, where only four High dose litters are included.

NS: Statistically not significant when compared to the vehicle control.

D: Dunn test

*= p<0.05

 

Summary table of the external abnormalities

Parameter			Dose (mg/kg bw/day)					HC data
			Control	40	60	80	100
Total number of examined litters			6	6	6	6	4	670
Total number of examined foetuses			61	48	61	57	38	6889
Total number of intact (normal) foetuses			61	47	61	67	25	--
Total number of foetuses / litters with malformations			0 / 0	0 / 0	0 / 0	0 / 0	13^**CH/ 1	--
Total number of foetuses / litters with variation			0 / 0	1 / 1	0 / 0	0 / 0	0 / 0	--
External variations
Oedema, localized	Litter incidence	n	0	1	0	0	0	--
		%	0.0	16.7	0.0	0.0	0.0	--
	Foetal incidence	n	0	1	0	0	0	--
		%	0.000	2.083	0.000	0.000	0.000	--
External malformation
Oedema, generalized	Litter incidence	n	0	0	0	0	1	3
		%	0.0	0.0	0.0	0.0	25.0	0.448
	Foetal incidence	n	0	0	0	0	13	3
		%	0.000	0.000	0.000	0.000	34.211	0.044

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

CH: Chi²test

**= p<0.01

 

Summary of the visceral abnormalities

Parameter			Dose (mg/kg bw/day)					HC data
			Control	40	60	80	100
Total number of examined litters			6	6	6	6	4	670
Total number of examined foetuses			30	258	30	34	18	3450
Total number of intact (normal) foetuses			30	25	30	34	16	--
Total number of foetuses / litters with malformations			0 / 0	0 / 0	0 / 0	0 / 0	2 / 10	--
Total number of foetuses / litters with variation			0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	--
Visceral variations
No visceral variations were observed.
Visceral malformations
Ovary, oviduct, Absent	Litter incidence	n	0	0	0	0	1	--
		%	0.0	0.0	0.0	0.0	25.0	--
	Foetal incidence	n	0	0	0	0	1	--
		%	0.000	0.000	0.000	0.000	5.556	--
Testis, Epididymis; Malpositioned	Litter incidence	n	0	0	0	0	1	--
		%	0.0	0.0	0.0	0.0	25.0	--
	Foetal incidence	n	0	0	0	0	1	--
		%	0.000	0.000	0.000	0.000	5.556	--

Notes: Number represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful).

No statistically significant differences were noted compared to the control group

 

Summary table of the skeletal abnormalities

Parameter	Dose (mg/kg bw/day)					HC data
	Control	40	60	80	100
Total number of examined litters	6	6	6	6	4	669
Total number of examined foetuses	31	23	31	33	20	3435
Total number of intact (normal) foetuses	29	20	27	28	9^**CH	--
Total number of foetuses / litters with malformations	0 / 0	0 / 0	1 / 1	0 / 0	3^*CH/ 1	--
Total number of number of foetuses / litters with variation	2 / 2	3 / 2	3 / 3	5 / 3	8^**CH/ 4	--

Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

CH: Chi²test

*= p <0.05

**= p <0.01

 

Details of the skeletal abnormalities

Parameter			Dose (mg/kg bw/day)					HC data
			Control	40	60	80	100
Total number of examined litters			6	6	6	6	4	669
Total number of examined foetuses			31	23	31	33	20	3435
Skeletal variations
Skull: 3 or More Bones, Incomplete Ossification	Litter incidence	n	0	0	0	0	1	123
		%	0.0	0.0	0.0	0.0	25.0	18.4
	Foetal incidence	n	0	0	0	0	2	153
		%	0.000	0.000	0.000	0.000	10.000	4.454
Skull: Unossified Bones	Litter incidence	n	0	0	0	0	1	4
		%	0.0	0.0	0.0	0.0	25.0	0.116
	Foetal incidence	n	0	0	0	0	3^*CH	3
		%	0.000	0.000	0.000	0.000	15.000	0.448
Skull: Fontanelle Large	Litter incidence	n	0	0	0	0	1	3
		%	0.0	0.0	0.0	0.0	25.0	0.087
	Foetal incidence	n	0	0	0	0	1	3
		%	0.000	0.000	0.000	0.000	5.000	0.448
Sternum: Ossified Sternebra (3 or less)	Litter incidence	n	0	0	0	1	3	57
		%	0.0	0.0	0.0	16.7	75.0	1.659
	Foetal incidence	n	0	0	0	1	9^**CH	48
		%	0.000	0.000	0.000	3.030	45.000	7.175
Ribs: Wavy, marked	Litter incidence	n	0	0	0	0	1	198
		%	0.0	0.0	0.0	0.0	25.0	29.6
	Foetal incidence	n	0	0	0	0	4^**CH	29.6
		%	0.000	0.000	0.000	0.000	20.000	9.054
Vertebra: Dumbbell or Asymmetric Ossification	Litter incidence	n	2	2	2	2	1	335
		%	33.3	33.3	33.3	33.3	25.0	50.1
	Foetal incidence	n	2	3	2	2	2	767
		%	6.452	13.043	6.452	6.061	10.000	22.329
Vertebra: Unossified Vertebrate(s)	Litter incidence	n	0	0	0	0	2	1
		%	0.0	0.0	0.0	0.0	50.0	0.149
	Foetal incidence	n	0	0	0	0	8^**CH	1
		%	0.000	0.000	0.000	0.000	40.000	0.058
Vertebra: Bipartite Ossification	Litter incidence	n	0	0	1	1	0	27
		%	0.0	0.0	16.7	16.7	0.0	4.036
	Foetal incidence	n	0	0	1	1	0	27
		%	0.000	0.000	3.226	3.030	0.000	0.786
Pubis unossified	Litter incidence	n	0	0	0	0	2	6
		%	0.0	0.0	0.0	0.0	50.0	0.897
	Foetal incidence	n	0	0	0	0	8^**CH	6
		%	0.000	0.000	0.000	0.000	40.000	0.175
Carpal ≤ 2.5	Litter incidence	n	0	0	0	0	2	5
		%	0.0	0.0	0.0	0.0	50.0	0.747
	Foetal incidence	n	0	0	0	0	7^**CH	5
		%	0.000	0.000	0.000	0.000	35.000	0.146
Tarsal ≤ 3	Litter incidence	n	0	0	0	1	2	34
		%	0.0	0.0	0.0	16.7	50.0	5.082
	Foetal incidence	n	0	0	0	1	8^**CH	49
		%	0.000	0.000	0.000	3.030	40.000	0.426
Malformed ribs#	Litter incidence	n	0	0	1	0	0	2
		%	0.0	0.0	16.7	0.0	0.0	0.058
	Foetal incidence	n	0	0	1	0	0	2
		%	0.000	0.000	3.226	0.000	0.000	0.299
Vertebrae Misshapen#	Litter incidence	n	0	0	1	0	0	3
		%	0.0	0.0	16.7	0.0	0.0	0.087
	Foetal incidence	n	0	0	1	0	0	3
		%	0.000	0.000	3.226	0.000	0.000	0.448
Radius, ulna, bent	Litter incidence		0	0	0	0	1	--
		%	0.0	0.0	0.0	0.0	25.0	--
	Foetal incidence	n	0	0	0	0	3^*CH	--
		%	0.000	0.000	0.000	0.000	15.000	--

Notes: Number represent the number (n) or ratio (%) of abnormalities

HC: historical control (data provided where considered useful).

#: Ribs, Costal Cartilage, fused and Multiple Malformed Vertebra are recorded in the historical control database.

CH: Chi²test

*= p <0.05; **= p <0.01

 

External discolouration effect

Parameter			Dose (mg/kg bw/day)
			Control	40	60	80	100
Total number of examined litters			6	6	6	6	4
Total number of examined foetuses			61	48	61	67	38
Total number of affected litters			0	1	5	5	4
Total number of affected foetuses			0	2	42	58	38
Foetus, pale	Litter incidence	n	0	1	5	1	0
		%	0.0	16.7	83.3	16.7	0.0
	Foetal incidence	n	0	2	42	14	0
		%	0.000	4.167	68.852	20.896	0.000
Foetus, yellowish discolouration	Litter incidence	n	0	0	0	4	4
		%	0.0	0.0	0.0	66.7	100.0
	Foetal incidence	n	0	0	0	44	38
		%	0.000	0.000	0.000	65.672	100.000

 

Conclusions:

In conclusion, C.I. Solvent Yellow 124, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19), was associated with the following findings:
One animal on Gestation Day 16 was found dead in the High (100 mg/kg bw/day) group.
Abnormal skin colour and piloerection was present in all Test Item treated groups, and hunched back in the Mid 2 and High dose animals (80 and 100 mg/kg bw/day, respectively) was present and considered to be a dose-dependent, Test Item related effect. At 80 and 100 mg/kg bw/day the extent of the findings were clear enough to class them as adverse.
Test item related adverse effect were observed on the body weight, body weight gain, and corrected body weight values of the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).
Additionally, a dose-dependent reduction of the food consumption was observed during the treatment, reaching significance in the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).
Diffuse yellow discoloration in the adipose tissue was present in all Test Item treated animals, demonstrating systemic exposure. Furthermore, similar discoloration was present in the non-glandular mucosal region in some of the Mid 2 and High dose animals (80 and 100 mg/kg bw and 3 out of 6, and 4 out of 5 animals, respectively).
Statistically significant increase of the late embryonic loss and, consequently, in the total intrauterine mortality was observed in the High dose group.
The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

The mean foetal weight per litter in the test item treated groups showed a dose-dependent decrease which reached significance in the Mid 2 and High dose group when compared to the control mean value. The total number of retarded foetuses (runts) were higher in the Mid 1 and High dose groups (60 and 100 mg/kg bw/day, respectively). The small group size prevents these numbers being classed as adverse or not.
In two out of four litters in the High dose group, placentas showed paleness or yellow discoloration. The rest of the placentas were considered to be normal (but the very dark nature of placenta tissue prevents the visualisation of colour changes that are not extreme).
Foetal findings in the High dose litters indicates an observable test item related effect. Malformations ascribed to test item were observed in the High dose at External, Visceral and Skeletal examination. A dose-dependent pale colour or yellow discoloration was observed across all treated groups, indicating the presence of the Test Item in the foetal tissues, and suggesting potential neuro- and developmental toxicity effects in the offspring’s generation. The colouration of foetuses is not necessarily a clear adverse effect in itself; however, the test item is clearly teratogenic at 100 mg/kg/day, and the test item is present in the tissues of the developing foetuses even at 40 mg/kg/day. Under these circumstances, the 40 mg/kg/day dose was considered to be the LOAEL. Classical foetal findings in the rest of the treated groups (40 to 80 mg/kg/day), in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment.

In conclusion, C.I. Solvent Yellow 124, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 100 mg/kg/day, was associated with maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity. The extent of the findings was sufficient to make a clear positive conclusion even with the group size of 5 litters. A potentially adverse test item related effects (discoloration of foetuses, indicating presence/accumulation of test item) was observed at all dose levels. Since the test item was positive, it is considered that because of this finding the LOAEL was 40 mg/kg.

The multiple adverse developmental effects observed at 100 mg/kg bw/day indicates that the test item is teratogenic. The extent of the findings in the group size of 5 litters is sufficient to classify the test item as having adverse effects on development of rat foetuses. It is not considered to be justified for classification purposes, on animal experiment ethical grounds, to repeat this study with a larger group size as in a full OECD 414 study design.

Executive summary:

The objective of the preliminary study was to determine the Maximum Tolerated Dose (MTD) and to assess the effects of the test item on pregnant females and on the developing conceptuses in order to set the dose levels for the main developmental toxicity study. Additionally, foetal examination was included in this preliminary study in order to identify if there were evident foetal effects.

 

Animals (one control and four test item treated groups) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

 

The dose levels were set by the Sponsor in agreement with the Study Director based on the available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats [2], with the aim of inducing toxic effects but no death or suffering at the highest dose (100 mg/kg bw/day).

 

Treatment solutions were analysed for test item concentration once during the treatment period.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically.

 

Thirty sperm positive females were included in the study, six in each group.

 

Results

All test item formulations were within the range of 97-100% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

 

One animal on Gestation Day 16 was found dead in the High (100 mg/kg bw/day) group.

 

Abnormal skin colour and piloerection was present in all Test Item treated groups, and hunched back in the Mid 2 and High dose animals (80 and 100 mg/kg bw/day, respectively) was present and considered to be a dose-dependent, Test Item related effect.

 

Test item related adverse effect were observed on the body weight, body weight gain, and corrected body weight values of the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).

 

Additionally, a dose-dependent reduction of the food consumption was observed during the treatment, reaching significance in the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).

 

Diffuse yellow discoloration in the adipose tissue was present in all Test Item treated animals. Furthermore, similar discoloration was present in the non-glandular mucosal region in some of the Mid 2 and High dose animals (80 and 100 mg/kg bw and 3 out of 6, and 4 out of 5 animals, respectively).

 

Statistically significant increase of the late embryonic loss and, consequently, in the total intrauterine mortality was observed in the High dose group.

 

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

 

The mean foetal weight per litter in the test item treated groups showed a dose-dependent decrease which reached significance in the Mid 2 and High dose group when compared to the control mean value. The total number of retarded foetuses (runts) were higher in the Mid 1 and High dose groups (60 and 100 mg/kg bw/day, respectively).

 

In two out of four litters in the High dose group, placentas showed paleness or yellow discoloration. The rest of the placentas were considered to be normal.

 

Foetal findings in the High dose litters indicates an observable test item related effect. Malformations ascribed to test item were observed in the High dose at External, Visceral and Skeletal examination. These finding were based on a group size of 5 litters, but even if the same number of findings had been seen in a group size of 16+ litters, it would still be considered as a positive foetal effect. A dose-dependent pale colour or yellow discoloration was observed across all treated groups, indicating the presence of the Test Item in the foetal tissues, and suggesting potential neuro- and developmental toxicity effects in the offspring’s generation. The colouration of foetuses is not necessarily a clear adverse effect in itself; however, the test item is clearly teratogenic at 100 mg/kg/day, and the test item is present in the tissues of the developing foetuses even at 40 mg/kg/day. Under these circumstances, the 40 mg/kg/day dose was considered to be the LOAEL. Classical foetal findings in the rest of the treated groups (40 to 80 mg/kg/day), in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment.

 

In conclusion, C.I. Solvent Yellow 124, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 100 mg/kg/day, was associated with maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity. The extent of the findings was sufficient to make a clear positive conclusion even with the group size of 5 litters. A potentially adverse test item related effects (discoloration of foetuses, indicating presence/accumulation of test item) was observed at all dose levels. Since the test item was positive, it is considered that because of this finding the LOAEL was 40 mg/kg.

 

The multiple adverse developmental effects observed at 100 mg/kg bw/day indicates that the test item is teratogenic. The extent of the findings in the group size of 5 litters is sufficient to classify the test item as having adverse effects on development of rat foetuses. It is not considered to be justified for classification purposes, on animal‑experiment ethical grounds, to repeat this study with a larger group size as in a full OECD 414 study design. The full DRF study is attached for ECHA's reference as an indication that the full test should not be considered. Classification as Reproduction Category 2 is applied on the basis of the findings in the DRF study conducted.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch score 1. The LOAEL was 40. For risk assessment purposes, a repeat dose 90 day study gave a NOAEL of 10 which will be used to derive the DNEL.

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

The objective of the preliminary study was to determine the Maximum Tolerated Dose (MTD) and to assess the effects of the test item on pregnant females and on the developing conceptuses in order to set the dose levels for the main developmental toxicity study. Additionally, foetal examination was included in this preliminary study in order to identify if there were evident foetal effects.

 

Animals (one control and four test item treated groups) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

 

The dose levels were set by the Sponsor in agreement with the Study Director based on the available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats, with the aim of inducing toxic effects but no death or suffering at the highest dose (100 mg/kg bw/day).

Treatment solutions were analysed for test item concentration once during the treatment period.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically.

 

Thirty sperm positive females were included in the study, six in each group.

 

Results 

One animal on Gestation Day 16 was found dead in the High (100 mg/kg bw/day) group.

Abnormal skin colour and piloerection was present in all Test Item treated groups, and hunched back in the Mid 2 and High dose animals (80 and 100 mg/kg bw/day, respectively) was present and considered to be a dose-dependent, Test Item related effect.

Test item related adverse effect were observed on the body weight, body weight gain, and corrected body weight values of the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).

Additionally, a dose-dependent reduction of the food consumption was observed during the treatment, reaching significance in the Mid 2 and High dose groups (80 and 100 mg/kg bw/day, respectively).

Diffuse yellow discoloration in the adipose tissue was present in all Test Item treated animals. Furthermore, similar discoloration was present in the non-glandular mucosal region in some of the Mid 2 and High dose animals (80 and 100 mg/kg bw and 3 out of 6, and 4 out of 5 animals, respectively).

Statistically significant increase of the late embryonic loss and, consequently, in the total intrauterine mortality was observed in the High dose group.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

 

The mean foetal weight per litter in the test item treated groups showed a dose-dependent decrease which reached significance in the Mid 2 and High dose group when compared to the control mean value. The total number of retarded foetuses (runts) were higher in the Mid 1 and High dose groups (60 and 100 mg/kg bw/day, respectively).

In two out of four litters in the High dose group, placentas showed paleness or yellow discoloration. The rest of the placentas were considered to be normal.

Foetal findings in the High dose litters indicates an observable test item related effect. Malformations ascribed to test item were observed in the High dose at External, Visceral and Skeletal examination. These finding were based on a group size of 5 litters, but even if the same number of findings had been seen in a group size of 16+ litters, it would still be considered as a positive foetal effect. A dose-dependent pale colour or yellow discoloration was observed across all treated groups, indicating the presence of the Test Item in the foetal tissues, and suggesting potential neuro- and developmental toxicity effects in the offspring’s generation. The colouration of foetuses is not necessarily a clear adverse effect in itself; however, the test item is clearly teratogenic at 100 mg/kg/day, and the test item is present in the tissues of the developing foetuses even at 40 mg/kg/day. Under these circumstances, the 40 mg/kg/day dose was considered to be the LOAEL. Classical foetal findings in the rest of the treated groups (40 to 80 mg/kg/day), in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment.

 

In conclusion, C.I. Solvent Yellow 124, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 100 mg/kg/day, was associated with maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity. The extent of the findings was sufficient to make a clear positive conclusion even with the group size of 5 litters. A potentially adverse test item related effects (discoloration of foetuses, indicating presence/accumulation of test item) was observed at all dose levels. Since the test item was positive, it is considered that because of this finding the LOAEL was 40 mg/kg.

The multiple adverse developmental effects observed at 100 mg/kg bw/day indicates that the test item is teratogenic. The extent of the findings in the group size of 5 litters is sufficient to classify the test item as having adverse effects on development of rat foetuses. It is not considered to be justified for classification purposes, on animal‑experiment ethical grounds, to repeat this study with a larger group size as in a full OECD 414 study design.

Justification for classification or non-classification

The multiple adverse developmental effects observed at 100 mg/kg bw/day indicates that the test item is teratogenic. The extent of the findings in the group size of 5 litters is sufficient to classify the test item as having adverse effects on development of rat foetuses. It is not considered to be justified for classification purposes, on animal‑experiment ethical grounds, to repeat this study with a larger group size as in a full OECD 414 study design. The full DRF study is attached for ECHA's reference as an indication that the full test should not be considered. Classification as Reproduction Category 1B is applied on the basis of the findings in the DRF study conducted.