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

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

252-021-1

EC Name:

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

Cas Number:

34432-92-3

Molecular formula:

C22H31N3O2

IUPAC Name:

N-ethyl-N-[2-(1-isobutoxyethoxy)ethyl]-4-(phenyldiazenyl)aniline

Test material form:

liquid: viscous

Details on test material:

Name: C.I. Solvent Yellow 124
Batch/Lot Number: S3424 / S3422
Description: Dark Brown Oily Viscous Liquid
Purity: 94.8 % / 94.5%
Expiry Date: 25 April 2018 / 06/04/2018
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 5

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

placenta

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

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.

Overall developmental toxicity

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.