4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The registered substance did not cause adverse effects after oral administration of up to 1000 mg/kg /day to rats in a screening study according to OECD TG 421.

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 JUL 2012 to 12 SEP 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421), GLP compliant

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within +/- 20% of the mean weight.
The range of the body weight was:
Females: 174-220 g, (mean: 199.10 +/- 20%= 39.82 g)
Males: 270-321 g, (mean: 292.90 g, +/- 20%= 58.58 g)
Number of animals: 10 Males and 10 females per group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

Housing and Feeding Conditions:
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.1151)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.300512)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Details on mating procedure:

Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) - (total 16 samples)
Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation in study week 1 and 5 (total 18 samples)
All samples were stored at -20°C until analysis. Sample quantity for all samples was 10 mL in a 15mL falcon tube.
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH after completion of in-life phase.

Duration of treatment / exposure:

Males- 28-31 days
Females- Approximately 54 days

Frequency of treatment:

7 days/week

Details on study schedule:

Date of Draft Study Plan: 05 July 2012
Date of Final Study Plan: 09 July 2012
Date of 1st amendment 24 July 2012
to study plan
Date of 2nd amendment 12 September 2012
to study plan

Date of Start of Experiment 19 July 2012
Date of End of Experiment: 12 Setpember 2012
Date of Draft Report (BSL): 31 January 2013
Date of Final Report (BSL): February 2013

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight/day
Basis:
other: content C.I.

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 100, 300, 1000 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C)
Dose concentration was based on the purity/content of the test item (98.2 %)
The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.
Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period and post mating period in males.

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Sperm Analysis:
At necropsy (one day after the last administration) one epididymis and one testis was separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count was evaluated in all male animals using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0 C).

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28-31 days) and females were sacrificed on respective post natal day 4 along with pups At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Positive control:

Not included

Parental animals: Observations and examinations:

Males and Females: Body Weight, Food Consumption, Clinical Signs, Mating behaviour, organ weight, Gross pathology and Histopathology.

Oestrous cyclicity (parental animals):

Not evaluated

Sperm parameters (parental animals):

Testicular sperm head count and Epididymal Sperm motility

Litter observations:

Number and sex of pups, still births, runts, gross abnormalities of the pups and litter weight

Postmortem examinations (parental animals):

Males- Day 29-32; Females: On PND 4

Postmortem examinations (offspring):

Gross external

Statistics:

A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

Copulation Index (%), Fertility Index (%) and Delivery Index (%)

Offspring viability indices:

Viability Index (%)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality:
No mortalities were observed at any dose levels of male and female animals.

Clinical Observation:
No test item related clinical signs were observed in male and female animals. Few spontaneous clinical signs observed occasionally in male and female animals were alopecia at forepaws (1/10 in Control males and 1/10 in LD, MD and HD females), red nasal discharge (2/10 in HD males), slight piloerection (1/10 in LD, HD males, 1/10 in LD females). Discoloured red faeces were observed in all male and female treatment group animals throughout the study. This discoluration of the faeces was attributed to the red colour of the test item and as such of no toxicological significance.

Body Weight and Body Weight Change:
In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls.
In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption:
In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in all groups.

Sperm Analysis:
Statistical analysis of sperm motility and testicular sperm head count data revealed no test item related effect on sperm parameters and all group mean and individual values from treatment groups were comparable with the controls.

Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were yellow spot on right epididymides (1/10 in MD males), cyst on left ovary (1/10 in LD females), cyst on right ovary (1/10 in LD and MD females), cyst on both ovaries (1/10 in MD females), white areas on lung (1/10 in MD females) and extra red tissue on thymus (1/10 in MD females).
These gross pathological findings were spontaneous in nature and as such not a systemic effect due to the test item administration.

Organ Weight:
In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology
At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect.
No test item-related histological findings were noted in the male and female reproductive organs. Female reproductive organs showed similar post-partum histomorphology in the control and high dose group. The number of large ovarian corpora lutea was not essentially different between control animals and animals treated at 1000 mg/kg/day.
Two control females, one female treated at 100 mg/kg/day and one female treated at 1000 mg/kg/day were found not to be pregnant at terminal sacrifice, but this was not considered to be test item-related.
As a conclusion, under the conditions of the present study and based on the histopathological evaluation as defined by the study plan, the NOAEL (No Observed Adverse Effect Level) for pathology is considered to be 1000 mg/kg/day.

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Sex:

male/female

Basis for effect level:

other: No Major signs of toxicity or mortality was observed up to 1000mg/kg body weight in both male and females

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Litter Weight Data:
Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls.

Precoital Interval and Duration of Gestation:
No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 8, 9, 10 and 9 pregnancies in the control, low, mid and high dose respectively

Pre and Post Natal Data:
Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Litter Data:
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.
All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Reproductive Indices:
No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C (80%), LD and HD (90 %) dose group as compared MD group (100 %).

Pup Survival Data:
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam. Since this incidence of cannibalism was observed in one female of the two intermediate groups and it was within the rat cannibalism rate, this incidence was not considered to be test item related.

Pup External Finding:
No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed and were not related to the treatment with test item

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Sex:

male/female

Basis for effect level:

other: No major signs of toxicity were observed up to 1000 mg/kg body weight.

Reproductive effects observed:

not specified

Conclusions:

In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.
Based on the data generated from this reproduction/ developmental toxicity screening test with the test item, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

The aim of this study was to assess the possible effect of the test item on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of test item at dose volume of 5 mL/kg body weight. The test item was formulated in sterile water. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups.

Doses evaluated were: 0, 100, 300, 1000  mg/kg body weight/day

Dose concentration was based on the purity/content (content C.I.) of the test item (98.2 %).

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females (45, 49, 57 and 74) were sacrificed on respective day 26 after the evidence of mating. The wet weight of male and female reproductive organs was taken and preserved in 10 % neutral buffered formalin except testes and epididymides which were initially fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin. Histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

 

Summary Results

Clinical Signs and Mortality: No test item related clinical signs and mortalities were observed in both males and females.

Body Weight Development: In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls. In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption: In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Litter Weight Data: Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls.

Precoital Interval and Duration of Gestation: No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data: Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Litter Data: No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Reproductive Indices: No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Pup Survival Data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam.

Pup External Findings: No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Sperm Analysis: Statistical analysis of sperm motility and testicular sperm head count data revealed no test item related effect on sperm parameters and all group mean and individual values from treatment groups were comparable with the controls.

Gross Pathology: At necropsy, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Fewspontaneous gross pathological findings were observed in male and female animals and as such not a systemic effect due to the test item administration.

Organ Weight:In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology: At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect. No test item-related histological findings were noted in the male and female reproductive organs.

Dose Formulation Analysis: Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

The NOAEL for toxicity to reproduction is 1000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliable without restriction

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

A close structural analogue of the registered substance did not cause any adverse effects after oral administration of up to 1000 mg/kg /day in two developmental toxicity studies according toOECD tG 414 in rats and rabbits.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study, Adopted on 25 June 2018

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

27 July 2021 to 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics & Coatings Deutschland
- Expiration date of the batch: 18-November-2027
- Purity test date: 95.5% (w/w)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: formulation in vehicle

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.15468 kg to 2.64927 kg;
Females: 2.00849 kg to 2.28642 kg
- Housing: stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches)
- Diet (e.g. ad libitum): Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: (Minimum of five days) Start: 05 August 2021; End: 10 August 2021

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 to 20.9oC
- Humidity (%): 44 to 61%
- Air changes (per hr): 12 hours fluorescent light and 12 hours dark cycle


IN-LIFE DATES: From: 05 August 2021 To: 05 October 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% w/v in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house suspendibility test results.
Hence, 1% w/v Carboxymethyl cellulose was used as vehicle for test item formulations. Carboxymethyl cellulose is a routinely used vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg
body weight
- Lot no. (if required): SLCH1520
- Purity: Analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations was performed during first week and last week of the treatment. Approximately, 5 mL of samples was collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Details on mating procedure:

- M/F ratio per cage: 1:1 (M:F)
- Length of cohabitation: Until visual confirmation of mating
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: [visual observation] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

The test item/vehicle was administered by oral (gavage) route to respective group of animals once daily from gestation day (GD) 6 to 28.

Frequency of treatment:

once daily

Duration of test:

22 days per animal starting from implantation to one day prior to scheduled delivery (GD 6 to GD 28)

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.
- Rationale for animal assignment (if not random):The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section).

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: all visceral organs along with uterus and ovaries

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,
Absolute / relative thyroid weights, Mean fetal weight per doe, Mean fetal crown rump length per doe

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per doe, No. of implantations per doe, Litter size per doe, No. of live fetuses per doe, Percent of live fetuses per doe, No. of early/late resorptions per doe,
Percent of early/late resorptions per doe, Sex ratio (m/f) per doe, Pre/Post implantation losses (%) per doe, Fetal external / visceral / skeletal anomalies per doe.

Frequencies Comparison: Cross Tabs - Chi-square test:
No. of Pregnant / Non-pregnant females (Pregnancy status), No. of does with / without live fetuses, No. of does with / without dead fetuses, No. of does with / without resorptions

Indices:

Corrected Body Weight (g) = (Gestation day 29 body weight - Gestation day 6 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100

Historical control data:

All the obtained Data within the historical control range

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group.
There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the does of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts in thyroid gland was noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rabbits (Elizabeth McInnes, 2012).

Details on results:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 13.78, 13.10, 13.58 and 15.76 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of pre-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
The mean percentage of post-implantation loss per litter was 0.00, 0.00, 0.00 and 4.05 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of early resorptions per litter was 0.00, 0.00, 0.00 and 0.10 and the percentage of early resorptions was 0.00, 0.00, 0.00 and 3.57 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of early resorptions per doe across dose groups when compared to the vehicle control group.
The mean number of late resorptions per litter was 0.00, 0.00, 0.00 and 0.05 and the percentage of late resorptions was 0.00, 0.00, 0.00 and 0.48 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of late resorptions per doe across dose groups when compared to the vehicle control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21, 20, 21 and 21 females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21(out of 25), 20(out of 25), 21(out of 25) and 21(out of 25) females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.

Details on maternal toxic effects:

The oral administration of Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No such incidences noted in any of the tested dose groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no changes or no statistically significant differences noted in all the tested dose groups
when compared with vehicle control group for mean litter size.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
General / developmental variations such as subcutaneous hemorrhagic spot/s on different regions of the body and noted external malformations such as hyperextension of forelimb and hyperextension of hindlimb across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group (refer table no. 10 in study report).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
These noted skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain (refer table no. 12 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
Developmental variations such as discoloured liver/thymus/lungs/spleen, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain.
developmental malformations lateral/third ventricular dilatation of brain, retinal fold, misshapen liver lobes and retrocaval ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain(refer table no. 11 in study report).

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in New Zealand White Rabbits, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant rabbits and in the developing organisms; this included the assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses.

A total of 100 mated female New Zealand White rabbits distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant rabbits. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 6 to 28. The end points of assessment for does were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification were performed during the first and last week of treatment. The prepared formulations were considered acceptable and the mean results were within the range of 85 to 115% of nominal concentration and the relative standard deviation (% RSD) is less than 10%(mean recovery was between 98.62% and 99.97% at all measurements).

 A total of 21, 20, 21 and 21 females from group G1, G2, G3 and G4 were found with implantations yielding pregnancy rates of 84%, 80%, 84% and 84% respectively.

All the animals from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 26, 26 to 28 and 28 to 29. All the animals were euthanized on gestation Day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the does was recorded on the day of caesarean section. Each doe was observed for number of live or dead fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the does were observed for number of corpora lutea. The pre-and post-implantation losses per doe were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the animals. All the does were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for their crown rump length. The mean fetal weight and mean crown rump length per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the fetuses from each litter were subjected for fresh soft tissue (visceral) examination and fixed head sections examination, except the heads of fetuses which were allotted for skeletal evaluation. The bodies of fetuses without heads and all other intact fetuses from each litter were double stained with alcian blue for cartilage and Alizarin Red S for bones and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted during the experimental period at all the tested dose and vehicle control group animals. No changes were noted in mean maternal body weight, percent change in mean maternal body weight gain and mean maternal feed consumption at all the tested dose groups. There were no gross pathological changes noted during caesarean section of all the tested dose and vehicle control group animals. There were no effects noted in pregnancy rate and mean gravid uterus weight in all the tested dose groups. There were no effects noted in mean number of live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutea at all the tested dose groups. The mean pre- and post-implantation losses per doe were unaffected at all the tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups. No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the does during histopathological examination from all the tested dose groups.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight and mean fetal crown rump length per litter in either sex at all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner.

Reference 2

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

27 July 2021 to 04 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 240.25 to 298.41 g, Females: 200.65 to 249.44 g
- Housing: Sterilized standard polypropylene cage of Size: L 430 × B 285 × H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Minimum of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.2
- Humidity (%): 49 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark cycle and 12 hours fluorescent light

IN-LIFE DATES: From:30 July 2021 To:23 September 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The test item was uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. 1% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle: 10 mL/kg
- Lot no.: SLCH1520
- Purity: Analytical grade.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10% (treatment day 1: mean recovery (%) is 99.15%, 99.17% and 99.06% with % RSD of 0.06, 0.06 and 0.03 for groups G2, G3 and G4, respectively; treatment day 38: mean recovery (%) is 100.10%, 100.16% and 100.09% with % RSD of 0.06, 0.05 and 0.09 for groups G2, G3 and G4, respectively).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:1:2 ratio
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: 14 days, Females not mated within 14 days of pairing with the first male were placed with a second proven male for one week
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.

Frequency of treatment:

Once Daily

Duration of test:

15 days per animal starting from implantation to one day prior to scheduled delivery (GD 5 to GD 19)

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproducti on toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs, along with uterus, ovaries and thyroid along with parathyroid.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]

Statistics:

Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,Absolute / relative thyroid weights, Mean fetal weight per dam, Mean fetal crown rump length per dam.

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison: Cross Tabs - Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions.

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100

Historical control data:

In-house historical data available for the same species and strain and referred to end points where required in the study report.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group. There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in group G3 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.

There were no changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 5.77, 7.29, 10.86 and 12.44 for groups G1, G2, G3 and G4 respectively.
There were no changes noted and no statistically significant differences noted for percentage of pre-implantation loss per litter in all the teste dose groups.

The mean percentage of post-implantation loss per litter was 9.61, 9.96, 6.94 and 11.62 for groups G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two incidental findings with total implantation loss (with total empty implantation sites/early resorptions) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions per litter was 0.71, 0.71, 0.62 and 0.95 with a percentage of early resorptions was 6.53, 9.20, 6.62 and 8.43 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

The mean number of late resorptions per litter was 0.14, 0.00, 0.00 and 0.00 and the percentage of late resorptions was 1.30, 0.00, 0.00 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of dead fetuses per litter was 0.05, 0.10, 0.05 and 0.00 with a percentage of 0.79, 0.84, 0.34 and 0.00. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted before caesarean sectioning.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively.

No female was littered during gestation period in all the dose groups and control group before caesarean sectioning.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Details on maternal toxic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.49, 1.26, 1.21 and 0.92 for groups G1, G2, G3 and G4 respectively. There were changes or no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 10.90, 10.05, 10.33 and 9.41 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group such as Subcutaneous hemorrhagic spot beneath the skin on different regions of the body, Skin discoloured / pale coloured, Bent tail, Kinked tail, Hyperextension (unilateral) of forelimb, Hyperextension (unilateral) of hind limb and Protrusion of tongue.These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant decrease in number of incidences of unossified sternebra no. 6 in group G3 when compared with vehicle control group is considered as incidental and toxicologically in-signficant. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (refer table no. 16 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal visceral/soft tissue alterations were noted across the dose groups and vehicle control group such as Pale colored liver, Renal pelvis dilation (bilateral) of kidneys, Dilatation of Ureters (bilateral), Dilated lateral ventricles of brain and Nasal conchae enlarged.These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal anogenital distance/mean fetal anogenital distance ratio and mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. In groups G1 and G3, 1 out of 25 females each was noted with total implantation loss with no evidence of live fetuses.

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO₂asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no differences noted between the groups in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam from all the tested dose groups. There were no differences noted between the dose groups in mean gravid uterus weight. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the in-house historical control range of this species and strain.

Reference 3

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study, Adopted on 25 June 2018

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2021 to 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics & Coatings Deutschland
- Expiration date of the batch: 18-November-2027
- Purity test date: 95.5% (w/w)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: formulation in vehicle

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.15468 kg to 2.64927 kg;
Females: 2.00849 kg to 2.28642 kg
- Housing: stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches)
- Diet (e.g. ad libitum): Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: (Minimum of five days) Start: 05 August 2021; End: 10 August 2021

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 to 20.9oC
- Humidity (%): 44 to 61%
- Air changes (per hr): 12 hours fluorescent light and 12 hours dark cycle


IN-LIFE DATES: From: 05 August 2021 To: 05 October 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% w/v in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house suspendibility test results.
Hence, 1% w/v Carboxymethyl cellulose was used as vehicle for test item formulations. Carboxymethyl cellulose is a routinely used vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg
body weight
- Lot no. (if required): SLCH1520
- Purity: Analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations was performed during first week and last week of the treatment. Approximately, 5 mL of samples was collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Details on mating procedure:

- M/F ratio per cage: 1:1 (M:F)
- Length of cohabitation: Until visual confirmation of mating
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: [visual observation] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

The test item/vehicle was administered by oral (gavage) route to respective group of animals once daily from gestation day (GD) 6 to 28.

Frequency of treatment:

once daily

Duration of test:

22 days per animal starting from implantation to one day prior to scheduled delivery (GD 6 to GD 28)

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.
- Rationale for animal assignment (if not random):The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section).

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: all visceral organs along with uterus and ovaries

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,
Absolute / relative thyroid weights, Mean fetal weight per doe, Mean fetal crown rump length per doe

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per doe, No. of implantations per doe, Litter size per doe, No. of live fetuses per doe, Percent of live fetuses per doe, No. of early/late resorptions per doe,
Percent of early/late resorptions per doe, Sex ratio (m/f) per doe, Pre/Post implantation losses (%) per doe, Fetal external / visceral / skeletal anomalies per doe.

Frequencies Comparison: Cross Tabs - Chi-square test:
No. of Pregnant / Non-pregnant females (Pregnancy status), No. of does with / without live fetuses, No. of does with / without dead fetuses, No. of does with / without resorptions

Indices:

Corrected Body Weight (g) = (Gestation day 29 body weight - Gestation day 6 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100

Historical control data:

All the obtained Data within the historical control range

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group.
There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the does of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts in thyroid gland was noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rabbits (Elizabeth McInnes, 2012).

Details on results:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 13.78, 13.10, 13.58 and 15.76 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of pre-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
The mean percentage of post-implantation loss per litter was 0.00, 0.00, 0.00 and 4.05 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of early resorptions per litter was 0.00, 0.00, 0.00 and 0.10 and the percentage of early resorptions was 0.00, 0.00, 0.00 and 3.57 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of early resorptions per doe across dose groups when compared to the vehicle control group.
The mean number of late resorptions per litter was 0.00, 0.00, 0.00 and 0.05 and the percentage of late resorptions was 0.00, 0.00, 0.00 and 0.48 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of late resorptions per doe across dose groups when compared to the vehicle control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21, 20, 21 and 21 females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21(out of 25), 20(out of 25), 21(out of 25) and 21(out of 25) females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.

Details on maternal toxic effects:

The oral administration of the test item by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No such incidences noted in any of the tested dose groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No such incidences noted in any of the tested dose groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no changes or no statistically significant differences noted in all the tested dose groups
when compared with vehicle control group for mean litter size.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
General / developmental variations such as subcutaneous hemorrhagic spot/s on different regions of the body and noted external malformations such as hyperextension of forelimb and hyperextension of hindlimb across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group (refer table no. 10 in study report).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
These noted skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain (refer table no. 12 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
Developmental variations such as discoloured liver/thymus/lungs/spleen, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain.
developmental malformations lateral/third ventricular dilatation of brain, retinal fold, misshapen liver lobes and retrocaval ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain(refer table no. 11 in study report).

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

oral administration of the test item by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, the oral administration of test item by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in New Zealand White Rabbits, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure of test item on the pregnant rabbits and in the developing organisms; this included the assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses.

 

A total of 100 mated female New Zealand White rabbits distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant rabbits. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 6 to 28. The end points of assessment for does were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

 

Homogeneity and dose formulation analysis for dose concentration verification were performed during the first and last week of treatment. The prepared formulations were considered acceptable and the mean results were within the range of 85 to 115% of nominal concentration and the relative standard deviation (% RSD) is less than 10%(mean recovery was between 98.62% and 99.97% at all measurements).

 

 A total of 21, 20, 21 and 21 females from group G1, G2, G3 and G4 were found with implantations yielding pregnancy rates of 84%, 80%, 84% and 84% respectively.

 

All the animals from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 26, 26 to 28 and 28 to 29. All the animals were euthanized on gestation Day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the does was recorded on the day of caesarean section. Each doe was observed for number of live or dead fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the does were observed for number of corpora lutea. The pre-and post-implantation losses per doe were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the animals. All the does were evaluated for histopathology of thyroid along with the parathyroid.

 

All the fetuses collected from each litter were weighed and measured for their crown rump length. The mean fetal weight and mean crown rump length per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the fetuses from each litter were subjected for fresh soft tissue (visceral) examination and fixed head sections examination, except the heads of fetuses which were allotted for skeletal evaluation. The bodies of fetuses without heads and all other intact fetuses from each litter were double stained with alcian blue for cartilage and Alizarin Red S for bones and subjected to skeletal examination.

 

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted during the experimental period at all the tested dose and vehicle control group animals. No changes were noted in mean maternal body weight, percent change in mean maternal body weight gain and mean maternal feed consumption at all the tested dose groups. There were no gross pathological changes noted during caesarean section of all the tested dose and vehicle control group animals. There were no effects noted in pregnancy rate and mean gravid uterus weight in all the tested dose groups. There were no effects noted in mean number of live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutea at all the tested dose groups. The mean pre- and post-implantation losses per doe were unaffected at all the tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups. No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the does during histopathological examination from all the tested dose groups.

 

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight and mean fetal crown rump length per litter in either sex at all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner.

Reference 4

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2021 to 04 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Plastics & Coatings Deutschland, Germany
- Expiration date of the batch: 18-November-2027

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature in 1% w/v Carboxymethyl cellulose with the concentrations of 5 mg/mL and
100 mg/mL as per analytical method validation study (Bioneeds study no.: BIO-ANM 1756.

- Solubility and stability of the test substance in the solvent/vehicle:

The test item was insoluble in distilled water and uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.

The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.: BIO-ANM 1756). The test item, Agrocer Red 112 was stable for 6 hours and 48 hours at room temperature in 1% w/v Carboxymethyl cellulose with the concentrations of 5 mg/mL and 100 mg/mL as per analytical method validation study (Bioneeds study no.:
BIO-ANM 1756).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: Liquid [formulation in vehicle]

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 240.25 to 298.41 g, Females: 200.65 to 249.44 g
- Housing: Sterilized standard polypropylene cage of Size: L 430 × B 285 × H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Minimum of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.2
- Humidity (%): 49 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark cycle and 12 hours fluorescent light

IN-LIFE DATES: From:30 July 2021 To:23 September 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The test item was uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. 1% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle: 10 mL/kg
- Lot no.: SLCH1520
- Purity: Analytical grade.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10% (treatment day 1: mean recovery (%) is 99.15%, 99.17% and 99.06% with % RSD of 0.06, 0.06 and 0.03 for groups G2, G3 and G4, respectively; treatment day 38: mean recovery (%) is 100.10%, 100.16% and 100.09% with % RSD of 0.06, 0.05 and 0.09 for groups G2, G3 and G4, respectively).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:1:2 ratio
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: 14 days, Females not mated within 14 days of pairing with the first male were placed with a second proven male for one week
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.

Frequency of treatment:

Once Daily

Duration of test:

15 days per animal starting from implantation to one day prior to scheduled delivery (GD 5 to GD 19)

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproducti on toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs, along with uterus, ovaries and thyroid along with parathyroid.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]

Statistics:

Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,Absolute / relative thyroid weights, Mean fetal weight per dam, Mean fetal crown rump length per dam.

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison: Cross Tabs - Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions.

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100

Historical control data:

In-house historical data available for the same species and strain and referred to end points where required in the study report.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period

Mortality:

no mortality observed

Description (incidence):

There were no morbidity/mortality noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group. There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in group G3 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.

There were no changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group.

Details on results:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of general toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions were noted

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean percentage of pre-implantation loss per litter was 5.77, 7.29, 10.86 and 12.44 for groups G1, G2, G3 and G4 respectively.
There were no changes noted and no statistically significant differences noted for percentage of pre-implantation loss per litter in all the teste dose groups.

The mean percentage of post-implantation loss per litter was 9.61, 9.96, 6.94 and 11.62 for groups G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two incidental findings with total implantation loss (with total empty implantation sites/early resorptions) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early resorptions per litter was 0.71, 0.71, 0.62 and 0.95 with a percentage of early resorptions was 6.53, 9.20, 6.62 and 8.43 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

The mean number of late resorptions per litter was 0.14, 0.00, 0.00 and 0.00 and the percentage of late resorptions was 1.30, 0.00, 0.00 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of dead fetuses per litter was 0.05, 0.10, 0.05 and 0.00 with a percentage of 0.79, 0.84, 0.34 and 0.00. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No early deliveries noted before caesarean sectioning.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively.

No female was littered during gestation period in all the dose groups and control group before caesarean sectioning.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.

Details on maternal toxic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.49, 1.26, 1.21 and 0.92 for groups G1, G2, G3 and G4 respectively. There were changes or no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 10.90, 10.05, 10.33 and 9.41 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group such as Subcutaneous hemorrhagic spot beneath the skin on different regions of the body, Skin discoloured / pale coloured, Bent tail, Kinked tail, Hyperextension (unilateral) of forelimb, Hyperextension (unilateral) of hind limb and Protrusion of tongue.These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant decrease in number of incidences of unossified sternebra no. 6 in group G3 when compared with vehicle control group is considered as incidental and toxicologically in-signficant. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (refer table no. 16 in study report).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal visceral/soft tissue alterations were noted across the dose groups and vehicle control group such as Pale colored liver, Renal pelvis dilation (bilateral) of kidneys, Dilatation of Ureters (bilateral), Dilated lateral ventricles of brain and Nasal conchae enlarged.These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Description (incidence and severity):

There were no changes noted in mean fetal anogenital distance/mean fetal anogenital distance ratio and mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.

Details on embryotoxic / teratogenic effects:

The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. In groups G1 and G3, 1 out of 25 females each was noted with total implantation loss with no evidence of live fetuses.

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO₂asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no differences noted between the groups in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam from all the tested dose groups. There were no differences noted between the dose groups in mean gravid uterus weight. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the in-house historical control range of this species and strain.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 19 JUL 2012 to 12 SEP 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421), GLP compliant

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, No. 421, “Reproduction/Developmental Toxicity Screening Test”adopted on 27 July 1995.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within +/- 20% of the mean weight.
The range of the body weight was:
Females: 174-220 g, (mean: 199.10 +/- 20%= 39.82 g)
Males: 270-321 g, (mean: 292.90 g, +/- 20%= 58.58 g)
Number of animals: 10 Males and 10 females per group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

Housing and Feeding Conditions:
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.1151)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.300512)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) - (total 16 samples)

Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation in study week 1 and 5 (total 18 samples)

All samples were stored at -20°C until analysis. Sample quantity for all samples was 10 mL in a 15mL falcon tube.

All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH after completion of in-life phase.

Details on mating procedure:

Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation.

Duration of treatment / exposure:

Males 28-31 days; Females: Approx. 54 days

Frequency of treatment:

7 days/week

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 100, 300, 1000 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C)
Dose concentration was based on the purity/content of the test item (98.2 %)
The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

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

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Sperm Analysis:
At necropsy (one day after the last administration) one epididymis and one testis was separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count was evaluated in all male animals using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0 C).

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28-31 days) and females were sacrificed on respective post natal day 4 along with pups At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Maternal examinations:

Body weight, food consumption, clinical signs,macropscopic findings, micropscopic findings, reproductive behaviour (copulation index, fertility index)

Ovaries and uterine content:

macropscopic and micropscopic examination.

Fetal examinations:

Number, weight and sex of pups, No. of still births, live births, runts and gross external abnormalities on PND 0 and 4.

Statistics:

A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Indices:

Copulation, Fertility, Delivery and Viability Indices

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality:
No mortalities were observed at any dose levels of male and female animals.

Clinical Observation:
No test item related clinical signs were observed in male and female animals. Few spontaneous clinical signs observed occasionally in male and female animals were alopecia at forepaws (1/10 in Control males and 1/10 in LD, MD and HD females), red nasal discharge (2/10 in HD males), slight piloerection (1/10 in LD, HD males, 1/10 in LD females). Discoloured red faeces were observed in all male and female treatment group animals throughout the study. This discoluration of the faeces was attributed to the red colour of the test item and as such of no toxicological significance.

Body Weight and Body Weight Change:
In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls.
In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption:
In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in all groups.

Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were yellow spot on right epididymides (1/10 in MD males), cyst on left ovary (1/10 in LD females), cyst on right ovary (1/10 in LD and MD females), cyst on both ovaries (1/10 in MD females), white areas on lung (1/10 in MD females) and extra red tissue on thymus (1/10 in MD females).
These gross pathological findings were spontaneous in nature and as such not a systemic effect due to the test item administration.

Organ Weight:
In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology
At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect.
No test item-related histological findings were noted in the male and female reproductive organs. Female reproductive organs showed similar post-partum histomorphology in the control and high dose group. The number of large ovarian corpora lutea was not essentially different between control animals and animals treated at 1000 mg/kg/day.
Two control females, one female treated at 100 mg/kg/day and one female treated at 1000 mg/kg/day were found not to be pregnant at terminal sacrifice, but this was not considered to be test item-related.
As a conclusion, under the conditions of the present study and based on the histopathological evaluation as defined by the study plan, the NOAEL (No Observed Adverse Effect Level) for pathology is considered to be 1000 mg/kg/day.

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Soft tissue and skeletal examinations were not made.

Abnormalities:

not specified

Developmental effects observed:

not specified

Litter Weight Data: Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls. Precoital Interval and Duration of Gestation: No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births. Successful mating resulted in 8, 9, 10 and 9 pregnancies in the control, low, mid and high dose respectively Pre and Post Natal Data: Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls. Litter Data: No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls. Reproductive Indices: No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls. Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C (80%), LD and HD (90 %) dose group as compared MD group (100 %). Pup Survival Data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam. Since this incidence of cannibalism was observed in one female of the two intermediate groups and it was within the rat cannibalism rate, this incidence was not considered to be test item related. Pup External Finding: No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed and were not related to the treatment with test item.

Conclusions:

In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 0, 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.
Based on the data generated from this reproduction/ developmental toxicity screening test, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

The aim of this study was to assess the possible effect of the test item on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of test item at dose volume of 5 mL/kg body weight. The test item was formulated in sterile water. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups.

Doses evaluated were: 0, 100, 300, 1000  mg/kg body weight/day

Dose concentration was based on the purity/content (content C.I.) of the test item (98.2 %).

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females (45, 49, 57 and 74) were sacrificed on respective day 26 after the evidence of mating. The wet weight of male and female reproductive organs was taken and preserved in 10 % neutral buffered formalin except testes and epididymides which were initially fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin. Histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

 

Summary Results

Clinical Signs and Mortality: No test item related clinical signs and mortalities were observed in both males and females.

Body Weight Development: In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls. In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption: In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Litter Weight Data: Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls.

Precoital Interval and Duration of Gestation: No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data: Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Litter Data: No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Reproductive Indices: No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Pup Survival Data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam.

Pup External Findings: No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Sperm Analysis: Statistical analysis of sperm motility and testicular sperm head count data revealed no test item related effect on sperm parameters and all group mean and individual values from treatment groups were comparable with the controls.

Gross Pathology: At necropsy, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Fewspontaneous gross pathological findings were observed in male and female animals and as such not a systemic effect due to the test item administration.

Organ Weight:In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology: At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect. No test item-related histological findings were noted in the male and female reproductive organs.

Dose Formulation Analysis: Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

 

The NOAEL for developmental/parental toxicity is1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliable (read across)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

Neither in fertility, developmental toxicity nor other repeated dose studies any adverse effects were observed regarding reproductive sucess or function of the reproductive system in rats and rabbits.