JAUNE REACTIF 181

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The NOAEL (No Observed Adverse Effect Level) is considered to be 1000 mg/kg/d for reproduction toxicity in males and female rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2020 to 24 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: FAT 40400/F TE
Batch No. BOP 01-20 (MC-66870600)
Expiry Date: June, 8th 2025
Purity as per Certificate of Analysis: 81.4 % all colored organic constituents; 70.9 % main constituent
Physical Appearance: red powder, solid at 20 °C
Storage Conditions: Deep Frozen (-10 to -25 °C)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology (India) Pvt. Ltd. 4B MN Park, Turkapally Village,
Shameerpet Mandal, Medchal Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 12 weeks.
- Weight at study initiation: Males: 320.69 to 394.49 g; Females: 217.93 to 262.77 g. At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group
- Fasting period before study:
- Housing:
Pre-mating:
Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless-steel sipper tubes.
Mating and post-mating:
During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage once a week.
- Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany ad libitum):
- Water: Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes
- Acclimation period: 7 day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 14
- Humidity (%): 49 - 66
- Air changes (per hr): 12.7 – 12.8
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 22 September 2020 to 19 November 2020

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 38 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 44-59 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed). The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only. The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Details on mating procedure:

Mating Procedure
One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within six from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they litter. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination). The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

100, 300 and 1000 mg/kg bw/day

Duration of treatment / exposure:

Males: once daily for 38 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 44-59 days),

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

Clinical Signs, Morbidity and Mortality:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. All rats were observed for clinical signs twice daily during the treatment period i.e. once prior to dosing and 1-2 hour after the dosing. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).
Body Weights:
i) Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till mating confirmation with males.
ii) All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.
Food Consumption:
The food consumption was measured at weekly intervals (± 1 day) during treatment. Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
c. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
d. After standardization, the individual pup body weight was recorded on Day 7 and 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
f. The number of nipples/areolae in male pups was counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

Postmortem examinations (parental animals):

Hormone Analysis:
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
• Two available surplus pups on Day 4 after birth.
• All dams prior to sacrifice and two available pups per litter on Day 13.
• All adult males, prior to sacrifice
Pups were lightly anaesthetized with isoflurane and incised at the jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labelled tubes and kept on the bench top for 20 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4 °C. The serum samples were placed in labelled plastic tubes and stored at about -70 °C until they were analyzed.
Blood samples were not collected from the dams in which all pups were dead/cannibalized during lactation.
Thyroid Profile Hormones:
The following thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) method for the serum samples using BIO-RAD microplate washer and BIO-RAD model 680 readers.
Serial No. Parameters Abbreviations Units
1 Rodent Thyroid Stimulating Hormone TSH ng/mL
2 Rodent Thyroxin T4 ng/mL
c: expansion of unit: ng/mL: nano grams/ per milli litre

The kits manufactured by Endocrine Technologies Inc., USA were used for the assay.
The following procedures were adopted for the analysis:
1. Samples and standards were loaded to the microplate wells where antibody against analyte hormone was available in solid phase. Antibody-enzyme conjugate was then added to the wells.
2. The mixture was incubated (3 h for TSH and 1 h for T4) to enable the binding of hormones with antibody and simultaneously with the antibody in the antibody-enzyme conjugate.
3. The incubated wells were washed, chromogen/ substrate was added followed by a brief incubation for 20 minutes.
4. Stop solution was added for stopping the colour development reaction. The colour development was read at wavelength (450nm).
5. Nonlinear Curve plot was derived using ODs from standards and calculation of unknowns using curve plot & sample OD values.

Gross Necropsy:
All adult animals and pups were examined macroscopically for any structural abnormalities or pathological changes. The adult animals were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. All animals were subjected to detailed necropsy by a veterinary pathologist and findings were recorded. All the surviving pups were necropsied on Lactation Day 13 and findings were recorded. The reproductive organs were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death. Organs listed in the table below were collected and preserved from all adult animals
For non-pregnant rats (Rx9091 and Rx9094), the uterus was stained with Salewski stain to identify the pre-implantation loss of the embryos. The number of implantation sites were recorded for all the dams.

Organ/Tissue Collection, Weighing and Preservation:
On completion of the gross pathology examination, the tissues and organs noted below were collected and weighed from all adult animals. The organ weight ratios (organ to body weight) as percentage of fasting body weight were determined and presented in the report. The paired organs were weighed together and combined weights were presented. The tissues were preserved in 10 % Neutral Buffered Formalin (NBF) except for the testes.
On Day 13, thyroid gland from available one male and female pup from each litter (randomly selected) was collected and preserved in 10 % NBF for the histopathological examination. The thyroid weight was determined after fixation. In addition, below mentioned tissues were collected from surviving animals on LD 14 and weighed for further possible analysis at sponsor designated site.
1. Piece (approximately 200 mg) of medial lobe of liver, glandular stomach and a portion of duodenal intestinal segment (washed) each were collected in separate pre-labelled tubes and transferred quickly into the liquid nitrogen and stored at -80 °C.
2. The collected tissues were washed in saline and the blotted tissues were transferred to tubes.
3. From liver, glandular stomach and duodenal segment, remaining tissues were preserved in 10 % NBF for possible histopathology evaluation. The microscopic evaluation of liver, stomach and duodenum will be performed in consultation with Sponsor. If not evaluated, will be discarded before finalization of report.
4. Acid rinsed autoclaved surgical instruments were used (separate sets for each group).
These tissue samples were collected from all terminally sacrificed males and females at necropsy except for the non-littered females (Rx9091 and Rx9094). Collected tissues will be shipped under frozen condition to the address provided by the Sponsor. The results generated from these samples were not included in this study report.

Histopathology:
Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs were identified at high dose.
The reproductive organs of two not littered females (Rx9091 and Rx9094) from high dose group were examined.
The tissues were processed for routine paraffin embedding and approximately 5 micron sections were stained with Haematoxylin Eosin stain. Additional testes sections (4µm) were stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS). Parameters such as body weight, body weight change, food consumption, organ weights, organ weight ratios (organ to body weight), oestrous cycle, ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (thyroid profile) data were evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of non-normal, data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences. Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square was significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day. All hypothesis testing were carried out at the 5 % (2-sided) significance level unless otherwise specified. Significant differences were designated throughout the report as below:
*: Statistically significant difference from the control group at p <0.05

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the treatment period in either sex at all the doses tested. Red (light to dark) colour faeces were observed at low and high dose groups in either sex. This could be due to physical nature of the test item.

Mortality:

not specified

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group. Incidence of significantly higher total absolute weight gain in females at mid dose was observed. This isolated occurrence was toxicologically not significant as the mean body weights were not altered by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption was significantly higher during days 1-8, 8-15, 22-29 and 29-36 in males and during days 1-8 in females at 1000 mg/kg/day high dose. These sporadic incidences of significant difference in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Thyroid profile parameters (TSH and T4) were not affected by test item administration in adults.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effect observed

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. Test item had no effects on the number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses.

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid profile parameters (TSH and T4) were not affected by test item administration.

Sexual maturation:

no effects observed

Description (incidence and severity):

Most of the rats showed diestrous prior to necropsy.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The male pups did not exhibit areola/nipple retention on PND 13.

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effect observed

Critical effects observed:

no

Reproductive effects observed:

no

Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day (Dam No. Rx9093, Pup No. M04 and M06). This gross finding in pups was considered to be the congenital development defect and not treatment related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides (Rx9042 and Rx9070); enlarged spleen (Rx9040 and Rx9041) noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. In testes, the staging of spermatogenesis did not reveal any stage specific changes and the spermatogenic cycles observed in the different seminiferous tubules were complete in all treated groups. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats. The thyroids from pups did not show any histologic alterations. The cause of infertility in two female rats (Rx9091 and Rx9094) at 1000 mg/kg/day dose group could not be ascertained as no corresponding gross and microscopic changes were observed in reproductive organs. All the observed findings were the spontaneous/incidental findings and the incidences at 1000 mg/kg/day were comparable to the concurrent control groups.

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for Reproduction Toxicity for FAT 40400/F TE is determined to be 1000 mg/kg bw/day.

Executive summary:

The Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage with FAT 40400/F was carried out according OECD guideline 421 to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was dissolved in Milli-Q water and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bw/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bw/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no alterations on target organs were identified at high dose. Under the experimental conditions employed, there were no effects on clinical signs except red (light to dark) colour faeces were observed at low and high dose groups in either sex and this could be due to physical nature of the test item. There wer no mortality mortality and effects on body weights, food consumption, maternal body weights and food consumption, fertility parameters, litter parameters, thyroid stimulating hormone (TSH) and thyroxine (T4) levels, terminal fasting body weights, organ weights and its ratios. There were no test item- related changes on gross/microscopic examination. The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested. No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses. Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day. This gross finding in pups was considered to be the congenital development defect and not treatment related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides; enlarged spleen noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. As there were no treatment-related effects on reproduction and fertility parameters up to and including 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for FAT 40400/F TE is determined to be 1000 mg/kg bw/day under the test conditions and doses employed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study, Klimisch score 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage with FAT 40400/F was carried out according OECD guideline 421 to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was dissolved in Milli-Q water and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs aterations were identified at high dose. Under the experimental conditions employed, there were no effects on clinical signs except red (light to dark) colour faeces were observed at low and high dose groups in either sex and this could be due to physical nature of the test item. There were no mortality and effects on body weights, food consumption, maternal body weights and food consumption, fertility parameters, litter parameters, thyroid stimulating hormone (TSH) and thyroxine (T4) levels, terminal fasting body weights, organ weights and its ratios recorded. There were no test item related changes on gross/microscopic examination. The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested. No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses. Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day. This gross finding in pups was considered to be the congenital development defect and not treatment-related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides; enlarged spleen noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. As there were no treatment-related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for FAT 40400/F is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Effects on developmental toxicity

Description of key information

The NOAEL (No Observed Adverse Effect Level) for developmental toxicity is therefore considered to be 1000 mg/kg/day under the conditions of this study.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

Reproduction/ Developmental Toxicity Screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2020 to 24 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Reproduction/ Developmental Toxicity Screening test (OECD 421)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: FAT 40400/F TE
Batch No. BOP 01-20 (MC-66870600)
Expiry Date: June, 8th 2025
Purity as per Certificate of Analysis: 81.4 % all colored organic constituents; 70.9 % main constituent
Physical Appearance: red powder, solid at 20 °C
Storage Conditions: Deep Frozen (-10 to -25 °C)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Hylasco Biotechnology (India) Pvt. Ltd. 4B MN Park, Turkapally Village,
Shameerpet Mandal, Medchal Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 12 weeks.
- Weight at study initiation: Males: 320.69 to 394.49 g; Females: 217.93 to 262.77 g. At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group

- Fasting period before study:
- Housing:
Pre-mating:
Two rats of the same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless-steel sipper tubes.
Mating and post-mating:
During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (Gestation Day 20).
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage once a week.
- Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany ad libitum):
- Water: Deep bore-well water passed through an activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes
- Acclimation period:
7 day
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21 to 14
- Humidity (%):
49 - 66
- Air changes (per hr):
12.7 – 12.8
- Photoperiod (hrs dark / hrs light):
12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 22 September 2020 to 19 November 2020

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 38 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 44-59 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed). The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only. The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

100, 300 and 1000 mg/kg bw/day

Details on mating procedure:

Mating Procedure
One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within six from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they litter. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination). The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.

Duration of treatment / exposure:

Males: once daily for 38 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 44-59 days),

Frequency of treatment:

Daily

Duration of test:

44-59 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Maternal examinations:

Clinical Signs, Morbidity and Mortality:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. All rats were observed for clinical signs twice daily during the treatment period i.e. once prior to dosing and 1-2 hour after the dosing. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).
Body Weights:
i) Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till mating confirmation with males.
ii) All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.
Food Consumption:
The food consumption was measured at weekly intervals (± 1 day) during treatment. Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

Fetal examinations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
c. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
d. After standardization, the individual pup body weight was recorded on Day 7 and 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
f. The number of nipples/areolae in male pups was counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the treatment period in either sex at all the doses tested. Red (light to dark) colour faeces were observed at low and high dose groups in either sex. This could be due to physical nature of the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group. Incidence of significantly higher total absolute weight gain in females at mid dose was observed. This isolated occurrence was toxicologically not significant as the mean body weights were not altered by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption was significantly higher during days 1-8, 8-15, 22-29 and 29-36 in males and during days 1-8 in females at 1000 mg/kg/day high dose. These sporadic incidences of significant difference in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

Food efficiency:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid profile parameters (TSH and T4) were not affected by test item administration.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Test item had no effects on the number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Study findings

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. Test item had no effects on the number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Other effects:

not specified

Description (incidence and severity):

There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effect observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day (Dam No. Rx9093, Pup No. M04 and M06). This gross finding in pups was considered to be the congenital development defect and not treatment related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides (Rx9042 and Rx9070); enlarged spleen (Rx9040 and Rx9041) noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. In testes, the staging of spermatogenesis did not reveal any stage specific changes and the spermatogenic cycles observed in the different seminiferous tubules were complete in all treated groups. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats. The thyroids from pups did not show any histologic alterations. The cause of infertility in two female rats (Rx9091 and Rx9094) at 1000 mg/kg/day dose group could not be ascertained as no corresponding gross and microscopic changes were observed in reproductive organs. All observed findings were the spontaneous/incidental findings and the incidences at 1000 mg/kg/day were comparable to the concurrent control groups.

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for developmental toxicity for FAT 40400/F is determined to be 1000 mg/kg bw/day.

Executive summary:

The Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage with FAT 40400/F was carried out according OECD guideline 421 to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was dissolved in Milli-Q water and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bwtday to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs alterations were identified at high dose. Under the experimental conditions employed, there were no effects on clinical signs except red (light to dark) colour faeces were observed at low and high dose groups in either sex and this could be due to physical nature of the test item. There wer no mortality mortality and effects on body weights, food consumption, maternal body weights and food consumption, fertility parameters, litter parameters, thyroid stimulating hormone (TSH) and thyroxine (T4) levels, terminal fasting body weights, organ weights and its ratios. There were no test item related changes on gross/microscopic examination. The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested. No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses. Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day. This gross finding in pups was considered to be the congenital development defect and not treatment related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides; enlarged spleen noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for FAT 40400/F TE is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study, Klimisch score 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage with FAT 40400/F was carried out according OECD guideline 421 to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The test item was dissolved in Milli-Q water and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period. All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs aterations were identified at high dose. Under the experimental conditions employed, there were no effects on clinical signs except red (light to dark) colour faeces were observed at low and high dose groups in either sex and this could be due to physical nature of the test item. There were no mortality and effects on body weights, food consumption, maternal body weights and food consumption, fertility parameters, litter parameters, thyroid stimulating hormone (TSH) and thyroxine (T4) levels, terminal fasting body weights, organ weights and its ratios recorded. There were no test item related changes on gross/microscopic examination. The mean litter and viable litter size was significantly lower at birth and on Day 1 at 300 mg/kg/day mid dose. This significant difference was toxicologically not significant as the litter at high dose was comparable to vehicle control. There were no treatment-related effects on the uterine/implantation data, mean and mean viable litter size and survival index. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested. No treatment-related changes were observed in live birth index, sex ratio and survival data of pups up to LD 4 at all the tested doses. Gross pathological examination of pups did not reveal any abnormalities at all the dose levels tested except for two incidences of hydrocephalus of brain in two pups at 1000 mg/kg/day. This gross finding in pups was considered to be the congenital development defect and not treatment-related. Solitary incidences of unilateral/bilateral small and flaccid testes and small epididymides; enlarged spleen noted across the treated groups were confirmed by histopathology and considered as spontaneous changes. As there were no treatment-related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for FAT 40400/F is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Justification for classification or non-classification

Based on the findings of the absence of adverse effect in the reproduction/developmental toxicity screening test, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.