Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August, 2020 - 25 March, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August, 2020 - 25 March, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Justification for study design:
The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further this study also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment.
Specific details on test material used for the study:
Batch No.: 1404301
Manufactured Date: 20.09.2014
Expiry Date: 08.04.2024
Physical Appearance: Black powder
Storage Conditions: Ambient (+15 to +25 ºC)
Physical-chemical properties: pH: 5.0, Density: 1.2654 g/cm³
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and 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:
Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 25°C and relative humidity between 49 and 66 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.8– 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 52 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period). Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13). Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.
Details on 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. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. 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 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:
For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 32) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19993. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
On Day 1(21 September 2020), the analysis results of G2 was out of specification. Hence, the backup samples were analysed and results were within the specified limit. This could be due to sampling or processing error.
During 2nd month (Day 32, 22 October 20202), the analysis results were out of specification in all the doses. Hence, the samples collected from the freshly prepared formulations on 23 October 2020 were sent for analysis. The analysed results were within the specified limit as per study plan except G4. Hence, freshly prepared formulation from G4 group was sent for analysis on 25 October 2020 and the results were within the specified limit.
On both these occasions, the error could happen while during sampling or processing. Throughout the study period, the same procedure for the formulation preparation was followed. The formulations were freshly prepared on daily basis prior to administration. Further, the degree of the deviation of results that failed to be within the given specification limits was rather low or high when compared to the dose spacing. Hence, this does not affect the integrity of the study results.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 52 days 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) for total of 43-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.
Frequency of treatment:
Daily
Details on study schedule:
Study initiation date: 28 August 2020
Experimental starting date: 31 August 2020
Acclimatization: Start: 31 August 2020 End: 06 September 2020
Pre-treatment period: Start: 07 September 2020 End: 20 September 2020
Treatment start: Start: 21 September 2020 End: 17 November 2020
Experiment completion Date: 22 December 2020
Submission of Draft report: 23 December 2020
Study completion: 25 March 2021
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 10 males and 10 females
Recovery groups: 5 males and 5 females
Control animals:
yes
Details on study design:
Group
No. Group Colour of cage card Dose (mg/kg bwt/day) Concentration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx8921 Rx8930
10 F Rx8931 Rx8940
G2 Low dose Yellow 100 10 10 10 M Rx8941 Rx8950
10 F Rx8951 Rx8960
G3 Mid dose Green 300 30 10 10 M Rx8961 Rx8970
10 F Rx8971 Rx8980
G4 High dose Pink 1000 100 10 10 M Rx8981 Rx8990
10 F Rx8991 Rx9000
Recovery Groups
G1R Vehicle Control recovery White 0 0 5 M Rx9001 Rx9005
5 F Rx9006 Rx9010
G4R High dose recovery Pink 1000 10 5 M Rx9011 Rx9015
5 F Rx9016 Rx9020
Parental animals: Observations and examinations:
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 intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.
The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13. The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13. Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 47 and towards the end of recovery period (Day 66) for the recovery group animals. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13. At sacrifice, the parental males (Day 52), parental females (LD14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development. Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD 13 pups. The reproductive organs were examined from the non-pregnant females (Rx8975, Rx8958 and Rx8995).
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 LDs 0 and 4 were recorded.
c. The ano-genital distance (AGD) of each pup was measured on LD 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.
d. On LD 4, 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.
e. After standardization, the individual pup body weight was measured on LD13.
f. The number of nipples/areolae in male pups was counted on LD 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, all pups were observed for any abnormal behaviour.
j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.
Postmortem examinations (parental animals):
All adult animals including one pre-terminally dead dam (Rx8951) and pups were subjected for detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. All the surviving pups were necropsied on lactation Day 13 and findings were recorded with particular attention to the external genitals for the altered development. Dead pups were examined for possible defects and/or cause of death. For apparently non-pregnant rats (Rx8975, Rx8958 and Rx8995), the uteri were stained with Salewski stain to identify the post implantation loss of the embryos. The number of implantation sites were recorded for all the dams.
Statistics:
Data was captured using the Provantis(TM) laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, 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 (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be non-homogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be 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 is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in Provantis(TM) built-in statistical tests.
Data captured outside of Provantis(TM): The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0.

*: Statistically significant difference from the control group at p <0.05
Reproductive indices:
11.1 Reproductive Performance Data of Parents
a. Male mating index (%)

Number of males with evidence of mating
= ------------------------------------------------------------------- x 100
Number of males cohabited

b. Male fertility index (%)

Number of males siring a litter/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

Number of females mated
= ------------------------------------------ x 100
Number of females cohabited

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of mating

e. Mean number of implantations/group

Total number of implantations
= ---------------------------------------
Total number of pregnant animals






f. Post implantation loss (%)

Number of implantations - Number of live pups
= ------------------------------------------------------------------- x 100
Number of implantations
Offspring viability indices:
11.2 Litter Data
a. Mean litter size per group

Total Number of pups born
= -------------------------------------------------
Total Number of littered animals

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

c. Live birth index (%)

No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)


d. Day 4 survival index (%)

Number of viable pups on lactation Day 4
= -------------------------------------------------------- x 100
Number of viable pups born

e. Sex Ratio/ Percentage of male offspring (%)

No. of male pups born
= -------------------------------- x 100
Total no. of pups born

f. Ano-genital Distance Ratio (mm/g1/3 )

Ano-genital distance
= --------------------------------
Cube root of body weight
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed throughout the treatment period in either sex at all the doses tested. The black colour faeces was observed at G3 and G4 of the tested doses in both the sexes which recovered by Day 2 of recovery period. This black coloured faecal matter is related to physical nature of the test item. One female rat (Rx8951) in low dose was died on GD 23. This rat did not show any clinical signs or reduction in the body weight and food consumption. The gross pathology examination revealed dead foetuses in the uterus/cervix. The cause of death could be due to dystocia though there were no apparent gross lesions.
There were no abnormalities observed in pups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the haematology and coagulation at all the doses tested in both sexes.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the clinical chemistry parameters at all the doses tested in both sexes.
Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urine parameters at all the doses tested in both sexes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related neurological abnormalities were observed at any of the doses tested.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The calculated mean oestrous cycle length was 4.8, 4.8, 4.7 and 4.8 days in vehicle control, low, mid and high dose groups, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects seen at any of the tested doses
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Food consumption and compound intake (if feeding study):
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No changes attributable to the test item were detected in the ano-genital distance and ano-genital ratio in either sex.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The male pups did not exhibit areolae/nipple retention on PND 13.
Description (incidence and severity):
Gross examination of pups on lactation day 13 did not reveal any gross lesions/developmental malformations.
There were no test item-related changes in thyroid weights and microscopic changes in thyroid gland of pups.
There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in pups on LD4 and LD13.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects seen upto the highest tested dose
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item FAT36038/J TE is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.
Executive summary:

FAT 36038/J was assessed in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. The test item was weighed and suspended in vehicle [0.5% sodium carboxymethyl cellulose with 0.5 % Tween 80 in Milli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bw/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats. The dose formulations were administered once daily to 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. In the control and high dose recovery groups, the treatment period was followed by a 14-day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams. During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2ndmonth (Day 31) of the treatment period.The results indicated that the analysed concentrations were within ± 15 % of variations from the claimed concentrationsthe relative standard deviation (%RSD) is equal to or less than 10 %. 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 intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20. The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13. The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13. Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 47 and towards the end of recovery period (Day 66) for the recovery group animals. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13. At sacrifice, the parental males (Day 52), parental females (LD14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development. Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females (Rx8975, Rx8958 and Rx8995). 


Under the experimental conditions employed, the following results were obtained:


Clinical signs and Mortality: There were no treatment-related clinical signs or mortality observed at any of the doses tested. The black-coloured faecal matters were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the black-coloured faecal matter was due to physical nature of the test item. There were no abnormalities observed in pups.


Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.


Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.


Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.


Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.


Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.


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


Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.


Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.


Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.  There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.


Gross and histopathology: There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item-associated findings in parental rats.


In view of the results observed:


As there were no treatment-related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item FAT36038/J is determined to be 1000 mg/kg bw/day under the test conditions and doses employed.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August, 2020 - 25 March, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Batch No.: 1404301
Manufactured Date: 20.09.2014
Expiry Date: 08.04.2024
Physical Appearance: Black powder
Storage Conditions: Ambient (+15 to +25 ºC)
Physical-chemical properties: pH: 5.0, Density: 1.2654 g/cm³
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 25°C and relative humidity between 49 and 66 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.8– 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 52 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period). Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13). Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 32) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19993. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
On Day 1(21 September 2020), the analysis results of G2 was out of specification. Hence, the backup samples were analysed and results were within the specified limit. This could be due to sampling or processing error.
During 2nd month (Day 32, 22 October 20202), the analysis results were out of specification in all the doses. Hence, the samples collected from the freshly prepared formulations on 23 October 2020 were sent for analysis. The analysed results were within the specified limit as per study plan except G4. Hence, freshly prepared formulation from G4 group was sent for analysis on 25 October 2020 and the results were within the specified limit.
On both these occasions, the error could happen while during sampling or processing. Throughout the study period, the same procedure for the formulation preparation was followed. The formulations were freshly prepared on daily basis prior to administration. Further, the degree of the deviation of results that failed to be within the given specification limits was rather low or high when compared to the dose spacing. Hence, this does not affect the integrity of the study results.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 52 days 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) for total of 43-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: 10 males and 10 females
Recovery groups: 5 males and 5 females
Control animals:
yes
Details on study design:
Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx8921 Rx8930
10 F Rx8931 Rx8940
G2 Low dose Yellow 100 10 10 10 M Rx8941 Rx8950
10 F Rx8951 Rx8960
G3 Mid dose Green 300 30 10 10 M Rx8961 Rx8970
10 F Rx8971 Rx8980
G4 High dose Pink 1000 100 10 10 M Rx8981 Rx8990
10 F Rx8991 Rx9000
Recovery Groups
G1R Vehicle Control recovery White 0 0 5 M Rx9001 Rx9005
5 F Rx9006 Rx9010
G4R High dose recovery Pink 1000 10 5 M Rx9011 Rx9015
5 F Rx9016 Rx9020
Observations and examinations performed and frequency:
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 intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.
The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13. The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13. Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 47 and towards the end of recovery period (Day 66) for the recovery group animals. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13. At sacrifice, the parental males (Day 52), parental females (LD14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development. Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD 13 pups. The reproductive organs were examined from the non-pregnant females (Rx8975, Rx8958 and Rx8995).
Sacrifice and pathology:
All adult animals including one pre-terminally dead dam (Rx8951) and pups were subjected for detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. All the surviving pups were necropsied on lactation Day 13 and findings were recorded with particular attention to the external genitals for the altered development. Dead pups were examined for possible defects and/or cause of death. For apparently non-pregnant rats (Rx8975, Rx8958 and Rx8995), the uteri were stained with Salewski stain to identify the post implantation loss of the embryos. The number of implantation sites were recorded for all the dams.
Statistics:
Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, 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 (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be 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 is found to be 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 ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0.

*: Statistically significant difference from the control group at p <0.05
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed throughout the treatment period in either sex at all the doses tested. The black colour faeces was observed at G3 and G4 of the tested doses in both the sexes which recovered by Day 2 of recovery period. This black coloured faecal matter is related to physical nature of the test item. One female rat (Rx8951) in low dose was died on GD 23. This rat did not show any clinical signs or reduction in the body weight and food consumption. The gross pathology examination revealed dead foetuses in the uterus/cervix. The cause of death could be due to dystocia though there were no apparent gross lesions.
There were no abnormalities observed in pups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the haematology and coagulation at all the doses tested in both sexes.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the clinical chemistry parameters at all the doses tested in both sexes.
Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urine parameters at all the doses tested in both sexes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related neurological abnormalities were observed at any of the doses tested.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group. There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects seen at any of the tested dose
Critical effects observed:
no
Conclusions:
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test itemFAT36038/J is determined to be 1000 mg/kg bw/day under the test conditions and doses employed.
Executive summary:

FAT 36038/J was assessed in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavag to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time.The test item was weighed and suspended in vehicle [0.5 % sodium carboxymethyl cellulose with 0.5 % Tween 80 inMilli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bw/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.The dose formulations were administered once daily to 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. In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams. During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2nd month (Day 31) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the claimed concentrationsthe relative standard deviation (%RSD) is equal to or less than 10 %. 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 intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumedGestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20. The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13.The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13. Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 47 and towards the end of recovery period (Day 66) for the recovery group animals.Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13. At sacrifice, the parental males (Day 52), parental females (LD14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development. Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females (Rx8975, Rx8958 and Rx8995). Under the experimental conditions employed, the following results were obtained:


Clinical signs and Mortality: There were no treatment related clinical signs or mortality observed at any of the doses tested. The black coloured faecal matters were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the black coloured faecal matter was due to physical nature of the test item.There were no abnormalities observed in pups.


Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.


Body weights: The mean body weights and body weight gainswere unaffected by the treatment at all the tested doses in both sexes.


Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.


Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.


Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.


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


Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.


Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.


Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.


Gross and histopathology: There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.


In view of the results observed:


As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item FAT36038/J is determined to be 1000 mg/kg bw/day under the test conditions and doses employed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-hydroxy-4-[[4-[(methylsulphonyl)oxy]phenyl]amino]anthraquinone
EC Number:
216-475-4
EC Name:
1-hydroxy-4-[[4-[(methylsulphonyl)oxy]phenyl]amino]anthraquinone
Cas Number:
1594-08-7
Molecular formula:
C21H15NO6S
IUPAC Name:
1-hydroxy-4-[[4-[(methylsulphonyl)oxy]phenyl]amino]anthraquinone
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch No.: 1404301
Manufactured Date: 20.09.2014
Expiry Date: 08.04.2024
Physical Appearance: Black powder
Storage Conditions: Ambient (+15 to +25 ºC)
Physical-chemical properties: pH: 5.0, Density: 1.2654 g/cm³

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 25 °C and relative humidity between 49 and 66 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.8– 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 52 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period). Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13). Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 32) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19993. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
On Day 1(21 September 2020), the analysis results of G2 was out of specification. Hence, the backup samples were analysed and results were within the specified limit. This could be due to sampling or processing error.
During 2nd month (Day 32, 22 October 20202), the analysis results were out of specification in all the doses. Hence, the samples collected from the freshly prepared formulations on 23 October 2020 were sent for analysis. The analysed results were within the specified limit as per study plan except G4. Hence, freshly prepared formulation from G4 group was sent for analysis on 25 October 2020 and the results were within the specified limit.
On both these occasions, the error could happen while during sampling or processing. Throughout the study period, the same procedure for the formulation preparation was followed. The formulations were freshly prepared on daily basis prior to administration. Further, the degree of the deviation of results that failed to be within the given specification limits was rather low or high when compared to the dose spacing. Hence, this does not affect the integrity of the study results.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %
Details on 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. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. 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 GD 0. The pre-coital time (days) was calculated for each female.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 52 days 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) for total of 43-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
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:
mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
Main groups: 10 males and 10 females
Recovery groups: 5 males and 5 females
Control animals:
yes
Details on study design:
Group
No. Group Colour of cage card Dose (mg/kg bwt/day) Concentration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx8921 Rx8930
10 F Rx8931 Rx8940
G2 Low dose Yellow 100 10 10 10 M Rx8941 Rx8950
10 F Rx8951 Rx8960
G3 Mid dose Green 300 30 10 10 M Rx8961 Rx8970
10 F Rx8971 Rx8980
G4 High dose Pink 1000 100 10 10 M Rx8981 Rx8990
10 F Rx8991 Rx9000
Recovery Groups
G1R Vehicle Control recovery White 0 0 5 M Rx9001 Rx9005
5 F Rx9006 Rx9010
G4R High dose recovery Pink 1000 10 5 M Rx9011 Rx9015
5 F Rx9016 Rx9020

Examinations

Maternal examinations:
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 intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.
The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 0, 4 and 13. Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 47 and towards the end of recovery period (Day 66) for the recovery group animals. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13. At sacrifice, the parental females (LD14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. Histopathology examination was carried out on the preserved organs from randomly selected 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Thyroids were examined from all adult females and in LD13 pups. The reproductive organs were examined from the non-pregnant females (Rx8975, Rx8958 and Rx8995).
Fetal examinations:
The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed throughout the treatment period in either sex at all the doses tested. The black colour faeces was observed at G3 and G4 of the tested doses in both the sexes which recovered by Day 2 of recovery period. This black coloured faecal matter is related to physical nature of the test item. One female rat (Rx8951) in low dose was died on GD 23. This rat did not show any clinical signs or reduction in the body weight and food consumption. The gross pathology examination revealed dead foetuses in the uterus/cervix. The cause of death could be due to dystocia though there were no apparent gross lesions.
There were no abnormalities observed in pups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the haematology and coagulation parameters at all the doses tested.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the clinical chemistry parameters at all the doses tested.
Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urine parameters at all the doses tested.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related neurological abnormalities were observed at any of the doses tested.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related gross and microscopic changes (including reproductive organs) in the females.
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Treatment had no effects on the maternal body weights and food consumption measured different intervals of the lactation period at all the doses when compared to the vehicle control.

There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested.

Test item had no treatment-related effects on the mean litter size, mean viable litter size and 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 the survival data of pups up to LD 4 at all the tested doses.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: no adverse effects seen at any of the tested doses

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Test item had no treatment-related effects on the mean litter size weights.
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities in live or dead pups in any of the groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities in live or dead pups in any of the groups.
Other effects:
no effects observed
Description (incidence and severity):
- No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex.
- The male pups did not exhibit areolae/nipple retention on PND 13.

Gross examination of pups on lactation day 13 did not reveal any gross lesions/developmental malformations.
There were no test item-related changes in thyroid weights and microscopic changes in thyroid gland of pups.
There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in pups on LD4 and LD13.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects seen at any of the tested doses

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
As there were no treatment-related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bw/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item FAT36038/J is determined to be 1000 mg/kg bw/day under the test conditions and doses employed.
Executive summary:

FAT 36038/J was assessed in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavag to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time.The test item was weighed and suspended in vehicle [0.5% sodium carboxymethyl cellulose with 0.5 % Tween 80 in Milli-Q water] and administered at the graduated dose levels of 100, 300 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. 


Under the experimental conditions employed, the following results were obtained:


Clinical signs and Mortality: There were no treatment-related clinical signs or mortality observed at any of the doses tested. The black-coloured faecal matters were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the black-coloured faecal matter was due to physical nature of the test item. There were no abnormalities observed in pups.


Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.


Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in maternal animals.


Food consumption: Treatment did not affect the food consumption at any of the tested doses in maternal animals.


Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.


Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.


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


Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.


Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.


Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.


Gross and histopathology: There were no test item-related adverse histopathological changes observed either in parents or the offspring.


F1 results: The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses. No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses. Test item had no treatment-related effects on the mean litter size weights. No changes attributable to the test item were detected in the ano-genital distance and ano-genital ratio in either sex. The male pups did not exhibit areolae/nipple retention on PND 13.


There were no external abnormalities in live or dead pups in any of the groups.


Gross examination of pups on lactation day 13 did not reveal any gross lesions/developmental malformations. There were no test item-related changes in thyroid weights and microscopic changes in thyroid gland of pups. There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in pups on LD4 and LD13.


In view of the results observed:


As there were no treatment-related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item FAT36038/J is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.