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Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicological effects from repeated oral-gavage administration of the an analogue sbstance Pigment Yellow 175 to rats were investigated in a study according to OECD 422 (GLP compliant). The test item was administered in vehicle at dosages of up to 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 13 post partum.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 13 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.


Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level used.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
13 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See read across justification Document in chapter 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
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
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 19 to 24°C and relative humidity between 49 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.5 – 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 57 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 23) 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.: G19468. 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.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
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 57 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 51-58 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: 27 May 2020
Experimental starting date: 28 May 2020
Acclimatization: Start: 28 May 2020, End: 01 June 2020
Pre-treatment period: Start : 02 June 2020, End: 15 June 2020
Treatment start: Start: 16 June 2020, End: 12 August 2020
Experiment completion Date: 31 August 2020
Submission of Draft report: 31 August 2020
Study completion: 13 November 2020
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 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 Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580
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 and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for defects and/or cause of death.
For apparently non-pregnant rats, the uteri was stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of the embryos (by staining the implantation sites) thereby, for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.
Statistics:
Parameters such as body weight, body weight change, food consumption, oestrous cycle, gestation length (days), mean litter size, mean viable litter size, ano-genital distance, sex ratio, survival index and pup body weight, laboratory Investigations – haematology, coagulation, clinical chemistry, urinalysis & thyroid hormone profile, organ weights, organ weight ratios (organ to body weight and organ to brain weight), no. of implantations, post implantation loss (%) were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was 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 was 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. All quantitative data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroscedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.

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 and mortality observed throughout the treatment period in either sex at all the doses tested, except for an incidental observation of dehydration, piloerection and weakness in a female rat (Rx7557) of high dose group on days 42 and 43. This dam was sacrificed on Day 43 due to total litter loss. The yellowish colour faeces was observed at all the tested doses in both the sexes which recovered by Day 2 of recovery period. This yellowish coloured faecal matter is related to physical nature of the test item.
There were no abnormalities observed in pups
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 treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
The significantly lower weekly absolute weight change during Days 52-59 in recovery group females was considered incidental 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 food consumption was not altered throughout treatment and recovery period when compared to the concurrent vehicle control.
Incidences of significantly decreased food consumption during Days 1-8 in 1000 mg/kg bwt/day recovery dose group males and during Days 8-14 in both 111 and 333 mg/kg bwt/day dosed females and significantly increased food consumption during Days 43-50 in 1000 mg/kg bwt/day recovery dose group males was observed. These isolated statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the hematology parameters including PT and APTT values in both males and females. A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical chemistry parameters were not affected by test item administration. Increased creatinine kinase activity in 1000 mg/kg bwt/day dose males was considered as incidental change as it was not associated with any microscopic changes in muscle tissues examined. Increased creatinine concentration in 1000 mg/kg bwt/day dose females was considered as incidental change as it was not associated microscopic changes in kidneys examined. A few other variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for an incidence of lower distance travelled at interval 1 and lower total distance travelled in recovery males treated at 1000 mg/kg bwt/day.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic changes at any of the dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Litter Date: 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.
Body Weight of Pups: 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.
Anogental Distance (AGD): No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex. An incidence of higher ano-genital distance in female pups at 111 mg/kg bwt/day was considered as not related to treatment as this difference was not dose related and was minimal (̴ 3%).
Uterine/Implantation Data: No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Areolae/Nipple Retention in Pups: The male pups did not exhibit areola/nipple retention on PND 13.
Oestrous Cycle Prior to Sacrifice: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed;
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
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. The lower gestation length at high dose was considered not treatment related as the difference was minimal (̴ 3%).
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Gross pathological findings:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
To summarize, oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean 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 and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item. Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
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 C. I. Pigment Yellow 175 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.
Executive summary:

 


The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.


The test item was suspended in vehicle [0.5 % (w/v) of carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q®water] and administered at the graduated dose levels of 111, 333 and 1000 mg/kg bwt/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 body weight/day. Each main group in the experiment comprised of 10 male and 10 female rats and each recovery group comprised of 5 male and 5 female rats.


The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.


The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19468 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 48 hours when stored at room temperature.


During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 4 (Day 23) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % variation from the claimed concentrations.


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 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 56 and towards the end of recovery period for the recovery group animals.


Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental 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 58), parental females (LD14) and the recovery animals (Day 65) 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.


Tissues/organs collected from randomly selected 5 males and females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups.In the absence of test item related histopathological changes in any of the suspected tissues in the high dose group (G4), tissues from the lower dose groups were not evaluated. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.


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 yellowish faeces 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 yellowish 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 areola/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:


Grossly noted yellow contents in intestinal segments ≥333 mg/kg bwt/day were attributed to the test item colour.


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 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 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
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
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 19 to 24°C and relative humidity between 49 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.5 – 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 57 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 23) 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.: G19468. 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.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
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 57 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 51-58 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: 27 May 2020
Experimental starting date: 28 May 2020
Acclimatization: Start: 28 May 2020, End: 01 June 2020
Pre-treatment period: Start : 02 June 2020, End: 15 June 2020
Treatment start: Start: 16 June 2020, End: 12 August 2020
Experiment completion Date: 31 August 2020
Submission of Draft report: 31 August 2020
Study completion: 13 November 2020
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 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 Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580
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 and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for defects and/or cause of death.
For apparently non-pregnant rats, the uteri was stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of the embryos (by staining the implantation sites) thereby, for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.
Statistics:
Parameters such as body weight, body weight change, food consumption, oestrous cycle, gestation length (days), mean litter size, mean viable litter size, ano-genital distance, sex ratio, survival index and pup body weight, laboratory Investigations – haematology, coagulation, clinical chemistry, urinalysis & thyroid hormone profile, organ weights, organ weight ratios (organ to body weight and organ to brain weight), no. of implantations, post implantation loss (%) were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was 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 was 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. All quantitative data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroscedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.

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 and mortality observed throughout the treatment period in either sex at all the doses tested, except for an incidental observation of dehydration, piloerection and weakness in a female rat (Rx7557) of high dose group on days 42 and 43. This dam was sacrificed on Day 43 due to total litter loss. The yellowish colour faeces was observed at all the tested doses in both the sexes which recovered by Day 2 of recovery period. This yellowish coloured faecal matter is related to physical nature of the test item.
There were no abnormalities observed in pups
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 treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
The significantly lower weekly absolute weight change during Days 52-59 in recovery group females was considered incidental 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 food consumption was not altered throughout treatment and recovery period when compared to the concurrent vehicle control.
Incidences of significantly decreased food consumption during Days 1-8 in 1000 mg/kg bwt/day recovery dose group males and during Days 8-14 in both 111 and 333 mg/kg bwt/day dosed females and significantly increased food consumption during Days 43-50 in 1000 mg/kg bwt/day recovery dose group males was observed. These isolated statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the hematology parameters including PT and APTT values in both males and females. A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical chemistry parameters were not affected by test item administration. Increased creatinine kinase activity in 1000 mg/kg bwt/day dose males was considered as incidental change as it was not associated with any microscopic changes in muscle tissues examined. Increased creatinine concentration in 1000 mg/kg bwt/day dose females was considered as incidental change as it was not associated microscopic changes in kidneys examined. A few other variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for an incidence of lower distance travelled at interval 1 and lower total distance travelled in recovery males treated at 1000 mg/kg bwt/day.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic changes at any of the dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Litter Date: 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.
Body Weight of Pups: 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.
Anogental Distance (AGD): No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex. An incidence of higher ano-genital distance in female pups at 111 mg/kg bwt/day was considered as not related to treatment as this difference was not dose related and was minimal (̴ 3%).
Uterine/Implantation Data: No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Areolae/Nipple Retention in Pups: The male pups did not exhibit areola/nipple retention on PND 13.
Oestrous Cycle Prior to Sacrifice: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed;
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
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. The lower gestation length at high dose was considered not treatment related as the difference was minimal (̴ 3%).
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Gross pathological findings:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
To summarize, oral (gavage) administration of the test item C. I. Pigment Yellow 175 to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean 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 and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item. Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
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 C. I. Pigment Yellow 175 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle [0.5 % (w/v) of carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q®water] and administered at the graduated dose levels of 111, 333 and 1000 mg/kg bwt/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 body weight/day. Each main group in the experiment comprised of 10 male and 10 female rats and each recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19468 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 4 (Day 23) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % variation from the claimed concentrations.

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 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 56 and towards the end of recovery period for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental 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 58), parental females (LD14) and the recovery animals (Day 65) 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.

Tissues/organs collected from randomly selected 5 males and females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups.In the absence of test item related histopathological changes in any of the suspected tissues in the high dose group (G4), tissues from the lower dose groups were not evaluated. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.

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 yellowish faeces 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 yellowish 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 areola/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:

Grossly noted yellow contents in intestinal segments ≥333 mg/kg bwt/day were attributed to the test item colour.

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 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 ratsfor the test itemHostaperm-Gelb H6G (C. I. Pigment Yellow 175) is determined to be 1000 mg/kg bwt/dayunder 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
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

no classification - No adverse effects observed at 1000 mg/kg bw/d

Additional information