N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

This study 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.

The test item, C.I. Pigment Yellow 194, was suspended in 0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®waterand administered by oral gavage at the dose levels of 111, 333 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period.All dams were weighed onGestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.

Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs were identified at high dose

Results:

Clinical signs and Mortality:There were no clinical signs or mortalities observed at all the tested doses. The clinical sign of yellow coloured faecal matters was observed at all the tested doses. This could be due to physical nature of the test item.

Body weights:The mean body weights and Total 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 all 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 pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

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 and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

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 and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194. 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.The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Effect Level (NOEL) forReproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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:

Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 13 weeks
Body weight range at the start of treatment: Males: 362.07 to 464.96 g & Females: 239.96 to 287.13C g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 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.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 37) of treatment 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.: G19479. One set of samples were analysed for concentration (a.i) analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup. These second set of samples were disposed, as the analysis results of first set of samples were within the accepted limit as per study plan.
Formulations were considered acceptable as the mean results (calculated using all the replicate values) of all the layers and mean of each layer were 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 for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Frequency of treatment:

Daily

Details on study schedule:

Experimental starting date: 27 May 2020
Acclimatization: Start: 27 May 2020 & End: 31 May 2020
Pre-treatment period : Start: 01 June 2020 & End: 14 June 2020
Treatment start: Start: 15 June 2020 & End: 10 August 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:

10

Control animals:

yes

Details on study design:

The test item was administered by oral gavage in graded doses to three groups of male and female rats. The males were dosed for six weeks, up to and including the day before scheduled sacrifice. This included a two-week period prior to mating, during the mating period and post mating.
Females were dosed throughout the treatment period. This included 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 13 days after delivery, up to and including the day before scheduled sacrifice.

Parental animals: Observations and examinations:

Parental animals: Observations and examinations
Clinical Signs, Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. All rats were observed for clinical signs once daily during the treatment period. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations forgeneral clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre be haviour like self-mutilation, walking backwards). On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.
Body Weights
i) Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals the reafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

Food Consumption: Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

Oestrous cyclicity (parental animals):

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

Litter observations:

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

Postmortem examinations (parental animals):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Statistics:

Data captured using ProvantisTM: The body weight, organ weight and ano-genital distance data captured using Provantis™ was analyzed using built-in statistical tests. Derived data like net body weight gain, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertil
ity and survival indices were analyzed using above mentioned methods. The statistical analysis of the hormonal data was carried out using licensed copies of SYSTAT Statistical package version 12.0. The data was tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA). The non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. ANOVA was done using log
transformation. Comparison of means between treatment groups and control group was done using Dunnett’s t-test test when the overall treatment, ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group

Reproductive indices:

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 cohabited
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 used for 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:

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
= -----------------------------------------
No. of females littered
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 (%)
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 mortalities observed throughout the treatment period in either sex at all the doses tested. Incidence of dehydration and wound (right flank) was observed in one female (Rx6840) at 111mg/kg. This was considered not related to treatment because of isolated occurrence. However, yellow colour faeces was observed at at all the tested doses in either sex. This could be due to physical nature of the test item.
There were no abnormalities observed in pups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Hormonal Assay: Test item did not have any effects on effect on the thyroid hormone profile.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

To summarize, daily oral (gavage) administration of the test item “Novoperm-Gelb F2G (C.I. Pigment Yellow 194)” 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, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or 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), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item Novoperm-Gelb F2G (C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for Novoperm-Gelb F2G (C.I. Pigment Yellow 194) is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

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:

other: No adverse effects were observed

Key result

Critical effects observed:

no

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:

other: No adverse effects were observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To summarize, daily oral (gavage) administration of the test item C.I. Pigment Yellow 194 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, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or 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), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this 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 provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

 

The test item, C.I. Pigment Yellow 194, was suspended in 0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®waterand administered by oral gavage at the dose levels of 111, 333 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

 

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. G19479 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 24 hours when stored at room temperature.

 

The dose formulations were analysed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2^nd(Day 37) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations 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 interval except during the cohabitation period.All dams were weighed onGestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.

 

Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs were identified at high dose

 

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

 

Clinical signs and Mortality:There were no clinical signs or mortalities observed at all the tested doses. The clinical sign of yellow coloured faecal matters was observed at all the tested doses. This could be due to physical nature of the test item.

Body weights:The mean body weights and Total 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 all 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 pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

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 and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

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 and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194. 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.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 reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Effect Level (NOEL) forReproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 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

Species:

rat

Quality of whole database:

reliable without restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Yellow 194 to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.

·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 

 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Yellow 194 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 

The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz121 Rz144
G2 Low dose 111 10 11.1 24 Rz145 Rz168
G3 Mid dose 333 10 33.3 24 Rz169 Rz192
G4 High dose 1000 10 100 24 Rz193 Rz216

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the
group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s
exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Gelb F2G (C.I. Pigment Yellow 194) up to the highest dose of 1000 mg/kg/day.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: There were no adverse effects at all

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

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:

other: There were no effects at all

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as
- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.
- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.
- the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Yellow 194 to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.

·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 

 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Yellow 194 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 

The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

reliable without restrictions

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

There were no adverse effects in a reproductive toxicity screening test (OECD 421. rat) and in an oral developmental toxicity study (OECD 414, rat).