Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from a study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Principles of method if other than guideline:
According to OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2-naphthyl ether
EC Number:
202-213-6
EC Name:
Methyl 2-naphthyl ether
Cas Number:
93-04-9
Molecular formula:
C11H10O
IUPAC Name:
2-methoxynaphthalene
Test material form:
solid: crystalline
Details on test material:
Name: Methyl-2-naphthyl ether
CAS No.: 93-04-9
IUPAC Name: 2-Methoxynaphthalene
Molecular Weight: 158.19 g/mol
Molecular Formula: C11-H10-O
SMILES: COc1ccc2ccccc2c1

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The test animals were procured from a CPCSEA approved vendor.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-14 weeks at the start of treatment wks; (F1) 11-12 weeks at the start of treatment wks
- Weight at study initiation: (P) Males: 291-413 g; Females: 198-277 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: No Data Available
- Housing: Two to four rats were housed in each polycarbonate cage. During mating, one male and one female rat were housed in a single cage. Pregnant and lactating females were housed individually. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): A conventional laboratory pellet diet from approved supplier was available ad libitum.
- Water (e.g. ad libitum): Aquaguard™ filtered drinking water was available ad libitum in regularly cleaned bottles.
- Acclimation period: Animals were acclimatized for at least 5 days prior to test item administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.60 to 24.80°C
- Humidity (%): 34.40 to 66.70 %
- Air changes (per hr): At least 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES:
From: August 8, 2019
To: March 1, 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle used for dose administration was corn oil. Required quantity for each concentration level of test chemical was weighed and triturated in a mortar-pestle, first without vehicle and then in small volume of the vehicle. After thorough trituration, the test chemical and vehicle were transferred quantitatively into a calibrated measuring cylinder and diluted using vehicle up to the required volume of the formulation, before being transferred to the final container. Formulations were prepared on the same day or one day prior to dose administration and stored at room temperature until usage as the test item stability in solution was proved up to 24 hours. At the time of dosing, dose formulations were kept on a magnetic stirrer for maintaining homogeneity of test formulation. The dose formulation samples [of doses viz; G1 (Control), G2 (Low dose), G3 (Mid dose), and G4 (High dose)] were analyzed for homogeneity and concentration. The dose formulation analysis was carried out once on Day 1 of dose administration to animals, on day 91 and on day 182.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil was selected as a vehicle because the test chemical was found to be insoluble in water whereas corn oil provided adequate suspensibility and the formulation was found stable in this vehicle. Further, corn oil is widely used as vehicle in oral toxicity study and the selected concentration is well below the acceptable limit.
- Concentration in vehicle: 0 mg/kg bw: 0 mg/ml; 62.5 mg/kg bw: 15.63 mg/ml; 125 mg/kg bw: 31.25 mg/ml and 250 mg/kg bw: 62.5 mg/ml
- Amount of vehicle (if gavage): 4 mL/ kg
- Lot/batch no. (if required): Batch No.A1906001, N2191405, N2192016, O21904007
- Purity: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated with respect to the parameters covering specificity, linearity, limit of quantification (LOQ), precision (% RSD), Accuracy (% recovery) and Homogeneity. The analytical method was validated with respect to the following parameters as below:

1. Specificity: The result of specificity was reported either no interference observed based on visual comparison or the degree of interference does not contribute more than 20 % peak area of the target analyte at the LOQ Level.

2. Limit of Quantification: The LOQ of the method was the minimum concentration/quantity of a component, which could be quantified precisely and accurately.
The LOQ was carried out at the lowest sensitivity of the device under the same instrumental parameters used for the qualified specificity.
The LOQ of a compound for a specific method was determined by injecting in duplicate the working solution (s) made in suitable solvent fortified with test chemical / reference standard, until it produces a minimum signal to noise ratio ≥ 10:1 for precise quantification.

3. Linearity: The linearity was carried out by preparing and analyzing minimum five linear concentrations of reference standard with at least one determination. The linear calibration curve was established by plotting the analyte peak response against concentration (mg/l). The linearity was given in the form of equation of the resulting curve and correlation coefficient (r). The value of correlation coefficient (r) ≥ 0.99 was considered as an acceptable value.

4. Precision (% RSD), Accuracy (% Recovery) and Homogeneity: Precision, Accuracy was established at the level of test concentration used i.e., at low, mid and high dose level along with control. The active ingredient was diluted using sample diluent and injected onto HPLC using validated HPLC method. Minimum six determinations were made at each fortification level. The homogeneity was performed by analysing Top, Middle and Bottom layers of each fortification level. The active ingredient concentration in each replicate of each fortification level, mean of active ingredient concentration, accuracy (% recovery), SD and % RSD was calculated and reported.
The active ingredient concentration of test item in dose formulation was calculated using the following formula:
Y = bX + a

"AI Concentration (mg/L) = " "Y - a" /"b" " × D"


Where,
Y = Peak area of the sample
a = Intercept
b = Slope of the line
D = Dilution factor (if applicable)
X = A.I. Concentration (mg/L)


The accuracy (% Recovery) will be calculated using the following formula:

"Accuracy " ("% Recovery" )"= " "Obtained Concentration" /"Fortified concentration" " ×100"
The precision (% RSD) will be calculated using the formula provided as below:
"Precision (% RSD)= " "Standard Deviation" /"Mean Recovered Concentration" " ×100"

All the paramaters were according to the acceptance criteria of the above test.
Details on mating procedure:
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: 2 weeks until evidence of copulation was observed.
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: If mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No Data Available
Duration of treatment / exposure:
Dosing in all animals was carried out on a 7-days/week basis from the treatment initiation until necropsy. All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Females were treated throughout gestation and lactation up to the day of termination after weaning. Males were treated in the same manner until termination. Dosing of selected F1 males and females begun at weaning and continue until scheduled necropsy, depending on cohort assignment.
Frequency of treatment:
Once Daily
Duration of test:
No Data Available
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group (G3)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
No. of animals per sex per dose:
30 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of previously performed GLP compliant repeated dose 28 days oral toxicity study. Two deaths were observed in the highest dose (500 mg/kg bw) unrelated to the gavage error of the OECD 407 study and therefore a subsequent lower dose of 250 mg/kg bw was selected as a highest dose in current study.
- Rationale for animal assignment (if not random): During the acclimatisation phase, male and female animals were randomized into four different experimental groups, based on the most recently recorded body weights, using the “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/) for MS Excel. After randomisation, it was ensured that individual body weights were within ± 20% of the respective group means. Details of the randomization were documented in the study raw data.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for cage size observation, throughout the acclimatization and study periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of rats from all the groups were made once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Detailed clinical examinations were carried out for P and F1 animals (after weaning) weekly, additionally P animals were examined once before the first treatment (to allow for within-subject comparisons).

BODY WEIGHT: Yes
- Time schedule for examinations: P animals were weighed once on receipt, once during randomization, once at the start of treatment and once every week thereafter. In addition, pregnant females were weighed during gestation (GD 0, GD 7, GD 14 and GD 20) and lactation on the same days as the weighing of the pups in their litters. All F1 animals were weighed individually on PND 0, 4, 7, 14, 21 and at least weekly thereafter. Body weight was also recorded on the day when they attained puberty (completion of preputial separation or vaginal patency). All animals were weighed at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, during the study, feed consumption was recorded weekly or on the same days as animal body weights (except during cohabitation).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No Data Available

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice was performed after 7 weeks.
- Organs examined: All visceral

OTHER: No Data Available
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: No Data Available
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: No data
- Head examinations: Yes
Statistics:
The findings of this study were evaluated in terms of the observed effects, necropsy and microscopic findings. The evaluation will included the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including fertility, clinical abnormalities, body weight changes, effects on mortality and any other toxic effects. Raw data was processed using statistical software. The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data were checked for their homogeneity using Shapiro Wilk test. All homogenous data were analyzed using ANOVA and data showing significance in their variances were subjected to Dunnett’s t-test. All heterogeneous data was analysed using Kruskal-Wallis ANOVA on ranks and data showing significance in their variances were subjected to Dunn’s Test. P values of less than or equal to 0.05 were accepted as being statistically significant.
Indices:
Maternal Index, Implantation Index, Resorption Index, Gestation Index, Pup Viability Index.
Historical control data:
No Data Available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in maternal animals.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. Also, the mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. However, this effects was neither dose dependent or treatment related.
Food efficiency:
no effects observed
Description (incidence and severity):
Among the parental females, no statistical differences were observed for feed efficiency in any of the groups.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No significant difference was observed in any of the measured haematology parameters in all the groups in maternal animals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All the clinical chemistry parameters in maternal females were found comparable in control and treatment groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine parameters tested did not show up any significant changes among the experimental groups of maternal animals.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was observed in absolute organ weight or organ weight relative to body weight for the entire set of measured organs in maternal females. However, significant reduction was observed in absolute and relative liver weight in G4 females (P<0.05) as compared to control animals. However, the decreased liver weights with respect to body weights in G4 females did not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test-chemical. Also, no difference was observed on the weights of uterus, ovaries and Thyroid & Parathyroid organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross anomalies were observed in the maternal animals in gross pathological examinations.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of maternal animals; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect.
Details on results:
No treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in maternal animals. No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment. Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. Also, the mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods. The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. However, this effects was neither dose dependent or treatment related. Among the parental females, no statistical differences were observed for feed efficiency in any of the groups. No significant difference was observed in any of the measured haematology parameters in all the groups in maternal animals. All the clinical chemistry parameters in maternal females were found comparable in control and treatment groups. The urine parameters tested did not show up any significant changes among the experimental groups of maternal animals. No significant difference was observed in absolute organ weight or organ weight relative to body weight for the entire set of measured organs in maternal females. However, significant reduction was observed in absolute and relative liver weight in G4 females (P<0.05) as compared to control animals. However, the decreased liver weights with respect to body weights in G4 females did not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test-chemical. Also, no difference was observed on the weights of uterus, ovaries and Thyroid & Parathyroid organs. No gross anomalies were observed in the maternal animals in gross pathological examinations. No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity. The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of maternal animals; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect.

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Out of 30 females 29, 28, 29 and 28 females delivered pups from G1, G2, G3 and G4 groups respectively. There were no treatment related effects on the number of abortions in maternal females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of pre and post implantation losses were comparable among females of control and treatment groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No cases of litter losses due to resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
No cases of early or late resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration.
Dead fetuses:
no effects observed
Description (incidence and severity):
No cases of dead fetuses were observed due to the test chemical administration.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The duration of pregnancy (days from insemination to parturition) / gestation length was comparable among females of all the experimental groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All the maternal animals remained pregnant throughout the gestation period and there were no changes observed number of pregnant females due to the test chemical administration.
Other effects:
no effects observed
Description (incidence and severity):
No changes in the number of corpora luteas were observed, no case of dystocia (difficulty in parturition) was observed in females of any of the groups and no unusual nesting or nursing behaviour was found as well, due to the test chemical administration.
Details on maternal toxic effects:
No treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in maternal animals. No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment. Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. Also, the mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods. The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. However, this effects was neither dose dependent or treatment related. Among the parental females, no statistical differences were observed for feed efficiency in any of the groups. No significant difference was observed in any of the measured haematology parameters in all the groups in maternal animals. All the clinical chemistry parameters in maternal females were found comparable in control and treatment groups. The urine parameters tested did not show up any significant changes among the experimental groups of maternal animals. No significant difference was observed in absolute organ weight or organ weight relative to body weight for the entire set of measured organs in maternal females. However, significant reduction was observed in absolute and relative liver weight in G4 females (P<0.05) as compared to control animals. However, the decreased liver weights with respect to body weights in G4 females did not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test-chemical. Also, no difference was observed on the weights of uterus, ovaries and Thyroid & Parathyroid organs. No gross anomalies were observed in the maternal animals in gross pathological examinations. No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity. The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of maternal animals; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect. Out of 30 females 29, 28, 29 and 28 females delivered pups from G1, G2, G3 and G4 groups respectively. There were no treatment related effects on the number of abortions in maternal females. The mean number of pre and post implantation losses were comparable among females of control and treatment groups. No cases of litter losses due to resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration. No cases of early or late resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration. No cases of dead fetuses were observed due to the test chemical administration. The duration of pregnancy (days from insemination to parturition) / gestation length was comparable among females of all the experimental groups. All the maternal animals remained pregnant throughout the gestation period and there were no changes observed number of pregnant females due to the test chemical administration. No changes in the number of corpora luteas were observed, no case of dystocia (difficulty in parturition) was observed in females of any of the groups and no unusual nesting or nursing behaviour was found as well, due to the test chemical administration.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Progesterone was found to be significantly reduced in G4 females (P<0.001) which was considered to be the test chemical administration related effect.
Remarks on result:
other: Changes in hormonal levels were the basis of the LOAEL.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical biochemistry
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
urinalysis
other: Hormone Analysis
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight of pups of both sexes combined on post-natal day (PND) 0 was found to be significantly lower in G4 (P≤0.05) as compared to controls. This difference, however, did not persist for any of the other days of observation. On the contrary, the G2 and G4 group pups returned significantly higher body weights on PND 21 as compared to control (P≤0.05 and P≤0.001 respectively). Sex-specific analysis of pup weights revealed statistically reduced weight of male pups of G3 groups on PNDs 4 (P≤0.01) and 7 (P≤0.05) as compared to control and an increased weight of male pups of G2 and G4 groups on PND 21 (P≤0.05) as compared to control. Interestingly, this trend was also observed for female pups of G2 and G4, whose weights were significantly higher than control on PND 21 (P≤0.05). Among the female pups, additionally, G2 pups showed reduced weights on PND 0 as compared to the control group (P≤0.01).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number and percent of live births remained comparable for all the groups of pregnant females.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Significant increase in male pups of G3 group (P≤0.001) and a statistically significant decrease in female pups of G2 group (P≤0.01) was observed. None of these observations were consistent with the dose levels and hence not attributable to test-chemical treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test chemical administration related changes were observed in the litter sizes and weight across all the experimental groups.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No changes in the postnatal survival was observed due to the test chemical administration across all the experimental groups.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations in the pups was observed from the maternal animals of all the groups. No changes in the ano-genital distances and no of the male pups showed presence of nipples/areolae on PND 13. Mean age and body weight at balanopreputial separation remained comparable for all groups of males. Similarly, the mean age and body weight at the time of vaginal opening remained statistically comparable for all groups of female rats. Further, no significant difference was observed from the day of vaginal opening to appearance of first cornified smear (estrous). No treatment-related effects were observed on male and female pup sexual development markers.
Skeletal malformations:
not specified
Visceral malformations:
no effects observed
Description (incidence and severity):
No test chemical related effects were observed in the visceral examinations of the pups of maternal animals from all the dose groups.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Mean body weight of pups of both sexes combined on post-natal day (PND) 0 was found to be significantly lower in G4 (P≤0.05) as compared to controls. This difference, however, did not persist for any of the other days of observation. On the contrary, the G2 and G4 group pups returned significantly higher body weights on PND 21 as compared to control (P≤0.05 and P≤0.001 respectively). Sex-specific analysis of pup weights revealed statistically reduced weight of male pups of G3 groups on PNDs 4 (P≤0.01) and 7 (P≤0.05) as compared to control and an increased weight of male pups of G2 and G4 groups on PND 21 (P≤0.05) as compared to control. Interestingly, this trend was also observed for female pups of G2 and G4, whose weights were significantly higher than control on PND 21 (P≤0.05). Among the female pups, additionally, G2 pups showed reduced weights on PND 0 as compared to the control group (P≤0.01). The number and percent of live births remained comparable for all the groups of pregnant females. Significant increase in male pups of G3 group (P≤0.001) and a statistically significant decrease in female pups of G2 group (P≤0.01) was observed. None of these observations were consistent with the dose levels and hence not attributable to test-chemical treatment. No test chemical administration related changes were observed in the litter sizes and weight across all the experimental groups. No changes in the postnatal survival was observed due to the test chemical administration across all the experimental groups. No external malformations in the pups was observed from the maternal animals of all the groups. No changes in the ano-genital distances and no of the male pups showed presence of nipples/areolae on PND 13. Mean age and body weight at balanopreputial separation remained comparable for all groups of males. Similarly, the mean age and body weight at the time of vaginal opening remained statistically comparable for all groups of female rats. Further, no significant difference was observed from the day of vaginal opening to appearance of first cornified smear (estrous). No treatment-related effects were observed on male and female pup sexual development markers. No test chemical related effects were observed in the visceral examinations of the pups of maternal animals from all the dose groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on all the available data, it was observed that the test chemical did not show any adverse effects in the maternal animals and fetuses except a few changes in the hormonal levels in the maternal animals at 250 mg/kg bw animals. Therefore, it is concluded that the LOAEL and NOAEL for the maternal toxicity was 250 mg/kg bw/day and 125 mg/kg bw/day, respectively, while the NOAEL for the fetal parameters was 250 mg/kg bw/ day.
Executive summary:

An Extended One-Generation Reproductive Toxicity Study was performed according to OECD TG 443 to evaluate the general, reproductive and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of the test chemical in Wistar rats. A total of 240 wistar rats (120 males and 120 females) were randomized into 4 experimental groups each containing 30 animals per sex per group. The groups, viz., G1, G2, G3 and G4 received 0, 62.5, 125 and 250 mg/kg body weight of test chemical respectively. Dose administration was through an oral gavage and the vehicle used in this study was corn oil. All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Females were treated throughout gestation and lactation up to the day of termination after weaning. Males were treated in the same manner until termination. Dosing of selected F1 males and females begun at weaning and continue until scheduled necropsy, depending on cohort assignment. To ensure the accurate administration of the test chemical, the concentrations and homogeneity of the dose formulation were verified through formulation analyses on day 1, day 91 and day 182 of treatment. The results of these were found within the acceptable limits in both occasions. In maternal parameter observations, it was observed that, no treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in maternal animals. No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment. Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. Also, the mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods. The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. However, this effects was neither dose dependent nor treatment related. Among the parental females, no statistical differences were observed for feed efficiency in any of the groups. No significant difference was observed in any of the measured haematology parameters in all the groups in maternal animals. All the clinical chemistry parameters in maternal females were found comparable in control and treatment groups. The urine parameters tested did not show up any significant changes among the experimental groups of maternal animals. No significant difference was observed in absolute organ weight or organ weight relative to body weight for the entire set of measured organs in maternal females. However, significant reduction was observed in absolute and relative liver weight in G4 females (P<0.05) as compared to control animals. However, the decreased liver weights with respect to body weights in G4 females did not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test-chemical. Also, no difference was observed on the weights of uterus, ovaries and Thyroid & Parathyroid organs. No gross anomalies were observed in the maternal animals in gross pathological examinations. No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity. The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of maternal animals; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect. Out of 30 females 29, 28, 29 and 28 females delivered pups from G1, G2, G3 and G4 groups respectively. There were no treatment related effects on the number of abortions in maternal females. The mean number of pre and post implantation losses were comparable among females of control and treatment groups. No cases of litter losses due to resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration. No cases of early or late resorptions were observed in all the maternal animals across all the experimental groups due to the test chemical administration. No cases of dead fetuses were observed due to the test chemical administration. The duration of pregnancy (days from insemination to parturition) / gestation length was comparable among females of all the experimental groups. All the maternal animals remained pregnant throughout the gestation period and there were no changes observed number of pregnant females due to the test chemical administration. No changes in the number of corpora lutea were observed, no case of dystocia (difficulty in parturition) was observed in females of any of the groups and no unusual nesting or nursing behaviour was found as well, due to the test chemical administration. In fetal examinations, it was observed that, mean body weight of pups of both sexes combined on post-natal day (PND) 0 was found to be significantly lower in G4 (P≤0.05) as compared to controls. This difference, however, did not persist for any of the other days of observation. On the contrary, the G2 and G4 group pups returned significantly higher body weights on PND 21 as compared to control (P≤0.05 and P≤0.001 respectively). Sex-specific analysis of pup weights revealed statistically reduced weight of male pups of G3 groups on PNDs 4 (P≤0.01) and 7 (P≤0.05) as compared to control and an increased weight of male pups of G2 and G4 groups on PND 21 (P≤0.05) as compared to control. Interestingly, this trend was also observed for female pups of G2 and G4, whose weights were significantly higher than control on PND 21 (P≤0.05). Among the female pups, additionally, G2 pups showed reduced weights on PND 0 as compared to the control group (P≤0.01). The number and percent of live births remained comparable for all the groups of pregnant females. Significant increase in male pups of G3 group (P≤0.001) and a statistically significant decrease in female pups of G2 group (P≤0.01) was observed. None of these observations were consistent with the dose levels and hence not attributable to test-chemical treatment. No test chemical administration related changes were observed in the litter sizes and weight across all the experimental groups. No changes in the postnatal survival was observed due to the test chemical administration across all the experimental groups. No external malformations in the pups was observed from the maternal animals of all the groups. No changes in the ano-genital distances and no of the male pups showed presence of nipples/areolae on PND 13. Mean age and body weight at balanopreputial separation remained comparable for all groups of males. Similarly, the mean age and body weight at the time of vaginal opening remained statistically comparable for all groups of female rats. Further, no significant difference was observed from the day of vaginal opening to appearance of first cornified smear (estrous). No treatment-related effects were observed on male and female pup sexual development markers. No test chemical related effects were observed in the visceral examinations of the pups of maternal animals from all the dose groups. Thus, based on all the available data, it was observed that the test chemical did not show any adverse effects in the maternal animals and fetuses except a few changes in the hormonal levels in the maternal animals at 250 mg/kg bw animals. Therefore, it is concluded that the LOAEL and NOAEL for the maternal toxicity was 250 mg/kg bw/day and 125 mg/kg bw/day, respectively, while the NOAEL for the fetal parameters was 250 mg/kg bw/ day.