Registration Dossier

Administrative data

Endpoint:
screening for reproductive / 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:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
According to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Name: Tetraethyl ammonium bromide
CAS No.: 71-91-0
Molecular Formula: C8-H20-N.Br
Molecular Weight: 210.157 g/mol
SMILES: [N+](CC)(CC)(CC)CC.[BrH-]
Purity: 99.18%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) x 9 wks
- Weight at study initiation: (P) Males: 248.07 g to 285.05 g; Females: 215.85 g to 244.57 g
- Fasting period before study: No Data Available
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
(i) Pre mating: Two animals of same sex and group per cage were housed.
(ii) Mating: During mating, two animals (one male and one female) of same group were housed.
(iii) Post-Mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy and young adult males and females were acclimatized for five days initially to experimental room conditions and observed for clinical signs once daily. The females were then evaluated for oestrus cyclicity for a period of fourteen days and females noted with normal oestrous cyclicity (4 to 5 days cycle) in a two weeks pre-treatment period were selected for the study. The females screened for oestrus cyclicity and males were observed for clinical signs once daily during this period. Veterinary examination of all the animals was performed on the day of receipt and on day of randomization and grouping.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 to 23.4˚C
- Humidity (%): 44 to 66%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES:
From: 09 January 2019
To: 02 March 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled Water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared before dose administration on each treatment day. The required quantity of test item was weighed into a clean beaker and small volume of the vehicle was added into the beaker, mixed well using glass rod and transferred into measuring cylinder. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 6.25 mg/mL, 12.5 mg/mL and 25 mg/ml for low, mid and high doses, respectively.

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): Distilled water was used as a vehicle. The test item was clearly soluble in distilled water at the concentration of 100 mg/mL as evidenced by the in-house solubility test results. Hence, distilled water was selected as a vehicle for test item formulation preparation. Also, distilled water is universally accepted vehicle used in pre-clinical toxicity studies.
- Concentration in vehicle: 0 mg/ml (Control), 6.25 mg/ml (Low Dose Group), 12.5 mg/ml (Mid Dose Group) and 25 mg/ml (High Dose Group)
- Amount of vehicle (if gavage): 10 ml
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: The female was placed with the same male until pregnancy occurs by evidence of sperm in vaginal smear until two weeks have elapsed.
- Proof of pregnancy: Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: All the females were found to be mated within 14 days of cohabitation period.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chromatographic Conditions:
Column: Zorbax Eclipse plus C18, 4.6 × 250 mm, 5 µm
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Oven temperature: 25º C
Run time: 15 minutes
Wavelength: 198 nm
Method validation parameters evaluated for determination of Tetraethyl ammonium bromide (CAS No. 71-91-0) met the acceptance criteria. The results obtained werewithin the specified limits. Thus, the method was suitable for the analysis of the Tetraethyl ammonium bromide (CAS No. 71-91-0) in milli-Q water for dose formulations in the study.
Duration of treatment / exposure:
Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 29 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).
Frequency of treatment:
Once Daily
Details on study schedule:
No Data Available
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
No. of animals per sex per dose:
Total Animals: 96 (48 males + 48 females)
Control Group: 12 Males and 12 Females
Low Dose Group: 12 Males and 12 Females
Mid Dose Group: 12 Males and 12 Females
High Dose Group: 12 Males and 12 Females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 62.5, 125 and 250 mg/kg body weight for low, mid and high dose were selected in consultation with the sponsor.
- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (-11.11% to +13.25% for males and -8.16% to +6.84% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
- Fasting period before blood sampling for clinical biochemistry: Yes, animals were fasted before sampling of bllod for clinical biochemistry.
- Other: No Data Available
Positive control:
No Data Available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption was measured for all animals once a week during premating and once for males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No Data Available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No Data Available

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

OTHER: No Data Available
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Testis weight, Epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of all pups/litter.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: [number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other. Particular attention was paid to the external reproductive genitals and was examined for signs of altered development; gross evaluation of external genitalia was performed.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No Data Available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No Data Available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, the males were sacrificed after completion of 29 days of treatment.
- Maternal animals: All surviving animals, females were sacrificed on lactation day 14 and pups were sacrificed on lactation day 13.

GROSS NECROPSY
- Gross necropsy consisted of Epididymides, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands, Glans penis, Ovaries, Prostate and, Seminal vesicles and coagulating glands, Testes, Uterus with cervix.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [ I ] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded.
Statistics:
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. Afterverification, the data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below: (Please Check Table II for Statistical Methods used). *Statistically significant (P<0.05) change than the vehicle control group.
Reproductive indices:
Copulatory interval, Gestation length, Absolute and relative organ weights, Anogenital distance ratio, Mean pup weight , Live birth Index, Pre/post implantation loss, Pre/post natal loss, No. of resorptions per dam, Corpora lutea per dam, Implantations per dam, Pregnancy rate and No. of live/dead pups/dam.
Offspring viability indices:
Pup survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex during the experimental period.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality/morbidity was observed at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex during the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in mean body weight and percent change in body weight with respect to day 1 at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex during the experimental period when compared with vehicle control group.
However, statistical significant increase in percent change in body weight on day 15 with respect to day 1 at group G4 [250 mg/kg] females was noted when compared with vehicle control group females, which were considered as incidental and not treatment related as there was no effect in mean body weight at this period, and also the change is not consistent during the experimental period.

Gestation Weights of Dams: There were no changes observed in the gestation body weight and percent change in body weight during gestation period at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group animals.

Lactation Weights of Dams: There were no changes observed in the lactation body weight and percent change in body weight during lactation period at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes in mean feed consumption at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex when compared with vehicle control group during the experimental period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum thyroxine hormone (T4) levels in adult males collected at termination and lactation day 13 pups at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group. However, statistical significant reduction in serum T4 levels of adult males at group G3 (125 mg/kg) and statistical significant reduction in serum T4 levels of lactation day 13 pups at group G4 (250 mg/kg) was noted when compared with vehicle control group levels. These changes were not considered as treatment related due to lack of dose dependency and also the obtained values are within historical control data.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related histopathological findings were noticed during microscopic examination conducted for epididymides, levator ani plus bulbocavernosus muscle complex, cowper’s glands, glans penis, prostate and seminal vesicles and coagulating glands, testes and thyroid along with parathyroid [in males]; ovaries, uterus with cervix and thyroid along with parathyroid [in females].
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no effects noted in the oestrus cyclicity of females at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] evaluated during pre-mating period, co-habitation period and also on the day of sacrifice (Lactation day 14).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
The observed finding like Interstitium, Mononuclear cells infiltrate prostate gland from group G4 males [one male with minimal focal and one male with moderate multifocal] is considered as spontaneous and incidental.
Reproductive performance:
no effects observed
Description (incidence and severity):
A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100.0% for all the tested dose groups.
A total of 11, 11, 11 and 10 males from vehicle control [0 mg/kg], low dose [62.5 mg/kg], mid dose [125 mg/kg] and high dose [250 mg/kg] groups respectively, were found to be fertile with a fertility rate (with evidence of implantations in females) of 91.7%, 91.7%, 91.7% and 83.3%.

Details on results (P0)

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex during treatment period. No treatment related changes in body weight, percent change in body weight with respect to day 1 and feed consumption of either sex was noted during the treatment period. No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose groups of either sex. A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 11, 11, 11 and 10 males from vehicle control [0 mg/kg], low dose [62.5 mg/kg], mid dose [125 mg/kg] and high dose [250 mg/kg] groups respectively, were found to be fertile with a fertility rate (with evidence of implantations in females) of 91.7%, 91.7%, 91.7% and 83.3%.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs and external anomalies observed in any of the pups at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] litters during lactation period observations.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The post-natal loss [loss of pups during lactation period] occurred at the tested dose group litters is considered as incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup (male and female) weight recorded on lactation day 1, 4, 7 and 13 at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum thyroxine hormone (T4) levels in adult males collected at termination and lactation day 13 pups at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group.
However, statistical significant reduction in serum T4 levels of adult males at group G3 (125 mg/kg) and statistical significant reduction in serum T4 levels of lactation day 13 pups at group G4 (250 mg/kg) was noted when compared with vehicle control group levels. These changes are not considered as treatment related due to lack of dose dependency and also the obtained values are within historical control data.
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no effects noted in pup ano-genital distance ratio of either sex recorded on lactation day 4 at all the tested dose groups [62.5 mg/kg, 125 mg/kg and 250 mg/kg] when compared with vehicle control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences of nipples in male pups at all the tested dose group litters [62.5 mg/kg, 125 mg/kg and 250 mg/kg] and also at vehicle control group litters observed on lactation day 13.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were noted during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND4 and 13] from all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] and vehicle control group litters.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. No gross pathological changes were noted during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND4 and 13] from all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] and vehicle control group litters.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no
Treatment related:
no

Any other information on results incl. tables

TABLE 1 (a): SUMMARY OF THE STUDY


Parameters ↓

Group & Dose (mg/kg body weight/day)

 

G1 &

0

G2 & 62.5

G3 & 125

G4 &

250

Reproductive Indices

Mating Indices 

Pairs started (No.)

 

12

12

12

12

Males showing evidence of mating (No.)

 

12

12

12

12

Females showing evidence of copulation (No.)

 

12

12

12

12

Male Mating Index (%)

 

100.00

100.00

100.00

100.00

Female Mating Index (%)

 

100.00

100.00

100.00

100.00

 

Fertility Indices

Females achieving pregnancy (No.)

 

11

11

11

10

Male Fertility Index (%)

 

100.00

100.00

100.00

100.00

Female Fertility Index (%)

 

91.67

91.67

91.67

83.33

 

Copulatory Indices

Conceiving days 1 to 5 (No.)

 

9

6

7

5

Conceiving days >6 (No.)

 

3

6

5

7

Precoital Interval (Days)

Mean

4.33

6.67

6.75

8.08

±SD

3.34

4.62

4.75

4.12

 

Gestation Indices

Pregnancy≤21 days (No.)

 

0

0

0

0

Pregnancy = 22 days (No.)

 

5

4

4

4

Pregnancy≥23 days (No.)

 

6

7

7

6

Gestation length (Days)

Mean

22.55

22.64

22.64

22.60

±SD

0.52

0.50

0.50

0.52

Gestation Index (%)

 

100.00

100.00

100.00

100.00

 

Dams with live young born (No.)

 

11

11

11

10

Dams with live young at day 4 post-partum (No.)

 

11

11

11

10

Dams with live young at day 13 post-partum (No.)

 

11

11

11

10

 

Implantation Index and Pre and Post implantation Losses 

Implants/dam

Mean

11.36

11.64

11.82

12.00

±SD

3.17

1.91

2.96

2.94

Corpora luetea/dam (Mean)

Mean

11.36

11.64

11.82

12.00

±SD

3.17

1.91

2.96

2.94

Implantation Index (%)

Mean

100.00

100.00

100.00

100.00

±SD

0.00

0.00

0.00

0.00

Pre-Implantation Loss (%)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

Post-Implantation Loss (%)

Mean

0.00

0.83

3.67

2.14

±SD

0.00

2.74

9.55

6.78


Parameters ↓

Group & Dose (mg/kg body weight/day)

 

G1 &

0

G2 & 62.5

G3 & 125

G4 &

250

 

Offspring Viability Indices

Live Birth Indices and Sex Ratio at Birth 

Live pups/dam at birth

Mean

11.36

11.55

11.27

11.70

±SD

3.17

1.97

2.65

2.87

Litter Size (Total No. of pups born/dam) at birth

Mean

11.36

11.64

11.45

12.00

±SD

3.17

1.91

2.62

2.94

 Live Birth Index/dam (%)

Mean

100.00

99.17

98.42

97.86

±SD

0.00

2.74

3.53

6.78

 Male Live pups/dam at birth

Mean

5.45

5.18

5.45

5.50

±SD

2.58

2.23

2.07

1.84

Female Live pups/dam at birth

Mean

5.91

6.36

5.82

6.20

±SD

2.63

1.91

2.09

3.36

Sex Ratio (male/female)

Mean

1.09

0.94

1.12

1.48

±SD

0.66

0.56

0.64

1.47

Pup Survival Indices and Sex Ratio during lactation  

Pups survived per dam ( LD1 to 4)

Mean

11.36

11.55

11.27

11.60

±SD

3.17

1.97

2.65

2.91

 Pups dead per dam ( LD1 to 4)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

 Pup Survival Index (%) per dam (LD1 to 4) 

Mean

100.00

100.00

100.00

99.09

±SD

0.00

0.00

0.00

2.87

Sex Ratio (male/female) per dam at LD 4

Mean

1.09

0.94

1.12

1.49

±SD

0.66

0.56

0.64

1.47

 Pups Sacrificed for Blood Collection on LD4

Mean

1.09

1.36

1.00

1.20

±SD

1.04

0.92

1.00

1.03

 Pups survived per dam (LD4 to 7)

Mean

10.27

10.18

10.27

10.40

±SD

2.28

1.40

1.85

2.12

Pups dead per dam (LD4 to 7)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

 Pup Survival Index (%) per dam (LD4 to 7)

Mean

100.00

100.00

100.00

100.00

±SD

0.00

0.00

0.00

0.00

Sex Ratio (male/female) per dam at LD 7

Mean

1.41

1.34

1.38

2.24

±SD

1.03

1.13

0.80

2.73

 Pups survived per dam (LD7 to 13)

Mean

10.27

10.18

10.27

10.40

±SD

2.28

1.40

1.85

2.12

 Pups dead per dam (LD7 to 13)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

 Pup Survival Index (%) per dam ( LD7 to 13)

Mean

100.00

100.00

100.00

100.00

±SD

0.00

0.00

0.00

0.00

Sex Ratio (male/female) per dam at LD 13

Mean

1.41

1.34

1.38

2.24

±SD

1.03

1.13

0.80

2.73

 

Pre and Postnatal loss 

Pre-natal (implantations minus live births) (No.)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

Females with 0 (No.)

 

11

11

11

10

Females with ≥ 1 (No.)

 

0

0

0

0

Post-natal (live births minus alive at post natal day 13)

Females with 0 (No.)

 

11

11

11

10

Females with ≥ 1 (No.)

 

0

0

0

0

 


Parameters ↓

Group & Dose (mg/kg body weight/day)

 

G1 &

0

G2 & 62.5

G3 & 125

G4 &

250

Pup Observations 

Male Pup weight at birth in gram

Mean

6.83

6.89

6.99

6.92

±SD

0.24

0.41

0.30

0.31

Female Pup weight at birth in gram

Mean

5.87

5.96

6.03

6.06

±SD

0.51

0.33

0.23

0.33

Male Pup weight on LD4 in gram

Mean

10.18

10.11

10.29

10.21

±SD

0.37

0.30

0.60

0.62

Female Pup weight on LD4 in gram

Mean

9.11

8.86

8.85

8.96

±SD

0.66

0.42

0.18

0.71

Male Pup weight on LD7 in gram

Mean

15.69

16.10

15.84

16.18

±SD

1.27

0.94

0.97

1.06

Female Pup weight on LD7 in gram

Mean

13.84

13.57

13.55

13.95

±SD

1.33

0.74

0.58

1.11

Male Pup weight on LD13 in gram

Mean

26.84

26.80

26.79

26.71

±SD

0.75

0.58

0.63

0.67

Female Pup weight on LD13 in gram

Mean

24.36

24.14

24.21

24.38

±SD

0.86

0.75

0.63

0.92

Male Pup Ano-genital distance ratio

Mean

2.04

2.09

2.11

2.08

±SD

0.05

0.09

0.07

0.11

Female Pup Ano-genital distance ratio

Mean

1.24

1.19

1.19

1.20

±SD

0.11

0.08

0.05

0.08

Male Pup Nipples (No.)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

Applicant's summary and conclusion

Conclusions:
Based on all the observations and results, it was concluded that the NOAEL for the test chemical based on no systemic and reproductive effects observed was considered to be 250 mg/kg bw/day.
Executive summary:

A reproductive and developmental toxicity study according to OECD TG 421 (Reproduction / Developmental Toxicity Screening Test) was performed using Sprague-Dawley rats. The study was performed to evaluate possible adverse effects following repeated oral dosing to males for 29 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle [distilled water], animals in G2, G3 and G4 groups were administered with test item at dose levels of62.5, 125 and 250 mg/kg body weight/day for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. All animals were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter were screened for Thyroxine hormone (T4) levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The ano-genital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 30 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy. All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] animals of either sex during treatment period. No treatment related changes in body weight, percent change in body weight with respect to day 1 and feed consumption of either sex was noted during the treatment period. No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose groups of either sex. A total of twelve pairs were started for mating from each group, all the females were confirmed with mating within 14 days of cohabitation period with the first male only. The mating index for the males and females was 100% for all the tested dose groups. A total of 11, 11, 11 and 10 males from vehicle control [0 mg/kg], low dose [62.5 mg/kg], mid dose [125 mg/kg] and high dose [250 mg/kg] groups respectively, were found to be fertile with a fertility rate (with evidence of implantations in females) of 91.7%, 91.7%, 91.7% and 83.3%. A total of 11, 11, 11 and 10 females from vehicle control [0 mg/kg], low dose [62.5 mg/kg], mid dose [125 mg/kg] and high dose [250 mg/kg] groups respectively, were found to be pregnant [with evidence of implantations] with a fertility rate of 91.7%, 91.7%, 91.7% and 83.3% and a total of 1, 1, 1 and 2 females from vehicle control [0 mg/kg], low dose [62.5 mg/kg], mid dose [125 mg/kg] and high dose [250 mg/kg] groups respectively, were confirmed as non-pregnant [with no evidence of implantations] at a percent of 8.3%, 8.3%, 8.3% and 16.7% were noted. Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weight and feed consumption during gestation and lactation at all the tested dose groups. All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group. There were no gross pathological changes noticed during conduct of necropsy in any of the adult animals of either sex at all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] and vehicle control group animals during conduct of necropsy. No gross pathological changes were noted during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND4 and 13] from all the tested dose group [62.5 mg/kg, 125 mg/kg and 250 mg/kg] and vehicle control group litters. During histopathological examination, no treatment related histopathological findings noticed in the microscopic evaluation of collected reproductive organs and thyroid along with parathyroid at high dose [250 mg/kg] group males and females. Thus, based on all the observations and results, it was concluded that the NOAEL for the test chemical based on no systemic and reproductive effects observed was considered to be 250 mg/kg bw/day.