Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available, it was concluded that the test chemical did not cause any adverse effect on in the tested animals and therefore according to the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
[As per OECD Guideline No. 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29 July 2016]
Deviations:
no
Principles of method if other than guideline:
According to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Justification for study design:
No Data Available
Specific details on test material used for the study:
Name of Test Item: Cinnamyl Alcohol
Chemical Name (IUPAC): (2E)-3-phenylprop-2-en-1-ol
CAS No.: 104-54-1
Physical Appearance (with colour): White to yellow solid
Lot No.: L241381803
Date of Manufacture : March 2018
Date of Expiry: February 2023
Purity (As per Certificate of Analysis): GC - 98.53%

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 250.25 to 297.51 g; Females: 200.85 to 249.90 g
- Fasting period before study: No Data Available
- Housing: Housing was done as follows:
- 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.
- During acclimatization, two animals of same sex were housed.
- Pre mating - Per cage two animals of the same sex and group were housed.
- Cohabitation Period (mating) - Per cage two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming presence of sperm in the vaginal smear and/or vaginal plugs (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 for mated females 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 (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 animals were acclimatized for five days to experimental room conditions and females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment and were observed for clinical signs once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 23.2 deg C
- Humidity (%): 46 to 65%
- 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: To:
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical formulations were prepared before dose administration and administered within stability period. The required quantity of test chemical was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar, and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 17.5, 35 and 70 mg/mL of test chemical for low, mid and high dose groups respectively. The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test chemical formulations.

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):
- Concentration in vehicle: 17.5, 35 and 70 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): L12017004 and L32011001
- Purity: No data available
Details on mating procedure:
- M/F ratio per cage: The males and females were placed in 1:1 ratio.
- Length of cohabitation: The female was placed with the same male until pregnancy occured by evidence of sperm in the vaginal smear until two weeks.
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Column: Zorbax Eclipse plus C18, 4.6×250 mm ,5µm
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Wavelength: 242 nm
Run time: 10 minutes
Column Oven Temperature: 25°C
Mobile Phase: Acetonitrile: Milli-Q Water (50:50 %, v/v)
Diluent: Acetonitrile
Method validation parameters evaluated for the test chemical met the acceptance criteria. The results obtained are within the specified limits. Thus, this method is suitable for the analysis of the test chemical for dose formulations in the proposed Toxicology Studies.
Duration of treatment / exposure:
Males: 34 days
Females: Approximately 70 days
Frequency of treatment:
Once Daily
Details on study schedule:
No Data Available
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group
Dose / conc.:
87.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based upon the available literature and previously performed studies of the test chemicals and similar source chemicals.
- Rationale for animal assignment (if not random): The animals were randomly assigned to the groups on the basis of their body weights.
- Fasting period before blood sampling for clinical biochemistry: No Data Available
- Other: No Data Available
Positive control:
No Data Available
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once Daily

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): Yes
- 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

OTHER: No Data Available
Oestrous cyclicity (parental animals):
Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (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, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
The day of littering was considered as lactation day 1 for the dam and postnatal day 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and the mean litter size is reported. The sex ratio (m/f) at birth were calculated per litter. The live birth index (%) per litter was calculated. The number of pups survived/dead per litter were recorded during lactation period and presented as pup survival index (%) per litter between lactation day 1 to 4, 4 to 7 and 7 to 13. The sex ratio on lactation day 4, 7 and 13 was calculated per litter.
Postmortem examinations (parental animals):
The males were sacrificed after completion of 35 days of treatment, females were sacrificed on lactation day 14 and pups were sacrificed on PND 13. The females confirmed with mating but not littered were sacrificed on gestation day 25. The animals were fasted overnight, but water was availableprovided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to necropsy. The vaginal smear of females on the day of necropsy (lactation dayLD 14) was performed and stage of oestrus cycle was recorded. The animals were humanely sacrificed using CO2 asphyxiation and subjected to gross necropsy, external and internal gross pathological examinations. Prior to necropsy, males from all the groups, were randomized and necropsy was conducted as per the randomized order to avoid sampling biasness.

Detailed histopathological examination was performed on the ovaries, testes and epididymides with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals from high dose group and the vehicle control group sacrificed at termination.
Postmortem examinations (offspring):
The dead pups and pups which were sacrificed on PNDs 4 and 13 were examined for gross abnormalities with particular attention to the external reproductive genitals and findings were recorded.
Statistics:
The statistical analysis includes One-way ANOVA with Dunnett’s post test, Kruskal-Wallis and Chi-square test. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, 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).
Reproductive indices:
Male Mating Index (%), Male Fertility Index (%), Female Mating Index (%), Female Fertility Index (%), Pre-coital Interval (Days), Pre-coital Interval (Days), Gestation Index (%), Parturition Index (%) and Mean number of Implantations (%),
Offspring viability indices:
Post-Implantation Loss, Post-implantation Loss/litter (No.), Post-natal Loss on lactation day 13 (No.), Post natal loss on lactation day 13 (%), Live Birth Index (%) per dam, Pup Survival index (%) on lactation day 4/7/13, Sex ratio (M/F), Percentage of male/female offspring (%) and Ano-genital Distance to cube root of the body weight Ratio (AGD ratio).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test chemical related clinical signs in either sex at all the dose groups, tested till 350 mg/kg bw (high dose groups).
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No case of mortality/morbidity was observed in the either sex at all the dose groups, tested till 350 mg/kg bw (high dose groups).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean body weight and percent change in mean body weight with respect to day 1 in either sex during the experimental period in dosed groups when compared with vehicle control group. Also, there were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose groups when compared with vehicle control group. There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the dosed groups when compared with vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes noted in mean feed consumption measured during pre-mating period (first 2 weeks) in either sex during the experimental period in dosed groups when compared with vehicle control group. However, statistically significant decreases in the mean feed consumption were noted during gestation day 7 to 14 and 14 to 20 in group G4 when compared with vehicle control group. There were no changes noted in mean feed consumption during lactation period in any of the the tested dosed groups when compared with vehicle control group.
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:
no effects observed
Description (incidence and severity):
There were no changes observed in serum Thyroxine (T4) hormone levels at all the dose groups in either sexes when compared with vehicle control group.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related histopathological findings noted in high dose group animals of both sexes. The minimal degeneration of seminiferous tubules in testes observed in two males of high dose group were considered as incidental finding as the changes were of minimal in severity, distributed in focal areas, unilateral in nature and/or commonly expected in laboratory rats.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no irregularities observed in the oestrus cyclicity of females at any of the groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in dosed groups when compared with vehicle control group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects on the weight of testes and epididymes were observed. Also, there were no effects on the testicular sperm generation due the administration of the test chemical.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect on the overal reproductive performance of the test animals due to the administration of the test chemical. There were no treatment related and adverse changes in the Male Mating Index (%), Male Fertility Index (%), Female Mating Index (%), Female Fertility Index (%), Pre-coital Interval (Days), Pre-coital Interval (Days), Gestation Index (%), Parturition Index (%) and Mean number of Implantations (%) when compared to the control group.
There were no test chemical related clinical signs in either sex at all the dose groups, tested till 350 mg/kg bw (high dose groups). No case of mortality/morbidity was observed in the either sex at all the dose groups, tested till 350 mg/kg bw (high dose groups). There were no changes noted in mean body weight and percent change in mean body weight with respect to day 1 in either sex during the experimental period in dosed groups when compared with vehicle control group. Also, there were no changes noted in mean body weight and percent change in mean body weight gain during gestation period in any of the tested dose groups when compared with vehicle control group. There were no test item-related changes noted in mean body weight and percent change in mean body weight gain during lactation period in any of the dosed groups when compared with vehicle control group. There were no changes noted in mean feed consumption measured during pre-mating period (first 2 weeks) in either sex during the experimental period in dosed groups when compared with vehicle control group. However, statistically significant decreases in the mean feed consumption were noted during gestation day 7 to 14 and 14 to 20 in group G4 when compared with vehicle control group. There were no changes noted in mean feed consumption during lactation period in any of the the tested dosed groups when compared with vehicle control group. There were no changes observed in serum Thyroxine (T4) hormone levels at all the dose groups in either sexes when compared with vehicle control group. There were no test item-related changes observed in both absolute and relative organ weights at any of the tested dose group animals of both the sexes when compared with concurrent vehicle control group. There were no gross pathological changes observed during necropsy in any of the adult animals and pups of both sexes the tested dose groups and vehicle control group. There were no test item-related histopathological findings noted in high dose group animals of both sexes. The minimal degeneration of seminiferous tubules in testes observed in two males of high dose group were considered as incidental finding as the changes were of minimal in severity, distributed in focal areas, unilateral in nature and/or commonly expected in laboratory rats. There were no irregularities observed in the oestrus cyclicity of females at any of the groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in dosed groups when compared with vehicle control group. No effects on the weight of testes and epididymes were observed. Also, there were no effects on the testicular sperm generation due the administration of the test chemical. There was no effect on the overal reproductive performance of the test animals due to the administration of the test chemical. There were no treatment related and adverse changes in the Male Mating Index (%), Male Fertility Index (%), Female Mating Index (%), Female Fertility Index (%), Pre-coital Interval (Days), Pre-coital Interval (Days), Gestation Index (%), Parturition Index (%) and Mean number of Implantations (%) when compared to the control group.
Dose descriptor:
NOAEL
Effect level:
350 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
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
All the pups were noted with normal behaviour during daily observations.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no external anomalies noted and no test item-related mortalities were noted during daily observation of pups from treated and vehicle control group litters during post-natal period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 in the tested dose group litters when compared with vehicle control group. However, statistically significant increase in the mean female pup weight on PND 7 was noted in tested groups. G2, G3 and G4 when compared with vehicle control group. These noted changes are considered as incidental and unrelated to treatment as there were no changes in daily pup observations and also no effects on mean pup weight on PND 1, 4 or 13 at any of the tested dose groups and the changes were not consistent.
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:
no effects observed
Description (incidence and severity):
There were no changes observed in serum Thyroxine (T4) hormone levels of PND 13 pups from all the tested dose group litters when compared with vehicle control group.
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
No effect on the sexual maturation of pups were observed.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup [both male and female] ano-genital distance measurement (mm) and ratio per litter recorded on PND 4 at all the tested dose groups 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 examined on PND 13 in any of the tested dose group litters and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during necropsy in any of the pups of both sexes at all the tested dose groups and vehicle control group.
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
All the pups were noted with normal behaviour during daily observations. There were no external anomalies noted and no test item-related mortalities were noted during daily observation of pups from treated and vehicle control group litters during post-natal period. There were no changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 in the tested dose group litters when compared with vehicle control group. However, statistically significant increase in the mean female pup weight on PND 7 was noted in tested groups. G2, G3 and G4 when compared with vehicle control group. These noted changes are considered as incidental and unrelated to treatment as there were no changes in daily pup observations and also no effects on mean pup weight on PND 1, 4 or 13 at any of the tested dose groups and the changes were not consistent. There were no changes observed in serum Thyroxine (T4) hormone levels of PND 13 pups from all the tested dose group litters when compared with vehicle control group. No effect on the sexual maturation of pups were observed. There were no changes observed in mean pup [both male and female] ano-genital distance measurement (mm) and ratio per litter recorded on PND 4 at all the tested dose groups when compared with vehicle control group. There were no occurrences of nipples in male pups examined on PND 13 in any of the tested dose group litters and vehicle control group litters. There were no gross pathological changes observed during necropsy in any of the pups of both sexes at all the tested dose groups and vehicle control group.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
other: Ano-genital distance, Nipple retention in male pups
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Treatment related:
no
Conclusions:
Based on all the available data, it was concluded that the test chemical did not induce any systemic or reproductive toxicity in all animals in the tested dose groups and therefore the NOAEL for the test chemical was considered to be 350 mg/kg bw/day viz. the highest dose tested for the study.
Executive summary:

A reproduction/developmental toxicity screening test as per the GLP compliance and in accordance with OECD TG 421 was performed using male and female Sprague Dawley rats for assessing the potential of the test chemical to cause adverse effect on the reproductive potential and effects on the developing fetuses. In this study 12 animals per sex per group were included which were grouped into 4 groups i.e. 0 mg/kg bw (Vehicle Control Group), 87.5 mg/kg bw (Low Dose Group), 175 mg/kg bw (Mid Dose Group) and 350 mg/kg bw (High Dose Group), respectively. Males were dosed for a period of 35 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period, while females were dosed for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 49 to day 67 ). The vehicle and test chemical formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. All the animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The weekly body weight and weekly feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. All the males were evaluated for reproductive performance such as, mating and fertility index. All the dams were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All the females were evaluated for oestrus cyclicity during premating, mating treatment periods and at termination. The body weight and feed consumption was recorded for all the females during gestation day 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. The terminal body weight for all the animals was measured on the day of the termination (LD 14). At birth/litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all the litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated. The pups were observed once daily for external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method. There were no clinical signs of toxicity and no mortality/morbidity noted at all in any of the tested dose groups of either sex animals. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all thein any of the tested dose groups of both the either sex. There were no changes noted in mean serum thyroxine hormone (T4) levels at all in any of the tested dose group of males. The absolute and relative organ weights did not reveal any changes at all thein any of the tested dose groups. No macroscopic changes noted during conduct of necropsy in at all the vehicle control and tested dosed groups of both either the sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test chemical related microscopic changes. For reproduction toxicity end points, there were no effects noted in male mating and fertility indexes of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length in at all tested all dosed groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption were observed all dosed groups during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by the test chemical at all the tested in all treated dose groups. For developmental toxicity end points, there were no external anomalies noted and no test chemical-related mortalities among pups noted were observed in pups during post-natal period at doses tested. at all the tested dose group litters. There were no test chemical-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups.at doses tested There were no occurrences of nipples in male pups of vehicle control and treated groups on PND 13 examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters at any doses tested. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose groups up litters. Thus, based on all the available data, it was concluded that the test chemical did not induce any systemic or reproductive toxicity in all animals in the tested dose groups and therefore the NOAEL for the test chemical was considered to be 350 mg/kg bw/day viz. the highest dose tested for the study.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is from a Klimisch 1 database and provides a robust study summary.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction:

Study 1:

A reproduction/developmental toxicity screening test as per the GLP compliance and in accordance with OECD TG 421 was performed using male and female Sprague Dawley rats for assessing the potential of the test chemical to cause adverse effect on the reproductive potential and effects on the developing fetuses. In this study 12 animals per sex per group were included which were grouped into 4 groups i.e. 0 mg/kg bw (Vehicle Control Group), 87.5 mg/kg bw (Low Dose Group), 175 mg/kg bw (Mid Dose Group) and 350 mg/kg bw (High Dose Group), respectively. Males were dosed for a period of 35 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period, while females were dosed for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 49 to day 67 ). The vehicle and test chemical formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. All the animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The weekly body weight and weekly feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. All the males were evaluated for reproductive performance such as, mating and fertility index. All the dams were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All the females were evaluated for oestrus cyclicity during premating, mating treatment periods and at termination. The body weight and feed consumption was recorded for all the females during gestation day 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. The terminal body weight for all the animals was measured on the day of the termination (LD 14). At birth/litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all the litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated. The pups were observed once daily for external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method. There were no clinical signs of toxicity and no mortality/morbidity noted at all in any of the tested dose groups of either sex animals. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all thein any of the tested dose groups of both the either sex. There were no changes noted in mean serum thyroxine hormone (T4) levels at all in any of the tested dose group of males. The absolute and relative organ weights did not reveal any changes at all thein any of the tested dose groups. No macroscopic changes noted during conduct of necropsy in at all the vehicle control and tested dosed groups of both either the sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test chemical related microscopic changes. For reproduction toxicity end points, there were no effects noted in male mating and fertility indexes of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length in at all tested all dosed groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption were observed all dosed groups during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by the test chemical at all the tested in all treated dose groups. For developmental toxicity end points, there were no external anomalies noted and no test chemical-related mortalities among pups noted were observed in pups during post-natal period at doses tested. at all the tested dose group litters. There were no test chemical-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups at doses tested. There were no occurrences of nipples in male pups of vehicle control and treated groups on PND 13 examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters at any doses tested. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose groups up litters. Thus, based on all the available data, it was concluded that the test chemical did not induce any systemic or reproductive toxicity in all animals in the tested dose groups and therefore the NOAEL for the test chemical was considered to be 350 mg/kg bw/day viz. the highest dose tested for the study.

Based on the data available from different studies, test material did not show any reproductive toxicity when male and female rats were treated with test chemical orally. Thus, according tothe criteria of CLP regulation, the test chemical is not likely to classify as reproductive toxicant.

Effects on developmental toxicity

Description of key information

Developmental toxicity study

Based on the various developmental toxicity studies available for the test chemical were reviewed to determine the developmental toxicity, it was concluded that the test chemical did not cause developmental toxicity, when rats were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive and developmental toxicant.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer-reviewed journal
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Route of administration:
oral: unspecified
Details on exposure:
No data available
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
No data available
Duration of treatment / exposure:
Entire course of pregnancy (days 22–23 of gestation)
Frequency of treatment:
Once Daily
Duration of test:
No Data Available
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
5.35 mg/kg bw/day
Dose / conc.:
53.5 mg/kg bw/day
No. of animals per sex per dose:
0 mg/kg bodyweight : 14–15 female rats
5.35 mg/kg bodyweight: 14–15 female rats
53.5 mg/kg bodyweight: 14–15 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: No data
- Time schedule for examinations:No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- 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

POST-MORTEM EXAMINATIONS: Yes
- On day 20 of gestation, 6–9 rats from each group were sacrificed and the fetuses removed for examination.
- Organs examined: Fetuses were examined.

OTHER
The remaining animals delivered normally on days 22–23 of gestation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No data
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
Statistics:
No data available
Indices:
No data available
Historical control data:
No data available
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
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:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
53.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No toxic effects were observed
Remarks on result:
other: No toxic effects were observed
Abnormalities:
not specified
Fetal body weight changes:
not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Measurements of general development at birth and at one month following birth revealed no significant differences between test and control animals.
Details on embryotoxic / teratogenic effects:
Measurements of offspring bodyweight, size, survival number and general development at birth and at one month following birth revealed no significant differences between test and control animals.
Dose descriptor:
NOAEL
Effect level:
53.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Measurements of offspring bodyweight, size, survival number and general development at birth and at one month following birth revealed no significant differences between test and control animals.
Remarks on result:
other: No developmental toxic effects were observed
Abnormalities:
no effects observed
Developmental effects observed:
not specified
Treatment related:
not specified
Conclusions:
Based on all the available data, it was concluded that under the condition of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 53.5 mg/kg bw/day.
Executive summary:

The present study was conducted to determine the developmental toxicity potential of test chemical when administered orally to rats. Groups of 14–15 female rats were orally administered either 0, 5.35 or 53.5 mg/kg body weight test chemical once daily for the entire course of pregnancy. On day 20 of gestation, 6–9 rats from each group were sacrificed and the fetuses removed for examination. The remaining animals delivered normally on days 22–23 of gestation. Measurements of offspring bodyweight, size, survival number and general development at birth and at one month following birth revealed no significant differences between test and control animals. Therefore, Based on all the available data, it was concluded that under the condition of this study, the no-observed-adverse-effect level (NOAEL) was considered to be  53.5 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
53.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from authoritative database
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Data available from different studies was reviewed to determine the developmental toxicity of test chemical. The studies are as mentioned below:

Study 1

The present study was conducted to determine the developmental toxicity potential of test chemical when administered orally to rats. Groups of 14–15 female rats were orally administered either 0, 5.35 or 53.5 mg/kg body weight test chemical once daily for the entire course of pregnancy. On day 20 of gestation, 6–9 rats from each group were sacrificed and the fetuses removed for examination. The remaining animals delivered normally on days 22–23 of gestation. Measurements of offspring bodyweight, size, survival number and general development at birth and at one month following birth revealed no significant differences between test and control animals. Therefore, Based on all the available data, it was concluded that under the condition of this study, the no-observed-adverse-effect level (NOAEL) was considered to be  53.5 mg/kg bw/day.

Study 2

A Combined repeated dose repro-developmental toxicity study was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of test chemical in Wistar rats. The animals were randomly allocated to the four main groups (13/sex/group) and two recovery groups (5/sex/group). The doses selected for main groups were; 0 (G1-control),308 mg/kg body weight (G2), 556 mg/kg body weight (G3) and 1000 mg/kg body weight (G4) daily for 64 days. The recovery groups G1-R and G4-R were dosed with similar doses of respective main groups.Vehicle corn oil to G1 and G1-R and test item to G2, G3, G4 and G4-R animals were administered by oral gavage route each day during the dosing period. No mortality and morbidity were observed any of the groups of animals throughout the study period. Animals of all dose groups were observed for Clinical signs/ symptoms daily once during the experimental period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements were comparable and no statistically significant changes were revealed in animals of treatment groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the repective control groups. Motor activity measurements were comparable and no changes were revealed in any of the animals of all treated groups of both the sexes as compared to control group. Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No test chemical related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. All hematological and clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups. No treatment related changes were observed in any of the treatment groups.  At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to the respective control group rats. External and visceral examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. From the patho-morphological results presented, it is concluded that, the treatment of Methyl Phenyl acetate at 308, 556 and 1000 mg/kg body weight in male and female rats did not affect adversely and no alteration of pathological significance was observed in any of the organs including reproductive organs. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed to the administration of the test chemical.Based on all the observations and results, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg for parental (P0) generation and 556 mg/kg bw for F1 generation, when male and female wistar rats were treated with test chemical  through oral gavage.

Study 3

In a prenatal developmental toxicity study, pregnant female Sprague-Dawley rats was orally given with 0, 5, 25 or 250 mg/kg/day of the test chemical on a daily basis for 10 days (day 7-17 of gestation). Decreased weight gain between days 7 and 20 was observed in the presence of decreased food intake at all doses. Developmental effects in the offspring was observed at low and high doses and included decreased ossification of the cranium and tympanic bulla, increased incidence of dilated pelvis/ reduced papilla in kidney, dilated ureter and incidences of hypoplastic/dysplastic kidneys. The observed effects were not dose dependent and thereforeBased on all the available data, it was concluded that the NOAEL of the test chemical in a 10 day study in Sprague-Dawley rats was considered to be 250 mg/kg/day.

Study 4

A prenatal developmental toxicity study of test chemicalwas performed onCrl:COBSCD(SD)BR female rats. The test material was microencapsulated in spray-dried gum Arabic before incorporation into the diet in dose concentration 0, 1000, 3000 or 10,000 ppm (calculated doses were 83, 266 and 799 mg/kg for the respective exposure groups). The concentrations tested were based on a pilot study in which dietary inclusion of up to 9000 ppm test material was well tolerated in pregnant rats and did not result in unacceptable loss of diet palatability. High performance liquid chromatographic (HPLC) analyses were used to validate concentration, homogeneity and stability of test material throughout the study. 28 female rats per dose group were exposed to test material on GDs 6 through 15, In all the animals Feed consumption was measured daily, and body weights were taken on GDs 1, 3, 6 and then on alternate days until necropsy on GD 20, when in utero development of the conceptuses was assessed by determination of litter values and examination of the fetuses for external, soft tissue and skeletal alterations (malformations and variations).No deaths occurred during the study.No obvious clinical signs of toxicity was noted. No effects were observed at 83mg/kg and 266mg/kg dose group . At 799 mg/kg dose group , reduced maternal feed consumption and slight weight loss occurred on treatment days 1 and 2 (GDs 6–7, 7–8). Calculated mean group intake of test material was 0, 83, 266 and 799 mg/kg/day for the pregnant rats in the 0, 1000, 3000 and 10,000 ppm groups, respectively. Mean values for embryo-fetal loss, litter sizes, sex ratios were equivalent in control and test group also the mean fetal body weights, was equivalent in control and test groups.The number, type and distribution of fetal malformations, anomalies and skeletal variations also were unaffected by maternal exposure to test material at concentrationsas high as 10,000 ppm (799 mg/kg/day), although at799mg/kg dose group there was a possible marginal delay in fetal ossification(a slightly higher incidence of fetuses with incomplete ossification of some sites, such as the sacro-caudal vertebral arches). HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 799 mg/kg. When femalerats were treated withtest material by orally for GDs 6 through 15.

Thus, based on the data available, it was concluded that the test chemical did not cause developmental toxicity when rats were treated with orally. Thus, comparing this value with the criteria of CLP regulationtest chemicalis not likely to classify as reproductive and developmental toxicant.

Justification for classification or non-classification

Based on all the available data, it was concluded that the test chemical did not cause adverse effects in both reproductive and developmental parameters and therefore comparing this value with the criteria of CLP regulation, the test chemical is not likely to classify as reproductive and developmental toxicant.

Additional information