4-phenylbutan-2-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

the testing was conducted according to the decision number TPE-D-2114440221-67-01/F issued by ECHA

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-phenylbutan-2-one

EC Number:

219-847-4

EC Name:

4-phenylbutan-2-one

Cas Number:

2550-26-7

Molecular formula:

C10H12O

IUPAC Name:

4-phenylbutan-2-one

Test material form:

liquid

Specific details on test material used for the study:

Name of the product: Benzyl acetone
Appearance: Colorless liquid
Odor: Floral, grass and sweet scent of jasmine odor
Specific Gravity (25/25℃): 0.986
Purity % (GC): 99.90
Storage Conditions: Room Temperature (24 ± 3⁰C)
Formula: C6C5CH2CH2COCH3
Formula Weight: 148.20

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Justification for Selection of Test System Rat is one of the recommended rodent species by regulatory guidelines for conduct of embryo-fetal developmental toxicity studies and owing to its proven suitability and availability of vast background data

Details on test animals or test system and environmental conditions:

Sex:Male and Female (nulliparous and non-pregnant) at the time of receipt
No. of animals: 90 (30 male and 60 female) Sprague Dawley rats
Age at receipt: 9 to 10 weeks.
Body Weight at the start of 255.71 to 340.02 grams
Source: Animal Facility (AFT), Vanta Bioscience Limited or any approved CPCSEA supplier
Health status: Prior to inclusion into the study, animals were examined by the Study Veterinarian for health status and their suitability for use in the experiment.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: 0.1% Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate in Type-1 Milli Q water) was used as vehicle in the present study.

Remarks:

Justification for Selection of Vehicle The test item, Benzyl acetone was soluble in 0.1% Tween 80 in Type 1 Milli Q water v/v which is also one of the most common vehicles used toxicity studies.

Details on exposure:

Environment
Rats were housed in an experimental room maintained at 21.0°C to 22.6°C and 51 to 66% relative humidity with adequate fresh air supply (minimum 12 air changes/hour). Light and dark cycles of 12 hours each will be maintained throughout the experimental phase.

Housing
Animals were housed in solid bottomed individually ventilated cages (IVC’s) having facilities for holding pelleted food and drinking water. The phytoestrogen free bedding material square paper bedding (PURE-O-CEL) was provided and changed periodically

Feed
Altromin maintenance diet (1324 P) was provided to the animals ad libitum throughout the study period.

Water
Animals had free access to Reverse Osmosis (RO) purified water throughout the study. RO water analysis were performed for parameters including total dissolved solids, hardness, specified microbiological content, and for selected environmental contaminants, at frequencies as per the in-house SOPs

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A limit dose of 1000 mg/kg body weight was selected for the present study based on the results of a preliminary dose range finding study in non-pregnant and pregnant females (Study No. 19-020-G) conducted at VBS.

Details on mating procedure:

After completion of acclimatization period, females were housed with males for pairing or cohabitation based on one-to-one ratio (one male: one female) for 14 days or until the evidence of mating, whichever is earlier. In order to manage the experimental activities and terminal procedures efficiently, the cohabitation was performed in different slots. The details of paring and slots for cohabitation was planned in such a way that on a given day similar number of females were available for allotment into different treatment groups. The details of slots including animal numbers were recorded and maintained in raw data file and reported in the study report.
Each day, the evidence of mating was confirmed by presence of sperms in the vaginal lavage. The day of presence of sperms in the vaginal lavage was considered as day ‘0’ of the pregnancy or ‘GD 0 – (Gestation Day – 0)’. Subsequently, post detection of sperms in the vaginal lavage, females were separated from the male partner and housed individually through gestation period until their euthanasia on day 20 of gestation (GD20).
The cohabitation or pairing was done till availability of sufficient number (50 female) of confirmed mating (sperm positive in vaginal lavage) females. After completion of successful cohabitation period, all the males were sent back to VBS animal holding/stock and extra females (both mated but negative for sperms and non-mated) was sent for euthanasia.

Duration of treatment / exposure:

On the day of confirmation of pregnancy, females were allotted into two groups. Based on the number of confirmed pregnant (sperm positive) females, they were allotted sequentially into G1 and G2. At same time, these females were given the permanent numbers based on allocation into different groups viz. 01 to 25 for G1 (Vehicle Control) and 26 to 50 for G2 (Limit Dose). The details of temporary number (prefixed with ‘PM’), allotment into the specific group and permanent numbers were maintained in the raw data and reported in the study report

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Justification for Dose Selection
A limit dose of 1000 mg/kg body weight was selected for the present study based on the results of a preliminary dose range finding study in non-pregnant and pregnant females (Study No. 19-020-G) conducted at VBS.

No. of animals per sex per dose:

A total number of 60 females and 30 male was received along with their health certificate for the study.

Randomization and Grouping
On the day of confirmation of pregnancy, females were allotted into two groups (refer Section 6.9.6). Based on the number of confirmed pregnant (sperm positive) females, they were allotted sequentially into G1 and G2. At same time, these females were given the permanent numbers based on allocation into different groups viz. 01 to 25 for G1 (Vehicle Control) and 26 to 50 for G2 (Limit Dose). The details of temporary number (prefixed with ‘PM’), allotment into the specific group and permanent numbers were maintained in the raw data and reported in the study report.

An outline of the Study design is presented in the following table:
Groups G1 G2
Dose Level 0.1% Tween 80 (Vehicle Control) Benzyl acetone (Limit Dose)
Dose (mg/kg b.wt.) 0 1000
Female 01 to 25 26 to 50
Male 61 to 90 (No Treatment)

Control animals:

yes, concurrent vehicle

Details on study design:

An outline of the Study design is presented in the following table:
Groups G1 G2
Dose Level 0.1% Tween 80(Vehicle Control) Benzyl acetone (Limit Dose)
Dose (mg/kg b.wt.) 0 1000
Female 01 to 25 26 to 50
Male 61 to 90 (No Treatment)



Dose Volume
A constant dose volume of 10 mL/kg body weight was utilized to deliver the required amount of dose throughout the treatment period. Amount of dose required for each animal was calculated based on the most recent body weights.

Route and Method of Administration
The vehicle (G1 - 0.1% Tween 80) and the test item (G2 – Benzyl Acetone) was administered to pregnant females through oral (gavage) route by using disposal syringes from a calibrated batch and reusable stainless steel ball tipped intubation needle (16 to 18 gauge).

Justification for Selection of Route
It is likely (to be expected) route of exposure in the target species.

Treatment/Administration Schedule
Starting from Gestation Day (GD) 5 till GD 19 of the respective pregnant females, the vehicle or test item, Benzyl Acetone (1000 mg/kg b.wt) was administered once daily to rats from G1 and G2, respectively.

Examinations

Maternal examinations:

Post necropsy and gross pathology examination, following (but not limited to) observations were performed on each female:  Pregnancy status (Pregnant/Non pregnant)
 Number of corpora lutea
 Weight of gravid uterus
 Number of implantations/sites
 Number of live and dead fetuses
 Number of early resorptions
 Number of late resorptions
For apparently non-pregnant females, ammonium supplied staining of the uterus were performed to identify the pre-implantation loss of the embryos.

Fetal examinations:

Post uterine observations, each fetus was identified individually as per in house procedure and placed along with its placenta in acrylic fetal trays filled with normal saline. Following (but not limited to) observations were performed on each fetus:
 Determination of sex
 Weight of individual fetus
 The anogenital distance (AGD)
 External abnormalities, if any
In addition, corrected AGD with respect to body weight was performed as per the method described by Gallavan et al. (1999).

Statistics:

The following list of data (but not limited to) were subjected to statistical analyses by using SigmaPlot® 12 software (Systat, San Jose, California).
 Feed consumption
 Body weights and body weight gains
 Organ weights (absolute and relative to body weight)
 Maternal data
 Fetal data
Data were subjected to Modified-Levene equal-variance test (Brown and Forsythe, 1974) for homogeneity and the Shapiro-Wilk test (Shapiro and Wilk, 1965) for normality. A level of significance of p< 0.05 was required for the Modified-Levene’s test to reject the assumption. A level of significance of p< 0.05 was required for the Shapiro-Wilk test to reject the assumption. Normally distributed and homogenous data were submitted to One- way Analysis of Variance (ANOVA) followed by Dunnett’s test (Dunnett, 1955; Dunnett, 1964) for post hoc comparison. If the data were heterogeneous, it was subjected to Mann Whitney test. Statistical significance was reported at the alpha level of 0.05. Data has been reported in terms of number of observations (N), group means and standard deviations (%, where applicable)

Historical control data:

NA

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs noticed in the vehicle control (G1- 0 mg/kg b.wt.) and Benzyl Acetone (G2 – 1000 mg/kg b.wt.) groups in the present study

Mortality:

no mortality observed

Description (incidence):

There was no mortality and/or morbidity in the present study and all rats survived till the end of experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related changes noticed in the body weights and/or body weight gain/loss in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to
vehicle control (G1-0 mg/kg b.wt.)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noticed in the feed consumption. However, female from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) during day 17 to 20 showed a significantly (p<0.05) increased feed consumption when compared to the vehicle control (G1). The increased feed consumption in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) during day 17 to 20 was considered as an incidental finding unrelated with the treatment.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noticed in T3 and TSH parameters. However, there was a slight but significant (p<0.05) decrease in the T4 levels observed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to vehicle control group (G1- 0 mg/kg b.wt.). In absence of any associated changes in the histopathology of thyroid from this group, the changes in T4 parameter have been considered as incidental finding unrelated with the treatment.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant changes noticed in the absolute and relative organ weights of thyroid gland in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to vehicle control group (G1- 0 mg/kg b.wt.)

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related gross pathology (external and internal) findings noticed in rats from the vehicle control (G1-0 mg/body weight) and the test item, Benzyl Acetone
group (G2 - 1000 mg/kg b.wt.)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathology evaluation of thyroid gland from the control group (G1- 0 mg/kg b.wt.) and the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) rats showed some spontaneous/incidental findings. There were no treatment related findings noticed in the thyroid histopathology.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.)

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.)

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.)

Early or late resorptions:

no effects observed

Description (incidence and severity):

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.)

Dead fetuses:

no effects observed

Description (incidence and severity):

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.)

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Weights Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (malefemale combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.). Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/incidental findings unrelated with the treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Weights Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (malefemale combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.). Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/incidental findings unrelated with the treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Weights Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (malefemale combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.). Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/incidental findings unrelated with the treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Weights Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (malefemale combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.). Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/incidental findings unrelated with the treatment.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Weights Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (malefemale combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.). Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/incidental findings unrelated with the treatment.

External malformations:

no effects observed

Description (incidence and severity):

There were no maternal-fetal abnormalities noticed in any of the pups/dam/placenta in the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.). External evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some external abnormalities in the male and female pups at comparable incidence.
There were no treatment related external abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group. Visceral, head-razor and skeletal evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some
abnormalities in the male and female pups at comparable incidence.
There were no treatment related visceral, head-razor and skeletal abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no maternal-fetal abnormalities noticed in any of the pups/dam/placenta in the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.). External evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some external abnormalities in the male and female pups at comparable incidence.
There were no treatment related external abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group. Visceral, head-razor and skeletal evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some
abnormalities in the male and female pups at comparable incidence.
There were no treatment related visceral, head-razor and skeletal abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no maternal-fetal abnormalities noticed in any of the pups/dam/placenta in the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.). External evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some external abnormalities in the male and female pups at comparable incidence.
There were no treatment related external abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group. Visceral, head-razor and skeletal evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some
abnormalities in the male and female pups at comparable incidence.
There were no treatment related visceral, head-razor and skeletal abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

To conclude, based on overall observations and under the circumstances of the present
study, the test item, Benzyl Acetone when administered at 1000 mg/kg body weight by
oral gavage route in Sprague Dawley rats during gestation day 5 to 19 produced -
no treatment related and abnormal clinical signs,
 no treatment related effect on the body weights or body weight gain/loss,  no treatment related effect on the feed consumption,
 no treatment related effect on the thyroid hormone profile,
 no treatment related maternal toxicity,
 no treatment related fetal toxicity,  no treatment related maternal-fetal abnormalities,
 no treatment related visceral and head-razor abnormalities and
 no treatment related skeletal abnormalities
Therefore, the dose of 1000 mg/kg body weight of the test item, Benzyl Acetone has been considered as No Observed Effect Level (NOEL) in Sprague Dawley rats in the present study

Executive summary:

The present study was conducted as ‘Limit Test’ at a single dose of 1000 mg/kg body weight based on the preliminary range finding study in non-pregnant and pregnant rats (Study No. 19-020-G). A total number of 90 (30 male and 60 female) Sprague Dawley rats of about 9-10 weeks age were received for conduct of this study. Animals were acclimatized to the experimental conditions for minimum 5 days prior to start of mating. During acclimatization period, animals were provided with Altromin maintenance diet (1324 P) rodent pellet feed ad libitum and had free access to RO purified drinking water. After completion of acclimatization period females were housed with males for cohabitation based on one-to-one ratio (one male: one female) for 14 days or until the evidence of mating, whichever was earlier. Each day, the evidence of mating was confirmed by presence of sperms in the vaginal lavage. The day of presence of sperms in the vaginal lavage was considered as day ‘0’ of the pregnancy or ‘GD 0 – (Gestation Day – 0)’ for the particular female. Subsequently, post detection of sperms in the vaginal lavage, females were separated from the male partner and housed individually through gestation period until their euthanasia on day 20 of gestation (GD20).

The cohabitation or pairing was done till availability of sufficient number (50 female) of confirmed mating (sperm positive in vaginal lavage) females. On the day of confirmation of mating, females were allotted into one of two groups. Based on the number of confirmed pregnant (successfully

mated) females, they was allotted sequentially into G1 and G2. At same time, these females were given the permanent numbers based on allocation into two groups viz. 01 to 25 for G1 (Vehicle Control) and 26 to 50 for G2 (Benzyl Acetone – 1000 mg/kg b.wt.), After completion of successful cohabitation period, all the males were sent back to VBS animal holding/stock and females (both mated but negative for sperms) were sent for euthanasia.

Test item formulation was freshly prepared daily. Required quantity of the Test Item (Benzyl Acetone) weighed and mixed in the vehicle (0.1% Tween 80 in Type 1 Milli Q water v/v) to attain the desired dose concentration in this study. The test item formulation was analyzed prior to first dosing and during last week of the treatment. The samples were analyzed by using Gas Chromatography (GC) method. The vehicle (G1 - 0.1% Tween 80) and the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) were administered through oral (gavage) route to pregnant females starting from Gestation Day 5 (GD 5) till GD 19 by using disposal syringes from a calibrated batch and reusable stainless steel ball tipped intubation needle (16 to 18 gauge).

During the course of study, all animals were observed twice daily for morbidity and mortality. Similarly, each animal was observed for signs of behavioral changes, reaction to treatment or illness once daily throughout the treatment period. General health condition and any clinically relevant signs were recorded for individual animals from each group. Measured quantity of feed was offered to each cage and feed left over was measured to calculate the feed consumption. Feed consumption per animal per day during different days were calculated based on the quantity of feed consumption in the particular intervals. Similarly, individual animal body weight were recorded on Day 0 (gestation) and once in three days throughout the gestation period. Body weight gain/loss was calculated for each animal at specific interval.

On gestation day 20 (before scheduled necropsy) of the respective female, approximately 1.5 ml of blood was collected in tubes without any anticoagulant for obtaining the serum samples for estimation of thyroid hormones. The serum samples were separated and collected in pre-labelled vials and stored below -20oC until estimation of the thyroid hormones. Serum levels of thyroid hormones were determined by using commercially available ELISA kits.

Before scheduled necropsy, body weights were recorded for determination of organ-body weight ratios. Animals were euthanized by Carbon Dioxide (CO2) asphyxiation followed by exsanguination. Complete necropsies were carried out and gross pathology findings were recorded

for each animal. Particular emphasis was given to the uterus, uterine contents including placenta, position of fetuses in the uterus and ovaries. The weight of the thyroid gland was taken from every dam.

At necropsy, the observations like pregnancy status (Pregnant/Non pregnant), number of corpora lutea, weight of gravid uterus, number of implantations/sites, number of live and dead fetuses, number of early resorptions and number of late resorptions were performed on each female. Post uterine observations, each fetus was identified individually and placed along with its placenta in acrylic fetal trays filled with normal saline and the observations like determination of sex of pup, weight of individual fetus, anogenital distance (AGD), and external evaluation were performed. In addition, corrected AGD with respect to body weight was performed for each pup.

From each dam, half number of fetuses were subjected for evaluation of soft tissue alterations (visceral and head-razor examination) and remaining half for skeletal evaluation. For this, fetuses with odd numbers were subjected for skeletal evaluation and fetuses with even numbers were evaluated for visceral examination.

RESULTS

The results from method validation parameters namely system suitability, linearity, precision, assay accuracy, stability and homogeneity data showed that the method followed was acceptable for the determination of Benzyl acetone in dose verification samples using GC conditions

Mean Recovery (%) of Benzyl acetone was within 70 -110 % of its nominal concentration or theoretical concentration of individual layers (top, middle and bottom) in the dose formulation samples with relative standard deviation ≤10% (RSD). Hence the concentration claimed in the dose formulation samples was supported and found homogeneous in nature.

There was no mortality and/or morbidity in the present study and all rats survived till the end of experimental period. There were no clinical signs noticed in females from the vehicle control (G1- 0 mg/kg b.wt.) and Benzyl Acetone (G2 – 1000 mg/kg b.wt.) groups in the present study.

There were no treatment related changes noticed in the feed consumption of pregnant female rats. However, rats from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) during day 17 to 20 showed a significantly (p<0.05) increased feed consumption when compared to the vehicle control (G1). The increased feed consumption in the test item, Benzyl Acetone group (G2 - 1000

mg/kg b.wt.) during day 17 to 20 was considered as an incidental finding unrelated with the treatment. There were no treatment related changes noticed in the body weights and/or body weight gain/loss of pregnant females from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to vehicle control (G1-0 mg/kg b.wt.).

There were no treatment related changes noticed in T3 and TSH parameters. However, there was a slight but significant (p<0.05) decrease in the T4 levels observed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to vehicle control group (G1- 0 mg/kg b.wt.). In absence of any associated changes in the histopathology of thyroid, the changes in T4 parameter have been considered as incidental finding unrelated with the treatment.

There were no treatment related gross pathology (external and internal) findings noticed in the vehicle control (G1-0 mg/body weight) and the test item, Benzyl Acetone groups (G2 - 1000 mg/kg b.wt.). There were no significant changes noticed in the absolute and relative organ weights of thyroid gland in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to vehicle control group (G1- 0 mg/kg b.wt.). Similarly, there were no treatment related findings noticed in the histopathology of thyroid.

Evaluation of maternal parameters viz. average weight of gravid uteri, number of corpora lutea, implantations, live fetuses, pre-implantation loss, early and late resorptions did not show any statistical significant difference in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 – 0 mg/kg b.wt.).

Evaluation of fetal parameters viz. average number of fetuses per dam, number of live fetus, number of male and female fetuses, anogenital distance and corrected anogentital distance did not show any statistical significant different in the pups from the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) with that of the pups from the vehicle control (G1 – 0 mg/kg b.wt). However, there was a very slight but statistical significant (p<0.05) decrease in the combined (male and female) and male alone pup body weight noticed in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) as compared to the vehicle control (G1 - 0 mg/kg b.wt.). In addition, average placental weight (male-female combined, male and female alone) in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) was also slightly but significantly (p<0.05) increased as compared to the vehicle control (G1 - 0 mg/kg b.wt.).

Although statistically significant, the slightly decrease in male and male-female combined fetuses and an increase in placental weights of male, female and male-female combined in the test item, Benzyl Acetone group (G2 - 1000 mg/kg b.wt.) did not correlate with any other parameters/observations. Therefore, these findings have been considered as spontaneous/ incidental findings unrelated with the treatment.

There were no maternal-fetal abnormalities noticed in any of the pups/dam/placenta in the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.).

External evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some external abnormalities in the male and female pups at comparable incidence. There were no treatment related external abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group.

Visceral, head-razor and skeletal evaluation of pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) and vehicle control (G1 – 0 mg/kg b.wt.) groups showed some abnormalities in the male and female pups at comparable incidence. There were no treatment related visceral, head-razor and skeletal abnormalities noticed in the pups from the test item, Benzyl Acetone (G2 - 1000 mg/kg b.wt.) group.