α,α-dimethylphenethyl butyrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Prenatal developmental toxicity study

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:

structural analogue

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Remarks:

Prenatal developmental toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

The required quantity of test item, Dimethyl Benzyl Carbinyl Acetate was weighed and mixed with 0.1% Tween 80 in water v/v in (G2- 1000 mg/kg body weight and 0.1% Tween 80 in water v/v vehicle control G1- 0 mg/kg body weight. The prepared dose formulation was used for 7 days based on the stability data .The vehicle control (G1-0 0 mg/kg body weight.) and test item (G2- 1000 mg/kg body weight, were administered to pregnant animals daily for GD 5 till GD 19 days by oral gavage once daily using calibrated syringe.. A constant dose volume of 10 mL/kg body weight was utilized to deliver the required amount of dose throughout the treatment period.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total number of 80 (30 male and 50 female) Sprague Dawley rats of about 12 to 13 weeks were received along with their health certificate for the Study.
Rats were housed in an experimental room maintained at 20.1 to 23.6 °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 were maintained throughout the experimental phase.

Route of administration:

oral: gavage

Vehicle:

other: 0.1% Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate) in water

Remarks:

As the test item, Dimethyl benzyl carbinyl acetate is soluble in 0.1% Tween 80 in water v/v which is also one of the most common vehicles used toxicity studies.

Details on exposure:

The vehicle (G1 - 0.1% Tween 80) and the test item (G2 – Dimethyl Benzyl Carbinyl Acetate) were administered to pregnant females using disposal syringes from a calibrated batch and reusable stainless steel ball tipped intubation needle (16 to 18 gauge) as specified in Section 6.7.11 by oral (gavage).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of Samples for Dose Concentration analysis on 27.04.2020:
An aliquot of 1000 μl was pipetted from each of the submitted samples (i.e., Top, Middle and Bottom layers), transferred separately into 100 ml volumetric flasks and the content was diluted up to mark using Acetonitrile and mixed thoroughly. From each of the above solutions 1000 μL aliquot was pipetted, transferred to the 10 mL of volumetric flask and diluted up to the mark using acetonitrile and filtered through 0.22 μm Nylon Syringe Filter to obtain samples for injection onto HPLC using the conditions described in section 3.1. All the samples were marked in terms of the respective layers from which they were prepared by the above mentioned procedure, vehicle also followed same procedure.
Preparation of Samples for Dose Concentration analysis on 21.05.2020:
An aliquot of 1500 μl was pipetted from each of the submitted samples (i.e., Top, Middle and Bottom layers), transferred separately into 100 ml volumetric flasks and the content was diluted up to mark using Acetonitrile and mixed thoroughly. From each of the above solutions 500 μL aliquot was pipetted, transferred to the 20 mL of volumetric flask and diluted up to the mark using acetonitrile and filtered through 0.22 μm Nylon Syringe Filter to obtain samples for injection onto HPLC using the conditions described in section 3.1. All the samples were marked in terms of the respective layers from which they were prepared by the above mentioned procedure, vehicle also followed same procedure.
The conditions in section 3.1:
Chromatographic Conditions
Dimethyl Benzyl Carbinyl Butyrate
Instrument: Agilent 1260 HPLC
HPLC Column: Waters Exterra , C18 - 4.6 mm x 250, 5 micron
Mobile phase (A): Water
Mobile phase (B): Acetonitrile
Mobile phase Ratio: A:B = 20:80 % v/v
Stop time: 12 min
Injection Volume: 10.0 μL
Column Oven Temperature: 25.0°C
Flow Rate: 1.0 mL/min
Wavelength: 210 nm
Signal BW: 4 nm
Auto Sampler Cooler Temperature: 22°C
Retention Time: ~ 4.4 minutes
Conclusion:
This method is applicable to determine the Dimethyl Benzyl Carbinyl Acetateat dose verification samples. The results Evaluated from the method validation parameters namely System suitability, specificity, linearity, Assay accuracy and precision data showed this method was acceptable for the determination of Dimethyl Benzyl Carbinyl Acetate in dose verification samples using HPLC conditions
Mean Recovery (%) of Dimethyl Benzyl Carbinyl Acetate was within 100 ± 15% % of its nominal concentration or theoretical concentration of individual layers (top, middle and bottom) in the dose formulation samples analyzed with relative standard deviation ≤10% (RSD). Hence the concentration claimed in the dose formulation samples was supported and it was found homogeneous in nature.

Details on mating procedure:

After completion of acclimatization period, females were housed with males for pairing or cohabitation based on 1:1 ratio (one male: one female) for 14 days or until the evidence of mating, whichever is 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. Sufficient number of confirmed mated females are achieved within 11 days.
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.
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 two groups viz. 01 to 25 for G1 (Vehicle Control 0- mg/kg body weight), 26 to 50 for G2 (Limit dose -1000 mg/kg body weight.The details of permanent numbers allotment into the specific group was maintained in the raw data and reported in the study report.

Frequency of treatment:

The vehicle (G1 - 0.1% Tween 80) and the test item, Dimethyl Benzyl Carbinyl Acetate (G2: 1000 mg/kg body weight) was administered orally gavage once daily to the pregnant females starting from Gestation Day 5 (GD 5) to till 19 (GD 19) of the respective females.

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

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.

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

A limit dose of 1000 mg/kg body weight has been selected based on the results of a dose range finding study in pregnant females (Study No. 19-598-G).

No. of animals per sex per dose:

An outline of the Study design is presented in the following table:
Treatment Details 0.1% Tween 80 (Vehicle Control) Dimethyl Benzyl Carbinyl Acetate (Limit Dose)
Groups G1 G2
Dose Level Control Limit Dose
Dose (mg/kg body weight) 0 1000
Female 1to 25 26 to 50
Male 30 (No Treatment)

Control animals:

yes

Details on study design:

An outline of the Study design is presented in the following table:
Treatment Details 0.1% Tween 80 (Vehicle Control) Dimethyl Benzyl Carbinyl Acetate (Limit Dose)
Groups G1 G2
Dose Level Control Limit Dose
Dose (mg/kg body weight) 0 1000
Female 1to 25 26 to 50
Male 30 (No Treatment)

Maternal examinations:

Post necropsy and gross pathology examination, following 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 sulphide staining of the uterus were performed to identify the pre-implantation loss of the embryos.

Ovaries and uterine content:

From each dam, half number of fetuses were subjected for evaluation of soft tissue alterations (visceral examination), 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 and head razor examination (Example, for a dam having 7 fetuses, fetus number 1, 3, 5 and 7 was processed for skeletal evaluation and fetus number 2, 4 and 6 was evaluated for visceral and head razor examination). However, if any fetus scheduled for visceral evaluation shows an abnormality that can best be confirmed and/or verified by skeletal examination and vice versa, the particular fetus were interchanged and subjected to particular type of evaluation. The details of such alterations were documented and reported in the study report

Fetal examinations:

Post uterine observations (as per Section 7.5.4), each fetus were identified individually as per in house procedure and placed along with its placenta in acrylic fetal trays or container filled with normal saline. Following observations were performed on each fetus:
 Determination of sex
 Weight of individual fetus
 External abnormalities if any
 The anogenital distance (AGD) was measured in all live fetuses from each dam.

Statistics:

The following list of data (but not limited to) was 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
 Thyroid Hormones analysis
Data was 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 was submitted to One-way Analysis of Variance (ANOVA) followed by Dunnett’s test (Dunnett, 1955; Dunnett, 1964) for post hoc comparison. If the data is heterogeneous, it will be subjected to Kruskal-Wallis test (Kruskal and Wallis, 1952) and the Multiple Fisher non-parametric tests (Agresti, 1992). Statistical significance was reported at the alpha level of 0.05. Data was reported in terms of number of observations (N), group means and standard deviations (%, where applicable).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs noticed in females of vehicle control (G1- 0
mg/kg body weight) and limit test Dose- G2 1000 mg/kg body weight throughout the
experiment period.

Mortality:

no mortality observed

Description (incidence):

There were no mortality and morbidity in the present study in vehicle control G1 and limit
test Dose- G2 1000 mg/kg body weight.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related statistical significant changes noticed in the body weights,
body weight gain and feed consumption on female from limit dose G2 group animals when
compared with G1 group animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistical significant changes were observed in female of G2 group when compared
with G1 group females.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

On completion of the gross pathology examination, no abnormalities were observed in any
of the organs in gross pathology examination and no organs were collected and preserved.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination of none of the terminally euthanized female animals belonging to the
control (G1) and limit dose groups G2) did not revealed any external and internal
abnormalities, during gross pathology observation.

Neuropathological findings:

no effects observed

Description (incidence and severity):

External examination of none of the terminally euthanized female animals belonging to the
control (G1) and limit dose groups G2) did not revealed any external and internal
abnormalities, during gross pathology observation.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Evaluation of maternal parameters viz: gravid uteri, total implantation, live fetus, number of corpora lutea, implantation sites, early and late resorptions, pre implantation loss, did not show any statistically significant difference in the treated groups as compared with vehicle control.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Description (incidence and severity):

Total placenta weight, did not show any statistically significant difference in the treated groups as compared with vehicle control. Whereas, statistical significance decrease in male and female combined fetuses weight, male fetuses weight and female fetus weight in G2 and increase in anogenital distance (AGD) in male and female pups in G2 group as compared with vehicle control G1. Although statistically significant, above these parameters did not correlate with any other parameters/observations. Therefore, these significances have been considered as spontaneous/ incidental findings unrelated with the test item treatment as compared to control group G1.

Details on embryotoxic / teratogenic effects:

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

no

Group G1 G2

Dose (mg/kg b.wt.) 0 1000

Number of Females 25 25

Number of Mated Females 25 25

Number of Pregnant Females 24 23

Average Weight of Gravid Uteri (Mean ± SD) 48.2616 ± 6.9434 45.6832 ± 12.7079

Corpora Lutea (Total Number) 285 268

Corpora Lutea (Mean ± SD) 11.88± 2.01 11.65± 1.75

Implantations / Implantation Sites (Total Number) 226 210

Implantations / Implantation Sites (Mean ± SD) 9.42±1.53 9.48 ± 2.48

Number of Fetuses (Total) 222 205

Average Number of Male and Female Fetuses / Dam (Mean ± SD) 5.31 ± 2.91 5.47 ± 3.13

Average Number of Male Fetuses / Dam (Mean ± SD) 5.46 ± 2.78 5.28 ± 3.17

Average Number of Female Fetuses / Dam (Mean ± SD) 5.07 ± 3.05 5.79 ± 3.19

Number of Live Fetuses (Total) 222 205

Average Number of Live Fetuses / Dam (Mean ± SD) 9.25± 1.29 8.91 ± 2.61

Number of Dead Fetuses (Total) 0 0

Average Number of Dead Fetuses / Dam (Mean ± SD) 0.00 ± 0.00 0.00 ± 0.00

Pre-implantation Loss (Mean ± SD) 2.46 ± 1.28 2.17 ± 1.67

Pre-implantation Loss (%) 20.05 ± 9.03 19.32 ± 17.02

Post-implantation Loss (Mean ± SD) 0.17 ± 0.48 0.57 ± 0.95

Post-implantation Loss (%) 1.40 ± 3.95 8.87± 21.52

Total implantation Loss (Mean ± SD) 2.63 ± 1.35 2.74 ± 1.84

Total implantation Loss (%) 21.23 ±9.09 24.44 ± 20.37

Early Resorptions (Total) 4 8

Early Resorptions (Mean ± SD) 0.17 ± 0.48 0.57 ± 0.95

Early Resorptions (%) 1.77 3.81

Late Resorptions (Total) 0 0

Late Resorptions (Mean ± SD) 0.00 ± 0.00 0.00 ± 0.00

Late Resorptions (%) 0.00 ± 0.00 0.00 ± 0.00

Average Weight (in g) of Fetuses (Male & Female) 3.4575± 0.3087 3.2885↓ ± 0.3239

Average Weight (in g) of Male Fetuses (Mean ± SD) 3.5312 ± 0.3079 3.3474 ↓ ± 0.3323

Average Weight (in g) of Female Fetuses (Mean ± SD) 3.3783 ± 0.2906 3.2340 ↓± 0.3076

Average placental Weight of Male and Female Fetuses (Mean ± SD) 0.5390 ± 0.0978 0.5555 ± 0.1060

Average placental Weight of Male Fetuses (Mean ± SD) 0.5426 ± 0.9992 0.5585 ± 0.1065

Average placental Weight of Female Fetuses (Mean ± SD) 0.5351 ± 0.0965 0.5528 ± 0.1059

Average of anogenital distance (AGD) Male+ Female Fetuses (Mean ± SD) 2.86 ±0.87 2.95 ±0.94

Average of anogenital distance (AGD) Male Fetuses (Mean ± SD) 3.60 ±0.46 3.81↑ ±0.52

Average of anogenital distance (AGD) Female Fetuses (Mean ± SD) 2.08 ±0.39 2.15↑ ±0.94

Key: Values are Mean ± SD, ↑ significantly (p<0.05) higher than control (G1) group. ↓significantly (p<0.05) lower than control (G1) group.

Conclusions:

To conclude, based on overall observations and under the circumstances of the present study, the test item, Dimethyl Benzyl Carbinyl Acetate when administered at 0, and 1000 mg/kg body weight by oral gavage route in Sprague Dawley rats for (GD 5-19) days did not produce any treatment related effects on the body weights or body weight gain/loss, feed consumption and fetal abnormalities. There were no treatment related clinical signs noticed in females all the dose levels. Based on the above observations, the dose of 1000 mg/kg body weight has been considered as No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo fetal toxicity of the test item, Dimethyl Benzyl Carbinyl Acetate in Sprague Dawley rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

α,α-dimethylphenethyl acetate

EC Number:

205-781-3

EC Name:

α,α-dimethylphenethyl acetate

Cas Number:

151-05-3

Molecular formula:

C12H16O2

IUPAC Name:

1,1-dimethyl-2-phenylethyl acetate

Test material form:

solid

Specific details on test material used for the study:

The required quantity of test item, Dimethyl Benzyl Carbinyl Acetate was weighed and mixed with 0.1% Tween 80 in water v/v in (G2- 1000 mg/kg body weight and 0.1% Tween 80 in water v/v vehicle control G1- 0 mg/kg body weight. The prepared dose formulation was used for 7 days based on the stability data .The vehicle control (G1-0 0 mg/kg body weight.) and test item (G2- 1000 mg/kg body weight, were administered to pregnant animals daily for GD 5 till GD 19 days by oral gavage once daily using calibrated syringe.. A constant dose volume of 10 mL/kg body weight was utilized to deliver the required amount of dose throughout the treatment period.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total number of 80 (30 male and 50 female) Sprague Dawley rats of about 12 to 13 weeks were received along with their health certificate for the Study.
Rats were housed in an experimental room maintained at 20.1 to 23.6 °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 were maintained throughout the experimental phase.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.1% Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate) in water

Remarks:

As the test item, Dimethyl benzyl carbinyl acetate is soluble in 0.1% Tween 80 in water v/v which is also one of the most common vehicles used toxicity studies.

Details on exposure:

The vehicle (G1 - 0.1% Tween 80) and the test item (G2 – Dimethyl Benzyl Carbinyl Acetate) were administered to pregnant females using disposal syringes from a calibrated batch and reusable stainless steel ball tipped intubation needle (16 to 18 gauge) as specified in Section 6.7.11 by oral (gavage).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of Samples for Dose Concentration analysis on 27.04.2020:
An aliquot of 1000 μl was pipetted from each of the submitted samples (i.e., Top, Middle and Bottom layers), transferred separately into 100 ml volumetric flasks and the content was diluted up to mark using Acetonitrile and mixed thoroughly. From each of the above solutions 1000 μL aliquot was pipetted, transferred to the 10 mL of volumetric flask and diluted up to the mark using acetonitrile and filtered through 0.22 μm Nylon Syringe Filter to obtain samples for injection onto HPLC using the conditions described in section 3.1. All the samples were marked in terms of the respective layers from which they were prepared by the above mentioned procedure, vehicle also followed same procedure.
Preparation of Samples for Dose Concentration analysis on 21.05.2020:
An aliquot of 1500 μl was pipetted from each of the submitted samples (i.e., Top, Middle and Bottom layers), transferred separately into 100 ml volumetric flasks and the content was diluted up to mark using Acetonitrile and mixed thoroughly. From each of the above solutions 500 μL aliquot was pipetted, transferred to the 20 mL of volumetric flask and diluted up to the mark using acetonitrile and filtered through 0.22 μm Nylon Syringe Filter to obtain samples for injection onto HPLC using the conditions described in section 3.1. All the samples were marked in terms of the respective layers from which they were prepared by the above mentioned procedure, vehicle also followed same procedure.
The conditions in section 3.1:
Chromatographic Conditions
Dimethyl Benzyl Carbinyl Butyrate
Instrument: Agilent 1260 HPLC
HPLC Column: Waters Exterra , C18 - 4.6 mm x 250, 5 micron
Mobile phase (A): Water
Mobile phase (B): Acetonitrile
Mobile phase Ratio: A:B = 20:80 % v/v
Stop time: 12 min
Injection Volume: 10.0 μL
Column Oven Temperature: 25.0°C
Flow Rate: 1.0 mL/min
Wavelength: 210 nm
Signal BW: 4 nm
Auto Sampler Cooler Temperature: 22°C
Retention Time: ~ 4.4 minutes
Conclusion:
This method is applicable to determine the Dimethyl Benzyl Carbinyl Acetateat dose verification samples. The results Evaluated from the method validation parameters namely System suitability, specificity, linearity, Assay accuracy and precision data showed this method was acceptable for the determination of Dimethyl Benzyl Carbinyl Acetate in dose verification samples using HPLC conditions
Mean Recovery (%) of Dimethyl Benzyl Carbinyl Acetate was within 100 ± 15% % of its nominal concentration or theoretical concentration of individual layers (top, middle and bottom) in the dose formulation samples analyzed with relative standard deviation ≤10% (RSD). Hence the concentration claimed in the dose formulation samples was supported and it was found homogeneous in nature.

Details on mating procedure:

After completion of acclimatization period, females were housed with males for pairing or cohabitation based on 1:1 ratio (one male: one female) for 14 days or until the evidence of mating, whichever is 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. Sufficient number of confirmed mated females are achieved within 11 days.
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.
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 two groups viz. 01 to 25 for G1 (Vehicle Control 0- mg/kg body weight), 26 to 50 for G2 (Limit dose -1000 mg/kg body weight.The details of permanent numbers allotment into the specific group was maintained in the raw data and reported in the study report.

Frequency of treatment:

The vehicle (G1 - 0.1% Tween 80) and the test item, Dimethyl Benzyl Carbinyl Acetate (G2: 1000 mg/kg body weight) was administered orally gavage once daily to the pregnant females starting from Gestation Day 5 (GD 5) to till 19 (GD 19) of the respective females.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

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.

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

A limit dose of 1000 mg/kg body weight has been selected based on the results of a dose range finding study in pregnant females (Study No. 19-598-G).

No. of animals per sex per dose:

An outline of the Study design is presented in the following table:
Treatment Details 0.1% Tween 80 (Vehicle Control) Dimethyl Benzyl Carbinyl Acetate (Limit Dose)
Groups G1 G2
Dose Level Control Limit Dose
Dose (mg/kg body weight) 0 1000
Female 1to 25 26 to 50
Male 30 (No Treatment)

Control animals:

yes

Details on study design:

An outline of the Study design is presented in the following table:
Treatment Details 0.1% Tween 80 (Vehicle Control) Dimethyl Benzyl Carbinyl Acetate (Limit Dose)
Groups G1 G2
Dose Level Control Limit Dose
Dose (mg/kg body weight) 0 1000
Female 1to 25 26 to 50
Male 30 (No Treatment)

Examinations

Maternal examinations:

Post necropsy and gross pathology examination, following 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 sulphide staining of the uterus were performed to identify the pre-implantation loss of the embryos.

Ovaries and uterine content:

From each dam, half number of fetuses were subjected for evaluation of soft tissue alterations (visceral examination), 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 and head razor examination (Example, for a dam having 7 fetuses, fetus number 1, 3, 5 and 7 was processed for skeletal evaluation and fetus number 2, 4 and 6 was evaluated for visceral and head razor examination). However, if any fetus scheduled for visceral evaluation shows an abnormality that can best be confirmed and/or verified by skeletal examination and vice versa, the particular fetus were interchanged and subjected to particular type of evaluation. The details of such alterations were documented and reported in the study report

Fetal examinations:

Post uterine observations (as per Section 7.5.4), each fetus were identified individually as per in house procedure and placed along with its placenta in acrylic fetal trays or container filled with normal saline. Following observations were performed on each fetus:
 Determination of sex
 Weight of individual fetus
 External abnormalities if any
 The anogenital distance (AGD) was measured in all live fetuses from each dam.

Statistics:

The following list of data (but not limited to) was 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
 Thyroid Hormones analysis
Data was 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 was submitted to One-way Analysis of Variance (ANOVA) followed by Dunnett’s test (Dunnett, 1955; Dunnett, 1964) for post hoc comparison. If the data is heterogeneous, it will be subjected to Kruskal-Wallis test (Kruskal and Wallis, 1952) and the Multiple Fisher non-parametric tests (Agresti, 1992). Statistical significance was reported at the alpha level of 0.05. Data was reported in terms of number of observations (N), group means and standard deviations (%, where applicable).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs noticed in females of vehicle control (G1- 0
mg/kg body weight) and limit test Dose- G2 1000 mg/kg body weight throughout the
experiment period.

Mortality:

no mortality observed

Description (incidence):

There were no mortality and morbidity in the present study in vehicle control G1 and limit
test Dose- G2 1000 mg/kg body weight.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related statistical significant changes noticed in the body weights,
body weight gain and feed consumption on female from limit dose G2 group animals when
compared with G1 group animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistical significant changes were observed in female of G2 group when compared
with G1 group females.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

On completion of the gross pathology examination, no abnormalities were observed in any
of the organs in gross pathology examination and no organs were collected and preserved.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination of none of the terminally euthanized female animals belonging to the
control (G1) and limit dose groups G2) did not revealed any external and internal
abnormalities, during gross pathology observation.

Neuropathological findings:

no effects observed

Description (incidence and severity):

External examination of none of the terminally euthanized female animals belonging to the
control (G1) and limit dose groups G2) did not revealed any external and internal
abnormalities, during gross pathology observation.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Evaluation of maternal parameters viz: gravid uteri, total implantation, live fetus, number of corpora lutea, implantation sites, early and late resorptions, pre implantation loss, did not show any statistically significant difference in the treated groups as compared with vehicle control.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

Results (fetuses)

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Description (incidence and severity):

Total placenta weight, did not show any statistically significant difference in the treated groups as compared with vehicle control. Whereas, statistical significance decrease in male and female combined fetuses weight, male fetuses weight and female fetus weight in G2 and increase in anogenital distance (AGD) in male and female pups in G2 group as compared with vehicle control G1. Although statistically significant, above these parameters did not correlate with any other parameters/observations. Therefore, these significances have been considered as spontaneous/ incidental findings unrelated with the test item treatment as compared to control group G1.

Details on embryotoxic / teratogenic effects:

There was no treatment related external, visceral, head- razor and skeletal abnormalities in the pups from the test item, Dimethyl Benzyl Carbinyl Acetate in control and treated groups. The abnormalities showed in the male and female pups were incidental and comparable.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Group G1 G2

Dose (mg/kg b.wt.) 0 1000

Number of Females 25 25

Number of Mated Females 25 25

Number of Pregnant Females 24 23

Average Weight of Gravid Uteri (Mean ± SD) 48.2616 ± 6.9434 45.6832 ± 12.7079

Corpora Lutea (Total Number) 285 268

Corpora Lutea (Mean ± SD) 11.88± 2.01 11.65± 1.75

Implantations / Implantation Sites (Total Number) 226 210

Implantations / Implantation Sites (Mean ± SD) 9.42±1.53 9.48 ± 2.48

Number of Fetuses (Total) 222 205

Average Number of Male and Female Fetuses / Dam (Mean ± SD) 5.31 ± 2.91 5.47 ± 3.13

Average Number of Male Fetuses / Dam (Mean ± SD) 5.46 ± 2.78 5.28 ± 3.17

Average Number of Female Fetuses / Dam (Mean ± SD) 5.07 ± 3.05 5.79 ± 3.19

Number of Live Fetuses (Total) 222 205

Average Number of Live Fetuses / Dam (Mean ± SD) 9.25± 1.29 8.91 ± 2.61

Number of Dead Fetuses (Total) 0 0

Average Number of Dead Fetuses / Dam (Mean ± SD) 0.00 ± 0.00 0.00 ± 0.00

Pre-implantation Loss (Mean ± SD) 2.46 ± 1.28 2.17 ± 1.67

Pre-implantation Loss (%) 20.05 ± 9.03 19.32 ± 17.02

Post-implantation Loss (Mean ± SD) 0.17 ± 0.48 0.57 ± 0.95

Post-implantation Loss (%) 1.40 ± 3.95 8.87± 21.52

Total implantation Loss (Mean ± SD) 2.63 ± 1.35 2.74 ± 1.84

Total implantation Loss (%) 21.23 ±9.09 24.44 ± 20.37

Early Resorptions (Total) 4 8

Early Resorptions (Mean ± SD) 0.17 ± 0.48 0.57 ± 0.95

Early Resorptions (%) 1.77 3.81

Late Resorptions (Total) 0 0

Late Resorptions (Mean ± SD) 0.00 ± 0.00 0.00 ± 0.00

Late Resorptions (%) 0.00 ± 0.00 0.00 ± 0.00

Average Weight (in g) of Fetuses (Male & Female) 3.4575± 0.3087 3.2885↓ ± 0.3239

Average Weight (in g) of Male Fetuses (Mean ± SD) 3.5312 ± 0.3079 3.3474 ↓ ± 0.3323

Average Weight (in g) of Female Fetuses (Mean ± SD) 3.3783 ± 0.2906 3.2340 ↓± 0.3076

Average placental Weight of Male and Female Fetuses (Mean ± SD) 0.5390 ± 0.0978 0.5555 ± 0.1060

Average placental Weight of Male Fetuses (Mean ± SD) 0.5426 ± 0.9992 0.5585 ± 0.1065

Average placental Weight of Female Fetuses (Mean ± SD) 0.5351 ± 0.0965 0.5528 ± 0.1059

Average of anogenital distance (AGD) Male+ Female Fetuses (Mean ± SD) 2.86 ±0.87 2.95 ±0.94

Average of anogenital distance (AGD) Male Fetuses (Mean ± SD) 3.60 ±0.46 3.81↑ ±0.52

Average of anogenital distance (AGD) Female Fetuses (Mean ± SD) 2.08 ±0.39 2.15↑ ±0.94

Key: Values are Mean ± SD, ↑ significantly (p<0.05) higher than control (G1) group. ↓significantly (p<0.05) lower than control (G1) group.

Applicant's summary and conclusion

Conclusions:

To conclude, based on overall observations and under the circumstances of the present study, the test item, Dimethyl Benzyl Carbinyl Acetate when administered at 0, and 1000 mg/kg body weight by oral gavage route in Sprague Dawley rats for (GD 5-19) days did not produce any treatment related effects on the body weights or body weight gain/loss, feed consumption and fetal abnormalities. There were no treatment related clinical signs noticed in females all the dose levels. Based on the above observations, the dose of 1000 mg/kg body weight has been considered as No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo fetal toxicity of the test item, Dimethyl Benzyl Carbinyl Acetate in Sprague Dawley rats.