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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.8. - 26.12.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
Clinical chemistry: Plasma separation protocol adapted. The deviation has no impact on the outcome of the study or the interpretation of results
GLP compliance:
yes (incl. QA statement)
Remarks:
National Good Laboratory Practice Compliance Monitoring Authority, Government of India.
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cinnamyl acetate
EC Number:
203-121-9
EC Name:
Cinnamyl acetate
Cas Number:
103-54-8
Molecular formula:
C11H12O2
IUPAC Name:
cinnamyl acetate

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
HsdHan: WIST rats. Conventionally bred (In-house random bred)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Wistar rats, strain HsdHan, conventionally bred (In-house random bred)
- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bengaluru 560 058, India
- Age at treatment: 11-12 weeks
- Body weight range at the start of treatment: Males: 330 to 410 g; Females: 200 to 240 g
- Housing: in a single experimental room in a barriered area: pre mating: two rats of same sex/cage; during mating: two rats/cage (one male and one female); post mating: males were housed with their former cage mates, females were housed individually
- Cages: standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill; the sterilised nesting material (paper shreds) was provided near-term
- Bedding: Steam sterilized clean Corn cob
- Diet: ad libitum; Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet
- Fasting (water allowed): overnight berfore blood collection for clinical pathology
- Water: ad libitum; Deep bore-well water passed through activated charcoal filter, exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier
- Acclimatization: five days before start of the treatment

The food and water provided to the animals were tested for contaminants

IN-LIFE DATES:
- Duration of test: males: 2 weeks prior to mating, continued during the mating period and approximately two weeks post mating.
Females: 2 weeks prior to the mating period, continued through mating, pregnancy and up to lactation day 4
- Duration of test: offspring: from birth to lactation day 4

ENVIRONMENTAL CONDITIONS:
Rats were housed in an environment controlled room
- Temperature between 20 and 25°C
- Relative humidity between 59 and 68%
- Photoperiod was 12 hours light and 12 hours dark cycle
- Rate of air exchange: 12 - 15 air changes/hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Concentration in vehicle: at an equivolume of 5 mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
The dose volume was adjusted based on the most recent body weight of individual rat.
- Concentrations of test material in vehicle: 65, 200 and 600 mg/kg bw/day
- Justification for choice of vehicle: corn oil was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study

Determination of stability of the test item in the dosing formulations was carried out at 1 and 200 mg/mL concentrations.
Based on the results, the test item was found to be stable for 24 hours at ambient condition at concentrations of 1 and 200 mg/mL in the vehicle.
Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most for two weeks, until proof of pregnancy was confirmed
- Proof of successful mating: evidence of sperms in the vaginal smear and /or vaginal plug
The day of confirmed mating was designated as Gestation Day 0 (GD0). The pre-coital time was calculated for each female.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- For the test item concentration analysis, prepared formulations were sampled on Day 1 and during 2nd month of the treatment period.
The prepared formulation was sampled in duplicate sets and analysed In-house. For each set, 3 replicates from composite sample was drawn and analyzed for the test item concentrations.
For the control, two replicates from composite sample was drawn.

Dose formulations were considered acceptable as the overall mean results were within ± 10.0% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10.0%.

- Determination of stability of the test item in the dosing formulations was carried out at 1 and 200 mg/mL concentrations.

Based on the results, the test item was found to be stable for 24 hours at ambient condition at concentrations of 1 and 200 mg/mL in the vehicle (corn oil).
Duration of treatment / exposure:
- Males: for a minimum of four weeks, two weeks prior to mating and continued during the mating period and approximately two weeks post mating
- Females: throughout the treatment period, two weeks prior to the mating period and continued through mating, pregnancy and up to lactation day 4
- Offspring were not dosed
- In the control and high dose recovery groups, the treatment period was followed by a 14-day no treatment (recovery) period. The recovery period of the study was started from the day of sacrifice of the first littered animals.
Frequency of treatment:
- Males: once daily at approximately the same time each day.
- Females: once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehivle control
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Recovery group (vehicle control)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Recovery group (high dose)
No. of animals per sex per dose:
- Main groups : 10 males +10 females
- Recovery groups : 5 males + 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study (OECD 422 test guideline).

The test item was administered in graduated doses to three groups of male and female rats. The males were dosed for a minimum of four weeks, females were dosed throughout the treatment period.

Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days post treatment and these animals were not mated and consequently not used for assessment of reproduction/developmental toxicity.
Animals were sacrificed after completion of recovery period.
The recovery period of the study was started from the day of sacrifice of the first littered animals.

- Dose selection rationale:
The dose levels were selected based on the results of a repeated 2-week dose range-finding study in Wistar rats in which doses of 100, 300, 600 and 1000 mg/kg bw/day have been tested. Animals of the vehicle control were administered with the vehicle alone (corn oil).
In this range-finding study, no mortality or clinical signs were observed in any of the groups during the experimental period. Mean body weights, body weight gains and food consumption of treated groups were comparable to the vehicle control group. There were no test item-related changes in haematology and coagulation. Clinical chemistry analysis revealed a test item-related increase in AST and ALP in male rats at 600 and 1000 mg/kg bw/day. There were organ weight changes observed that were considered treatment-related: an increase of liver weights in males at 600 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day; a decrease in thymus weights in both sexes at 1000 mg/kg bw/day. Gross and histopathology did not reveal any test item-related effects.
Based on the results of this dose range-finding study, 600 mg/kg bw/day was selected as the high-dose, 200 mg/kg bw/day as the mid-dose and 65 mg/kg bw/day as the low-dose, respectively.




Positive control:
not included in the study

Examinations

Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical Signs and Mortality: all the rats were observed at least once daily (post-dose) for changes in appearance, behaviour and clinical/toxic signs and twice daily for morbidity and mortality
- Clinical Examination: at weekly intervals during treatment and recovery periods.
- Functional observation battery tests (FOB): for randomly selected 5 males and 5 females in each group at termination i.e. towards the end of the dosing period for males (shortly prior to their scheduled kill) and during lactation period for females (shortly before the scheduled kill).
For recovery groups, neurological examination was carried out towards the end of recovery period.
- Body Weight: individual body weights were recorded initially and at weekly intervals thereafter (till mating confirmation for females)
All dams were weighed on GD 0, 7, 14 and 20 and on lactation days 0 and 4
- Food consumption: at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 of lactation period.

Haematology:
- Haematological observations (at the end of the pre-mating period, randomly selected 5 males and 5 females of main group animals and at the end of recovery period all animals of recovery groups): red blood corpuscles, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, white blood corpuscles, differential leukocyte count (absolute), platelets

Clinical chemistry:
- Blood plasma chemical observations (at the end of the pre-mating period, randomly selected 5 males and 5 females of main group animals and at the end of recovery period all animals of recovery groups): alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, albumin/globulin ratio (calculated values), blood urea nitrogen, bile acids, calcium, chloride, creatinine, gamma glutamyl transferase, glucose, globulin (calculated values), inorganic phosphorous, potassium, sodium, total bilirubin, total cholesterol, total plasma protein, triglycerides

Sacrifices and pathology:
Terminal sacrifice:
- All rats were sacrificed at term after overnight fasting by anaesthesy with isoflurane, weighed and exsanguinated and subjected for detailed necropsy.
- All surviving pups were necropsied on lactation Day 4.
- Dead and moribund pups were examined for possible defects and/or cause of death.

Gross pathology: Yes

Weighing of organs: Yes

Histopathology:
- Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. The reproductive organs of infertile males and females across the groups were examined microscopically. The tissues were processed for routine paraffin embedding and 4-5 micron thickness sections were stained with Mayer's Haematoxylin Eosin stain. In addition, testes was sectioned at 3-4 µm and stained with PAS reagent and haematoxilin to aid in qualitative assessment of spermatogenesis.
Litter observations:
At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities was recorded.
The number of pups born (litter size), sex and individual pup body weight of male and female pups on days 0 and 4 post-partum were recorded.
The litters were observed daily in order to note the number of pups alive, dead and cannibalized pups.
Postmortem examinations (parental animals):
All the rats in this study were sacrificed at term after overnight fasting. These rats were anaesthetized with isoflurane, weighed and exsanguinated and subjected for detailed necropsy by a veterinary pathologist and findings were recorded.

For apparently non-pregnant rats, the uteri were stained with ammonium sulphide solution to identify the pre-implantation loss of the embryos.
The number of implantation sites and corpora lutea was recorded for all the dams.
Postmortem examinations (offspring):
All the surviving pups were necropsied on lactation Day 4 and findings were recorded.
Dead and moribund pups were examined for possible defects and/or cause of death.
Statistics:
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like body weight, food intake, haematology, clinical chemistry, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found significant.

All analyses and comparisons were evaluated at the 5% (P≤0.05) level.
Reproductive indices:
The male mating index (%), the male fertility index (%), the female mating index (%), the female fertility index (%), the mean number of corpora lutea (CL)/group, the mean number of implantations/group, the implantation index, the percentage of pre-implantation loss per group and the post implantation loss (%)were determined.
Offspring viability indices:
The mean litter size per group and the day 4 survival index (%) were determined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in any of the groups during the experimental period.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any of the groups during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights were not affected by the treatment at all the doses tested when compared to vehicle control in both sexes. In the high dose recovery group, the body weights were unaffected both during the treatment and recovery periods.

Maternal Body Weights and Food Intake during the Gestation Period:
Treatment had no effects on the body weights and food intake during different intervals of the gestation period at all the tested doses.

Maternal Body Weights and Food Intake during the Lactation Period:
Treatment had no effects on the body weights and food intake during different intervals of the lactation period at all the doses tested when compared to vehicle control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control.
In the high dose recovery group, the food consumption was not affected both during the treatment and recovery periods.
Treatment had no effects on the food intake during different intervals of the gestation period at all the tested doses.
Treatment had no effects on the food intake during different intervals of the lactation period at all the doses tested when compared to vehicle
control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The test item administration did not reveal any treatment related changes in the haematology and coagulation of both male and female rats.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both male and female rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Home cage and Handling observations: No treatment-related abnormalities were observed in all the tested dose groups in both sexes

- Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.

- Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.

- Motor Activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:

Males:
Lower : Stereotypic time at interval 3 at the high dose, horizontal counts at interval 3 at the high dose, ambulatory counts at interval 2 at the high dose in the main groups..
Higher : Ambulatory time at interval 1 at the mid dose, ambulatory counts at interval 1 at the mid dose.
The above observed statistical variations in the motor activity measurement is considered to be incidental as the observed changes did not follow the dose proportion and there were no changes observed in the home cage or open field observations.

In females no significant changes observed at all doses tested.

- Neuromuscular observation: landing hind limb footsplay: No significant changes were observed at all the tested doses in both sexes.

- Grip strength: no significant changes were observed at all the tested doses in both sexes.

- Physiological observation: body temperature: the physiological observation of body temperature was unaffected in both sexes.
However an incidence of significantly higher body temperature was observed at mid and high dose main group males.
This change was considered incidental as there were no clinical signs observed during daily observation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed at high dose group. histopathological evaluation was not carried out for lower and recovery dose groups.
All the alterations observed at 600 mg/kg bw/d were considered incidental as the changes were comparable to the concurrent control groups and/or commonly present in this age group of animals.

The reproductive organs of the male and female rats that failed to mate were examined microscopically. The cause of infertility for these animals could not be ascertained as no significant microscopic changes were observed in the reproductive organs examined.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on the mean pre-coital time and gestation length at all the tested doses.

The male and female mating and fertility indices were significantly lower at 65 and 600 mg/kg doses, when compared to vehicle control.

The changes observed at 65 mg/kg/day dose was considered incidental as the observed change was within the historical control data (HD range: male mating index 80 to 100%, female fertility index 80 to 100%).

However, the lower male and female mating and fertility indices at 600 mg/kg/day dose was considered treatment related as the changes were lower than the historical control data (HD range: male mating index 80 to 100%, female fertility index 80 to 100%).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related effects.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no treatment-related effects and the day 4 survival index was not altered by the treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male pups on Day 4 was significantly lower at the mid and high doses as compared to vehicle control.
This change was considered incidental as the mean weight of combined sex was not altered.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination of pups on lactation day 4 did not reveal any gross changes.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The “No Observed Adverse Effect Level (NOAEL)” for reproductive toxicity is considered to be 200 mg/kg bw/day.
Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test in Wistar Rats was performed in accordance to OECD guideline 422 and in compliance with GLP regulations.

The test item was suspended in corn oil and administered orally at the dose levels of 65, 200 and 600 mg/kg bw/day as low, mid and high dose/ high dose recovery group rats, respectively. A concurrent control and a control recovery group of rats received vehicle (corn oil) alone. The dose volume administered was 5 mL/kg body weight. The main groups consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The prepared dose formulations were administered once daily to specific group of rats prior to mating , during mating and post mating periods (for males), during pregnancy and up to lactation day 4 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14-day no treatment (recovery) period. The recovery period of the study was started from the day of sacrifice of the first littered animals.

The stability and homogeneity of test item in the vehicle was tested. Based on the results, the test item was found to be stable for up to 24 hours at 1 and 200 mg/mL concentrations when stored at room temperature. The dose formulations were analysed for the test item concentration on Day 1 and during month 2 of the treatment period. The results indicated that the analysed concentrations were within ± 10 % of variations from the nominal concentrations.

All animals were observed for clinical signs, physical abnormalities and mortality. The body weight and food consumption were measured at periodic intervals. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group.

For recovery groups the functional observation battery was performed prior to sacrifice.

Laboratory investigations such as haematology and clinical chemistry were performed in randomly selected 5 males and 5 females from each group at the end of the premating period for main groups and at the end of recovery period from all animals of recovery groups.

The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected.

Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes.

There were no test item-related changes observed at high dose group; hence, histopathological evaluation was not carried out for lower (65 and 200 mg/kg bw/day) and recovery dose groups. Gross lesion was examined microscopically. The reproductive organs of infertile males and females across the groups were examined microscopically.

Under the experimental conditions, the following results were obtained:

- Treatment had no effect on pre-coital time and gestation length at all the tested doses.

- No treatment-related changes were observed in the fertility indices of sires and dams at 65 and 200 mg/kg bw/day doses. At 600 mg/kg bw/day dose, the treatment resulted in significantly reduced male and female mating and fertility indices as compared to control group. The survival index was not altered by the treatment at all the doses tested.

No Observed Adverse Effect Level:

The “No Observed Adverse Effect Level (NOAEL)” for reproductive toxicity is considered to be 200 mg/kg bw/day.