Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

As in the Reproscreen study - OECD TG 421

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 6 May 2008 and 20 November 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols and GLP. Reliability 2 is assigned because the results from an analogue substance is used for read across.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

EC Number:

911-369-0

Molecular formula:

C12H16O2

IUPAC Name:

Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-5-yl acetate and 3a,4,5,6,7,7a-hexahydro-4,7-methanoinden-6-yl acetate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon

- Age at study initiation:
approximately twelve weeks old

- Weight at study initiation:
320 to 355g (male), 184 to 225g (female)

- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Diet:
A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK) was used (ad libitum).

- Water (e.g. ad libitum):
ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water was considered not to have any level of contaminant.

- Acclimation period:
twelve days

ENVIRONMENTAL CONDITIONS

- Temperature:
: (°C): 21 ± 2

- Humidity (%):
: 55 ± 15

- Air changes (per hr):
: At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
: 12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
From: Day1 To:Day 46

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

arachis oil

Details on exposure:

Method of administration:
Gavage
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP


DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not applicable.


VEHICLE
- Justification for use and choice of vehicle(if other than water): Most suitable

- Concentration in vehicle:
25, 75 and 250 mg/ml.

- Amount of vehicle (if gavage):
4 ml/kg bodyweight

- Lot/batch no. (if required):
Unknown

- Purity:
Unknown

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Lithium Nickel Cobalt Aluminium Oxide in the test material formulations was determined using a gravimetric technique. The test material formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test material residue. The samples were then dried in an oven at approximately 105oC before allowing to cool over silica gel in a desiccator and re-weighed.
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed within three days of preparation.
The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.

Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- Proof of pregnancy:
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)

- Further matings Not applicable:
No data

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable

Duration of treatment / exposure:

Animals were treated for at least 41 consecutive days.

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day (25 mg/ml)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day (75 mg/ml)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day (100 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of previous toxicity work (Harlan Project Number 1543-0230).
- Rationale for animal assignment (if not random):
Random

- Section schedule rationale (if not random):
Random

Examinations

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis,followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal- Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and
landmark developmental markers.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
In addition, histopathological findings were analysed using the following methods:

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:

1. Chi-squared analysis for differences in the incidence of lesions occurring with an
overall frequency of 1 or greater.

2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of
severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p≥0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs
indicate negative differences.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The oral administration of Cyclacet to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg/day did not result in any treatment-related reproductive effects.

Applicant's summary and conclusion

Conclusions:

The oral administration of Cyclacet to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg/day did not result in any treatment-related developmental effects.
The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be >=1000 mg/kg/day.

Executive summary:

Cyclacet was screened for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). In the present summary only the maternal and developmental toxicity will be discussed. The repeated dose and the reproductive toxicity is presented at the respective sections.

Methods: The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Clinical signs, bodyweight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of the tissues was performed.

Results: The oral administration of Cyclacet to rats by gavage, for a period of up to forty six consecutive days at dose levels of 100, 300 and 1000 mg/kg/day did not result in any treatment-related reproductive effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be >=1000 mg/kg/day.