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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 04 November 2009 and 18 March 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Cyclabute

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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

Sex:

male/female

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:

Vehicle:

arachis oil

Details on exposure:

Method of administration:

Oral 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

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)

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
No data

- Further matings after two unsuccessful attempts:
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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were sampled and analysis showed stability for at least twenty-one days. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark.


See attached appendix 15 - Chemical Analysis of Test Material Formulations, Methods and Results

Duration of treatment / exposure:

Oral administration of the test substance to all rats for a period of up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Dosing regime: 7 days/week

Details on study schedule:

Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of four days.

Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli was performed.

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

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

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

- Cage side observations checked in table [2] please see attached tables

DETAILED CLINICAL OBSERVATIONS: Yes see above
- Time schedule: As above

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Terminal bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY:
- Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:
Water intake was observed daily by visual inspection of water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: No data:
Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable


HAEMATOLOGY: Yes
Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable

URINALYSIS: No
- Time schedule for collection of urine: Not applicable
- Metabolism cages used for collection of urine: Not applicable
- Animals fasted: Not applicable

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:testis During histopathology, the male epididymides were examined for spermatocoel granuloma formation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE

- Male animals:
Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.

- Maternal animals:
Adult surviving females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY / ORGAN WEIGHTS

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each born was recorded. This procedurewas enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnantfemales were counted at necropsy. All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY

The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.

Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated.

Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi)(lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammarygland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), mid thoracic and lumbar, spleen, stomach, thyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.

The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to Harlan Laboratories Ltd, Switzerland (Principal Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control, 150 and 500 mg/kg/day were also processed. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney and spleen from five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 14 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following statistical procedures were used for males and females during the pre-mating phase, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring 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 also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100
Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and
parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of
mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of pregnant females ÷ Number of females delivering live offspring) x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY.

There were no unscheduled deaths

CLINICAL SIGNS (PARENTAL ANIMALS)

No clinically observable signs of toxicity were detected.

Episodes of increased salivation were evident in males from all treatment groups and in females treated with 1000 and 300 mg/kg/day throughout the treatment period. Three males treated with 300 mg/kg/day also showed instances of a red/brown stained snout or mouth. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test material formulation and in isolation are considered not to be of toxicological importance. One female treated with 300 mg/kg/day had generalised fur loss between Days 44 and 46. An observation of this nature is commonly observed during the lactation phase and is considered not to be toxicologically significant. One male treated with 100 mg/kg/day had a mass from Day 36 onwards, was lethargic on Days 33 and 34 and had hunched posture between Days 33 and 39. A female from this treatment also had a mass from Day 33 onwards. In the absence of a true dose related response these observations were not considered to be related to test material toxicity.

BODY WEIGHT (PARENTAL ANIMALS)

Males - No toxicologically significant effect on bodyweight development was detected. Males treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in bodyweight gain during Week 3. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.

Females - No adverse effect on bodyweight development was detected.

Statistical analysis did not reveal any significant intergroup differences.

FOOD CONSUMPTION (PARENTAL ANIMALS)

Males - No adverse effect on food consumption or food efficiency was detected in treated males throughout the treatment period.

Females - No adverse effect on food consumption or food efficiency was detected in treated females throughout the two week maturation period or during the gestation or lactation phases of the study.

Reproductive Performance

Mating

There were no treatment related effects on mating performance. The distribution of precoital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity). One female treated with 100 mg/kg/day did not show any positive evidence of mating but subsequently gave birth to live offspring.

Fertility

There were no treatment-related effects detected in conception rates.
One female treated with 300 mg/kg/day was non pregnant.

Gestation Length

There were no significant intergroup differences in gestation lengths and no effects upon parturition for treated females, with all of the females giving birth following 22 to 24 days of gestation. The distribution for treated females was comparable to controls.

Litter Response

In total all control females, all females from the 100 mg/kg/day dose group and nine females from the 300 and 1000 mg/kg/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 1000 mg/kg/day had a total litter loss on Day 2 post partum.

The following assessment of litter response is based on litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size and Viability

The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg/day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls. Sex ratio (percent of males) at birth, on Day 1 and on Day 4 was also unaffected.
Litter from females treated with 300 mg/kg/day did show a statistically significant reduction in the percent of males at birth, Day 1 and Day 4. In the absence of a similar effect in litters from females treated with 1000 mg/kg/day the intergroup difference was considered not to be of toxicological importance.

Offspring Growth and Development

Mean litter weights and bodyweight gains were considered to have been unaffected by maternal exposure. The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of agewere unaffected by maternal exposure.

ORGAN WEIGHTS (PARENTAL ANIMALS)

There were no treatment-related effects detected in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)

Offspring - Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

Adults - No toxicologically significant macroscopic abnormalities were detected.

One male and one female treated with 100 mg/kg/day had a mass observed at necropsy. In the absence of any histology correlates or similar effectspresent in 300 or 1000 mg/kg/day animals the intergroup differences were considered not to be of toxicological importance. One control male had a small right testis (left testis not present) and left epididymis at necropsy. A female from the control group showed the liver protruding through the diaphragm. In view of the fact these macroscopic observations were present in control animals only they were considered unrelated to treatment. One female treated with 300 mg/kg/day had generalised fur loss at necropsy. Observations of this nature are commonly observed following lactation and are considered not to be toxicologically significant. One male treated with 1000 mg/kg/day had a mottled appearance of the kidneys at necropsy. In the absence of any histopathological correlates the intergroup difference was considered of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)

No treatment-related microscopic observations were detected.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive and development toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)

The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 100, 300 or 1000 mg/kg/day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls. Sex ratio (percent of males) at birth, on Day 1 and on Day 4 was also unaffected.

Litter from females treated with 300 mg/kg/day did show a statistically significant reduction in the percent of males at birth, Day 1 and Day 4. In the absence of a similar effect in litters from females treated with 1000 mg/kg/day the intergroup difference was considered not to be of toxicological importance.

CLINICAL SIGNS (OFFSPRING)

The incidence and distribution of clinical signs in the offspring to termination on Day 5 of age were unaffected by maternal exposure

BODY WEIGHT (OFFSPRING)

Bodyweight gains were considered to have been unaffected by maternal exposure.

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

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 of100, 300 and 1000 mg/kg/day did not result in any treatment-related reproductive effects. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg/day.

Executive summary:

Introduction: The study was performed to screen 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).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

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 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results and conclusion: 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 Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be >=1000 mg/kg/day.