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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2010 - october 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The Sponsor supplied all data on the test item.

- Name of test material (as cited in study report): 2-Norbornene (Bicyclo(2.2.1)hept-2-ene)
- Physical state: solid at room temperature
- Analytical purity: 99,06% (GC analysis)
- Batch No.: 119702
- Storage condition of test material: Refrigerated (5°C ± 3°C) in Argon atmosphere

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: males: 289 g; females: 186 g(means)

- Housing:
- Diet: ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 to 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: pre-mating phase To: termination

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

A stock solution containing 125 mg/L vehicle was prepared in a water bath (60°C).Two serial dilutions (1+1, v/v) were prepared from the high dose with equal amounts of vehicle. The solutions were stored refrigerated (5°C± 3°C) under agron until application.

VEHICLE
- Justification for use and choice of vehicle (if other than water): very low water solubility of the test substance
- Concentration in vehicle: 125, 52.5, and 31.25 g test substance/L
- Amount of vehicle (if gavage): dose volume was 4 mL/kg bw
- Supplier: Sigma
- Purity: 100%
Details on mating procedure:
During the mating phase (up to 14 days), the female were placed with the same male until indications for mating were detected. Each morning, the females were examined for the presence of sperm or a vaginal plug. Day 0 of pregnancy was defined as the day a vaginal plug or sperm was
found. Females showing no evidence of copulation were regrouped with proven sires for a second or third mating phase.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
61 ± 10.0 days (because a second and third mating was necessary; report, page 24/36)
Frequency of treatment:
7 per week
Details on study schedule:
Males were dosed daily for a minimum of four weeks, including the day before the scheduled termination of the in-life phase. This included a minimum of two weeks of dosing prior to mating and continued throughout the mating period until the study was terminated. Females were dosed daily, including two weeks prior to mating, covering at least two complete oestrous cycles, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation depended on the female performance: 14 days pre-mating, up to 14 days until mating, an average of 21 days
of gestation, and a minimum of 4 days of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250, 500 mg/kg bw and day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Males were dosed daily for a minimum of four weeks, including the day before the scheduled termination of the in-life phase. This included a minimum of two weeks of dosing prior to mating and continued throughout the mating period until the study was terminated.
Females were dosed two weeks prior to mating, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase. Females showing no evidence of copulation were re-grouped with proven sires for further mating phases. Dosing was continued in both sexes during the mating period.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before beginning of treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before beginning of treatment and at least weekly thereafter. Females: dyas 0, 7, 14, 20, within 24 h post parturition and 4 days post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No; not required.
Food consumption was determined per cage at least one weakly.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No; not required.
Food consumption was determined per cage twice weakly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: all
- Parameters checked in table No. 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 14
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: all
- Parameters checked in table No. 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: once before beginning of application and once during the last week of exposure.
- Dose groups that were examined: no data
- Battery of functions tested: sensory activity / grip strength (cf. report section 3.2.4; results Appendix, pages 14-15)
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in male parental control and high dose groups :
testis weight, epididymis weight, sperm count in epididymides
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain days 0 to 4
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

GROSS NECROPSY
- Gross necropsy consisted of


RTable: Parameters examined at necropsy

No. Tissue/organ Procedure1
1 Gross lesions P 12 Adrenal gland P, W
2 Oesophagus P 13 Urinary bladder P
3 Trachea and thyroid P 14 Testes P, W
4 Stomach P 15 Epididymides P, W
5 Thymus P, W 16 Prostate / uterus / ovary P
6 Liver P, W 17 Heart P, W
7 Spleen P, W 18 Lung P
8 Duodenum P 19 Peripheral nerve P
9 Jejunum P 20 Bone marrow P
10 Ileum (with Peyer’s
patches) P 21 Sternum P
11 Cecum P 22 Spinal cord P
12 Adrenal gland P, W
13 Urinary bladder P
14 Testes P, W
16 Prostate / uterus / ovary P
17 Heart P, W
18 Lung P
19 Peripheral nerve P
20 Bone marrow P
21 Sternum P
22 Spinal cord P
23 Colon P
24 Rectum P
25 Lymph nodes (intestinal area, axillary) P
26 Kidney P, W
27 Whole brain W
28 Cerebrum P
29 Cerebellum P
30 Pons P

1: P = preservation, W = weight determination


HISTOPATHOLOGY / ORGAN WEIGHTS
Cf. table above
Postmortem examinations (offspring):
SACRIFICE
Offspring were sacrificed at 4 days of age. These animals were subjected to external postmortem examinations.

Statistics:
Spreadsheet calculations were performed using Microsoft® Excel® 2004 for Mac, Version 11.5.8. In addition, the statistical software Graph Pad Prism for Mac, Version 5.01c was used to calculate detailed column statistics (minimum / maximum data, 75% percentiles, standard error, upper and lower confidence interval 95%).

The significance of differences between the vehicle only and treated groups was analysed using various methods which included Bartlett's test for variances, followed either by a One-Way ANOVA (Analysis of variance), followed by a Dunnett's t-test, or followed by a Bartlett’s test for equal variances and a Kruskall-Wallis test. See the report section 3.5.2 for a full description of the statistical methods used. A decision tree is depicted on page 33 of the Appendix.
Reproductive indices:
Not calculated
Offspring viability indices:
Not calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Tendency of slightly decreased body weights in male and female high dose groups.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyaline droplet formation was noted in the renal proximal tubular epithelium of almost all male rats from the high dose group, and to a lesser degree in most male rats of the low and intermediate dose groups. No effects on male and female reproduction or
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

cf. attached document

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no change compared to controls

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

cf. attached document

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
reproduction, viability
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related change compared to controls

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

All relevant raw data and calculated data were documented within the appendix.

During first mating (Tab. 5, attached document), evidence of copulation in total numbers was similar between all groups and was found within the first five days for all but one female of the low dose group. During second mating, no apparent differences occurred between the dose groups regarding the time point for evidence of copulation, which was within the first five days in all cases.

Pregnancy lasted 22 days (n=22) or 23 days (n=20) and no apparent differences occurred between the dose groups. No differences between the dose groups were detectable regarding the number of dams with live young born at day zero (d0, day of birth), the mean number of alive pups at day four (d4) of lactation, the mean numbers per dam of corpora lutea, the mean numbers per dam of implantations, as well as live pups. The mean litter mass at d0 and d4 were normal for Wistar rats. A statistically significant increase of corpora lutea was detected in dams of the medium dose group. This effect was estimated to be not test item related but within the normal biological range. Of the five stillborns found in total, two were delivered by dams of the low dose group and three by dams of the medium dose group (Tab. 6). No bias was observed in the sex-ratio data. In general, mean pup body mass of rats born in the course of the present study was within normal range for Wistar rats and no statistically significant differences were detected between the dose groups. Data of the numbers of abnormal pups born, or the loss of offspring (pre-implantation, pre-natal and post-natal loss) were normal for rats of this strain and age.

Applicant's summary and conclusion

Conclusions:
In a repeated dose/reproduction screening study, norbornene had no effect on parental and progeny reproduction parameters of male and female rats. The NOAEL for toxicity to reproduction was therefore 500 mg/kg bw and day, the highest tested dose.
Executive summary:

In a combined repeated dose/reproduction toxicity study (performed according to OECD TG 422 and under GLP conditions), male and female Wistar rats (12 per sex and dose) received norbornene at dose levels of 125, 250, and 500 mg/kg bw and day by oral gavage. Vehicle controls received corn oil only.

There were no mortalities or clinical signs of toxicity that were attributable to the test substance. The NOAEL for local and systemic toxicity was 500 mg/kg bw and day for both sexes in this study (cf. section 7.5.1).

 

Regarding reproduction parameters, norbornene treatment had no effect on the following parameters:

Duration of pregnancy; the number of dams with live pups; the mean number of alive pups at day zero and at day four post partum; the mean numbers per dam of corpora lutea, the mean numbers per dam of implantations, as well as live pups. The mean litter mass at day o and day 4 were normal for Wistar rats. A statistically significant increase of corpora lutea was detected in dams of the medium dose group but this was within the normal biological range. Of the five stillborns found in total, two were delivered by dams of the low dose group and three by dams of the medium dose group (Tab. 6, attached document); this was considered to be incidental. The sex-ratio of pups was not affected. The mean pup body weight was within the normal range for Wistar rats, and no statistically significant differences were detected between the dose groups. Data of the numbers of abnormal pups born, or the loss of offspring (pre-implantation, pre-natal and post-natal loss) were normal for rats of this strain and age.

 

Thus, norbornene had no effect on reproduction and development of male and female Wistar rats (12 rats per sex and dose) exposed to the test substance at 125, 250, and 500 mg/kg bw and day under the conditions of this combined repeated dose/reproduction toxicity screening test which was performed according to OECD TG 422 and under GLP conditions (Vivo, 2010).

 

This study is considered to be valid and suitable for assessment. The NOAEL for local and systemic toxicity was therefore 500 mg/kg bw and day for both sexes. The NOAEL for toxicity to reproduction and development was also 500 mg/kg bw and day under the conditions of this study.