Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
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
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
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.
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
cf. attached document
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
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
cf. attached document
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
Reproductive effects observed:
not specified

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.

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.

 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality screening study (OECD 422 under GLP conditions)
Additional information

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.

 


Short description of key information:
The NOAEL for reproduction toxicity was 500 mg/kg bw and day, the highest tested dose, in male and female parental rats in a screening study (OECD TG 422, GLP, oral gavage).

Justification for selection of Effect on fertility via oral route:
High quality screening study (OECD 422 under GLP conditions)

Effects on developmental toxicity

Description of key information
The NOAEL of Norbornene in an OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Nov 2014 - 07 April2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Adopted 22 January 2001)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: approx. 200 g
- Fasting period before study: no data
- Housing: in groups of four animals in macrolon cages
- Diet (e.g. ad libitum): Altromin, No 1324 TPF, maintenance diet for rats and mice, breeding diet No. 1314 TPF, ad libitum
- Water: sterilised community tab water, ad libitum
- Acclimation period:

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

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Heat corn oil (measured amount) and test item to 60°C for 15 minutes. Pour test item into corn oil to produce the test solution, determine net weight and volume of the test item, calculate concentration (mg/mL) of the stock solution. Add required corn oil to produce the highest application dose for the 600 mg/kg dose level. Heat for one minute and vortex for 15 seconds. Then, produce two serial dilutions (1:1) for the mid and the low dose level, respectively. Store test materials at 5°C under argon until application.

DIET PREPARATION
n. a.

VEHICLE
- Justification for use and choice of vehicle: corn oil was used because of the low solubility of Norbornene in water
- Concentration in vehicle: 150/75/36.5 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Source: Sigma, catalogue No. C8267
- Purity: 100% (:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A validated GC/FID method was used for the determination of Norbornene in corn oil. Samples were diluted with i-hexane, and toluene was used as internal standard. 1 µL was injected split less. Instruments: column: ZB-624, ID 0.32 mm, 1.8µm; Temperature 40°C, 13 min isothermal,. Gas: hydrogen. Inlet: 250°C split less; Detector FID, 260°C; Flow 2 mL/min. The ratio of the areas test item/toluene was used for quantification. Retention times were 5.72 min for Norbornene and 9.21 min for toluene. Recoveries were in the range 103 - 112 % for solutions containing 37,5, 75, and 150 mg/mL. Linear calibration curve; y=0.00886x + 0.00731; r²= 0.99998. Further details: Appendix B
Details on mating procedure:
Animals were mated at the breeding facility; no details reported (section 3.1 of the study report)
Duration of treatment / exposure:
gestational days 6 through 20
Frequency of treatment:
daily
Duration of test:
24 days, including acclimatisation
Remarks:
Doses / Concentrations:
0, 150, 300, and 600 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
Planned: 24/group
Successfully mated: 18/23/21/21 in controls/low/mid/high dose group, respectively
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: dose levels were selected based on the results as follows:
-OECD 422 combined repeated dose and reproduction toxicity study, oral gavage: minor effects but no clear toxicity pattern at the highest tested dose of 500 mg/kg bw.
-OECD 413 90-day inhalation study: treatment related changes in the ovaries of females exposed to 4.98 mg/m³. No effects at 2.02 gm³, i.e. the NOAEL was 582 mg/kg bw in this study.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were not included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 3, 6, 9, 12, 15, 18, and 21

FOOD CONSUMPTION: Yes
- Food consumption for each animal group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: days 3, 6, 9, 12, 15, 18, and 21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: macroscopical examination following sacrifice. Uterine weight (not obtained from animals found dead during the study). Uterine content was examined for numbers of embryonic or foetal deaths, viable fetuses, and scars. In the ovaries, the number of corpora lutea was determined for pregnant animals.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Descriptive statistics: the arithmetic mean and the standard deviation was calculated for all grouped numerical data originating from monitoring the body weight, food consumption and gross pathology (fetal and placenta weights). Where appropriate, detailed column statistics were applied (minimum / maximum data, 25% quantiles, standard error, upper and lower confidence interval 95%).

Inductive statistics: for basic analysis the respective test item groups were compared to the vehicle group by assessing of statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level was set to 0.05. In case the basic analysis returned a statistical significance additional inductive statistical analysis was applied as outlined in the main decision tree (see appendix). Most statistical hypotheses in this study were best characterised as “many to one”– comparing a vehicle control vs. three treatment groups, respectively. Therefore the adequate analysis method was a one-way ANOVA (Analysis of variance), followed by a post hoc Dunnett´s t-test. Analysis of body weight data includes the influence of two different categorical independent variables – time and dose groups. Therefore, analysis by two-way ANOVA, followed by Bonferroni post-hoc test, was assumed as the most adequate analysis method for this set of data. For all calculations, the significance level was set to 0.05. These further inductive statistics were performed using Graph Pad Prism for Mac, Version 5. All statistical data are documented in the appendix of the study report.
Indices:
mean pre- and post-implantation loss
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs: salivation was seen in all treated groups. The incidence and severity increased with the dose. 2two rats were occasionally affected in the low dose group; in the mid dose group, single animals showed this sign, starting from day 9, and all animals were affected on day 20; at the high dose, 3 animals showed this signs on day 9, and almost all animals from day 11 onwards.
Mortalities: two mid dose rats dies immediately after dosing due to an application error. One high dose rat was found dead more than 8 hours after death, the cause of death could not be established.
Body weights: treatment had no effect on the gravid rat body weight of either test group
Food consumption: food consumption was comparable across all dose groups, i.e. no treatment-related effect was noted
Necropsy: a pale pancreas was noted in 19/20 high dose and in 2/20 mid dose dams. This observation was not noted in the vehicle and in the low dose groups. The macroscopic examination revealed no other pathological findings.
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: developmental effects

Details on embryotoxic / teratogenic effects:
Uterus contents: the mean gravid uterus weight (with ovaries) was comparable across all groups.
Ovaries: the number of corpora lutea was comparable across all groups.
Implantations: the number was comparable across all groups.
Number of live fetuses: comparable across all groups.
Dead fetuses: no dead foetus seen in any group.
Early resorptions: mean number increased at the high dose level (0.8 versus 0.2 in the control), but not statistically significantly increased.
Pre- and post-implantation losses: minor differences lacking statistical significance. Two of the control animals showed an unusual high incidence of pre-implantation losses (10 and 12). Re-calculation without these two animals did also not reveal significant differences between the animal groups.
Foetal weight: weight at birth was significantly (p<0.001) reduced in male and female fetuses in all dose groups. The effect was dose-dependent and most prominent at the high dose [-11.4% (males and -11.8% (females)]. Birth weight reductions >10% are considered to be adverse.
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Mean number of corpora lutea

 

Corpora lutea (n)

 

Left ovary

Right ovary

Total

Dose group

Mean

Mean

Mean

SD

600 mg/kg

6.9

6.6

13.5

1.7

300 mg/kg

6.8

6.6

13.3

1.7

150 mg/kg

6.3

6.9

11.1

2.4

Vehicle

6.5

6.7

13.2

1.9

 

Table 2: Mean number of implantations

 

Corpora lutea (n)

 

Left uterus horn

Right uterus horn

Total

Dose group

Mean

Mean

Mean

SD

600 mg/kg

6.1

6.2

12.3

1.6

300 mg/kg

5.7

6.2

11.9

1.6

150 mg/kg

5.7

6.1

11.8

1.2

Vehicle

5.5

5.3

10.8

3.1

 

Table 3: Mean number of foetuses and resorptions

 

Corpora lutea

Implantation sites

Live fetuses

Early resorptions

Scar

Dead fetuses

Dose group

Mean

Mean

Mean

Mean

Mean

Mean

600 mg/kg

13.5

12.3

11.5

0.8

0.0

0.0

300 mg/kg

13.3

11.9

11.5

0.2

0.0

0.0

150 mg/kg

11.1

11.8

11.5

0.2

0.0

0.0

Vehicle

13.2

10.8

10.2

0.2

0.0

0.0

Vehicle *

13.1

11.8

11.1

0.2

0.0

 0.0

 

Vehicle*: calculation that excludes two dams with unusually high number of pre-implantation losses (10 and 12, respectively)

Table 4: Indices of implantation losses

 

Pre-implantation loss (CL-IS)

 

Post-implantation loss (IS-LF)

Pre-implantation loss per group

Post-implantation loss per group

Dose group

Mean

Mean

Mean

Mean

600 mg/kg

1.2

0.8

9.76

7.05

300 mg/kg

1.4

0.4

11.76

3.73

150 mg/kg

1.3

0.3

11.02

2.64

Vehicle

2.4

0.7

22.22

6.55

Vehicle *

1.3

0.7

11.1

6.55

 

Legend:

Vehicle*: calculation that excludes two dams with unusually high number of pre-implantation losses (10 and 12, respectively)

CL = Corpora lutea
IS = Implantation site
LF = Life fetuses

Indices were calculated as follows:

Pre implantation loss: mean no. of corpora lutea – mean no. of implantations

Mean no. of resorptions: No. of resorptions / no. of litters

Mean pre implantation loss per group: (mean no. of corpora lutea – mean no. of implantations) * 100 /mean no. of implantations

Mean post implantation loss (%): (Total no. resorptions + total no. of dead fetuses) * 100 / no. of fetuses.

All calculations and statistical tests are documented within the appendix of the report.

 

Table 5: mean fetal birth weight and weight changes

 

 

Mean weight [g]

 

Both sexes

Males

Females

Weight change [%]

Versus controls

Dose group

Males

Females

600 mg/kg

4.76

4.91

4.65

-11.74

-11.8

300 mg/kg

5.01

5.11

4.90

-7.8

-7.0

150 mg/kg

506

5.19

4.94

-6.3

-6.3

Vehicle

5.38

5.54

5.27

 

 

 

Conclusions:
The NOAEL of Norbornene in an OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group.
Executive summary:

The developmental toxicity and teratogenicity of Norbornene was studied in time-mated Wistar rats according to the OECD 414 test guideline and under GLP conditions. Norbornene, dissolved in corn oil, was administered by oral gave at dose levels of 0, 150, 300 and 600 mg/kg bw, based on the results of preceding studies [OECD 422 (oral) and 413 (inhalation)] which gave NOAEL values of 500 and 582 mg/kg bw, respectively.

 

The NOAEL of Norbornene in the OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group. A significant increase of supernummary ribs was seen in fetuses of the high dose group (40% versus 8% in the control) and on litter basis (90% vs. 39% in the vehicle control. Therefore, this finding is considered to be test-item related, but not teratogen.

Overall the NOAEL of Norbornene in the Wistar rat was 600 mg/kg bw for maternal toxicity and teratogenicity, and 300 mg/kg bw for developmental toxicity.

 

The study is considered valid and suitable for assessment.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Valid OECD 414 guideline study (reliabiliy 1) in Wistar rats
Additional information

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.

The developmental toxicity and teratogenicity of Norbornene was studied in time-mated Wistar rats according to the OECD 414 test guideline and under GLP conditions. Norbornene, dissolved in corn oil, was administered by oral gave at dose levels of 0, 150, 300 and 600 mg/kg bw, based on the results of preceding studies [OECD 422 (oral) and 413 (inhalation)] which gave NOAEL values of 500 and 582 mg/kg bw, respectively.

 

The NOAEL of Norbornene in the OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group. A significant increase of supernummary ribs was seen in fetuses of the high dose group (40% versus 8% in the control) and on litter basis (90% vs. 39% in the vehicle control. Therefore, this finding is considered to be test-item related, but not teratogen.

Overall the NOAEL of Norbornene in the Wistar rat was 600 mg/kg bw for maternal toxicity and teratogenicity, and 300 mg/kg bw for developmental toxicity in the OECD 414 study, which is considered valid and suitable for assessment.

It is noteworthy that, in contrast to the 90 -day inhalation study, in the OECD 414 oral study no effect on the ovaries was seen (no increased weight or increased number of corpora lutea). The reason is presently unclear but may be related to the short exposure period (gestational day 6-20) compared to the 90 day exposure period of the OECD 413 study. Additionally, toxicokinetic differences are expected depending of the route of exposure. Further, as no effect on the foetal birth weight was seen in the OECD 422 study in contrast to the OECD 414 study, it may be speculated that induction of the xenobiotics metabolising enzymes in the OECD 422 study might have led to an increased metabolism and excretion

 

Justification for selection of Effect on developmental toxicity: via oral route:
Valid OECD 414 guideline study (reliability 1)

Justification for classification or non-classification

The single adverse effect observed in the OECD 414 study was reduced fetal body weight, potentially indicating delayed development. This effect has not been observed in fetuses of the earlier OECD 422 study conducted on the same strain of rats and by the same laboratory, introducing some element of doubt on the significance of the observed effect.

As a precautionary apporach the following classification according to 1272/2008/EC is proposed:

Reproductive toxicity category 2, H361