Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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 oral 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%
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:
daily, 7/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 250, and 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:
- Dose selection rationale:
At the time point when this study was conducted, data about the toxicity of the test item on vertebrates were poor. In an acute toxicological assessment quoted in the material safety sheet of the test item Norbonene (CAS498-66-8), 11300 mg/kg was determined as the LD50. The substance was therefore classified acute non-toxic. In accordance with the Sponsor, 1000 mg/kg and two graduated (1:3 v/v) serial dilutions thereof (250 mg/kg, 50 mg/kg) were determined as high, medium and low doses for a toxicity dose range finding assay.

Results from that dose range finding study for the present toxicity test indicated that the dose of 1000 mg/kg induced strong toxic effects within the first two weeks of oral administration to non-gravid rats of both sexes. However, animals of the medium dose group (250 mg/kg) remained unaffected throughout the entire in-life phase, including the gestation and delivery phase of the dams.

Based on those results and in accordance with the Sponsor, 500 mg/kg and two graduated (1:1 v/v) serial dilutions thereof (250 mg/kg, 125 mg/kg) were determined as high, medium and low doses for the present toxicity assay.
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
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)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see report, table 4. Results: Appendix, pages 34 - 54)
HISTOPATHOLOGY: Yes (see report, table 4. Results: Appendix, pages 34 - 54)
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Tendency of slightly decreased body weights in male and female high dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
high dose males showed increased water consumption in the post mating period (weeks 4-7); statistical significance (p<0.05) was reached in weeks 5 and 7
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
though some changes were observed, there was no clear pattern of toxicity
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significantly (p<0.05) increased organ weights in high dose groups: liver (males); kidney (females)
Gross pathological findings:
no effects observed
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities except from one animal that died due to the administration procedure.

BODY WEIGHT AND WEIGHT GAIN
Tendency of slightly decreased body weights in male and female high dose groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
No change.

FOOD EFFICIENCY
No change.

WATER CONSUMPTION AND COMPOUND INTAKE
High dose males showed increased water consumption in the post mating period (weeks 4-7); statistical significance (p<0.05) was reached in weeks 5 and 7.

HAEMATOLOGY
Data from haematology, serum biochemistry and blood coagulation of all animals from the vehicle control groups as well as all dose groups were within normal range for rats of this strain and age. Regarding haematology and blood clotting time, no statistically significant or toxicologically relevant differences between the control groups and the dose groups were found.

CLINICAL CHEMISTRY
Few mild to moderate changes (creatinine, urea, cholesterol affected) observed mainly in rats from the high dose groups were noted but lacked a clear pattern of toxicity.

NEUROBEHAVIOUR
No changes noted conmpared to the controls (see Appendix, pages 14-15).

ORGAN WEIGHTS
significantly (p<0.05) increased organ weights in high dose groups:
liver (males): 16.46 g versus 14.31g in controls
kidney (females): 1.26 g versus 1.11 g in controls

GROSS PATHOLOGY
No effects in treated groups compared to controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
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. This finding is related to the a2-microglobulin nephropathy that is only seen in male, but not in female, rats.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
local toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No changes in stomach or intestine that would indicate irritation.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no relevant adverse effects in male and female rats receiving norbornene (125, 250, 500 mg/kg bw and day) by oral gavage in a combined repeated dose/reproduction toxicity study (OECD 422). The NOAEL for local and systemic toxicity was therefore 500 mg/kg bw and day.
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. Body weight development, food and water intake, neurofunctions (grip strength and sensory functions), haematology, and clinical parameters were all comparable to controls. At termination there no findings at necropsy and microscopy in any organ including male and female reproduction organs, with the exception of isolated increased organ weights (liver weights in high dose males (p<0.05); kidney weights in high dose females (p<0.05), and hyaline droplets in the kidneys of males from all dose levels.The systemic toxicity NOAEL in rats was thus below 125 mg/kg body mass and day in males, based on effects in kidneys at all tested dose levels, and 500 mg/kg body mass and day in females (Vivo, 2010).

 

However, hyaline droplet formation in the renal proximal epithelium is related to alpha2-microglobulin agglomeration, and this finding is known to be specific for the male rat, i.e. this effect is not relevant for humans. Taking this into consideration, the NOAEL may be set at 500 mg/kg bw and day for both sexes for assessment.

 

To summarise, there were no adverse effects relevant for humans in male and female rats receiving norbornene (125, 250, 500 mg/kg bw and day) by oral gavage in a combined repeated dose/reproduction toxicity study (OECD 422). The NOAEL for local and systemic toxicity was therefore 500 mg/kg bw and day for both sexes.