8,9,10-trinorborn-2-ene

Endpoint summary

Description of key information

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, GLP). The NOAEL for local and systemic toxicity was therefore 500 mg/kg bw and day. A target organ was not identified. 
An inhalative 90d (600, 2000 and 5000 mg/m³) showed increased organ weights (ovaries) leading to an NOAEL for systemic toxicity of 2020 mg/m³.corresponding to 582 mg/kg bw/day. Inhalative irritation observed was considered not toxicollogically significant. Hyaline droplet formation observed in male rats both in the 28d and 90d study was identifed immunohistochemically as alpha-2µ-globulin nephropathy not significant to human.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 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

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

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.

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.

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.

Critical effects observed:

not specified

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.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good: GLP guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-10 through 2014-07-14 (includes range finder, main study and pathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: main study: 269 and 181 grams for male and female animals, respectively.
- Fasting period before study: no
- Housing: in groups of 5
- Diet: cereal-based rodent diet (Rat&Mouse No. 3 Breeding Diet, RM3), ad libitum
- Water: tap water ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°C
- Humidity (%): 30-70
- Air changes (per hr): not stated; airflow was min. 60 L/h per animal
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: n.a. (vapours only)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a schematic diagram is presented in Fig 1 of the study report. Briefly, a known amount of test material was placed in a glass flask and heated in a water bath to a temperature slightly above the melting point. A controlled flow of nitrogen was bubbled through the liquid test material to evaporate a known amount of test material. The stream of test material vapour (in nitrogen) was mixed with a mass flow controlled stream of humidified air after leaving the glass flask, ensuring that the test material in the flask was kept under nitrogen all time. This pre-mixture was subsequently diluted with mass flow controlled streams of humidified air to obtain the low-, mid- and high-concentration test atmospheres. For the low- and mid-concentration, parts of the pre-mixture were extracted using vacuum mass flow controllers; the extracted stream was subsequently diluted with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). The remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration test atmosphere. Each atmosphere was directed to the top inlet of an exposure unit, led to the noses of the animals and exhausted at the bottom of the unit.

- Method of holding animals in test chamber: animals were placed in animal holders, the nose protruding to a common cylinder for nose-only exposure
- Source and rate of air: compressed dry air
- Method of conditioning air: humidifier
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 20-24 L/min (report, Table 1.2)
- Method of particle size determination: n.a.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: total carbon analyser (also: calculations from amount of test material consumed and air flow; cf, report, Table 1.2)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test material in the test atmospheres was measured every minute by total carbon analysis (Sick Maihak EuroFID; Sick Instruments Benelux, Hedel, the Netherlands) using a calibration curve. The calibration of each target concentration was checked about weekly. The measured concentrations were compared with the nominal concentration that was calculated from the amount of consumed test material and the airflow.

Duration of treatment / exposure:

13 weeks; 65 exposures

Frequency of treatment:

5 per week

Remarks:

Doses / Concentrations:
0, 600, 2000, and 5000 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 600, 2020, and 4980 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a preceding 14-range finding study with target concentrations of 0, 500, 1500, and 5000 mg/m³; 5 rats/sex and dose

Positive control:

no needed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: cage side twice daily on exposure days; once per day during weekends and public holidays. Once during exposure
- Cage side observations listed in Annex 6 were checked

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: once pre-test, on the first exposure day, twice weekly during the study, and at days 93 and 94 (termination)
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. Food consumption was measured over successive 7-day periods per cage; expressed as g/animal/day over the respective period (Table 5)

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to exposure in all rats, and in week 13 in the rats of the control and the high dose groups
- Dose groups that were examined: see above

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes; pentobarbital
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters examined (section 4.11.5): red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV), reticulocytes, total white blood cells (WBC), differential white blood cells (Lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time (PT), thrombocytes.

The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters examined (section 4.11.6): alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), bilirubin (total), total protein, albumin, ratio albumin to globulin (calculated), glucose, cholesterol (total), phospholipids, triglycerides, creatinine, urea, inorganic phosphate (PO4), calcium (Ca), chloride (Cl), potassium (K), sodium (Na)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- Metabolism cages used for collection of urine: yes; all surviving animals; one animal per cage
- Animals fasted: Yes
- Parameters examined (section 4.11.7):

volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (redand white blood cells)


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Examinations (section 4.11.8): gross pathology; organ weights (adrenals, heart, kidneys, liver, lung with trachea and larynx, spleen)

HISTOPATHOLOGY: Yes
Fixation: Tissues were preserved in 10% formalin in neutral phosphate buffer.
Staining: after embedding in paraffin wax, slices of 5µm were stained with haematoxylin and eosin (HE). Renal tissue was also stained immunohistochemically with an antibody to alpha2u-microglobulin. Tissues of all control and high dose animals were processed.
Animals examined: all gross lesions; all control and high dose animals; additionally ovaries of all low and mid-dose females
Parameters examined (section 4.11.8):

all gross lesions pancreas
adrenals parathyroids
aorta parotid salivary glands
axillary lymph nodes pharynx
brain1 pituitary
caecum prostate
colon rectum
duodenum seminal vesicles / coagulating glands
epididymides skeletal muscle (thigh)
exorbital lachrymal glands skin (flank)
eyes (with optic nerve) spinal cord
femur with joint spleen
heart sternum with bone marrow
ileum stomach
jejunum sublingual salivary glands
kidneys submaxillary salivary glands
larynx testes
liver thymus
lung thyroid
mammary gland (females) tongue
mandibular (cervical) lymph nodes trachea
nasopharyngeal tissues (with teeth) tracheobronchial (mediastinal) lymph nodes
nerve-peripheral (sciatic) ureter
oesophagus urethra
olfactory bulb urinary bladder
ovaries uterus (with cervix)


Further details:
Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
Lung: Each lung lobe was examined microscopically at one level, including main bronchi.
Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]). An illustration of these levels is shown in in the report, Annex 10.
Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
Stomach: Non-glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.
Trachea: Three transverse sections and three longitudinal sections (at least one through the carina of the bifurcation of the bronchi) were examined microscopically.

Statistics:

A thorough statistical analysis of teh results was performed as detailed in section 4.1 2of the report. Methods used included ANCOVA& Dunnett`s test (body weights), Kruskal-Wallius&Dunnett's test (urinalysis), Dunnett's test (food consumption), Fisher's exact probability test (histopathology).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

statistically significant changes: increased kidney and liver weights in both sexes at the high dose. In high dose females higher weights of spleen, thymus, and ovaries

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

males: kidneys; females: ovaries. Respiratory tract: no changes in the nose; inflammation was seen in the larynx (m:6/10; f: 5/10), trachea (m: 5/10, f: 1/10), and the lungs (m: 6/10; f: 3/10; alveolar macrophages 5/10))

Histopathological findings: neoplastic:

not examined

Details on results:

For details see table in the section "any other information on results incl. tables" below

CLINICAL SIGNS AND MORTALITY
no treatment related effect; all animals survived except one control male No. 10 that was killed for humane reasons on Day 88.

BODY WEIGHT AND WEIGHT GAIN
The body weight of low- and mid-dose animals was comparable to that of controls; significant increases (males +5%; females +7%) were seen in high dose animals (note: no significance in males when the control male No. 10 (marked retarded body weight) was taken out).

FOOD CONSUMPTION
Increased in high dose groups; coincides with increased body weights.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects noted.

HAEMATOLOGY
No treatment-related effects noted.

CLINICAL CHEMISTRY
Differences were generally small except statisticalls significantly increased levels of GGT, cholesterol, and lipids in high dose males, and fasting glucose in females.The toxicological significance of the latter is unclear whereas the effects in males might be related to the observed nephropathy.

URINALYSIS
No treatment-related effects noted; includes high-dose males.

ORGAN WEIGHTS
Statistically significant changes: increased kidney and liver weights in both sexes at the high dose. In high dose females also higher weights of spleen, thymus, and ovaries.

GROSS PATHOLOGY
No treatment related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Two major advrese findings were noted:
1. Hyaline droplet nephropathy in all high-dose males (10/10), identified as alpha2u-globulin nephropathy which is known to be specific for the male rat. Low-and mid-dose rats were not examined because the effect is not relevant for human risk assessment. Hence, no NOAEL was established for this effect.
2. Ovaries of the high-dose females appeared to be quite large (weight +50%), which was attributed to a high number of corpora lutea in 9/10 females. No such change was seen in low- and mid-dose females.

Dose descriptor:

NOAEL

Effect level:

ca. 2 020 mg/m³ air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: hyaline droplet formation observed in high dose male rats was not considered relevant for human health considerations

Critical effects observed:

not specified

Summary of key results of 13-week inhalation study withNorborneneHP (Bicyclo(2.2.1)hept-2-ene) in rats

 

Parameter

Low conc. (0.6 g/m^3)

Mid conc. (2 g/m^3)

High conc. (5 g/m^3)

Remarks

Mortality, clinical observations, ophthalmoscopy

No treatment-related findings

♂

♀

♂

♀

♂

♀

Body weight

ic

ic

Mean terminal body weight 5% (♂) or 7% (♀) higher than control. Not adverse.

Food consumption

i

i

Haematology& Urinalysis

No treatment-related findings

Clinical chemistry

Gamma glutamyl  transferase (GGT)

ic

Toxicological significance not clear.

Cholesterol

ic

Phospholipids

ic

Fasting glucose

ic

Organ weights

Kidneys

ic^r

ic^a,r

ic^a,r

♂ DR;likely to be related to α2u-globulin nephropathy. ♀ Not adverse (modest increase, no corroborative changes in clinical pathology or histopathology).

Liver

ic^r

ic^a,r

ic^a,r

♂ and ♀ Not adverse (modest, no corroborative changes in clinical pathology or histopathology); ♀ DR

Spleen

ic^a,r

♀ Not adverse (modest increase, no corroborative changes in clinical pathology or histopathology).

Thymus

ic^a,r

Ovaries

ic^a,r

♀ Adverse effect. Mean relative weight 41% higher than control. Related to histopathological change.

Necropsy

No treatment-related findings

Histopathology

Ovaries: larger appearance and increased number of corpora lutea

0/10

0/10

9/10

♀ Adverse effect.

Kidneys: α2u-globulin nephropathy

NE

NE

10/10

♂ Adverse effect in rats but specific for male rats and therefore not relevant for human risk assessment.

Conclusion

NOAEL

Adverse effect level

DR = dose-related;i= increased but not statistically significantly;ic= statistically significantly increased;

^a =absolute organ weight;^r = organ weight relative to body weight; NE = not examinedmicrosocopically.

Conclusions:

The NOAEC was 2020 mg/m³ in an OECD 413 study (90 day inahaltion study, 10 Wistar rats per sex and dose); advers eeffct was only seen in the ovaries of high-dose females (+50% organ weight increase, with histopathological correlate, a high number of corpora lutea). The NOAEL corresponds to 582 mg/kg bw and day, using the assumptions in ECHA Guidance R8, Table R-8.2

Executive summary:

The repeated dose toxicity of Norbornene was examined in the rat (Wistar, 10/sex and dose) according to the OECD 413 test guideline and under GLP conditions (90 day inhalation, 5 days per week, 6 hours/day). The nominal dose levels (0, 600, 200, and 5000 mg/m³) were selected based on the results of a range-finding study (5 rats/sex and dose, 20 days). Examinations were performed according to the test guideline, and included, amongst others, histopathology of the respiratory tract and reproductive organs.

The analytical vapour concentrations (0, 600, 2020, and 4980 mg/m³) were very close to the target concentrations. Most examinations revealed no effect, i.e.

- no mortalities or clinical signs were noted;
-body weights comparable with the controls, albeit increased 5-7% at the high-dose level which was paralleled by a higher food intake of the high-dose animals;
- clinical chemistry, haematology, urinalysis and ophthalmology revealed no adverse effects;
- most organ weights were comparable with controls;
-histopathology revealed no changes in most tissues; of those of the respiratory tract, no changes were seen in the nose whereas inflammation was seen in the larynx (m:6/10; f: 5/10), trachea (m: 5/10, f: 1/10), and the lungs (m: 6/10; f: 3/10; alveolar macrophages 5/10) at the high dose. The inflammation was, however, minimal to mild and was not considered to be of toxicological relevance.

 

There were, however, two adverse effects:

1. Hyaline droplet nephropathy was seen in all high-dose males (10/10), identified immunohistochemically as alpha2u-globulin nephropathy which is known to be specific for the male rat. Low-and mid-dose rats were not examined because the effect is not relevant for human risk assessment. Hence, no NOAEL was established for this effect.

2. Ovaries of the high-dose females appeared to be quite large (mean weight +50%), which was attributed to a high number of corpora lutea that was seen in histopathology in 9/10 females. No such change was seen in low- and mid-dose females.

Hence, two adverse effects were seen in this study, i.e. alpha2u-globulin nephropathy in male rats (LOAEL: 4980 mg/m³) which is not relevant for human risk assessment, and adverse effects on the female ovary (NOAEL 2020 mg/m³). The latter corresponds to 582 mg/kg bw and day, based on the assumptions made by ECHA (Guidance R8 : absorption by the inhalation route 100%, and default values in Table R.8-2) (TNO, 2014).

 

The study is considered valid for assessment. The toxicological relevance of the changes observed in the ovary is not known. The absolute and relative weights of thymus, thyroid, and testes (males), and of relative thymus, thyroid weights (females) were not affected. Further, histopathology revealed no effects (high dose animals) in the following organs (selection):

males: testes, epididymides, seminal vesicles; thyroid gland.

females: mammary gland, thyroid gland, pituitary gland

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2 020 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good: GLP guideline study. Target organ: ovary

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-10 through 2014-07-14 (includes range finder, main study and pathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: main study: 269 and 181 grams for male and female animals, respectively.
- Fasting period before study: no
- Housing: in groups of 5
- Diet: cereal-based rodent diet (Rat&Mouse No. 3 Breeding Diet, RM3), ad libitum
- Water: tap water ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°C
- Humidity (%): 30-70
- Air changes (per hr): not stated; airflow was min. 60 L/h per animal
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: n.a. (vapours only)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a schematic diagram is presented in Fig 1 of the study report. Briefly, a known amount of test material was placed in a glass flask and heated in a water bath to a temperature slightly above the melting point. A controlled flow of nitrogen was bubbled through the liquid test material to evaporate a known amount of test material. The stream of test material vapour (in nitrogen) was mixed with a mass flow controlled stream of humidified air after leaving the glass flask, ensuring that the test material in the flask was kept under nitrogen all time. This pre-mixture was subsequently diluted with mass flow controlled streams of humidified air to obtain the low-, mid- and high-concentration test atmospheres. For the low- and mid-concentration, parts of the pre-mixture were extracted using vacuum mass flow controllers; the extracted stream was subsequently diluted with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). The remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration test atmosphere. Each atmosphere was directed to the top inlet of an exposure unit, led to the noses of the animals and exhausted at the bottom of the unit.

- Method of holding animals in test chamber: animals were placed in animal holders, the nose protruding to a common cylinder for nose-only exposure
- Source and rate of air: compressed dry air
- Method of conditioning air: humidifier
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 20-24 L/min (report, Table 1.2)
- Method of particle size determination: n.a.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: total carbon analyser (also: calculations from amount of test material consumed and air flow; cf, report, Table 1.2)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test material in the test atmospheres was measured every minute by total carbon analysis (Sick Maihak EuroFID; Sick Instruments Benelux, Hedel, the Netherlands) using a calibration curve. The calibration of each target concentration was checked about weekly. The measured concentrations were compared with the nominal concentration that was calculated from the amount of consumed test material and the airflow.

Duration of treatment / exposure:

13 weeks; 65 exposures

Frequency of treatment:

5 per week

Remarks:

Doses / Concentrations:
0, 600, 2000, and 5000 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 600, 2020, and 4980 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a preceding 14-range finding study with target concentrations of 0, 500, 1500, and 5000 mg/m³; 5 rats/sex and dose

Positive control:

no needed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: cage side twice daily on exposure days; once per day during weekends and public holidays. Once during exposure
- Cage side observations listed in Annex 6 were checked

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: once pre-test, on the first exposure day, twice weekly during the study, and at days 93 and 94 (termination)
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. Food consumption was measured over successive 7-day periods per cage; expressed as g/animal/day over the respective period (Table 5)

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to exposure in all rats, and in week 13 in the rats of the control and the high dose groups
- Dose groups that were examined: see above

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes; pentobarbital
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters examined (section 4.11.5): red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV), reticulocytes, total white blood cells (WBC), differential white blood cells (Lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time (PT), thrombocytes.

The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes
- How many animals: all surviving rats
- Parameters examined (section 4.11.6): alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), bilirubin (total), total protein, albumin, ratio albumin to globulin (calculated), glucose, cholesterol (total), phospholipids, triglycerides, creatinine, urea, inorganic phosphate (PO4), calcium (Ca), chloride (Cl), potassium (K), sodium (Na)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- Metabolism cages used for collection of urine: yes; all surviving animals; one animal per cage
- Animals fasted: Yes
- Parameters examined (section 4.11.7):

volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (redand white blood cells)


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Examinations (section 4.11.8): gross pathology; organ weights (adrenals, heart, kidneys, liver, lung with trachea and larynx, spleen)

HISTOPATHOLOGY: Yes
Fixation: Tissues were preserved in 10% formalin in neutral phosphate buffer.
Staining: after embedding in paraffin wax, slices of 5µm were stained with haematoxylin and eosin (HE). Renal tissue was also stained immunohistochemically with an antibody to alpha2u-microglobulin. Tissues of all control and high dose animals were processed.
Animals examined: all gross lesions; all control and high dose animals; additionally ovaries of all low and mid-dose females
Parameters examined (section 4.11.8):

all gross lesions pancreas
adrenals parathyroids
aorta parotid salivary glands
axillary lymph nodes pharynx
brain1 pituitary
caecum prostate
colon rectum
duodenum seminal vesicles / coagulating glands
epididymides skeletal muscle (thigh)
exorbital lachrymal glands skin (flank)
eyes (with optic nerve) spinal cord
femur with joint spleen
heart sternum with bone marrow
ileum stomach
jejunum sublingual salivary glands
kidneys submaxillary salivary glands
larynx testes
liver thymus
lung thyroid
mammary gland (females) tongue
mandibular (cervical) lymph nodes trachea
nasopharyngeal tissues (with teeth) tracheobronchial (mediastinal) lymph nodes
nerve-peripheral (sciatic) ureter
oesophagus urethra
olfactory bulb urinary bladder
ovaries uterus (with cervix)


Further details:
Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
Lung: Each lung lobe was examined microscopically at one level, including main bronchi.
Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]). An illustration of these levels is shown in in the report, Annex 10.
Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
Stomach: Non-glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.
Trachea: Three transverse sections and three longitudinal sections (at least one through the carina of the bifurcation of the bronchi) were examined microscopically.

Statistics:

A thorough statistical analysis of teh results was performed as detailed in section 4.1 2of the report. Methods used included ANCOVA& Dunnett`s test (body weights), Kruskal-Wallius&Dunnett's test (urinalysis), Dunnett's test (food consumption), Fisher's exact probability test (histopathology).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

statistically significant changes: increased kidney and liver weights in both sexes at the high dose. In high dose females higher weights of spleen, thymus, and ovaries

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

males: kidneys; females: ovaries. Respiratory tract: no changes in the nose; inflammation was seen in the larynx (m:6/10; f: 5/10), trachea (m: 5/10, f: 1/10), and the lungs (m: 6/10; f: 3/10; alveolar macrophages 5/10))

Histopathological findings: neoplastic:

not examined

Details on results:

For details see table in the section "any other information on results incl. tables" below

CLINICAL SIGNS AND MORTALITY
no treatment related effect; all animals survived except one control male No. 10 that was killed for humane reasons on Day 88.

BODY WEIGHT AND WEIGHT GAIN
The body weight of low- and mid-dose animals was comparable to that of controls; significant increases (males +5%; females +7%) were seen in high dose animals (note: no significance in males when the control male No. 10 (marked retarded body weight) was taken out).

FOOD CONSUMPTION
Increased in high dose groups; coincides with increased body weights.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects noted.

HAEMATOLOGY
No treatment-related effects noted.

CLINICAL CHEMISTRY
Differences were generally small except statisticalls significantly increased levels of GGT, cholesterol, and lipids in high dose males, and fasting glucose in females.The toxicological significance of the latter is unclear whereas the effects in males might be related to the observed nephropathy.

URINALYSIS
No treatment-related effects noted; includes high-dose males.

ORGAN WEIGHTS
Statistically significant changes: increased kidney and liver weights in both sexes at the high dose. In high dose females also higher weights of spleen, thymus, and ovaries.

GROSS PATHOLOGY
No treatment related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Two major advrese findings were noted:
1. Hyaline droplet nephropathy in all high-dose males (10/10), identified as alpha2u-globulin nephropathy which is known to be specific for the male rat. Low-and mid-dose rats were not examined because the effect is not relevant for human risk assessment. Hence, no NOAEL was established for this effect.
2. Ovaries of the high-dose females appeared to be quite large (weight +50%), which was attributed to a high number of corpora lutea in 9/10 females. No such change was seen in low- and mid-dose females.

Dose descriptor:

NOAEL

Effect level:

ca. 2 020 mg/m³ air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: hyaline droplet formation observed in high dose male rats was not considered relevant for human health considerations

Critical effects observed:

not specified

Summary of key results of 13-week inhalation study withNorborneneHP (Bicyclo(2.2.1)hept-2-ene) in rats

 

Parameter

Low conc. (0.6 g/m^3)

Mid conc. (2 g/m^3)

High conc. (5 g/m^3)

Remarks

Mortality, clinical observations, ophthalmoscopy

No treatment-related findings

♂

♀

♂

♀

♂

♀

Body weight

ic

ic

Mean terminal body weight 5% (♂) or 7% (♀) higher than control. Not adverse.

Food consumption

i

i

Haematology& Urinalysis

No treatment-related findings

Clinical chemistry

Gamma glutamyl  transferase (GGT)

ic

Toxicological significance not clear.

Cholesterol

ic

Phospholipids

ic

Fasting glucose

ic

Organ weights

Kidneys

ic^r

ic^a,r

ic^a,r

♂ DR;likely to be related to α2u-globulin nephropathy. ♀ Not adverse (modest increase, no corroborative changes in clinical pathology or histopathology).

Liver

ic^r

ic^a,r

ic^a,r

♂ and ♀ Not adverse (modest, no corroborative changes in clinical pathology or histopathology); ♀ DR

Spleen

ic^a,r

♀ Not adverse (modest increase, no corroborative changes in clinical pathology or histopathology).

Thymus

ic^a,r

Ovaries

ic^a,r

♀ Adverse effect. Mean relative weight 41% higher than control. Related to histopathological change.

Necropsy

No treatment-related findings

Histopathology

Ovaries: larger appearance and increased number of corpora lutea

0/10

0/10

9/10

♀ Adverse effect.

Kidneys: α2u-globulin nephropathy

NE

NE

10/10

♂ Adverse effect in rats but specific for male rats and therefore not relevant for human risk assessment.

Conclusion

NOAEL

Adverse effect level

DR = dose-related;i= increased but not statistically significantly;ic= statistically significantly increased;

^a =absolute organ weight;^r = organ weight relative to body weight; NE = not examinedmicrosocopically.

Conclusions:

The NOAEC was 2020 mg/m³ in an OECD 413 study (90 day inahaltion study, 10 Wistar rats per sex and dose); advers eeffct was only seen in the ovaries of high-dose females (+50% organ weight increase, with histopathological correlate, a high number of corpora lutea). The NOAEL corresponds to 582 mg/kg bw and day, using the assumptions in ECHA Guidance R8, Table R-8.2

Executive summary:

The repeated dose toxicity of Norbornene was examined in the rat (Wistar, 10/sex and dose) according to the OECD 413 test guideline and under GLP conditions (90 day inhalation, 5 days per week, 6 hours/day). The nominal dose levels (0, 600, 200, and 5000 mg/m³) were selected based on the results of a range-finding study (5 rats/sex and dose, 20 days). Examinations were performed according to the test guideline, and included, amongst others, histopathology of the respiratory tract and reproductive organs.

The analytical vapour concentrations (0, 600, 2020, and 4980 mg/m³) were very close to the target concentrations. Most examinations revealed no effect, i.e.

- no mortalities or clinical signs were noted;
-body weights comparable with the controls, albeit increased 5-7% at the high-dose level which was paralleled by a higher food intake of the high-dose animals;
- clinical chemistry, haematology, urinalysis and ophthalmology revealed no adverse effects;
- most organ weights were comparable with controls;
-histopathology revealed no changes in most tissues; of those of the respiratory tract, no changes were seen in the nose whereas inflammation was seen in the larynx (m:6/10; f: 5/10), trachea (m: 5/10, f: 1/10), and the lungs (m: 6/10; f: 3/10; alveolar macrophages 5/10) at the high dose. The inflammation was, however, minimal to mild and was not considered to be of toxicological relevance.

 

There were, however, two adverse effects:

1. Hyaline droplet nephropathy was seen in all high-dose males (10/10), identified immunohistochemically as alpha2u-globulin nephropathy which is known to be specific for the male rat. Low-and mid-dose rats were not examined because the effect is not relevant for human risk assessment. Hence, no NOAEL was established for this effect.

2. Ovaries of the high-dose females appeared to be quite large (mean weight +50%), which was attributed to a high number of corpora lutea that was seen in histopathology in 9/10 females. No such change was seen in low- and mid-dose females.

Hence, two adverse effects were seen in this study, i.e. alpha2u-globulin nephropathy in male rats (LOAEL: 4980 mg/m³) which is not relevant for human risk assessment, and adverse effects on the female ovary (NOAEL 2020 mg/m³). The latter corresponds to 582 mg/kg bw and day, based on the assumptions made by ECHA (Guidance R8 : absorption by the inhalation route 100%, and default values in Table R.8-2) (TNO, 2014).

 

The study is considered valid for assessment. The toxicological relevance of the changes observed in the ovary is not known. The absolute and relative weights of thymus, thyroid, and testes (males), and of relative thymus, thyroid weights (females) were not affected. Further, histopathology revealed no effects (high dose animals) in the following organs (selection):

males: testes, epididymides, seminal vesicles; thyroid gland.

females: mammary gland, thyroid gland, pituitary gland

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

4 980 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good: GLP guideline study with histopathology

Additional information

Screening study

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.

 

90 day inhalation study

The repeated dose toxicity of Norbornene was examined in the rat (Wistar, 10/sex and dose) according to the OECD 413 test guideline and under GLP conditions (90 day inhalation, 5 days per week, 6 hours/day). The nominal dose levels (0, 600, 2000, and 5000 mg/m³) were selected based on the results of a range-finding study (5 rats/sex and dose, 20 days). Examinations were performed according to the test guideline, and included, amongst others, histopathology of the respiratory tract and reproductive organs.

The analytical vapour concentrations (0, 600, 2020, and 4980 mg/m³) were very close to the target concentrations. Most examinations revealed no effect, i.e.

- no mortalities or clinical signs were noted;
-body weights comparable with the controls, albeit increased 5-7% at the high-dose level which was paralleled by a higher food intake of the high-dose animals;
- clinical chemistry, haematology, urinalysis and ophthalmology revealed no adverse effects;
- most organ weights were comparable with controls;

-histopathology revealed no changes in most tissues; of those of the respiratory tract, no changes were seen in the nose whereas inflammation was seen in the larynx (m:6/10; f: 5/10), trachea (m: 5/10, f: 1/10), and the lungs (m: 6/10; f: 3/10; alveolar macrophages 5/10) at the high dose. The inflammation was, however, minimal to mild and was not considered to be of toxicological relevance.

 

There were, however, two adverse effects:

1. Hyaline droplet nephropathy was seen in all high-dose males (10/10), identified immunohistochemically as alpha2u-globulin nephropathy which is known to be specific for the male rat. Low-and mid-dose rats were not examined because the effect is not relevant for human risk assessment. Hence, no NOAEL was established for this effect.

2. Ovaries of the high-dose females appeared to be quite large (mean weight +50%), which was attributed to a high number of corpora lutea that was seen in histopathology in 9/10 females. No such change was seen in low- and mid-dose females.

Hence, two adverse effects were seen in this study, i.e. alpha2u-globulin nephropathy in male rats (LOAEL: 4980 mg/m³) which is not relevant for human risk assessment, and adverse effects on the female ovary (NOAEL 2020 mg/m³). The latter corresponds to 582 mg/kg bw and day, based on the assumptions made by ECHA (Guidance R8 : absorption by the inhalation route 100%, and default values in Table R.8-2) (TNO, 2014).

 

The study is considered valid for assessment.

Discussion

The toxicological relevance of the changes observed in the ovary in the 90 -day inhalation study is not known. The absolute and relative weights of thymus, thyroid, and testes (males), and of relative thymus, thyroid weights (females) were not affected. Further, histopathology revealed no effects (high dose animals) in the following organs (selection):

males: testes, epididymides, seminal vesicles; thyroid gland.

females: mammary gland, thyroid gland, pituitary gland

The NOAEL(rat, oral) was 500 mg/kg bw and day in the screening study, whereas in the inhalation study the NOAEL (rat, inhal, 90d) was 2020 mg/m³ which corresponds to approx. 582 mg/kg bw and day.

 

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
High quality 90-day. Histopathology reveals irritation in the lower respiratory tract (larynx, trachea, lungs) in up to 50% of the high dose animals (4980 mg/m³). The nose was, however, not affected. The inflammation observed was minimlal to mild and not considered to be toxicologically significant.

Repeated dose toxicity: inhalation - systemic effects (target organ) urogenital: other

Justification for classification or non-classification

No classification according to regulations 1272/2008/EC and 67/548/EEC required.