Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Norbornene HP (Bicyclo(2.2.1)hept-2-ene)
- Substance type: bridged cyclic hydrocarbon
- Physical state: solid
- Analytical purity: 99.79% (GC analysis; Annex 2)
- Date of analysis: 2014-03-14 (Annex 2)
- Batch No.: 119710-001 to 119710-015
- Expiration date of the batch: 7 October 2015
- Stability under test conditions: no data; stability in water: stable over at least 96 hours at ambient temperature
- Storage condition of test material: at 2-10°C, under nitrogen

Other:
Molecular weight: 94.16 g/mol
Vapour pressure: 301 hPa at 59°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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/m3)

Mid conc.

(2 g/m3)

High conc.

(5 g/m3)

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

 

 

icr

 

ica,r

ica,r

♂ DR;likely to be related to α2u-globulin nephropathy.

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

  Liver

 

 

 

icr

ica,r

ica,r

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

♀ DR

  Spleen

 

 

 

 

 

ica,r

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

  Thymus

 

 

 

 

 

ica,r

  Ovaries

 

 

 

 

 

ica,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.

Applicant's summary and conclusion

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