Registration Dossier

Administrative data

Description of key information

The test item cyclohexylvinylether was tested for repeated dose inhalation toxicity in a GLP study with rats according  to OECD 413. The NOAEC for systemic effects was determined to be 500 mg/m³ bw . For local effects a LOEC of 100 mg/m³ was determined. No oral and dermal repeated dose study was conducted since inhalation is the relevant route of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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:
2011-08-23 to 2012-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline. The study is scientifically well documented and complete in any parts.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
adopted : 2009-09-07
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Version / remarks:
2008-05-31
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
adopted: 1998-08
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 6 weeks
- Weight at study initiation:
- male animals mean group 0= 221.4g; group 1= 225.5g; group 2 = 221.8 g, group 3 222.8 g.
-female animals mean group 0= 181.8g; group 1= 179.1g; group 2= 182.4g; group 3= 178.5g
- Assigned to test groups randomly: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program
- Housing:Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet : mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water : tab water
- Acclimation period: 21 day; For acclimatize the animals to the exposure conditions they were exposed to fresh air on three days before start of the exposure period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark (06.00 p.m. - 06.00 a.m. dark) and 12 hours light (06.00 a.m. - 06.00 p.m.)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE
The test substance was used unchanged.
For each concentration, the test substance was supplied to the two-component atomizer of a tempered vaporizer at a constant rate by means of the metering pump. The vapor / air mixture was generated by spraying the test substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system. The control group was exposed to conditioned air.

CHAMBER DESCRIPTION
- Exposure apparatus: Head-nose exposure system: INA 60, volume V=90L; BASF SE
The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.

- Temperature, humidity, pressure in air chamber: 50% +/- 20% humidity, 22°C+/-2°C
- Air flow rate: 5.3-5.9 m³/h
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by online gas chromatography (GC). Due to unforeseeable technical problem of the on-line analyses the data from 08 to 20 Sep 2011 were not valid. Therefore, absorption samples were taken from the atmospheres and the samples were analyzed off-line by GC from the very beginning of the study until including 07 Oct 2011. The results of the online analyses were used from 10 Oct 2011 onwards (08 and 09 Oct 2011 were exposure-free). Both online measurement and offline analyses were performed during the time between 21 Sep and 07 Oct 2011. This was to ensure the reliability of the online system.

The sample volumes were adjusted to achieve amounts of test substance in the samples within the calibration range of the analytical method.
• Sampling site: immediately adjacent to the animals' noses at a separate spare port
• Sampling frequency: 2 samples for test group 1 per exposure 3 samples for test groups 2 and 3 per exposure
The samples were drawn through the three absorption vessels connected in series, each of which was filled with 2-propanol as absorption solvent. After the sampling, the content of the probe and the first two absorption vessels was eluted and pooled into a 50 mL graduated flask for individual analysis. After the final sampling of each exposure, the content of the last absorption vessel was transferred to a 50 mL graduated flask and analyzed separately to check for the absorbing efficiency of sampling. For the online analyses, daily means were calculated based on 3 to 6 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The measured values over one day give information about the constancy of the concentrations over the exposure time.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week for 90 days
Remarks:
Doses / Concentrations:
100 mg/m³, 500 mg/m³; 1500 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
131.5 mg/m³; 617.5 mg/m³; 1814.5 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale:
In a previous range finding study, groups of five time-mated female male rats were exposed nose-only to the vapor of cyclohexylvinylether for 6 hours per day on 14 consecutive days, from gestation day 6 through to gestation day 19. The target concentrations were 2000, 5000 and 10000 mg/m³. A concurrent control group was exposed to clean air. Clinical signs of toxicity comprised apathy, unsteady gate and poor general condition at the high concentration group. Retarded (net) body weight development and reduced food consumption were observed at 5000 mg/m3 and higher. Thus the mid and high concentrations (5000 and 10000 mg/m³) clearly exceed the maximal tolerated concentration for subsequent screening study according to OECD 413. The histopathological investigation revealed centrilobular hepatocellular degeneration, centrilobular hepatocellular hyperplasia and degeneration of the olfactory epithelium in level I and III of the nasal cavity. The incidence and severity of these findings are related to exposure concentration and were still present at the low concentration of 2000 mg/m³. At cesarean section the uterus weight was significantly decreased in the high concentration group (10 % of the control) and in the medium group (44 % of the control). The decreased uteri weight was only observed in presence of clear maternal toxicity as indicated by reduced (net) body weight and food consumption. Based on available data, considering the longer exposure time the following concentrations were selected for the present study:
1500 mg/m³: high concentration causing toxic effects
500 mg/m³: medium concentration
100 mg/m³: low concentration and expected NOAEC
Positive control:
Not available
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait

Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and
observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nasal discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements/ stereotypies
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (amount/color) within two minutes
18. number of rearings within two minutes

DETAILED CLINICAL OBSERVATIONS:
A clinical inspection was performed on each animal at least three times on exposure days and once a day during pre-exposure and post exposure observation days. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.
All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0), at midterm on study day 49 for females and on study day 53 for males, against the end of the exposure period on study day 84 for females and on study day 88 for males. As a rule, the examination was performed in the morning. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed were based on the current index of findings in PDS ToxData® software including but not limited to the following parameters listed:
1. Abnormal behavior in handling
2. Condition of fur
3. Condition of skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the study
17. Pupil size

BODY WEIGHT:
The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter (Monday and Friday) until the day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.

FOOD CONSUMPTION:
Food consumption was determined weekly (e.g. Monday-Friday) and calculated as mean food consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION:
Before the start of the exposure period (day -2 males; -6 females) the eyes of all animals, and against the end of the exposure period (study day 90 males and study day 85 females) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY:
In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Parameters and methods:
1. Leukocyte count (WBC)
2. Erythrocyte count (RBC)
3. Hemoglobin (HGB)
4. Hematocrit (HCT)
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelet count (PLT)
9. Differential blood count
10. Reticulocytes
11. Clotting tests were carried out using a ball coagulometer: Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
Enzyme (systematic name andsystem number) :
1. Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.)
2. Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)
3. Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.)
4. γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase; EC 2.3.2.2.)
Blood Chemistry Parameter:
1. Sodium
2. Potassium
3. Chloride
4. Inorganic phosphate
5. Calcium
6. Urea
7. Creatinine (CREA)
8. Glucose (GLUC)
9. Total bilirubin (TBIL)
10 . total protein (TPROT)
11. Albumin (ALB)
12. Globulins (GLOB)
13. Triglycerides (TRIG)
14. Cholesterol (CHOL)
15. Magnesium
Sacrifice and pathology:
GROSS PATHOLOGY:
The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology. The animal (female animal no. 57) that died intercurrently was necropsied as soon as possible after death and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Spleen
11. Testes
12. Thymus
13. Thyroid glands
14. Uterus

Organ / Tissue preservation list
The following organs or tissues were fixed in 4% formaldehyde solution or modified
Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain with olfactory bulb
6. Cecum
7. Colon
8. Duodenum
9. Epididymides (fixed in modified Davidson 's solution)
10. Esophagus
11. Extraorbital lacrimal gland
12. Eyes with optic nerve and eyelid
13. Femur with knee joint
14. Harderian glands
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Larynx
20. Liver
21. Lung
22. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
23. Mammary gland (male + female)
24. Nose (nasal cavity)
25. Ovaries
26. Pharynx
27. Pancreas
28. Parathyroid glands
29. Pituitary gland
30. Prostate
31. Rectum
32. Salivary glands (mandibular and sublingual glands)
33. Sciatic nerve
34. Seminal vesicles
35. Skeletal muscle
36. Skin
37. Spinal cord (cervical, thoracic and lumbar cord)
38. Spleen
39. Sternum with marrow
40. Stomach (forestomach and glandular stomach)
41. Teeth
42. Testes (fixed in modified Davidson 's solution)
43. Thymus
44. Thyroid glands
45. Tongue
46. Trachea
47. Ureter
48. Urethra
49. Urinary bladder
50. Uterus

HISTOPATHOLOGY:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings
Please refer to table in any other information on materials and methods
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Brain
5. Bone marrow (femur)
6. Cecum
7. Colon
8. Duodenum
9. Esophagus
10. Femur with knee joint
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Larynx (3 levels)
16. Liver
17. Lung
18. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
19. Mammary gland (female)
20. Nasal cavity (4 levels)
21. Ovaries
22. Pancreas
23. Pharynx
24. Parathyroid glands
25. Pituitary gland
26. Prostate
27. Rectum
28. Salivary glands (mandibular and sublingual glands)
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Skin
33. Spinal cord (cervical, thoracic and lumbar cord)
34. Spleen
35. Sternum with marrow
36. Stomach (forestomach and glandular stomach)
37. Teeth
38. Testes
39. Thymus
40. Thyroid glands
41. Trachea
42. Urinary bladder
43. Uterus
44. Epididymides
45. Eyes with optic nerve
46. Extraorbital lacrimal glands
47. Harderian glands
Other examinations:
NA
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
Body weight, body weight change :
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
References: DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096 – 1121
Fecal pellets, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
References:SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
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):
increase of liver and kidney weights in 500 mg/m³ and 1500 mg/m³ test groups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male and female animals at all concentration levels including control.

MORTALITY
One female animal of test group 1 died on study day 7 after exposure. Because there were no other death, it was considered to be not substance-related.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the male and female animals of test groups 1 and 2 were comparable to the concurrent control group, as well as the mean body weights of test group 3 females. The mean body weights of test group 3 males tended to be lower than the concurrent control especially from mid-term onward. However, on most of the measurement days the mean body weights were statistically not different to the control, with exception of day 81 (p < 0.05). In general the mean body weight changes of all test groups were statistically not different to the control during the study period. The exceptions were:
• Male, test group 2 (day 25->29)
• Female, test group 3 (day 56 ->60)
Both changes were regarded as incidental, because there was no concentration-response relationship in males, no relation to exposure time in females of test group 3.

FOOD CONSUMPTION
Due to social housing of the animals, food consumption was determined for cages of 5 animals. As there were only two cages for each concentration group and sex, no statistical evaluation for food consumption can be performed. Food consumption of the male and female animals in test groups 1-3 (100, 500 or 1500 mg/m³) was comparable to the concurrent control group throughout the entire study.

OPHTHALMOSCOPIC EXAMINATION
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

HAEMATOLOGY
No treatment-related, adverse changes among hematological parameters were observed. In males of test groups 2 and 3 (500 and 1500 mg/m3) hemoglobin and hematocrit values were lower compared to controls. The decreases of the means in the high test group were marginal (hemoglobin – 3.3%, hematocrit -2.9%), so that the alterations were regarded as treatment-related, but not adverse (Müller et al, 2006). In males of test group 1 (100 mg/m3) relative reticulocyte counts were lower compared to controls, but they were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
In rats of both sexes in test group 3 (1500 mg/m3) alkaline phosphatase (ALP) activities were increased. Additionally, in females of the same test group triglyceride levels were increased. In males of test group 2 (500 mg/m3) alanine aminotransferase (ALT) activities were lower compared to controls but the means were not dose-dependently altered. In males of test groups 2 and 3 (500 and 1500 mg/m3) potassium levels were decreased and in females of the same test groups chloride concentrations were lower compared to controls. All electrolyte level means were within historical control ranges (males, potassium 4.35-4.95 mmol/L; females, chloride 100.2-107.8 mmol/L, PART III, Supplement). Therefore, the mentioned alterations of ALT activities and both electrolytes levels were regarded as incidental and not adverse.

NEUROBEHAVIOUR
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substance. Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Observations on day 90 males:
Home cage observations: No substance-related findings were observed. Open field observation: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed.

Observations on day 87 females:
Home cage observations: No substance-related findings were observed. Open field observations: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed.

There were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
Absolute organ weights
When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased: please refere to table 1 in section any other information on results
Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased or decreased : please refere to table 2 in section any other information on results

Reduction of heart weights in male (relative weights) and female (relative and absolute weights) animals in test group 1 (100 mg/m³) were regarded as incidental as there was no clear dose response and a morphological correlate was missing. A histopathological correlate was also not detected in lung and spleen of male animals of test group 3 (1500 mg/m³), a treatment related effect is unlikely and considered secondary to the slightly but not significantly decreased body weights (-6%) in this test group. The increase of liver and kidney weights in females of test group 2 (500 mg/m³) and in animals of both sexes in test group 3 (1500 mg/m³) was considered to be related to treatment.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no remarkable gross lesions in female animal 57 which died prematurely.


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment - related findings were noted in liver and nasal cavity (levels I, II and III) with incidences and grading according to the table 3-5 in section any other information on results

In the nasal cavity, eosinophilic globules in the olfactory epithelium were seen in male and female animals of test groups 1 to 3 (100, 500, 1500 ppm) in level I and in test group 3 (1500 mg/m³) animals of both sexes in level II; one single male animal of test group 1 (100 mg/m³) also showed minimal eosinophilic globules in level II. In level III of the nasal cavity, eosinophilic globules were noted only in 2/10 female test group 3 (1500 mg/m³) animals. Degeneration/ regeneration of the olfactory epithelium were observed in level I of the nasal cavity in a few female animals of all treated test groups (100, 500, 1500 ppm). In level II, degeneration/ regeneration of the olfactory epithelium of the nasal cavity were observed in a few animals of both sexes of test group 3 (1500 mg/m³).

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no histopathological findings in animal 57 to explain its death.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
500 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Remarks:
local effect
Effect level:
100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased (printed in bold):

 

Table 1 absolute organ weight (%) compared to controls

 

Male animals

Female animals

Test group

(mg/m³)

1
(100)

2

(500)

3

(1500)

1
(100)

2

(500)

3

(1500)

Heart

 

 

 

91%**

104%

101%

Kidneys

 

 

 

97%

108%*

109%

Liver

101%

101%

110%

95%

111%*

122%**

*p <= 0.05; **p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

 

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased or decreased (printed in bold):

Table 2 relative organ weights (%) compared to controls

 

Male animals

Female animals

Test group

(mg/m³)

1
(100)

2

(500)

3

(1500)

1
(100)

2

(500)

3

(1500)

Heart

89%

94%

97%

94%*

103%

104%

Kidneys

100%

105%

115%**

100%

107%**

111%*

Liver

100%

103%

117%**

98%

110%**

124%**

Lungs

100%

103%

108%*

 

 

 

Spleen

95%

105%

113%*

 

 

 

*p <= 0.05; **p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

 

Histopathology

 

Table 3 Incidence and severity of the histological findings in liver

 

Liver

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

Hypertrophy, centrilobular

 

 

 

10

• Grade 1

 

 

 

5

• Grade 2

 

 

 

5

Necrosis, centrilobular

 

 

 

4

• Grade 1

 

 

 

4

 

Table 4 Incidence and severity of the histological findings in nasal cavity level I

Nasal cavity, level I

Male animals

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

10

9*

10

10

Globules, eosinophilic,

multifocal

 

1

4

4

 

5

5

3

• Grade 1

 

1

4

4

 

5

4

 

• Grade 2

 

 

 

 

 

 

1

3

Degeneration/

regeneration, olfactory

epithelium

 

 

 

 

 

3

5

3

• Grade 1

 

 

 

 

 

3

5

 

• Grade 2

 

 

 

 

 

 

 

3

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

 

Table 5 Incidence and severity of the histological findings in nasal cavity level II

Nasal cavity, level II

Male animals

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

10

9*

10

10

Globules, eosinophilic,

multifocal

 

1

 

4

 

 

 

4

• Grade 1

 

1

 

4

 

 

 

3

• Grade 2

 

 

 

 

 

 

 

1

Degeneration/

regeneration, olfactory

epithelium

 

 

 

4

 

 

 

3

• Grade 1

 

 

 

4

 

 

 

3

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

 

Table 6 Incidence and severity of the histological findings in nasal cavity level III

Nasal cavity, level III

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

9*

10

10

Globules, eosinophilic,

multifocal

 

 

 

2

• Grade 1

 

 

 

2

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no histopathological findings in animal 57 to explain its death.

 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline conform study.

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:
2011-08-23 to 2012-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline. The study is scientifically well documented and complete in any parts.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
adopted : 2009-09-07
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Version / remarks:
2008-05-31
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
adopted: 1998-08
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 6 weeks
- Weight at study initiation:
- male animals mean group 0= 221.4g; group 1= 225.5g; group 2 = 221.8 g, group 3 222.8 g.
-female animals mean group 0= 181.8g; group 1= 179.1g; group 2= 182.4g; group 3= 178.5g
- Assigned to test groups randomly: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program
- Housing:Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet : mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water : tab water
- Acclimation period: 21 day; For acclimatize the animals to the exposure conditions they were exposed to fresh air on three days before start of the exposure period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark (06.00 p.m. - 06.00 a.m. dark) and 12 hours light (06.00 a.m. - 06.00 p.m.)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE
The test substance was used unchanged.
For each concentration, the test substance was supplied to the two-component atomizer of a tempered vaporizer at a constant rate by means of the metering pump. The vapor / air mixture was generated by spraying the test substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system. The control group was exposed to conditioned air.

CHAMBER DESCRIPTION
- Exposure apparatus: Head-nose exposure system: INA 60, volume V=90L; BASF SE
The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.

- Temperature, humidity, pressure in air chamber: 50% +/- 20% humidity, 22°C+/-2°C
- Air flow rate: 5.3-5.9 m³/h
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by online gas chromatography (GC). Due to unforeseeable technical problem of the on-line analyses the data from 08 to 20 Sep 2011 were not valid. Therefore, absorption samples were taken from the atmospheres and the samples were analyzed off-line by GC from the very beginning of the study until including 07 Oct 2011. The results of the online analyses were used from 10 Oct 2011 onwards (08 and 09 Oct 2011 were exposure-free). Both online measurement and offline analyses were performed during the time between 21 Sep and 07 Oct 2011. This was to ensure the reliability of the online system.

The sample volumes were adjusted to achieve amounts of test substance in the samples within the calibration range of the analytical method.
• Sampling site: immediately adjacent to the animals' noses at a separate spare port
• Sampling frequency: 2 samples for test group 1 per exposure 3 samples for test groups 2 and 3 per exposure
The samples were drawn through the three absorption vessels connected in series, each of which was filled with 2-propanol as absorption solvent. After the sampling, the content of the probe and the first two absorption vessels was eluted and pooled into a 50 mL graduated flask for individual analysis. After the final sampling of each exposure, the content of the last absorption vessel was transferred to a 50 mL graduated flask and analyzed separately to check for the absorbing efficiency of sampling. For the online analyses, daily means were calculated based on 3 to 6 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The measured values over one day give information about the constancy of the concentrations over the exposure time.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week for 90 days
Remarks:
Doses / Concentrations:
100 mg/m³, 500 mg/m³; 1500 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
131.5 mg/m³; 617.5 mg/m³; 1814.5 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale:
In a previous range finding study, groups of five time-mated female male rats were exposed nose-only to the vapor of cyclohexylvinylether for 6 hours per day on 14 consecutive days, from gestation day 6 through to gestation day 19. The target concentrations were 2000, 5000 and 10000 mg/m³. A concurrent control group was exposed to clean air. Clinical signs of toxicity comprised apathy, unsteady gate and poor general condition at the high concentration group. Retarded (net) body weight development and reduced food consumption were observed at 5000 mg/m3 and higher. Thus the mid and high concentrations (5000 and 10000 mg/m³) clearly exceed the maximal tolerated concentration for subsequent screening study according to OECD 413. The histopathological investigation revealed centrilobular hepatocellular degeneration, centrilobular hepatocellular hyperplasia and degeneration of the olfactory epithelium in level I and III of the nasal cavity. The incidence and severity of these findings are related to exposure concentration and were still present at the low concentration of 2000 mg/m³. At cesarean section the uterus weight was significantly decreased in the high concentration group (10 % of the control) and in the medium group (44 % of the control). The decreased uteri weight was only observed in presence of clear maternal toxicity as indicated by reduced (net) body weight and food consumption. Based on available data, considering the longer exposure time the following concentrations were selected for the present study:
1500 mg/m³: high concentration causing toxic effects
500 mg/m³: medium concentration
100 mg/m³: low concentration and expected NOAEC
Positive control:
Not available
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait

Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and
observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nasal discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements/ stereotypies
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (amount/color) within two minutes
18. number of rearings within two minutes

DETAILED CLINICAL OBSERVATIONS:
A clinical inspection was performed on each animal at least three times on exposure days and once a day during pre-exposure and post exposure observation days. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.
All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0), at midterm on study day 49 for females and on study day 53 for males, against the end of the exposure period on study day 84 for females and on study day 88 for males. As a rule, the examination was performed in the morning. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed were based on the current index of findings in PDS ToxData® software including but not limited to the following parameters listed:
1. Abnormal behavior in handling
2. Condition of fur
3. Condition of skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the study
17. Pupil size

BODY WEIGHT:
The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter (Monday and Friday) until the day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.

FOOD CONSUMPTION:
Food consumption was determined weekly (e.g. Monday-Friday) and calculated as mean food consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION:
Before the start of the exposure period (day -2 males; -6 females) the eyes of all animals, and against the end of the exposure period (study day 90 males and study day 85 females) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY:
In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Parameters and methods:
1. Leukocyte count (WBC)
2. Erythrocyte count (RBC)
3. Hemoglobin (HGB)
4. Hematocrit (HCT)
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelet count (PLT)
9. Differential blood count
10. Reticulocytes
11. Clotting tests were carried out using a ball coagulometer: Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
Enzyme (systematic name andsystem number) :
1. Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.)
2. Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)
3. Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.)
4. γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase; EC 2.3.2.2.)
Blood Chemistry Parameter:
1. Sodium
2. Potassium
3. Chloride
4. Inorganic phosphate
5. Calcium
6. Urea
7. Creatinine (CREA)
8. Glucose (GLUC)
9. Total bilirubin (TBIL)
10 . total protein (TPROT)
11. Albumin (ALB)
12. Globulins (GLOB)
13. Triglycerides (TRIG)
14. Cholesterol (CHOL)
15. Magnesium
Sacrifice and pathology:
GROSS PATHOLOGY:
The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology. The animal (female animal no. 57) that died intercurrently was necropsied as soon as possible after death and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Spleen
11. Testes
12. Thymus
13. Thyroid glands
14. Uterus

Organ / Tissue preservation list
The following organs or tissues were fixed in 4% formaldehyde solution or modified
Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain with olfactory bulb
6. Cecum
7. Colon
8. Duodenum
9. Epididymides (fixed in modified Davidson 's solution)
10. Esophagus
11. Extraorbital lacrimal gland
12. Eyes with optic nerve and eyelid
13. Femur with knee joint
14. Harderian glands
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Larynx
20. Liver
21. Lung
22. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
23. Mammary gland (male + female)
24. Nose (nasal cavity)
25. Ovaries
26. Pharynx
27. Pancreas
28. Parathyroid glands
29. Pituitary gland
30. Prostate
31. Rectum
32. Salivary glands (mandibular and sublingual glands)
33. Sciatic nerve
34. Seminal vesicles
35. Skeletal muscle
36. Skin
37. Spinal cord (cervical, thoracic and lumbar cord)
38. Spleen
39. Sternum with marrow
40. Stomach (forestomach and glandular stomach)
41. Teeth
42. Testes (fixed in modified Davidson 's solution)
43. Thymus
44. Thyroid glands
45. Tongue
46. Trachea
47. Ureter
48. Urethra
49. Urinary bladder
50. Uterus

HISTOPATHOLOGY:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings
Please refer to table in any other information on materials and methods
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Brain
5. Bone marrow (femur)
6. Cecum
7. Colon
8. Duodenum
9. Esophagus
10. Femur with knee joint
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Larynx (3 levels)
16. Liver
17. Lung
18. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
19. Mammary gland (female)
20. Nasal cavity (4 levels)
21. Ovaries
22. Pancreas
23. Pharynx
24. Parathyroid glands
25. Pituitary gland
26. Prostate
27. Rectum
28. Salivary glands (mandibular and sublingual glands)
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Skin
33. Spinal cord (cervical, thoracic and lumbar cord)
34. Spleen
35. Sternum with marrow
36. Stomach (forestomach and glandular stomach)
37. Teeth
38. Testes
39. Thymus
40. Thyroid glands
41. Trachea
42. Urinary bladder
43. Uterus
44. Epididymides
45. Eyes with optic nerve
46. Extraorbital lacrimal glands
47. Harderian glands
Other examinations:
NA
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
Body weight, body weight change :
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
References: DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096 – 1121
Fecal pellets, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
References:SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
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):
increase of liver and kidney weights in 500 mg/m³ and 1500 mg/m³ test groups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male and female animals at all concentration levels including control.

MORTALITY
One female animal of test group 1 died on study day 7 after exposure. Because there were no other death, it was considered to be not substance-related.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the male and female animals of test groups 1 and 2 were comparable to the concurrent control group, as well as the mean body weights of test group 3 females. The mean body weights of test group 3 males tended to be lower than the concurrent control especially from mid-term onward. However, on most of the measurement days the mean body weights were statistically not different to the control, with exception of day 81 (p < 0.05). In general the mean body weight changes of all test groups were statistically not different to the control during the study period. The exceptions were:
• Male, test group 2 (day 25->29)
• Female, test group 3 (day 56 ->60)
Both changes were regarded as incidental, because there was no concentration-response relationship in males, no relation to exposure time in females of test group 3.

FOOD CONSUMPTION
Due to social housing of the animals, food consumption was determined for cages of 5 animals. As there were only two cages for each concentration group and sex, no statistical evaluation for food consumption can be performed. Food consumption of the male and female animals in test groups 1-3 (100, 500 or 1500 mg/m³) was comparable to the concurrent control group throughout the entire study.

OPHTHALMOSCOPIC EXAMINATION
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

HAEMATOLOGY
No treatment-related, adverse changes among hematological parameters were observed. In males of test groups 2 and 3 (500 and 1500 mg/m3) hemoglobin and hematocrit values were lower compared to controls. The decreases of the means in the high test group were marginal (hemoglobin – 3.3%, hematocrit -2.9%), so that the alterations were regarded as treatment-related, but not adverse (Müller et al, 2006). In males of test group 1 (100 mg/m3) relative reticulocyte counts were lower compared to controls, but they were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
In rats of both sexes in test group 3 (1500 mg/m3) alkaline phosphatase (ALP) activities were increased. Additionally, in females of the same test group triglyceride levels were increased. In males of test group 2 (500 mg/m3) alanine aminotransferase (ALT) activities were lower compared to controls but the means were not dose-dependently altered. In males of test groups 2 and 3 (500 and 1500 mg/m3) potassium levels were decreased and in females of the same test groups chloride concentrations were lower compared to controls. All electrolyte level means were within historical control ranges (males, potassium 4.35-4.95 mmol/L; females, chloride 100.2-107.8 mmol/L, PART III, Supplement). Therefore, the mentioned alterations of ALT activities and both electrolytes levels were regarded as incidental and not adverse.

NEUROBEHAVIOUR
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substance. Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Observations on day 90 males:
Home cage observations: No substance-related findings were observed. Open field observation: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed.

Observations on day 87 females:
Home cage observations: No substance-related findings were observed. Open field observations: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed.

There were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
Absolute organ weights
When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased: please refere to table 1 in section any other information on results
Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased or decreased : please refere to table 2 in section any other information on results

Reduction of heart weights in male (relative weights) and female (relative and absolute weights) animals in test group 1 (100 mg/m³) were regarded as incidental as there was no clear dose response and a morphological correlate was missing. A histopathological correlate was also not detected in lung and spleen of male animals of test group 3 (1500 mg/m³), a treatment related effect is unlikely and considered secondary to the slightly but not significantly decreased body weights (-6%) in this test group. The increase of liver and kidney weights in females of test group 2 (500 mg/m³) and in animals of both sexes in test group 3 (1500 mg/m³) was considered to be related to treatment.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no remarkable gross lesions in female animal 57 which died prematurely.


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment - related findings were noted in liver and nasal cavity (levels I, II and III) with incidences and grading according to the table 3-5 in section any other information on results

In the nasal cavity, eosinophilic globules in the olfactory epithelium were seen in male and female animals of test groups 1 to 3 (100, 500, 1500 ppm) in level I and in test group 3 (1500 mg/m³) animals of both sexes in level II; one single male animal of test group 1 (100 mg/m³) also showed minimal eosinophilic globules in level II. In level III of the nasal cavity, eosinophilic globules were noted only in 2/10 female test group 3 (1500 mg/m³) animals. Degeneration/ regeneration of the olfactory epithelium were observed in level I of the nasal cavity in a few female animals of all treated test groups (100, 500, 1500 ppm). In level II, degeneration/ regeneration of the olfactory epithelium of the nasal cavity were observed in a few animals of both sexes of test group 3 (1500 mg/m³).

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no histopathological findings in animal 57 to explain its death.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
500 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Remarks:
local effect
Effect level:
100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased (printed in bold):

 

Table 1 absolute organ weight (%) compared to controls

 

Male animals

Female animals

Test group

(mg/m³)

1
(100)

2

(500)

3

(1500)

1
(100)

2

(500)

3

(1500)

Heart

 

 

 

91%**

104%

101%

Kidneys

 

 

 

97%

108%*

109%

Liver

101%

101%

110%

95%

111%*

122%**

*p <= 0.05; **p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

 

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased or decreased (printed in bold):

Table 2 relative organ weights (%) compared to controls

 

Male animals

Female animals

Test group

(mg/m³)

1
(100)

2

(500)

3

(1500)

1
(100)

2

(500)

3

(1500)

Heart

89%

94%

97%

94%*

103%

104%

Kidneys

100%

105%

115%**

100%

107%**

111%*

Liver

100%

103%

117%**

98%

110%**

124%**

Lungs

100%

103%

108%*

 

 

 

Spleen

95%

105%

113%*

 

 

 

*p <= 0.05; **p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

 

Histopathology

 

Table 3 Incidence and severity of the histological findings in liver

 

Liver

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

Hypertrophy, centrilobular

 

 

 

10

• Grade 1

 

 

 

5

• Grade 2

 

 

 

5

Necrosis, centrilobular

 

 

 

4

• Grade 1

 

 

 

4

 

Table 4 Incidence and severity of the histological findings in nasal cavity level I

Nasal cavity, level I

Male animals

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

10

9*

10

10

Globules, eosinophilic,

multifocal

 

1

4

4

 

5

5

3

• Grade 1

 

1

4

4

 

5

4

 

• Grade 2

 

 

 

 

 

 

1

3

Degeneration/

regeneration, olfactory

epithelium

 

 

 

 

 

3

5

3

• Grade 1

 

 

 

 

 

3

5

 

• Grade 2

 

 

 

 

 

 

 

3

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

 

Table 5 Incidence and severity of the histological findings in nasal cavity level II

Nasal cavity, level II

Male animals

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

10

10

10

10

9*

10

10

Globules, eosinophilic,

multifocal

 

1

 

4

 

 

 

4

• Grade 1

 

1

 

4

 

 

 

3

• Grade 2

 

 

 

 

 

 

 

1

Degeneration/

regeneration, olfactory

epithelium

 

 

 

4

 

 

 

3

• Grade 1

 

 

 

4

 

 

 

3

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

 

Table 6 Incidence and severity of the histological findings in nasal cavity level III

Nasal cavity, level III

Female animals

Test group

(mg/m³)

0
(0)

1
(100)

2

(500)

3

(1500)

No. of animals

10

9*

10

10

Globules, eosinophilic,

multifocal

 

 

 

2

• Grade 1

 

 

 

2

*The nasal cavity of animal 57 (female group 1 (100 mg/m³)) was autolytic and could not be evaluated

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. There were no histopathological findings in animal 57 to explain its death.

 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
100 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline conform study.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Inhalation:

A 90-day inhalation study was conducted according to OECD TG 413, Regulation (EC) No 440/2008 method B.29 and EPA OPPTS 870.3465. Groups of ten male and ten female Wistar rats were head-nose exposed to dynamic inhalation atmosphere of cyclohexylvinylether for 6 hours on 5 days per week for 90 days. The targeted concentrations were 100 mg/m³, 500 mg/m³ and 1500 mg/m³. A concurrent control group of ten female and ten male animals was exposed to conditioned air. Clinical observations and body weight determinations were performed for all animals. The animals were examined at least three times on exposure days and once a day on the preflow period. Ophthalmoscopy was performed before (all test groups) and against the end (test groups 0 (control) and 3 (high dose)) of the exposure period. Functional observation battery (FOB) including reflex test and motor activity (MA) was examined against the end of exposure period in the first 5 animals of each group. Food consumption was determined weekly from Monday to Friday. After the last exposure blood samples were collected from the retroorbital venous plexus in the morning from fasted animals using isoflurane anesthesia. Clinical pathology examination of numerous parameters according to the corresponding test guidelines was performed.

 

Results

 

CLINICAL SIGNS

No clinical signs or changes of general behaviour, which may be attributed to the test substance, were detected in any male and female animals at all concentration levels including control. 

MORTALITY

One female animal of test group 1 (low dose) died on study day 7 after exposure. Because there were no other deaths, it was considered to be not substance-related. 

BODY WEIGHT AND WEIGHT GAIN

Mean body weights of the male and female animals of test groups 1 (low dose) and 2 (mid dose) were comparable to the concurrent control group, as well as the mean body weights of test group 3 females. The mean body weights of test group 3 males tended to be lower than the concurrent control especially from mid-term onward. Both changes were regarded as incidental, because there was no concentration-response relationship in males, no relation to exposure time in females of test group 3. 

FOOD CONSUMPTION

Food consumption of the male and female animals in test groups 1-3 (100, 500 or 1500 mg/m³) was comparable to the concurrent control group throughout the entire study.

 

OPHTHALMOSCOPIC EXAMINATION

Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

 

HAEMATOLOGY

No treatment-related, adverse changes among hematological parameters were observed.

 

CLINICAL CHEMISTRY

In rats of both sexes in test group 3 (1500 mg/m³) alkaline phosphatase (ALP) activities were increased. Additionally, in females of the same test group triglyceride levels were increased. In males of test group 2 (500 mg/m³) alanine aminotransferase (ALT) activities were lower compared to controls but the means were not dose-dependently altered. The alterations of ALT activities and both electrolytes levels were regarded as incidental and not adverse.

 

NEUROBEHAVIOUR

There were no statistically significant deviations from the control group.

 

ORGAN WEIGHTS

Reduction of heart weights in male (relative weights) and female (relative and absolute weights) animals in test group 1 (100 mg/m³) were regarded as incidental as there was no clear dose response and a morphological correlate was missing. A histopathological correlate was also not detected in lung and spleen of male animals of test group 3 (1500 mg/m³), a treatment related effect is unlikely and considered secondary to the slightly but not significantly decreased body weights (-6%) in this test group. The increase of liver and kidney weights in females of test group 2 (500 mg/m³) and in animals of both sexes in test group 3 (1500 mg/m³) was considered to be related to treatment.

 

GROSS PATHOLOGY

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

HISTOPATHOLOGY: NON-NEOPLASTIC

Treatment - related findings were noted in liver and nasal cavity (levels I, II and III).

 

Conclusion

The exposure of rats to cyclohexylvinylether did not cause any clinical signs of toxicity. No abnormalities were detected during FOB, MA and ophthalmology. A slightly decreased body weight, though not significant, was observed in group 3 male animals, while food consumption was not impaired. Histological examination revealed centrilobular hypertrophy and centrilobular necrosis in liver of females of test group 3. The morphological changes in liver were consistent with increased alkaline phosphatase activity and increased triglycerin level in blood. With regards of liver toxicity males were less susceptible than females, as only increased liver weight and increased alkaline phosphatase was observed in group 3. Thus, considering the findings in the liver, the No Observed Adverse Effect Concentration for systemic toxicity of cyclohexylvinylether was 500 mg/m³. With regard of local effect, degeneration of the olfactory epithelium was observed in nasal cavity I and II in all animals. The incidence and severity was related to the exposure concentration. Based on the findings in nasal cavitiy a NOAEC could not be derived for local effect under the current study condition.

In a 14 -day dose finding inhalation study covering the prenatal developmental toxicity in time-mated female Wistar rats the animals were exposed to 2000, 5000 and 10000 mg/m³ of the test item during gestation day 6 and 19. Maternal toxicity including clinical signs of toxicity (respiration, salivation, apathy, unsteady gate and poor general condition), reduced body weight development, food consumption, reduced spleen and thymus weights, increased liver, kidney and lung weights reduced size of the thymus on gross pathology and histological findings in liver, larynx and nasal cavity were observed. In histopathology, larynx, liver and nasal cavity are target organs and showed lesions still at the low target concentration. With regard to systemic and local toxicity, no NOAEC could be established.

Oral:

No oral repeated dose toxicity study was performed since exposure by inhalation is the most appropriate exposure route in humans. However, a 5 -day oral toxicity study was performed in rabbits (BASF,1964). A 5 % aqueous emulsion of the test item was administered in a dose of 0.5 mL/kg bw/day by oral gavage to two male and two female rabbits. The test item was applied once daily at five consecutive days.

One female animal died on day 2 and one male animal died on day 4 after daily application. The second female rabbit died on day 28 and the male rabbit died on day 39 within the post-application observation period. No clinical and pathology examinations were performed.

Dermal:

No dermal repeated dose toxicity study was performed since exposure by inhalation is the most appropriate exposure route for humans.

 

 


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one GLP and guideline study available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Only one GLP and guideline study available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Local effects on the skin were observed in a non-GLP acute irritation/corrosion study in rabbits performed in equivalence to OECD 404.

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Based on the results of the available studies, the test item was not classified and labelled according to Directive 67/548/EEC (DSD) and to Regulation 1272/2008 (CLP).