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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-18 to 2012-05-29
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(27 Jul 1995)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 413 (inhalation exposure) (adopted 07 Sep 2009)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.29
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

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: 10 to 12 weeks, male and female
- Weight at study initiation: Males: 286.44-305.01 g; Females: 196.47- 216.51g
- 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: Makrolon type M III
- Mating: 1 male and female were housed individually
- Pregnat females were housed individually
- Diet: milled mouse/rat laboratory diet "GLP" (Provimi Kliba SA, Kaiseraugust; Basel Switzerland)
- Water: tab water
- Acclimation period: 5 days before study

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. to 06.00 a.m.) and 12 hours light (06.00 a.m. to 06.00 p.m.)

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
nose/head only
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / 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.

Source and rate of air: 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 vapour / air mixture was generated by spraying the substance with compressed air into a counter current of activated charcoal filtered supply air. Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: 50% +/- 20% humidity, 22°C +/-2°C
- Air flow rate: 5.3-5.9 m³/h

TEST ATMOSPHERE
Brief description of analytical method used: The test atmosphere of 4 chambers in which rats were exposed to the test item was fed to gas chromatograph via a stream selector. Sequential chromatographic separations were performed on a non-polar stationary phase with flame ionization detection. It was quantified with external standard.
Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: mating was discontinued as soon as sperm was detected in the vaginal smear.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations. The concentrations of the inhalation atmospheres in test groups 1-3 were analyzed by online gas chromatography. 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 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 / exposure
Frequency of treatment:
Each workday over a time period suitable to 40 exposures
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/m³, 131.5 mg/m³; 674.2 mg/m³; 1752.1 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 mg/m³; 100 mg/m³; 500 mg/m³; 1500 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex in the control and dose groups
Control animals:
yes, concurrent no treatment
Details on study design:
In a previous range finding study, groups of five time-mated female male rats were exposed nose-only to the vapour 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 exceeded the maximal tolerated concentration for subsequent study according to OECD 421. 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 cesarean section the uterus weight was significantly decreased in the high concentration uteri weight was only observed in presence of clear maternal toxicity as indicated by reduced body weight and food consumption. Based on the available data, considering the longer exposure time the following concentrations were selected for the 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 applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
On non exposure days a cage side examination was conducted at least once daily for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented for each affected animal. The parturition and lactation behaviour of the dams was generally evaluated in the mornings in combination with the daily inspection of the dams. Only particular findings were documented on an individual dam basis. On weekdays the parturition behaviour of the dams was inspected in the afternoon in addition to the evaluations in the morning.

DETAILED CLINICAL OBSERVATIONS:
On exposure days, a clinical inspection was performed in each animal at least three times a day (before, during and after exposure).

BODY WEIGHT:
In general the body weight of the male and female parental animal was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight changes of the animals were calculated from these results. The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD0) and on GD7, 14 and 20
- Females with litter were weighed on the day of parturition (PND0) and on PND 1 and 4.

Body weight was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation periods.


FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals with the following exceptions:
-Food consumption was not determined after 2nd premating week and during the mating period
-Food consumption of the F0 females with evidence of sperm was determined for gestation days (GD) 0-7, 7-14, 14-20
-Food consumption of F0 females, which gave birth to a litter, was determined for determined for postnatal days (PND) 1-4.
Food consumption was not determined in females without positive evidence of sperm and females without litter.
Sperm parameters (parental animals):
Not applicable
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
All parental 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, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Liver
4. Lung
5. Testes

Organ/tissue fixation
The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Cervix
3. Coagulating glands
4. Epididymides (fixed in modified Davidson 's solution)
5. Larynx
6. Liver
7. Lung
8. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes)
9. Nose (nasal cavity)
10. Ovaries (modified Davidson’s solution)
11. Oviducts
12. Prostate gland
13. Seminal vesicles
14. Trachea
15. Testes (modified Davidson’s solution)
16. Vagina
17. Uterus

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings
Postmortem examinations (offspring):
The body weigth and clinical sign were examined.
Statistics:
Food consumption
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means
Reference : Dunnett, C.W . (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096¬ 1121

Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy :

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
Reference : Siegel, S . (1956): Non-parametric statistics for behavioral sciences. McGraw-Hill New York

Proportions of affected pups per litter with necropsy observations:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
Reference : Nijenhuis, A.; Wilf, H.S. (1978): Combinatorial Algorithms. Academic Press New York, 32-33

Weight parameters:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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
Reference : HETTMANNSPERGER, T.P. (1984): Statistical Inference based on Ranks, John Wiley & Sons New York, 132-140. International Mathematical and Statistical Libraries, Inc., 2500 Park West Tower One, Houston, Texas 77042-3020, USA, nakl-1 - nakl-3
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Offspring viability indices:
The viability index indicating pup mortality during lactation (PND 0-4) varied between 99% (test groups 0 and 1) and 100% (test groups 2 and 3) without showing any association to the treatment.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY.
There were no test substance-related or spontaneous mortalities in the male and female F0 parental animals of all test groups.
No clinical signs or changes of general behaviour, which may be attributed to the test substance, were detected in any male F0 generation parental animals at all dose levels including control. There were also no test substance-related clinical findings in female F0 animals in any test group during premating, gestation and lactation periods.

During gestation, out of 10 animals respectively, 3 females of the control, 5 females in the test group 1, 5 females in the test group 2 and 7 females in the test group 3 showed a brown-reddish vaginal discharge or vaginal haemorrhage during or after exposure. These findings occurred between GD13 and 15. Due to the lack of dose-response relationship and the fact, that animals in the control group were also affected, a test substance-relation could be excluded. Furthermore, for one control female piloerection before, during and after treatment on GD 14-20 was recorded.

Food consumption:
Food consumption of the male and female F0 parental animal in the test groups 1-3 was comparable to the concurrent control group throughout the entire study, covering premating, gestation and lactation periods.

Body weight
Mean Body weight and body weight changes of the male and female F0 parental animals in test groups 1-3 were comparable to the concurrent control group during premating, gestation and lactation periods.

Regarding pathology, treatment - related findings were observed in the liver and nasal cavity, level I in male rats.In the liver, minimal centrilobular hypertrophy in 9/10 male test group 3 (1702.0 mg/m³) animals was noted and degeneration of the olfactory epithelium in the nasal cavity, level I was seen in a few male test group 2 and 3 (631.0 and 1702.0 mg/m³, respectively) animals. The lung showed a mild weight increase in male test group 3 (1702.0 mg/m³) animals, histopathological correlate could, however, not be detected. The finding in liver was regarded as adaptive, while the degeneration of the olfactory epithelium in level I of the nasal cavity was regarded as adverse. In female animals, no any adverse effect was observed. As the exposure of the females continued to GD 19 and the gross necropsy was several days after lactation day 4, the effect may have recovered within the exposure-free period of 5 to 6 days.

Reproductive Indices
For all F0 parental males, which were placed with females to generate F1 pubs, copulation was confirmed. Thus, the males mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One control male, one low-dose male, three mid-dose males and one high dose male did not generate F1 pubs. No histomorphological correlate was found to explain these apparent infertilities. Thus, the male fertility index ranged between 70% and 90% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD0) varied between 2.3 and 3.1 days without any relation to dosing.
All sperm positive rats delivered pups or had implants in utero with the following exceptions:
- One control female, one low-dose female, three mid doses female and one high-dose female did not become pregnant. The fertility index varied between 70% in test group 2 and 90% in test group 0,1 and 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant female rats in all test groups did not show a histomorphological correlate to explain these apparent infertilities.
The mean duration of gestation was similar in all test groups (between 22.3 and 22.0 days). The gestation index was 100% in all test groups.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
118 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: In histopathology degeneration of olfactory epithelia was observed in animals exposed to 631.0 and 1702.0 mg/m³ test item.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY
The viability index indicating pup mortality during lactation (PND 0-4) varied between 99% (test group 0 and 1) and 100% (test groups 2 and 3) without showing any association to the treatment.

The mean number of delivered F1 pubs per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain of rats used in this study.

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between the test groups.

Clinical observations
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Body weight
Mean pup body weights and pup body weight changes of all test-substance-treated groups were generally comparable to the concurrent control group throughout the lactation period. Runts were seen in the following test groups:
One female in the control group, 2 males and 2 females in test group 1.

Necropsy observation
None of the F1 pups showed any test substance-related spontaneous findings at gross necropsy.

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
> 1 702 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion