Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
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
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.)
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
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
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.
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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.
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
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.
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'
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 702 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline conform study.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproduction toxicity

A study was conducted according to OECD TG 421 reproduction/developmental toxicity screening test in wistar rats administration via Inhalation exposure. Therefore, three test groups of 10 animals per sex were exposed to 118.0, 631.0 and 1702.0 mg/m³ test substance (100, 500 and 1500 mg/m³ target concentration). The test substance was exposed using a head-nose exposure system.Inhalation exposure of Cyclohexylvinylether was conducted during 2 weeks premating, mating, and gestation period. Generally, no toxicologically relevant reproductive or developmental difference was observed between animals exposed to measured concentrations of 118.0, 631.0 and 1702.0 mg/m³ Cyclohexylvinylether and controls. No relevant clinical signs of toxicity were observed in exposed parental animals during the premating and mating phases. These concentrations of Cyclohexylvinylether did not adversely impact the reproduction of these rats, nor did treatment impact delivery and pup viability. Furthermore, none of the F1 generation pups showed any evidence of developmental toxicity in response to 118.0, 631.0 and 1702.0 mg/m³ Cyclohexylvinylether. In histopathology degeneration of olfactory epithelia was observed in animals exposed to 631.0 and 1702.0 mg/m³ test item. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for Cyclohexylvinylether was determined to be >1702.0 mg/m³. Considering histopathological changes the NOAEC for Cyclohexylvinylether was 118.0 mg/m³ for parental local toxicity.

Waiving argumentation for 2 -generation reproduction toxixicty study:

In accordance with column 1 of REACH Annex IX, the two-generation study has to be performed if adverse effects on reproductive organs were observed in a 28 day or 90 day repeated dose study. Since there are no hints for adverse effects on reproduction in repeated dose toxicity studies (please refer to IUCLID section 7.5) and a reproduction toxicity screening test (inhalation exposure) according to OECD 421 the two-generation study (required in section 8.7.3 of Annex IX) does not need to be conducted for animal welfare reasons.


Short description of key information:
The test item cyclohexylvinylether was tested for reproduction toxicity in a GLP study according to OECD 421 via inhalation in rats. The NOAEC was determined to be 1702 mg/m³ bw in rats. No adverse effects on reproduction and development were noted up to the highest dose tested.

Justification for selection of Effect on fertility via oral route:
In accordance with column 2 of REACH Annex IX, the reproduction toxicity study has to be performed for the most approriate exposure route in humans (one route only). Since long-term exposure by inhalation is expected to be the most relevant route, a reproduction toxicity screening study by inhalation was conducted. Thus, the test reproduction toxicity after oral application (required in section 8.7 of Annex IX) is not needed. Risk assessment is based on the inhalation study.

Justification for selection of Effect on fertility via inhalation route:
Only one study available.

Justification for selection of Effect on fertility via dermal route:
In accordance with column 2 of REACH Annex IX, the reproduction toxicity study has to be performed for the most approriate exposure route in humans (one route only). Since long-term exposure by inhalation is expected to be the most relevant route, a reproduction toxicity screening study by inhalation was conducted. Thus, the test reproduction toxicity after dermal application (required in section 8.7 of Annex IX) is not needed. Risk assessment is based on the inhalation study.

Effects on developmental toxicity

Description of key information
In a read-across approach, the test item methylvinylether was tested for developmental toxicity/teratogenicity according  to OECD 414 via inhalation in rats. Based on these results the LOAEC for maternal toxicity was 5000 ppm. The NOAEC for teratogenicity/embryolethality was 19500 ppm and the NOAEC for developmental toxicity was 5000 ppm.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with acceptable restrictions (tabulated results not available; exposure at gestation day 6-15). Read across to Methylvinylether (CAS 107-25-5) was done. Both substances are vinylethers only differing in the side chain. Thus, read across is justified.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
Bushy Run Research Center
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Source: Charles River Lab
- Animals per dose: 25 time-pregnant rats per dose level
- Acclimatisation: 2 weeks prior to mating
- Mean body weight range at gestation day 0 225-227 g
- Food and water: Tap water and certified diet ad libitum (not during exposure)
- Housind: Dams housed singly
- Temperature: 66-77°F
- Light: day/night cycle: 12/12 h
- Humidity: rel. air humidity: 40-70%
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
The rats were exposed to vinyl methyl ether vapour or filtered air for 6 h/d on gestational days 6 through 15 in stainless steel inhalation chambers. The chamber volume was 900 l, the airflow 200 l/min (13 changes/h). Target concentrations were 0; 5000; 10000 or 19500 ppm. Temperature and humidity were recorded.
MVE vapour introduced directly into the chambers from MVE cylinder (flowmeter control) after dilution with air.
Temperature: ranged from 21.1 to 22.4°C
Relative humidity: ranged from 37.4 to 46.2%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of MVE in each chamber was monitored twice per hour during each 6-hour exposure by gas chromatography.
Details on mating procedure:
One male mated with one female; vaginal plugs controlled daily; gestation day (GD) 0 was the day the vaginal plug was detected.
Duration of treatment / exposure:
GD 6-15
Frequency of treatment:
Once daily, 6 hours/day
Duration of test:
GD 21
Remarks:
Doses / Concentrations:
0; 5000; 10000; 19500 ppm (12; 24; 47 mg/L)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 4946+-216, 10249+-465, and 19407+-796 ppm
Basis:
analytical conc.
average +- standard deviation
No. of animals per sex per dose:
25 pregnant dams
Control animals:
yes, concurrent vehicle
Details on study design:
Randomisation based on body weight at GD0
Maternal examinations:
Twice daily clinical observations
Body weight GD 0, 6, 9, 12, 15, 18, 21
Food and water consumption measured GD 0-21 in 3-day intervals
The dams were necropsied on gestational day (gd) 21 and evaluated for body weight. Maternal liver, kidneys, and lungs were weighed and retained in fixative.
Ovaries and uterine content:
Gravid uterine weight and pregnancy status determined;
Fetal examinations:
All fetuses were dissected from the uterus, counted, weighed, examined for determination of gender and for external variations and malformations (including cleft palate). Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the fetuses were examined for skeletal malformations and variations after staining with alizarin red S.
Statistics:
The data for quantitative continuous variables were intercompared for the exposure groups and the control group by use of Levene's test homogeneity of variance, by ANOVA, and by t-tests. T-tests were used if the ANOVA was significant to delineate which groups differed from the controls. If Levene's test indicated homogeneous variances, the groups were compared by ANOVA for equal variances. If Levene's test indicated heterogeneous variances, groups were compared by an ANOVA for unequal variances, followed, when appropriate, by t-tests. Frequency data were compared using Fisher's exact test. All statistical tests, except the frequency comparisons, were performed using computer software. The probability value of p<0.05 (two-tailed) was used as the critical level of significance for all tests.
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: No mortality occurred during the study. Pregnancy rate was equivalent for all groups and ranged form 96 - 100 %. No females aborted or delivered early. At scheduled necropsy, one female each from 5000 and 10000 ppm group was not pregnant.
Clinical signs of toxicity: There were no exposure-related clinical signs observed in any exposure group.
Body weights: body weight decreases were noted as described below. p<0.05 indicates statistically significant differences compared to the control animals group. 19500 ppm group: slightly decreased on gd 9; (p<0.05) on gd 12 and 15; slightly on gd 18 and 21. 10000 ppm group: slightly decreased on gd 12; (p<0.05) on gd 15; 5000 ppm group: slightly decreased on gd 12; (p<0.05) on gd 15; Body weight gain was significantly reduced in the 19500 ppm group on most of the 3-days intervals, and in the 10000 and 5000 ppm groups on some of the 3-day intervals. Following the exposure period, body weight gain was comparable or slightly higher in the exposure groups when compared to the controls.
Food consumption was decreased in the 195000 and 10000 ppm groups during the exposure period. Food consumption was also reduced in the 5000 ppm group early in the exposure period (gd 6-9).
Maternal water consumption was increased, but the effect was not dose-related.
Terminal maternal body weight were slightly reduced, and corrected weight change was clearly reduced in all treatment groups. Gravid uterine weight and absolute and relative weights of lungs and kidneys were equivalent across groups. Absolute liver weights were slightly increased (2, 3, and 6% for the 5000m, 10000, and 19500 ppm groups, respectively. Increased relative liver weight was noted in all exposure groups.
Dose descriptor:
LOAEC
Effect level:
5 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
5 000 ppm
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
Effect level:
10 000 ppm
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Reproductive parameters: there were no effect of exposure on gestational parameters including resorptions, pre- and postimplantation losses, percentages of live fetuses, and sex ratios. There was no effect of exposure on fetal body weight/litter.
Pups
There were no significant effects of exposure on the incidence of visceral, skeletal, or external malformations by category or in total malformations. An increased incidence of one common external variation, ecchymosis in the trunk region, was noted in the 19500 ppm group. In addition, several skeletal variations involving various regions of the skeleton were observed for the 19500 and 10000 ppm groups. These included: unossified and poorly ossified cervical centra, reduced number of (ossifying) caudal segments, unossified anterior arch of the atlas, and poorly ossified sternebra. In addition increased incidences of all unossified proximal phalanges (forelimb and hindlimb), and some unossified metatarsals (hindlimb) occurred only in the 19500 ppm group. Overall, maternal toxicity was observed at all concentration levels used in this study. No embryolethality or teratogenic effects were observed. However, a concentration-dependent profile of delayed skeletal developmental was observed in all dose groups. Based on these results a NOAEL for maternal toxicity could not be established. Fetotoxicity was noted at all dose levels, thus a NOAEC for fetotoxicity could not be established in the main study. The NOAEC for teratogenicity/ embryolethality was 5000 ppm.
Re-analysis of this NOAEC for developmental toxicity see "Overall Remarks" (below).
Dose descriptor:
NOAEC
Effect level:
19 500 ppm
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Variations of the anterior arch of the atlas

Re-analysis of available data (see Overall Remarks)

Combined incidence of all 3 arch variations

% of litters at 0, 5000, 10000, 19500 ppm was 100, 100, 100, 100, respectively

% of fetuses was 95.2, 88.6, 95.8, 97.0 at 0, 5000, 10000, 19500 ppm, respectively

Conclusion: no statistically or biologically significant effects

Conclusions:
Maternal toxicity: LOAEC = 5000 ppm or 12 mg/L.
Developmental toxicity: NOAEC = 5000 ppm or 12 mg/L.
Teratogenicity: NOAEC = 19500 ppm or 47 mg/L.
Executive summary:

Guideline study with acceptable restrictions (tabulated results not available).

In this developmental toxicity study 25 pregnant Sprague-Dawley rats were exposed (whole body) 6 h/day to 0, 5000, 10000, or 19500 ppm (12; 24; 47 mg/L) at gestation day 6 -15. The dams were sacrificed at gestation day 21. Maternal toxicity was noted at all dose levels. Signs of toxicity included decreased maternal body weights, body weight gain, and food consumption during the exposure period. In addition, relative maternal liver weight was increased in treated animals (questionable relevance). Reproductive parameters were not affected at any dose level. Developmental toxicity was noted at the mid and high dose levels, taking into account the above cited re-evaluation of the skeletal findings, substantiated by the increased incidence of skeletal variations, mostly resulting from retarded ossification. The incidence of malformations was not increased, thus no teratogenicity was noted.

Conclusion: Maternal toxicity: LOAEC = 5000 ppm or 12 mg/L. Developmental toxicity: NOAEC = 5000 ppm or 12 mg/L. Teratogenicity: NOAEC = 19500 ppm or 47 mg/L.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
12 000 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study with acceptable restrictions (tabulated results not available; exposure at gestation day 6-15).
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No developmental toxicity study with cyclohexylvinylether is available. The performed OECD 422 study showed neither developmental toxicity nor teratogenic effects. A read across to the structural analogue methylvinylether (CAS 107 -25 -5) was conducted to fulfill the data requirements of developmental toxicity. Both substances are vinylethers that are exclusively used in industrial settings and are only differing in the side chain. Since exposure in industrial settings is strictly controlled, no further OECD 414 study was proposed due to animal welfare reasons. During manufacture and processing of cyclohexylvinylether, worker exposure is controlled by the use of closed systems, industrial hygiene controls, and personal protective equipment. Any risk of accumulation of cyclohexylvinylether or its impurities is minimized by natural ventilation, as the chemical is produced in closed systems installed in open air. At processing sites, the exposure of workers is minimized by vapor abstraction. At the production and processing sites, workers were personal protective equipment which includes gloves, face shields and safety googles in view of the low pH during processing. During repair and maintenance operations, respiratory protective equipment is aditionally used.

In a developmental toxicity study with methylvinylether, 25 pregnant Sprague-Dawley rats were exposed (whole body) 6 h/day to 0, 5000, 10000, or 19500 ppm (12; 24; 47 mg/L) at gestation day 6 -15. The dams were sacrificed at gestation day 21. Maternal toxicity was noted at all dose levels. Signs of toxicity included decreased maternal body weights, body weight gain, and food consumption during the exposure period. In addition, relative maternal liver weight was increased in treated animals (questionable relevance). Reproductive parameters were not affected at any dose level. The incidence of malformations was not increased, thus no teratogenicity was noted.

Conclusion: Maternal toxicity: LOAEC = 5000 ppm or 12 mg/L. Developmental toxicity: NOAEC = 5000 ppm or 12 mg/L. Teratogenicity: NOAEC = 19500 ppm or 47 mg/L.


Justification for selection of Effect on developmental toxicity: via oral route:
In accordance with column 2 of REACH Annex IX, the developmental toxicity study has to be performed for the most approriate exposure route in humans (one route only). Since long-term exposure by inhalation is expected to be the most relevant route, a developmental inhalation toxicity study was used to fulfill the data requirements. Thus, the test developmental toxicity after oral application (required in section 8.7.2 of Annex IX) is not needed. Risk assessment is based on the inhalation study.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Only one study available.

Justification for selection of Effect on developmental toxicity: via dermal route:
In accordance with column 2 of REACH Annex IX, the developmental toxicity study has to be performed for the most approriate exposure route in humans (one route only). Since long-term exposure by inhalation is expected to be the most relevant route, a developmental inhalation toxicity study was used to fulfill the data requirements. Thus, the test developmental toxicity after dermal application (required in section 8.7.2 of Annex IX) is not needed. Risk assessment is based on the inhalation study.

Justification for classification or non-classification

Based on the available data, the test item was not classified and labelled according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP).