Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Vinylcyclohexane was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). Dosing was based on the results of a 14-day dose range finding study. Adverse effects were found after oral administration of the test item in male and female Sprague Dawley rats in the high dose group. In this group, treatment-related changes were seen in the liver of parental animals. In the F1 generation, an increased presence of clinical signs, missing pups and found dead pups was noted in the high dose group. A relevant increase in post-natal loss on Day 4 post-partum and a reduction in mean pup weight was recorded post-partum in the same group. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-08 to 2018-04-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted: 29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 x 38 x 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material were provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5 x 26.6 x 18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5 x 26.6 x 18.5 cm).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The vehicle used was corn oil. All doses were administered at a constant volume of 5 mL/kg bw. Doses volumes were adjusted once per week for each animal according to the last recorded body weight up tp mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days have elapsed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared during the study were analysed for homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department at RTC.
Duration of treatment / exposure:
Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days.
Females: 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for approximately 51 days.
Frequency of treatment:
Once a day, 7 days a week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
High
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from a preliminary dose range finding study.
Positive control:
n.a.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
- Functional Observation Battery Test: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.
BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams and pups were also weighed on Days 1, 4, 7 and 13 post partum and just prior to necropsy.

FOOD CONSUMPTION: Yes
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

CLINICAL PATHOLOGY INVESTIGATIONS
Blood collection was taken only for animals at term under condition of food deprivation. No samples were taken for animals sacrificed for humane reasons.
Males: Blood samples were collected under isofluorane anaesthesia from the retro-orbital sinus for haematology, clinical chemistry and hormone determination. Blood samples for coagulation test were collected at necropsy from vena cava under isofluorane anaesthesia.
Females: As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava. The blood samples collected from 5 males and 5 females (females with viable litters, if possible) were divided into tubes as follows:
- EDTA anticoagulant for haematological investigations
- Heparin anticoagulant for biochemical tests
- Citrate anticoagulant for coagulation tests

HAEMATOLOGY: Yes
Parameters checked in Table 1 in box "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes
Parameters checked in Table 2 in box "Any other information on materials and methods incl. tables" were examined.

THYROID HORMONE DETERMINATION
Blood samples from all parental males were assayed to determine the levels of Total triiodothyronine (T3), Total thyroxine (T4) and thyroid stimulating hormone (TSH) by a multiplex assay.

(NEURO)BEHAVIOURAL EXAMINATION: Yes
Sensory reactivity to stimuli :
Time schedule: Once during the study, towards the end of treatment (Day 12 post partum for females which littered, where possible), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order.
Motor activity assessment:
Time schedule: Once during the study, towards the end of treatment (Day 12 post partum where possible), 5 males and 5 females were randomly selected from each group and the motor activity were measured (for approximately 5 minutes) by an automated activity recording device.

Oestrous cyclicity (parental animals):
Vaginal smears and oestrous cycle:
Stock females: Oestrus cycle was monitored by vaginal smears in all stock females for at least 1 week before allocation in order to exclude from the study females with irregular cycle.
Females allocated to groups: Vaginal smears were taken in the morning from the Day 1 of dosing up to positive identification of mating.
The vaginal smear data was examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating);
Vaginal smears were also taken from all females, before despatch to necropsy.
No vaginal smears were taken from females sacrificed for humane reasons
Sperm parameters (parental animals):
Seminiferous tubules were evaluated with respect to their stage in spermatogenic cycle and to the integrity of the various cell types within the different stages.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter); partial adjustment is acceptable.

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, anogenital distance (AGD), presence of nipples/areolae in male pups, Thyroid hormone determination in pups on Day 14 post partum.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities. Sex were determination by internal gonads inspection.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Parental animals in extremis and those that have completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia.
Animals sacrificed for humane reasons were killed with carbon dioxide.
Pups: Pups killed for humane reasons or those that have completed the scheduled test period (14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
Parental males: The males were killed after the mating of all females or after at least 28 days of treatment period.
Parental females: The females with live pups were killed on Day 14 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after.
The females showing no evidence of copulation were killed after 25 days of the last day of the mating session.

Necropsy
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination were conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females: All females were examined for number of visible implantation sites (pregnant animals) and number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation. All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities.

Organ weights
Parental animals: From all animals completing the scheduled test period, the organs indicated in Annex 1 were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
At the discretion of the pathologist, organs may be weighed from animals dying or killed prior to terminal kill.

Tissues fixed and preserved
Samples of all the tissues listed in Table 3 under "Any other information on materials and methods incl. tables" were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination
Histopathological evaluation was performed in the first instance on five randomly selected control and high dose males and females, as well as on all abnormalities detected during post mortem observation. Subsequently, the histopathological evaluation was extended to the target organs of the remaining control, mid- and high in box "Any other information on materials and methods incl. tables". After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Postmortem examinations (offspring):
SACRIFICE
Pups killed for human reasons or those that have completed the scheduled test period (day 14 post partum) will be euthanised by intraperitoneal injection of sodium thiopental.

GROSS NECROPSY
- All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities
- All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
- All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection.
- All pups with abnormalities were retained in 10% neutral buffered formalin.

ORGAN WEIGTHS
Thyroid was weighed from one male and female from each litter.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p< 0.05.
Reproductive indices:
Copulation, fertility, delivery and viability
Offspring viability indices:
Viability
Clinical signs:
no effects observed
Description (incidence and severity):
Salivation was noted in animals of both sexes treated at the dosages of 300 and 800 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences were found in body weight and body weight gain between control and treated males and females throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences were observed in both sexes between treated and control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant changes were recorded. The statistically significant decrease of platelets and eosinophils recorded in males dosed at 100 and 300 mg/kg bw/day, respectively, were not dose related, therefore they were considered unrelated to treatment. Moreover, the statistically significant decrease of prothrombin time recorded in females dosed at 800 mg/kg bw/day was considered to be of no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A significant increase of aspartate aminotransferase was recorded in a number of males dosed at 300 and 800 mg/kg bw/day. Mean group values were approximately 50% above controls, with no dose-relation. In addition, urea and creatinine were decreased in females from all treated groups with no clear dose-relation. The magnitude and/or the direction of these findings were not considered to be suggestive of organ/tissue injury. Moroever, treated males showed an increase of thyroxine. Compared to controls the increases were 1.9 to 2.3 fold, with no clear dose-relation. Since the thyroxine value was also observed in two control males and no other related changes were recorded in treated animals, the finding was considered to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes of toxicological significance were noted in the motor activity and landing foot splay.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were seen in the liver of some high (800 mg/kg bw/day) dose males and females characterised by minimal periportal and or/midzonal hepatocytic vacuolation of micro and macrovesicular form (fatty change like). In addition, increased hyaline droplet accumulation in the kidneys of all high dose males from moderate to marked degree and with less severity in all mid-dose males was observed, when compared to controls. However, the treatment-related changes in the kidneys were considered male rat specific and no renal degenerative changes were seen.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in spermatogenic cycle and the integrity of the various cell types within the different stages. Regular layering in the geminal epithelium was noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulatory and fertility index and pre-coital intervals were unaffected by treatment. Moreover, no significant differences were observed for implantation, pre-birth loss and gestation length of females.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
An increased presence of clinical signs, missing pups and found dead pups was noted in the high dose group.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A relevant increase in post-natal loss on Day 4 post partum was recorded in the high dose group of females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group a reduction in mean pup weight was recorded on days 1 and 4 post partum with respect to the control. Statistically significant decrease in litter weight and mean pup weight was detected in the high dose group on Day 13 post-partum.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No relevant changes in thyroid hormone levels were described in pups at day 14 post partum.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences were noted in thyroid weight between control and treated pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted in pups which died or in pups sacrificed on Day 4 and Day 14 post partum.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Anogenital distance: No relevant differences in the anogenital distance (normalised value) were seen between the control and the treated groups both for male and female pups.
- Litter data: No differences of toxicological relevance were detected in sex ratio between treated and control groups.
- Nipples were observed in one pup of the high dose group.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422, adverse effects were found after oral administration of the test item in male and female Sprague Dawley rats in the high dose group. In this group, treatment-related changes were seen in the liver of parental animals. A relevant increase in post-natal loss on Day 4 post partum was recorded in the high dose group of females. In the same group, a reduction in mean pup weight was recorded post-partum. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the test item Vinylcyclohexane (99.8 % purity) was administered orally to 10 male and 10 female Sprague Dawley rats/dose in corn oil by gavage at dose levels of 0, 100, 300 and 800 mg/kg bw/day. The animals were treated daily with the test item formulation on 7 days per week for a period of 51 days, i.e. during 14 days of pre-mating and during pairing and throughout the gestation and lactation periods until day 13 post-partum. Males were treated for two weeks prior to pairing with females until the day before necropsy, for a total of 32 days. No adverse effects of test item were found on male and female mortality, clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, haematology and coagulation and clinical biochemistry. An increase, statistically significant in males, in the liver absolute and relative organ mean weight was recorded in high dose animals of both sexes. Gross macroscopically examination showed a swollen liver in one high dose male. No other relevant treatment-related macroscopic changes were observed. Histopathologically, at 800 mg/kg bw/day, treatment-related changes were seen in the liver of some males and females characterized by minimal periportal and/or midzonal hepatocytic vacuolation of micro and macrovesicular form (fatty change like). In addition, increased hyaline droplet accumulation in the kidneys of all high dose males from moderate to marked degree and with less severity, in all mid dose males was observed, when compared to controls. However, the treatment-related changes in the kidneys were considered male rat specific and no renal degenerative changes were seen.

The estrous cycle, copulatory and fertility index, the pre-coital interval, implantation, pre-birth loss and gestation length was unaffected by the treatment. Also, the anogenital distance, the sex ratio of litters, the external and internal examination of pups, the pup thyroid weight and the thyroid hormone levels of pups were not affected after treatment with the test item. But, an increased presence of clinical signs, missing pups and found dead pups was noted in the high dose group. Moreover, a relevant increase in post-natal loss on day 4 post-partum was recorded in the high dose group of females. In the same group a reduction in mean pup weight was recorded on days 1, 4 and 13 post-partum in comparison to the control. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 300 mg/kg bw/day.

This study is classified as acceptable and satisfies the guideline requirement for an oral repeated dose toxicity study in rats. 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats according to OECD 422, a NOAEL of 300 mg/kg bw/day was derived for parental and embryo-fetal effects. Adverse effects on the offspring in the high dose group are considered secondary to maternal toxicity. Based on the available data, classification for reproductive toxicity is not warranted in accordance with CLP Regulation 1272/2008.