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Description of key information

The target substance Vinylcyclohexane was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422). An increase, statistically significant in males, in the liver absolute and relative organ mean weight was recorded in high dose animals of both sexes treated with 800 mg/kg bw/day. 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). Based on the results, the NOAEL is considered to be 300 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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
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 oral 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.
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.
Observations and examinations performed and frequency:
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 and pups on Day 14 post-partum 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.

LITTER OBSERVATIONS:
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Observation were performed once daily for all litters. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. The anogenital distance (AGD) of each pup were measured on Day 1 post partum. The AGD was normalized to a measure of pup size, preferably the cube root of body weight collected on Day 1 post partum.
On Day 13 post partum nipple areolas were counted and recorded.
Sacrifice and pathology:
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.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex were 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.
Nipples retention at Day 14 post partum: The nipples/areolae in male pups, where present, were retained in 10% neutral buffered formalin.

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.
Pups at Day 14 post partum: Thyroid were weighed from one male and female from each litter and preserved for possible histopathological examination (if required, additional cost). The thyroid weight were determined after fixation.

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.
Other examinations:
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
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.
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.
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. Moreover, observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal any changes attributable to the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase, statistically significant in males, in the liver absolute and relative mean weight, was recorded in high dose animals of both sexes.
No relevant changes were observed in terminal body weight, when compared to the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Swollen liver was noted in one high dose male. No other relevant treatment-related macroscopic changes were observed.
Neuropathological findings:
not examined
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. 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.
Histopathological findings: neoplastic:
not examined
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
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. Based on the results, the NOAEL is considered to be 300 mg/kg bw/day for both sexes.
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. Based on the results of this study, the NOAEL for repeated dose 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. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guidelien study
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

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. Based on the results of this study, the NOAEL for repeated dose toxicity is considered to be 300 mg/kg bw/day.

No reliable study is available for the inhalation route. Two poorly documented studies report adverse effects of Vinylcyclohexane after repeated inhalation at a concentration of 420 and 1.000 mg/m³ for up to 3.5 months. Repeated exposure caused disturbances in several of the animal’s functions, mainly in the central nervous and cardiovascular systems. However, as the methodology and the results of the described sub-chronic repeated dose toxicity study via the inhalation route were limited, the study by Savchenkov (1965) and Bandman (1990) were not considered reliable for risk assessment.

Justification for classification or non-classification

Based on the available data, the target substance does not warrant classification for specific target organ toxicity in accordance with CLP regulation (EC) No 1272/2008.