Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Principles of method if other than guideline:
Subchronic toxicity by subcutaneous injection with focus on potential effects on reproductive system
GLP compliance:
not specified
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): methylcyclohexane
- Substance type: pure substance
- Physical state: liquid
- Physical state: 98.9%
- Source: Sigma-Aldrich’s (St. Louis, MO, USA)
- Lot/batch No.: 01144TH

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Central Lab. Animal Inc., Korea
- Age at study initiation: 6 weeks
- Housing: animals were housed in groups of 5 per sex in polysulfone rearing boxes (W 235 × L 380 × H 175 mm)
- Diet: Hard food (LabDiet Picolab 5053 PMI), sterilised with radiation (ORIENT BIO Inc.), ad libitum
- Water: service water, sterilised with a UV sterilizer and microfiltration, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
olive oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
single injections
Frequency of treatment:
once daily, 5 days/week
Duration of test:
13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: data from a previous toxicity study were used to determine suitable levels for subcutaneous injection of the test substance. The low dose of 10 mg/kg bw/day should be equivalent to a level that does not induce toxicity, whereas the high dose of 100 mg/kg bw/day was selected as a dose inducing toxicity in the rat.

EXAMINATIONS

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and clinical signs were observed daily during the study period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: skin, hair, eye irritation, respiratory system, movement and behaviour pattern, and others were examined daily during the study period.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were measured prior to test substance administration, twice a week during the study and prior to necropsy at the end of the study period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- How many animals: all surviving animals
- Parameters examined: white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red blood cell distribution width, platelets and mean platelet volume

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- How many animals: all surviving aniamls
- Parameters examined: total protein, albumin, urea nitrogen in blood, creatinine, total bilirubin, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, glucose, total cholesterol, triglyceride, creatine phosphokinase and inorganic phosphorus

OTHER:
SPERMATOGENIC CELL COUNT AND ABNORMALITY TEST:
- To study the effect of the substance on spermatogenesis, the spermatogenic cell counts were performed. Each animal’s peritubular hyalinization was classified into four stages according to the spermatogenic cells’ characteristics. The sperm generation cycle method was used to determine the germ cell number and normality in each stage of generation within the seminiferous tubule.

HORMONE ANALYSIS:
Whole blood was sampled from the abdominal aorta of males. A centrifugal separator (2000 rpm, 10 min) was used to separate the serum from plasma and levels of the hormones estradiol, prolactin, testosterone, follicle stimulating hormone, progesterone, luteinizing hormone, were quantified using Vitros Eci (Johnson & Johnson, USA).

OESTRUS CYCLE:
A vaginal smear of the females was microscopically evaluated 4 and 13 weeks after the subcutaneous injection of the test substance.

SACRIFICE AND PATHOLOGY

GROSS PATHOLOGY: Yes, all organs and tissues.
HISTOPATHOLOGY: Yes, histopathological examinations were performed in liver, kidney and heart.
At necropsy, organ weights of thymus, adrenal gland, lungs, kidney, spleen, liver, brain, and ovary were determined.
Statistics:
Mean values and standard deviation were calculated for body weights, haematological and clinical chemistry parameters, organ weights, hormones and duration of the oestrus cycle. Statistical analyses were performed by analysis of variance (ANOVA) test using SPSS version 18 (SPSS Inc., Chicago, IL, USA) at a significance level of p < 0.01. If ANOVA showed a significant result, Dunnett’s or Duncan’s multiple range test for multiple comparison of control and experimental groups was performed.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
effects on reproductive organs
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no (adverse) effects on spermatogenic cell count and morphology, sex hormones levels, histopathology of testes, estrous cycle

Observed effects

CLINICAL SIGNS AND MORTALITY
No mortality occurred in animals of the 10 and 100 mg/kg bw/day groups. Slowed movement was observed in these animals.
At 1000 mg/kg bw/day, 4/5 males and females died between Day 5 and 60 after start of injections. No particular abnormalities were found in the surviving animals.

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight were observed in males and females dosed 10 mg/kg bw/day and in females given 100 mg/kg bw/day. A very slight and not statistically significant increase in body weight was observed in males at 100 mg/kg bw/day.
At 1000 mg/kg bw/day, a statistically significant decrease (by ca. 16%) in body was observed in males and females from the day after the start of injections until death of the animals.

HAEMATOLOGY
No effects on haematological parameters were seen in male animals dosed 10 and 100 mg/kg bw/day. In females of the 100 mg/kg bw/day group, monocytes were statistically significantly decreased. However, this change was not considered to be of biological relevance, since it was within the normal range of this parameter in rats.
The number of surviving animals in the 1000 mg/kg bw/day male and female groups was too small for assessment of statistical significance in the observed changes. These included a marked increase in the counts of white blood cells, neutrophils, eosinophils and platelets observed in the surviving male, whereas the red blood cell count, haemoglobin, and haematocrit were decreased. In contrast to the finding in the surviving male of the 1000 mg/kg bw/day group, all these haematological parameters were decreased in the surviving female rat of this group. The changes in animals at 1000 mg/kg bw/day were considered to be treatment-related.

CLINICAL CHEMISTRY
Inorganic phosphorus (IP) concentrations were slightly but statistically significantly and dose-dependently increased in the 10 and 100 mg/kg bw/day male groups. At 1000 mg/kg bw/day, IP concentration was also higher than the mean concentration in the control group. In females, no significant changes in IP concentration were observed, but a dose-dependent tendency to decrease.
Males in the 100 mg/kg bw/day showed a very slight but statistically significant increase in total protein and albumin in serum. These values decreased again in the surviving male dosed 1000 mg/kg bw/day. These changes were considered not to be treatment-related as they were slight and there was no dose dependency.
Urea nitrogen in blood (BUN), creatinine and alkaline phosphatase were markedly increased in females of the 100 mg/kg bw/day group. However, due to high interindividual variability, these changes were not statistically significant.
At 1000 mg/kg bw/day, the low number of surviving animals did not allow statistical analysis. Male animals showed an increase in ALP, creatine phosphokinase, and IP. Female animals showed an increase in lactate dehydrogenase and ALP and a decrease in BUN, alanine aminotransferase, and IP.

ORGAN WEIGHTS
No changes in organ weights were observed in male and female animals dosed at 10 and 100 mg/kg bw/day.
The weight of thymus, adrenal gland, lungs, kidney, spleen, liver and brain was decreased in males of the 1000 mg/kg bw/day group. In females of the same group, a decrease was observed in the weight of thymus, adrenal gland, ovary, lungs, kidney, spleen and liver.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microgranuloma and bile duct proliferation were observed in each 1/5 males at 10 mg/kg bw/day. Hepatic cytoplasmic vacuolation was observed in 1/5 males at 100 mg/kg bw/day. At 1000 mg/kg bw/day, liver microgranuloma, bile duct proliferation, cardiac hyperemia/hemorrhage and myocardial necrosis were observed (incidence not reported). Some of the animals showed renal protein casts and hyperemia/haemorrhage (incidence not reported). No effects were observed in the diameter of the seminiferous tubules and thickness of gametes.
In females of the 100 mg/kg bw/day, liver microgranuloma and bile duct proliferation were observed in 1/5 and 2/5 animals, respectively. At 1000 mg/kg bw/day, bile duct proliferation of the liver, hyperemia, and renal hyperemia/hemorrhage were seen, but the incidence was not reported.
Due to the low incidence of liver changes in the 10 and 100 mg/kg bw/day groups and the fact that such changes are commonly found also in untreated rats, these findings were considered to be spontaneous and not treatment-related. According to the authors, due to the severe level of abnormalities seen at 1000 mg/kg bw/day, findings organs of male and female animals seen at this dose level were considered to be treatment-related.

REPRODUCTIVE SYSTEM
- Spermatogenic cell count and morphology: no effects were observed at any dose level.
- Sex hormone levels: in males, no effects were seen on the levels of prolactin, testosterone, follicle stimulating hormone and luteinizing hormone. A very slight but statistically significant and dose-dependent increase in estradiol levels was observed at 10 and 100 mg/kg bw/day. At the same dose levels, a significant decrease in progesterone levels was noted. The toxicological relevance of this finding is unclear, since no corresponding alterations were observed in testes at microscopic examination.
No female data were reported. According to the authors, the hormone levels in females were dramatically changed in accordance with the estrous cycle.
- Estrous cycle: the menstrual cycle period was statistically significantly increased in the 4th week in the 10 mg/kg bw/day group, but this change was not dose-dependent. After the 13th week, no effects were observed.

Applicant's summary and conclusion

Conclusions:
Methyl cyclohexane was tested in a subchronic toxicity study focusing on potential compound-related effects on the reproductive system (see Repeated dose toxicity). Groups of 5 male and female rats were exposed to methylcyclohexane at 10, 100 and 1000 mg/kg bw/day by subcutaneous injections, 5 days/week for 13 weeks. Concurrent vehicle control animals were treated with olive oil.
In regard to effects on reproductive organs, no changes in organ weights were observed in male and female animals dosed at 10, 100 and 1000 mg/kg bw/day, except for a decrease in the weight of ovaries in the high-dose female group.
No effects were observed in spermatogenic cell count and morphology at any dose level. In males, no effects were seen on the levels of prolactin, testosterone, follicle stimulating hormone and luteinizing hormone. A very slight but statistically significant and dose-dependent increase in estradiol levels was observed at 10 and 100 mg/kg bw/day. At the same dose levels, a significant decrease in progesterone levels was noted. The toxicological relevance of this finding is unclear, since no corresponding alterations were observed in testes at microscopic examination. No female data were reported. According to the authors, the hormone levels in females were dramatically changed in accordance with the estrous cycle. The menstrual cycle period was statistically significantly increased in the 4th week in the 10 mg/kg bw/day group, but this change was not dose-dependent. After the 13th week, no effects were observed.
The NOAEL for systemic toxicity was 100 mg/kg bw/day (see Repeated dose toxicity), while the NOAEL for effects on reproductive organs was 1000 mg/kg bw/day based on the reported findings.