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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Sep 2010 - 01 Dec 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

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: clear colourless liquid with characteristic odour
- Analytical purity: 99.9%
- Lot/batch No.: KWH4629
- Stability under test conditions: Verified after study completion
- Storage condition of test material: Temperature: 22 - 25°C, Humidity: 40.8 - 67%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 wks
- Weight at study initiation: 319 - 378 g (males); 246 - 276 g (females)
- Housing: individual in stainless steel cages
- Diet: CRF-1, pelleted, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 - 23.8
- Humidity (%): 41.4 - 63.3
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 Sep 2010 To: 01 Dec 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared more than once a week. Concentration of stock solution was 200 mg/mL and stored at 4 °C in a refrigerator. The stock solution was diluted with corn oil to achieve the concentration of the dosing solutions.

VEHICLE
- Concentration in vehicle: 12.5, 50 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: V8N8411 and V03H3633
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the dosing solutions was verified by GC. The results were 94.2 - 99.4% of target concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 250 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
6 males, 5 females per dose (main study)
6 males per dose (satellite control and treatment groups)
5 females per dose (satellite control and high-dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 0, 200, 500 and 1000 mg/kg bw/day for 14 days. Decrease in weight of thymus and increase in liver weight in both sexes, increase in platelet, decrease in leucocytes, increase in total protein and total cholesterol in males were observed at 1000 mg/kg bw/day. Therefore, 62.5, 250 and 1000 were selected as the dose levels for the main study.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during administration period, daily during recovery period, and once before necropsy

DETAILED CLINICAL OBSERVATIONS: Yes (including FOB)
- Time schedule: Day 0, 7, 14, 21 and 27

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week (Day 1, 4, 8, 11, 15, 18, 22, 25 and 28 during administration period, Day 1, 4, 8, 11, 14 and 15 during recovery period)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations:
Males: on Day 2, 5, 9 and 12 of administration, on Day 2, 5, 9 and 12 of recovery
Females: on Day 2, 5, 9, 12, 16, 19, 23 and 26 of administration, on Day 2, 5, 9 and 12 of recovery

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day after last administration or Day 15 of recovery period
- Anaesthetic used for blood collection: Yes (Pentobarbital-Na)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, Fibrinogen, WBC, Differential leukocytes: Lymphocytes, Neutrophils, Eosinophils, Basophils, Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day after last administration or Day 15 of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters examined: AST, ALT, ALP, gamma-GTP, T-potein, Albumin, A/G, T-bilirubin, Urea nitrogen, Creatinine, Glucose, T-cholesterol, Triglycerides, Na, K, Cl, Ca, Inorganic-P, Fe

URINALYSIS: Yes
- Time schedule for collection of urine: before administration on Day 22 in males, Day 24 in females, Day 12 of recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: Color, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Urinary sediments, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

HORMONE CONCENTRATION
- Time schedule for collection of blood: on day after last administration or day 15 of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters examined: T3, T4 and TSH
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Organ weight: Brain, pituitary, salivary glands, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ventral prostate, seminal vesicles, ovaries and uterus.
HISTOPATHOLOGY: Yes.
- Tissues examined: Heart, lung, trachea, liver, pancreas, sublingual gland, oesophagus, stomach, duodenum, jejunum, ileum, Payer's patch, caecum, colon, rectum, thymus, spleen, mandibular lymph node, mesenteric lymph node, kidney, urinary bladder, testis, epididymis, ventral prostate, seminal vesicle, coagulating gland, pituitary, adrenal, thyroid, parathyroid, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, eyeball, harderian gland, sternal bone, gemoral bone, sternal bone marrow, femoral bone marrow, muscle (rectus femoris), mammary gland, ovary, uterus, vagina and mammary gland.
Statistics:
Differences in body weight, food consumption, water consumtion, clinical observation (frequency of urination, defecation rearing and grooming), grip strength, spontaneous motor activity, volume of urine, specific gravity of urine, hematological findings, clinical biochemistry findings, blood concetration of hormone (T3, T4 and TSH) and organ weight were tested by Bartlett's test. In case of homogeneity of variance or heteroscedasticity, Dunnett's test or Steel's test was performed between administration groups and control groups, respectively.

For differences in body weight, food consumption, water consumption, volume of urine, specific gravity of urine, hematological findings, clinical biochemistry findings and organ weight of female during recovery period, F test was applied. In case of homogeneity of variance or heteroscedasticity, Student' t-test or Aspin-Welch t-test was performed between administration groups and control groups, respectively.

For differences in clinical observation (except for frequency of urination, defecation, rearing and grooming) and sensory reactivity, Steel test was applied.

For differences in histopathological findings, Steel test was performed. In case of significant difference, Cochran-Armitage test was applied to test dose response relationship.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (male and female): transient salivation following dosing
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day (male and female): transient salivation following dosing
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
250 and 1000 mg/kg bw/day (recovery males): slight decrease in MCHC and WBC; 1000 mg/kg bw/day (females): increase in reticulocytes and monocytes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
all groups (males): increase in total cholesterol, reversible except for high-dose group; 250 mg/kg bw/day (males): slight decrease in Cl, reversible; 1000 mg/kg bw/day (males): increase in ALT (non-reversible), gamma-GT (reversible)
Urinalysis findings:
no effects observed
Description (incidence and severity):
1000 mg/kg bw/day (males): non-adverse transient increase in urine volume
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day (males): increased liver weight, reversible; 1000 mg/kg bw/day (males): increased liver and kidney weights, reversible; 1000 mg/kg bw/day (females): increased liver, kidney, adrenals and uterus weights, reversible except for liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
250 and 1000 mg/kg bw/day (males): slight bilateral hyaline droplet in the renal tubule, reversible
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
11 males in the 1000 mg/kg bw/day group showed transient salivation. No abnormalities and no mortalities were observed in other groups during administration and recovery periods.
9 females in the 1000 mg/kg bw/day group showed transient salivation. No abnormalities and no mortalities were observed in other groups both during administration period and recovery period.

BODY WEIGHT AND FOOD CONSUMPTION
No changes were found between control and administration groups in both sexes during the study period.

WATER CONSUMPTION
Males in the 62.5 mg/kg bw/day group showed a significant decrease in water consumption on Day 12 of administration. However, no changes were found in higher dose groups. These changes were not regarded as relevant toxicological effects. On the other hand, no changes were observed in females during administration and recovery period.

DETAILED CLINICAL OBSERVATION
6 males, 4 males and 3 males showed slight salivation on Day 14, 21 and 27 of administration, respectively. Frequency of defecation was increased at dose of 62.5 and 1000 mg/kg bw/day on Day 14 of administration. However, these changes were not considered toxicologically relevant since observations were transient. Frequency of defecation was significantly increased at 62.5 mg/kg bw/day on Day 21 of administration. However, this was not considered a toxicological effect, since the change was not found in higher dose groups.
One female at 1000/kg bw/day showed slight salivation on Day 14 and 21 of administration. A significant increase in frequency of defecation was observed on Day 21 of administration in the 250 mg/kg bw/day group. This change was not due to the test item, since there was no dose-response relationship.

SENSORY REACTIVITY, GRIP STRENGTH, SPONTANEOUS MOTOR ACTIVITY
No changes were found in both sexes during the study period.

URINALYSIS
A significant increase in urine volume was observed at 1000 mg/kg bw/day in males towards the end of the administration period on Day 22. This change was not considered toxicologically relevant, since the urine volume in the control group (8.7 mL) was apparently less than usual. Thus, the urine volume of the control group was 13.4 mL after the recovery period.
No changes were found in females.

HEMATOLOGICAL FINDINGS
No changes were found in males after the administration period. After the recovery period, MCHC values were slightly (ca. 1.5%) but significantly decreased in males dosed 250 and 1000 mg/kg bw/day. WBC values were decreased in all administration groups, changes being significant in males at 62.5 and 1000 mg/kg bw/day. The toxicological relevance of this finding is questionable since such a tendency was not observed at the end of the administration period and the corresponding control value (68.3E02/µL) was clearly lower than the one after the recovery period (79.0E02/µL). Basophils seemed to be also significantly lower but this change was within the range of historical data of the test facility. Therefore, it was not considered to be test item-related.
A significant increase in reticulocytes and monocytes was observed in females of the 1000 mg/kg bw/day group after the administration period. This effect was not seen after the recovery period.

CLINICAL BIOCHEMISTRY FINDINGS
In comparison with the control group, total cholesterol was significantly increased in males of all treated groups after the administration period without clear dose-response relationship. ALT, gamma-GT, total protein and Ca values were significantly increased, and Cl significantly decreased, in males at 1000 mg/kg bw/day after the administration period. Cl values were slightly but significantly decreased at 250 mg/kg bw/day. ALT and total cholesterol were still significantly increased in males of the satellite recovery group dosed 1000 mg/kg bw/day. Glucose was significantly decreased in males of the satellite recovery group dosed 250 mg/kg bw/day. This last change was not regarded as toxicologically relevant since no dose-response relationship was observed and no significant changes were seen after the administration period.
Glucose was significantly decreased in females of the 1000 mg/kg bw/day group after the administration period. No changes were found in females of the satellite recovery groups.

HORMONE CONCENTRATION
No differences were found between control and administration groups after the administration period.

ORGAN WEIGHTS
In males at the dose level of 1000 mg/kg bw/day, absolute and relative weight of liver and kidney were significantly increased in comparison with the control group after the administration period. Relative liver weight was slightly (by 8.5%) but significantly increased in males dosed 250 mg/kg bw/day after the administration period. The significant increase in relative weight of kidney which was observed in males at 62.5 mg/kg bw/day after the administration period was not considered as test compound-related, since this change was within the historical background data of the test facility. No differences in liver and kidney weights were observed after the recovery period at any dose level.
In males of the 1000 mg/kg bw/day group, relative testis weight was significantly increased compared with the control group after the recovery period. However, this change was not considered as test item-related, since this was not observed after the administration period.
Based on the results of clinical chemistry examinations and organ weights, the test item may affect the liver. However, no changes were found at histopathological examinations in the liver. The test item did not cause any organic change. Changes observed in kidney weight and at histopathological examination suggest that the test item or its metabolites impaired lesions which occurred naturally. However, these changes in kidneys were reversible since they disappeared after the recovery period.
In females dosed 1000 mg/kg bw/day, absolute and relative weights of adrenals as well as relative weight of liver, kidney and uterus were significantly increased compared with the control group after the administration period. The increase in relative uterus weight was not considered test item-related, since this change was within the historical background data of the test facility. After the recovery period, the absolute, but not the relative, liver weight was significantly increased and no further changes were found.

GROSS PATHOLOGY
One male in the control group showed bilateral small size of testis and bilateral softness of testis after the administration period. One male in the 62.5 mg/kg bw/day showed lateral small size of testis and lateral small size of epididymis after the administration period. One male in the 1000 mg/kg bw/day showed bilateral small size of testis and bilateral small size of epididymis after the administration period. Due to the low incidence and lack of dose-response relationship, these changes were considered accidental.
No abnormalities were found in females after the administration period and in both sexes after the recovery period.

HISTOPATHOLOGY
After the administration period, one male in the control group showed slight cellular infiltration in the heart. One male in the control groups showed slight extramedullary haematopoiesis in the spleen. Slight bilateral hyaline droplet in renal tubule was observed in all males at 1000 mg/kg bw/day and 4 males at 250 mg/kg bw/day. Changes at 1000 mg/kg bw/day were statistically significant and dose-dependent. Slight bilateral basophilic change in renal tubules was observed in one male at 250 mg/kg bw/day. One male in the control group showed lateral cyst in kidney. Atrophy of the seminiferous tubule was observed in one male each at 62.5 and 1000 mg/kg bw/day. These animals showed also small size or softness of testis at gross pathology. One animal in the control, 62.5 and 1000 mg/kg bw/day group, respectively, showed slight cell debris in the lumen of epididymis. These animals showed also small size or softness of epididymis at gross pathology. Cell infiltration in the ventral prostate was observed in one male of the 1000 mg/kg bw/day group. All these changes except for slight bilateral hyaline droplet in the renal tubule at 250 and 1000 mg/kg bw/day were considered accidental, since they are usually observed in untreated animals and the incidence was not statistically significant. Hyaline droplet formation was likely related to alpha2µ-globulin accumulation, which is an effect specific for male rats and not relevant for humans.
After the recovery period, two males in the 1000 mg/kg bw/day group and one male in the control group showed bilateral/lateral basophilic change in the renal tubules. One male in the control group showed lateral cyst in the kidney. These changes were considered accidental, since they are usually observed in untreated animals and the incidence was not statistically significant.
One female of the control and 1000 mg/kg bw/day groups, respectively, showed slight lateral retinal dysplasia in the eyeball. This change was not considered test item-related, since it was also observed in control group and the incidence was not statistically significant.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: non-adverse/reversible overall effects
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. General clinical signs (salivation)

Administration period

Recovery period

Dose level (mg/kg bw/day)

No. of animals with transient salivation/No. of animals examined

No. of animals with transient salivation/No. of animals examined

males

Control

0/12

0/6

62.5

0/12

0/6

250

0/12

0/6

1000

11/12

0/6

females

Control

0/12

0/5

62.5

0/12

-

250

0/12

-

1000

9/10

0/5

 

Table 2. Water consumption (g/day) during administration period (males, mean ±SD)

Dose level (mg/kg bw/day)

Control

62.5

250

1000

males

Days of administration

2

33 ± 10

33 ± 4

33 ± 14

31 ± 5

5

34 ± 5

33 ± 5

32 ± 6

37 ± 5

9

37 ± 9

35 ± 6

32 ± 5

38 ± 5

12

37 ± 5

30 ± 6*

36 ± 6

39 ± 5

* p < 0.05 by Dunnett’s test

 

Table 3. Urinary volume (mL) after administration period (males, mean ± SD)

Dose level (mg/kg bw/day)

Day 22 of administration

Day 12 of recovery

Control

8.7 ± 2.1

13.4 ± 3.9

62.5

15.5 ± 14.6

17.0 ± 7.0

250

16.1 ± 10.3

13.1 ± 6.0

1000

16.3 ± 4.9*

15.4 ± 5.6

* p < 0.05 by Steel’s test

 

Table 4. Haematological and clinical chemistry findings (males, mean ± SD)

Parameter

Dose level (mg/kg bw/day)

Control

62.5

250

1000

males (administration period)

MCHC (g/dL)

35.7 ± 0.3

36.0 ± 0.6

36.1 ± 0.6

36.1 ± 0.6

WBC (1E02/µL)

68.3 ± 9.8

63.2 ± 13.6

65.2 ± 11.4

65.1 ± 24.3

Basophil (%)

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

ALT (IU/L)

25.5 ± 1.1

28.4 ± 3.9

35.6 ± 16.2

44.3 ± 17.6***

Gamma-GT

0.61 ± 0.08

0.51 ± 0.11

0.65 ± 0.17

1.18 ± 0.33***

Total protein

5.59 ± 0.22

5.48 ± 0.18

5.55 ± 0.15

6.25 ± 0.28**

Glucose (mg/dL)

107.9 ± 7.5

112.1 ± 9.2

103.2 ± 6.7

97.9 ± 10.1

Total cholesterol (mg/dL)

45.1 ± 5.9

60.8 ± 8.4*

60.5 ± 9.3*

81.2 ± 12.6*

Cl (mEq/L)

108.6 ± 1.0

108.2 ± 1.0

107.2 ± 1.1*

105.7 ± 0.5**

Ca (mg/dL)

9.6 ± 0.2

9.5 ± 0.2

9.5 ± 0.2

10.0 ± 0.2**

males (recovery period)

MCHC (g/dL)

36.4 ± 0.2

36.1 ± 0.4

35.9 ± 0.3*

35.8 ± 0.3**

WBC (1E02/µL)

79.0 ± 22.2

52.1 ± 14.3*

56.7 ± 18.3

50.2 ± 6.4*

Basophil (%)

0.1 ± 0.1

0.0 ± 0.0*

0.0 ± 0.0*

0.0 ± 0.0*

ALT (IU/L)

29.5 ± 6.2

27.6 ± 2.9

30.7 ± 6.4

39.6 ± 8.8*

Gamma-GT

0.44 ± 0.11

0.41 ± 0.06

0.43 ± 0.06

0.47 ± 0.12

Total protein

5.64 ± 0.13

5.65 ± 0.33

5.62 ± 0.19

5.87 ± 0.47

Glucose (mg/dL)

116.1 ± 7.6

109.9 ± 8.2

103.3 ± 8.8*

109.5 ± 9.4

Total cholesterol (mg/dL)

45.3 ± 6.3

51.6 ± 15.4

52.8 ± 10.4

73.1 ± 13.5**

Cl (mEq/L)

107.5 ± 1.1

108.0 ± 1.4

108.7 ± 1.3

106.8 ± 1.1

Ca (mg/dL)

9.5 ± 0.4

9.4 ± 0.2

9.6 ± 0.2

9.7 ± 0.2

* p < 0.05 by Dunnett’s test

** p < 0.01 by Dunnett’s test

*** p < 0.05 by Steel’s test

 

Table 5. Haematological and clinical chemistry findings (females, mean ± SD)

Parameter

Dose level (mg/kg bw/day)

Control

62.5

250

1000

females (administration period)

Reticulocytes (%)

2.76 ± 0.19

2.81 ± 0.28

2.71 ± 0.47

3.48 ± 0.48*

Monocytes (%)

1.7 ± 0.3

2.1 ± 0.7

2.5 ± 0.9

2.7 ± 0.4*

Glucose (mg/dL)

119.2 ± 10.6

110.1 ± 2.7

110.2 ± 9.0

105.9 ± 7.2*

females (recovery period)

Reticulocytes (%)

2.50 ± 0.67

-

-

2.23 ± 0.49

Monocytes (%)

3.8 ± 1.2

-

-

3.1 ± 1.0

Glucose (mg/dL)

118.3. ± 9.2

-

-

120.5 ± 8.3

* p < 0.05 by Dunnett’s test

 

Table 6. Organ weights (males , mean ± SD)

Organ

absolute/relative weight

Dose level (mg/kg bw/day)

Control

62.5

250

1000

males (administration period)

Liver

g

11.00 ± 0.32

10.95 ± 0.98

11.65 ± 0.57

15.18 ± 0.94**

g%

2.59 ± 0.08

2.64 ± 0.18

2.81 ± 0.13*

3.82 ± 0.13**

Kidney

g

2.75 ± 0.16

2.99 ± 0.31

2.92 ± 0.14

3.14 ± 0.20*

g%

0.65 ± 0.03

0.72 ± 0.07*

0.70 ± 0.03

0.79 ± 0.04**

Testes

g

2.85 ± 0.81

3.06 ± 0.47

3.16 ± 0.35

2.79 ± 0.98

g%

0.67 ± 0.19

0.75 ± 0.16

0.76 ± 0.08

0.70 ± 0.25

males (recovery period)

Liver

g

11.16 ± 1.42

10.97 ± 0.38

11.55 ± 0.97

12.25 ± 1.52

g%

2.50 ± 0.26

2.49 ± 0.07

2.57 ± 0.10

2.83 ± 0.17

Kidney

g

3.04 ± 0.18

302 ± 0.25

2.96 ± 0.38

3.15 ± 0.22

g%

0.68 ± 0.01

0.69 ± 0.05

0.66 ± 0.08

0.73 ± 0.05

Testes

g

3.05 ± 0.15

3.06 ± 0.18

3.23 ± 0.25

3.40 ± 0.46

g%

0.68 ± 0.04

0.69 ± 0.04

0.72 ± 0.08

0.79 ± 0.09*

* p < 0.05 by Dunnett’s test

** p < 0.01 by Dunnett’s test

 

Table 7. Organ weights (females, mean ± SD)

Organ

absolute/relative weight

Dose level (mg/kg bw/day)

Control

62.5

250

1000

females (administration period)

Liver

g

7.58 ± 0.60

7.84 ± 0.47

7.77 ± 0.48

8.29 ± 0.88

g%

2.23 ± 0.19

2.75 ± 0.14

2.71 ± 0.12

3.02 ± 0.28*

Kidney

g

1.83 ± 0.11

1.87 ± 0.09

1.95 ± 0.08

2.04 ± 0.25

g%

0.64 ± 0.04

0.66 ± 0.03

0.68 ± 0.03

0.74 ± 0.09*

Adrenals

g

65.3 ± 5.6

75.3 ± 8.7

74.7 ± 6.5

84.1 ± 8.5**

g%

22.7 ± 2.6

26.5 ± 3.5

26.1 ± 2.7

30.6 ± 2.4**

Uterus

g

499 ± 69

542 ± 87

568 ± 148

665 ± 108

g%

173 ± 23

190 ± 33

199 ± 54

243 ± 38*

females (recovery period)

Liver

g

7.41 ± 0.18

-

-

7.86 ± 0.37***

g%

2.51 ± 0.07

-

-

2.69 ± 0.23

Kidney

g

1.94 ± 0.12

-

-

1.92 ± 0.18

g%

0.66 ± 0.04

-

-

0.66 ± 0.06

Adrenals

g

78.2 ± 6.5

-

-

80.5 ± 10.0

g%

26.5 ± 2.3

-

-

27.7 ± 4.5

Uterus

g

581 ± 165

-

-

595 ± 109

g%

196 ± 56

-

-

203 ± 36

* p < 0.05 by Dunnett’s test

** p < 0.01 by Dunnett’s test

*** p < 0.05 by Student’s t-test

 

Table 8. Gross necropsy findings after administration period (males)

Finding

Dose level (mg/kg bw/day)

Control

62.5

250

1000

males (No. with findings/No. of animals examined)

Testis, small in size, bilateral

1/6

0/6

0/6

1/6

Testis, small in size, lateral

0/6

1/6

0/6

0/6

Testis, softness, bilateral

1/6

0/6

0/6

0/6

Epididymis, small in size, bilateral

0/6

0/6

0/6

1/6

Epididymis, small in size, lateral

0/6

1/6

0/6

0/6

 

Table 9. Summary of histopathological findings after administration (males and females) and recovery period (males)

Finding

Dose level (mg/kg bw/day)

Control

62.5

250

1000

males after administration period (No. with findings/No. of animals examined)

Heart

cellular infiltration, slight

1/6

-

-

0/6

Spleen

extramedullary haematopoiesis, slight

1/6

-

-

0/6

Kidney

Hyaline droplet, renal tubule, bilateral, slight

0/6

0/6

4/6

6/6*

Basophilic change, renal tubule, bilateral, slight

0/6

0/6

1/6

0/6

Cyst, lateral, slight

1/6

0/6

0/6

0/6

Testis

Atrophy, seminiferous tubule, bi-/lateral, marked

1/6

1/1

-

1/6

Epididymis

Atrophy, bi-/lateral, slight

1/6

0/1

-

1/6

Atrophy, bi-/lateral, moderate

0/6

1/1

-

0/6

Cell debris, lumen, bi-/lateral, mild

0/6

1/1

-

0/6

Cell debris, lumen, bi-/lateral, marked

1/6

0/1

-

1/6

Ventral prostate

cellular infiltration, slight

0/6

-

-

1/6

males after recovery period (No. with findings/No. of animals examined)

Kidney

Basophilic change, renal tubule, bilateral, slight

1/6

0/6

0/6

2/6

Cyst, lateral, slight

1/6

0/6

0/6

0/6

females after administration period (No. with findings/No. of animals examined)

Eyeball

retinal dysplasia, lateral, slight

1/5

-

-

1/5

* p < 0.01 by Steel’s test (compared to control)

* p < 0.01 by Cochran-Armitage test (dose-response)

Applicant's summary and conclusion

Conclusions:
A GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422) was conducted with methylcyclohexane. Groups of 12 male and 10 female rats per dose were given methylcyclohexane by oral gavage at 62.5, 250 and 1000 mg/kg bw/day, daily for 28 days. Concurrent male and female vehicle (corn oil) control groups were included. Satellite control and treatment groups were included for a 14-day recovery period. These groups comprised 6/12 males in the control and all dose groups, and 5/10 females in the control and high-dose groups, respectively.
At 1000 mg/kg bw/day, the observed effects in males included increased transient salivation after dosing, slight decrease in MCHC and WBC (only after recovery period), increased ALT, gamma-GT, total cholesterol and Ca values, decreased Cl values (all reversible except for ALT and total cholesterol), increased absolute and relative liver and kidney weights (reversible), increased relative testes weight (only after recovery period) and slight bilateral hyaline droplet in the renal tubules (reversible; alpha-2-microglubin-related and thus not relevant for humans). In females dosed 1000 mg/kg bw/day, the observed effects comprised increased transient salivation after dosing, increased reticulocytes and monocytes values (reversible), decreased glucose (reversible), increased absolute and relative adrenals and relative kidney weights (reversible) as well as increased relative liver weight (non-reversible).
In males of the 250 mg/kg bw/day group the observed effects were limited to a slight decrease in MCHC and WBC (only after recovery period), reversible increase in total cholesterol, slight reversible decrease in Cl and slight reversible bilateral hyaline droplet in the renal tubules (alpha-2-microglubin-related and thus not relevant for humans). No effects were observed in females dosed 250 mg/kg bw/day.
Based on the effects observed at 1000 mg/kg bw/day, in particular transient salivation after dosing (males and females) and non-reversible increases in ALT and total cholesterol values in males and relative liver weight in females, the oral NOAEL for males and female rats was 250 mg/kg bw/day.

Based on the study results, methylcyclohexane does not fulfil the classification criteria for toxicity after repeated exposure according to Regulation (EC) No 1272/2008 and Directive 67/548/EEC.

CLP: not classified
DSD: not classified