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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from J-CHECK

Data source

Reference
Reference Type:
secondary source
Title:
Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of Cyclohexene by Oral Administration in Rats
Author:
Ministry of Health, Labour and Welfare", "Ministry of the Environment" and "National Institute of Technology and Evaluation
Year:
2010
Bibliographic source:
J-CHECK, 2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of Cyclohexene in Rats
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
Cyclohexene
Cas Number:
110-83-8
Molecular formula:
C6H10
IUPAC Name:
Cyclohexene
Details on test material:
- Name of test material (as cited in study report): Cyclohexene
- Molecular formula (if other than submission substance): C6H10
- Molecular weight (if other than submission substance): 82.14 g/mole
- Substance type: Organic
- Physical state: Colorless transparent liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cyclohexene
- Molecular formula (if other than submission substance): C6H10
- Molecular weight (if other than submission substance): 82.14 g/mole
- Substance type: Organic
- Physical state: Colorless transparent liquid
- Impurities (identity and concentrations): 3.4%

Test animals

Species:
rat
Strain:
other: Crj: CD (SD) IGS , SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River KK (Kanagawa)
- Age at study initiation: 10 weeks
- Weight at study initiation:
341 to 406 g for males and 207 to 252 g for females.
- Fasting period before study: No data
- Housing: Animals were housed individually in an aluminum front and floor stainless steel mesh breeding cage in a breeding room. Maternal animals after gestation 18 days were kept in an aluminum front and floor stainless steel mesh breeding cage with nursery trays and nest material (sunflake, manufactured by Charles River Japan Co., Ltd.) placed until nursing 4th.
- Diet (e.g. ad libitum): CRF-1 solid feed (radiation sterilized feed) , ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 3 ° C
- Humidity (%):55 ± 20 %
- Air changes (per hr): 15 times / hour, an illuminance of 150 to 300 lux,
- Photoperiod (hrs dark / hrs light): 12
hours (7 am lights on, 7 p.m. off)

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on mating procedure:
PREPARATION OF DOSING SOLUTIONS: Each test substance was diluted in cotton-seed oil in a concentration sufficient to provide the predetermined dosage in 2% of the diet. The oil solutions were incorporated into a nutritionally adequate basal ration.

DIET PREPARATION-No data
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE-
- Justification for use and choice of vehicle (if other than water): Corn oil was used as test substances in corn oil were stable for 24 hours at room temperature (about 25 ° C.)
- Concentration in vehicle: 0 (vehicle), 50, 150, 500 mg/kg/day
- Amount of vehicle (if gavage): 0.5 mL per 100 g body weight
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
For male: 48 consecutive days
For female : 53 days
Frequency of treatment:
Dailly
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
Total: 96
0 mg/kg bw: 12 male, 12 female
50 mg/kg bw: 12 male, 12 female
150 mg/kg: 12 male, 12 female
500 mg/kg bw: 12 male, 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose were selected on the basis of 2 weeks preliminary test, death occurred in both males and females at 1000 mg / kg administration, and obvious toxic effects such as salivation, lacrimation, suppression of weight gain, decrease in food intake were confirmed as symptoms after administration. However, at 300 mg / kg administration, although salivation was observed as a symptom after administration, it was not a sufficient toxicity expression amount. Therefore, considering that the administration period will be extended in this study compared to the preliminary test, high doses of 500 mg / kg were divided by the common ratio of about 3 and 150 and 50 mg / kg were set.
- Rationale for animal assignment (if not random): Animals were stratified based on the body weight at the start of administration, and 12 animals per group were sorted by random sampling method.
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included.: General conditions, and the presence or absence of abnormalities and deaths was recorded.

BODY WEIGHT: Yes
Time schedule for examinations
On 1 (administration start date), 8, 15, 22, 29, 36, 43 and 49 days (autopsy day), and the body weight gain from 1st to 43th administration was calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, feed consumption was calculated and cumulative food intake was calculated from 1 to 15 days of administration and from 22 to 48 days of administration.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At necropsy
- Anaesthetic used for blood collection: yes, ether was used.
- Animals fasted: Yes, Animals fasted for
about 16 hours
- How many animals: All 96 animals were examined.
- Parameters checked in table [No.?] were examined.: Number of white blood cells (WBC: flow cytometry) was measured using a general hematological examination apparatus ADVIA 120 (Calculated by HCT: RBC, MCV), average red blood cell volume (MCV: dark field plate method), mean (red blood cell count) (RBC: dark field plate method),
hemoglobin amount (HGB: cyanomethemoglobin method) (Calculated from MCH: HGB, RBC), average red blood cell pigment concentration (calculated from MCHC: HGB, HCT), platelet count (PLT: dark field plate method), leukocyte percentage (flow cytometry method) and reticulocyte rate, Prothrombin time (PT: Quick single step method) and activated partial thromboplastin time (APTT: clot method) were measured using a blood coagulation measuring apparatus KC - 40 (Amelung) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At necropsy
- Anaesthetic used for blood collection: yes, ether was used.
- Animals fasted: Yes, Animals fasted for
about 16 hours
- How many animals: All 96 animals were examined.
- Parameters checked in table [No.?] were examined.: Total protein (T. protein: Biuret method), albumin (Albumin: BCG
method), A / G (calculated value), blood glucose (Glucose: (TK cholesterol: cholesterol oxidase • HDAOS method), urea nitrogen (BUN: urease / GLDH method), neutral fat (Triglyceride: GK-GPO free glycerol elimination method), total cholesterol Allyl aminotransferase (ALT: enzyme-UV method), alkaline phosphatase (ALP (enzyme-UV method)), aspartate aminotransferase (AST: enzyme-UV method), alanine aminotransferase (P. nitro phenyl phosphate substrate method), calcium (Calcium: MXB method), inorganic phosphorus (I. phosphorus: PNP-XDH method) and total bile acid (T. bile acid: enzyme cycling method) Sodium (Sodium: ion selective electrode method) using EA 06 R (A & T Co., Ltd.) Potassium (Potassium: ion selective electrode method) and chlorine (Chloride Useful: ion selective electrode method) were measured.

URINALYSIS: Yes
- Time schedule for collection of urine: Until the end of the administration period, 3 urine and 24 hour urine were collected
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.?] were examined.: pH, occult blood, sugar and protein, urine volume (weighing) and color tone (visual inspection) and Urine osmotic pressure were examined.

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Oestrous cyclicity (parental animals):
Yes
Litter observations:
numbers of offspring or live offspring, the sex ratio, live birth index, viability index or body weights were examined.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
At autopsy brain, thymus, liver, kidney, spleen, adrenal glands, testis and epididymis and ovary weights were recorded.
Macroscopic observation of the organs / tissues was performed and the skin, mammary gland, lymph node (mesenteric lymph node, mandibular lymph node), sternum, femur, marrow (sternum, femur), thymus, trachea, lung and bronchus , Heart, thyroid, parathyroid gland, tongue, esophagus, stomach and duodenum, small intestine (including Peyer's patch), large intestine (including Peyer's patch), liver, pancreas,
spleen, kidney, adrenal gland, bladder, seminal vesicle, A 10 vol% neutral buffered formalin solution was added
to the eyeball, Harder's gland, brain (including cerebrum, cerebellum, bridge), pituitary gland, spinal cord (neck, chest, lumbar region), sciatic nerve (including muscle proximal end) Testis and epididymis were fixed in Bouin's solution.

HISTOPATHOLOGY: Yes
Five fixed organs in each of the control group and high dose group were performed on all the fixed organs, and abnormal lesion tissues observed in all groups at autopsy were performed.
Postmortem examinations (offspring):
Gross pathology were examined.
Statistics:
Statistical analysis were performed by using automatic discrimination method for Weight, body weight gain, food intake, cumulative food intake, average sex cycle, number of corpus luteums, number of implantation traces, gestation period, number of births, number of stillbirth children, sex ratio, implantation rate, birth rate and by using Bartlett 's equidistance test abnormal appearance rate of external table, survival rate of newborn on 4th, hematology examination value, blood biochemical examination value, urinalysis value (urine volume and osmotic pressure), organ weight and relative weight were analysis. In the case of equal variance, a significant difference between the control group and each administration group was tested in Dunnett's multiple comparison test. In the case of unequal variance in Bartlett's isomorphic test, a significant difference between the control group and each administration group was tested by Steel's test. For the birth rate, mating rate and conception rate, χ2test was used. The abnormal periodic incidence rate, necropsy findings, and incidence of histopathological findings were tested
by Fisher's exact test. The significance level was 5% for Bartlett's equidistance test and 2 and 5% for other tests. However, when the number of animals to be tested was 2 cases or less per group, no significant difference test was conducted. In addition, the results pertaining to the babies during the nursing period were tabulated with the average per mother as one sample.
Reproductive indices:
copulation index, fertility index, live birth index, viability index, implantation index, gestation index and delivery index were examined.
Offspring viability indices:
Yes, on day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
When treated with 50 mg/kg bw, , nasal secretions in 2 cases, loose stools in 1 cases of male and depilation and crusts in 2 cases were observed.

When treated with 150 mg/kg bw, salivation in 3 cases, nasal secretions in 1 cases, loose stools in 2 cases, crust 1 cases in male rats. Most salivation observed in the 150 mg / kg group was a transient change from
immediately after administration to about 5 minutes after administration.
In female rats, salivation in 2 cases, lacrimation before mating, mating, pregnancy and nursing period, depilation and crusts in 2 cases were observed. Salivation was a transient change from immediately after administration to about 5 minutes after administration in the 150 mg / kg group.


When treated with 500 mg/kg bw, salivation in 12 cases, lacrimation in 2 cases, loose stools in 2 cases, crust in 4 cases, trauma 1 case in male and salivation in continued from 30 to 60 minutes after administration.
In female, lacrimation in all 12 cases, nasal secretions and ocular secretion in 1 case, depilation and crusts in 2 cases were observed. several animals continuing up to 6 hours after administration were also observed.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A dead case was males in the 50 mg / kg group and one case was observed on the 33th day. However, this animal was accidental death that was mistakenly caught in a breeding cage and died, and it was not related to administration of the test substance.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant change in body weight were observed in treated rats as compared to control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
When treated with 500 mg/kg bw in male rat, significant increase in food consumption were observed during the administration of 43 to 48 days. but it was a slight change and was not judged as the influence of administration of the test substance. There was no difference in the cumulative food consumption between 1 and 15 days after administration and the control group in any of the test substance administration groups.
In females, no significant difference was observed between the control group and the test substance administered group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When treated with 500 mg/kg in males , significant increase in reticulocyte rate, significantly shorter in activated partial thromboplastin time in the blood clotting ability were observed.
In female, significant increase in large unstained cell ratio and significantly prolonged prothrombin time were observed, but it was a minor change and was not judged as the influence of administration of the test substance.

No changes were observed at 50 and 150 mg/kg bw treated male and female rats as compared to control.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
When treated with 500 mg/kg in males, increase in A / G ratios and total bile acids level but not significant as compared to control. Low neutral fat Value were observed. Total bilirubin level was significantly increased but, it was a minor change and was not judged as the influence of administration of the test substance.
In female rats, significant decrease in ALT value was observed. Significant increase in total bile acids value although there was no significant or statistically significant difference in all test substance administered groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Slight Yellow urine were observed in 3, 2, 1 and 0 cases in the control group, 50, 150 and 500 mg / kg group, respectively.
No significant differences were found between the control group and the test substance-administered group in
other test items.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No significant histopathological changes were observed in treated male and female rats as compared to control.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant on estrous cycle of trreated female rats were observed as compared to control.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect on Copulation, pregnancy period, the number of corpus luteums, thenumber of implantation traces, the number of births and the number of births, implantation rate, birth rate and sex ratio of treated female as compared to control.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
other: NO effect observed

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effect on 4-day survival rate of treated pups were observed as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant change in body weight at 0 and 4 days of treated pups were as compared to control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
When treated with 150 mg/kg bw, pelvic dilation were observed in one male pup during the nursing period.

At necropsy on 4th day of nursing, thymus neck residues were 1 in 150 males and 500 mg / kg male group, 1 in each case in liver white spots of 500 males and 50 mg / kg females, 1 kidney 4, 3, 2 and 1 cases respectively in the 4, 3 and 1 cases, 50, 150 and 500 mg / kg group in the 50, 150 and 500 mg / kg male groups, respectively Tubular expansion was 1, 3, 5 and 4 cases respectively in 4, 3 and 1 cases, 50, 150 and 500 mg / kg group in males, control group in females, 50, 150 and 500 mg / kg group, respectively Crust was found in 150 mg / kg group of male and female, respectively 3 cases, gangrene of the tail was 1 in case of female in control group, 1 case in female with mucosa, ankle and liver whitening in 500 mg / kg group and skin Crust was observed in one case in a 500 mg / kg group males.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology
other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table No 1 Hematology and Coagulation of rats trated orally with Cyclohexane in the combined repeat dose and reproductive/developemental toxicity screening test

Dose Level (mg/kg)

0

50

150

500

Male

No. of animals

12

11

12

12

APTT (sec.)

22.0 ± 2.1

20.4 ± 1.2

20.6 ± 2.0

19.4 ± 1.9**

Reticulocyte

2.1 ± 0.3

2.3 ± 0.3

2.4 ± 0.3

2.5 ± 0.4*

Female

No. of animals

10

10

10

10

LUC

0 ± 0

0 ± 0

0 ± 1

1 ± 0*

PT (sec.)

14.5 ± 0.5

15.2 ± 0.6

15.1 ± 0.6

15.4 ± 0.6**

NEUT: Neutrophil, LYMPH: Lymphocyte, MONO: Monocyte, EOSN: Eosinophil, BASO: Basophil, LUC: Large Unstained cells,

Values are expressed as Mean ± S.D.

Significant different from control group: *p≤ 0.05, **p≤ 0.01

 

Table No 2 Blood Chemical examination of rats treated orally with Cyclohexane in the combined repeat dose and reproductive/developmental toxicity screening test

Dose Level (mg/kg)

10

50

150

500

Male

No. of animals

12

11

12

12

T. billirubin (mg/dL)

0.03 ± 0.01

0.04 ± 0.01

0.05 ± 0.01

0.05 ± 0.01*

Female

No. of animals

10

10

10

10

ALT (U/L)

61 ± 71

56 ± 18

51 ± 9

45 ± 10*

T. bile acid (µmol/L)

19.3 ± 8.6

49.2 ± 28.8*

31.2 ± 19.7

82.2 ± 81.1*

Values are expressed as Mean ± S.D.

Significant different from control group: *p≤ 0.05, **p≤ 0.01

 

Table No 4 Absolute and relative organ weights of rats treated orally with Cyclohexane in the combined repeat dose and reproductive/developmental toxicity screening test

Dose Level (mg/kg)

0

50

150

500

Male

No. of animals examined

12

11

12

12

Absolute organ weight

Testes (g)

3.10 ± 0.72

3.15 ± 0.68

3.28 ± 0.36

3.19 ± 0.62

Relative organ weight

Kidneys (g%)

0.625 ± 0.057

0.619 ± 0.031

0.667 ± 0.059

0.705 ± 0.053*

Female

No. of animals examined

10

10

10

10

Absolute organ weight

Ovaries (mg)

104 ± 11

95 ± 12

105 ± 9

99 ± 10

Relative organ weight

Ovaries (mg%)

36.099 ± 3.119

35.550 ± 4.599

36.061 ± 3.939

34.688 ± 3.495

Values are expressed as Mean ± S.D.

Significant different from control group: *p≤ 0.05, **p≤ 0.01

 

Table No 5 Summary of histological findings with statistical analysis in rats treated orally with Cyclohexane in the combined repeat dose and reproductive/developmental toxicity screening test

Dose Level (mg/kg)

No. of animals examined histologically

 

Male

Female

0

50

150

500

0

50

150

500

     Organ

findings

7

2

2

7

5

3

1

6

Cardiovascular system

Heart

 

(5)

 

 

(5)

 

 

 

(5)

 

Cellular infiltration

4

-

-

3

0

-

-

0

Hematopoietic system

Spleen

 

(5)

 

 

(5)

 

 

 

(5)

 

Deposit, pigment

1

-

-

1

0

-

-

0

 

Hematopiesis, extramedullary

0

-

-

0

4

-

-

4

Thymus

 

5

 

 

5

 

1

 

 

 

Hamorrhage

0

-

-

0

0

0

-

1

 

Atrophy

0

-

-

0

1

1

-

0

 

Cyst

2

-

-

0

2

0

-

1

Respiratory system

Lung

 

(5)

 

 

(5)

 

 

 

(5)

 

Accumulation of macrophage

1

-

-

1

1

-

-

2

 

Hypertrophy, media

Artery

1

-

-

1

1

-

-

2

Trachea

 

(5)

 

 

(5)

 

 

 

(5)

 

Dilatation, gland

0

-

-

0

2

-

-

1

Digestive system

Exocrine pancreas

 

(5)

 

 

(5)

 

 

 

(5)

 

Degeneration, vacuolar

1

-

-

0

0

-

-

0

 

Cellular infiltration,

lymphocyte

1

-

-

0

0

-

-

0

Small intestine

 

(5)

 

 

(5)

 

 

 

(5)

 

Diverticula

1

-

-

1

0

-

-

0

 

 

Hyperplasia, lymphoid tissue

1

-

-

1

2

-

-

1

Large intestine

 

(5)

 

 

(5)

 

 

 

(5)

 

Hyperplasia, lymphoid tissue

0

-

-

0

2

-

-

2

Liver

 

(5)

 

(1)

(5)

 

(2)

 

(5)

 

Fatty change

4

-

0

5

4

0

-

5

 

Cellular infiltration,

lymphocyte

2

-

0

1

0

0

-

0

 

Microgranuloma

5

-

0

5

5

2

-

4

 

Hematopiesis, extramedullary

0

-

0

0

3

1

-

2

 

Hepatodiaphragmatic nodule

0

-

1

0

0

0

-

0

Urinary system

Kidney

 

(5)

 

 

(5)

 

(5)

 

(5)

 

Basophilic tubules

5

-

-

3

3

1

-

1

 

Cast, hyaline

1

-

-

0

0

1

-

0

 

Cell debris, lumen

0

-

-

0

0

1

-

0

 

Degeneration

0

-

-

0

0

1

-

0

 

Degeneration, vacuolar

0

-

-

0

0

1

-

0

 

Dilatation, tubules

1

-

-

2

0

0

-

0

 

Eosinophilic body

2

-

-

3

0

0

-

0

 

Hyaline droplet

3

-

-

4

0

0

-

0

 

Mineralization

0

-

-

0

1

0

-

1

 

Necrosis, tubular epithelium

0

-

-

0

0

1

-

0

 

Cellular infiltration,

lymphocyte

2

-

-

1

0

0

-

0

Reproductive system

Mammary glands

 

(5)

 

 

(5)

 

 

 

(5)

 

Hyperplasia

0

-

-

0

5

-

-

5

Testis

 

(5)

 

 

(5)

 

 

 

 

 

Atrophy, seminiferous tubule

1

-

-

1

-

-

-

-

Epididymis

 

 

 

 

(6)

 

 

 

 

 

Spermatic granuloma

2

2

-

1

-

-

-

-

Prostate

 

(5)

 

 

(5)

 

 

 

 

 

Cellular infiltration,

Lymphocyte

3

-

-

3

-

-

-

-

Uterus

 

 

 

 

 

 

 

 

(5)

 

Post delivery lesion

-

-

-

-

4

-

-

4

Endocrine system

Thyroid gland

 

(5)

 

 

(5)

 

 

 

(5)

 

Ultimobranchial remnant

0

-

-

0

1

-

-

1

Adrenal gland

 

(5)

 

 

(5)

 

 

 

(5)

 

Degeneration, vacuolar

1

-

-

2

0

-

-

0

Special Sense System

Harderian gland

 

(5)

 

 

(5)

 

 

 

(5)

 

Cellular infiltration,

Lymphocyte

1

-

-

1

0

-

-

0

Integumentary System

Skin

 

(5)

 

(1)

(1)

 

(1)

 

(5)

 

Ulcer

0

-

0

0

0

1

0

0

( ): Number of animals examined microscopically at this site.

-: Not applicable

 

Table no 6 Summary of reproductive performance in rats treated orally with Cyclohexane in the combined repeat dose and reproductive/developmental toxicity screening test

Dose level (mg/kg)

0

50

150

500

No. of pairs mated

12

12

12

12

No. of pairs copulated

12

11

12

12

No. of pregnant females

11

10

10

10

Copulation index(%)a

100.0

91.7

100.0

100.0

Fertility index (%)b

91.7

90.9

83.3

83.3

Estrous cycle

No. of animals examined

12

12

12

12

Mean Estrous cycle (Days, mean ± S.D.)

4.1 ± 0.1

4.1 ± 0.3

4.1 ± 0.4 (11)

4.1 ± 0.3

Irregular cyclec(%)d

0(0.0)

0(0.0)

1(8.3)

2(16.7)

a)     (No. of animals with successful copulation / no of animals mated ) x 100

a)     (No. of pregnant animals/ no. of animals with successful copulation) x 100

b)     (No. of animals having irregular estrous cycle)

c)      (No. animals having irregular estrous cycle / no. of animals examined ) x 100

Values in parentheses are expressed no. of animals observed

Applicant's summary and conclusion

Conclusions:
No adverse effect level(NOAEL) was considered to be 500 mg/kg for P and F1 generation when when Crj:CD(SD)IGS male and female rats were treated with Cyclohexene orally by gavage for 48 consecutive days in male and 53 days in female respectively.
Executive summary:

In a Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test, Crj:CD(SD)IGS male and female rats were treated with Cyclohexenein the concentration of 0, 50, 150 and 500 mg/kg/day orally by gavage in corn oil for 48 consecutive days in male and 53 days in female respectively as per OECD 422. Nasal secretions in 2 cases, loose stools in 1 cases of male and depilation and crusts in 2 cases were observed at 50 mg/kg bw, salivation in 3 cases, nasal secretions in 1 cases, loose stools in 2 cases, crust 1 cases in male rats at 150 mg/kg bw. Most salivation observed was a transient change from immediately after administration to about 5 minutes after administration. In female rats, salivation in 2 cases, lacrimation before mating, mating, pregnancy and nursing period, depilation and crusts in 2 cases were observed at 150 mg/kg bw. Salivation was a transient change from immediately after administration to about 5 minutes after administration in the 150 mg / kg group. Salivation in 12 cases, lacrimation in 2 cases, loose stools in 2 cases, crust in 4 cases, trauma 1 case in male at 500 mg/kg bw and salivation in continued from 30 to 60 minutes after administration. In female, lacrimation in all 12 cases, nasal secretions and ocular secretion in 1 case, depilation and crusts in 2 cases were observed. several animals continuing up to 6 hours after administration were also observed at 500 mg/kg bw. A dead case was males in the 50 mg / kg group and one case was observed on the 33th day. However, this animal was accidental death that was mistakenly caught in a breeding cage and died, and it was not related to administration of the test substance. No significant change in body weight was observed in treated rats as compared to control. In male rat, significant increase in food consumption were observed during the administration of 43 to 48 days at 500 mg/kg bw, but it was a slight change and was not judged as the influence of administration of the test substance. There was no difference in the cumulative food consumption between 1 and 15 days after administration and the control group in any of the test substance administration groups. In females, no significant difference was observed between the control group and the test substance administered group. In male rats, significant increase in reticulocyte rate, significantly shorter in activated partial thromboplastin time in the blood clotting ability were observed at 500 mg/kg. In female, significant increase in large unstained cell ratio and significantly prolonged prothrombin time were observed at 500 mg/kg, but it was a minor change and was not judged as the influence of administration of the test substance. No changes were observed at 50 and 150 mg/kg bw treated male and female rats as compared to control. In males, increase in A / G ratios and total bile acids level at 500 mg/kg, but not significant as compared to control. Low neutral fat Value were observed. Total bilirubin level was significantly increased at 500 mg/kg but, it was a minor change and was not judged as the influence of administration of the test substance. In female rats, significant decrease in ALT value was observed at 500 mg/kg. Significant increase in total bile acids value although there was no significant or statistically significant difference in all test substance administered groups. Slight Yellow urine were observed in 3, 2, 1 and 0 cases in the control group, 50, 150 and 500 mg / kg group, respectively. No significant differences were found between the control group and the test substance-administered group in other treated groups. Similarly, no significant effect on reproductive parameters of treated rats were observed as compared to control such as estrous cycle, copulation index, fertility index, gestation length, numbers of corpora lutea or implantations, implantation index, gestation index, delivery index, purturition or maternal behavior, numbers of offspring or live offspring, the sex ratio and live birth index. Scar of the liver and red spots of the brain of dead were observed at 50 mg / kg in male rat. No gross pathological and histopathological changes were observed in P treated male and female rats as compared to control. In addition, No effect on 4-day survival rate and body weight of treated pups were observed as compared to control. When treated with 150 mg/kg bw, pelvic dilation were observed in one male pup during the nursing period. At necropsy on 4th day of nursing, thymus neck residues were 1 in 150 males and 500 mg / kg male group, 1 in each case in liver white spots of 500 males and 50 mg / kg females, 1 kidney 4, 3, 2 and 1 cases respectively in the 4, 3 and 1 cases, 50, 150 and 500 mg / kg group in the 50, 150 and 500 mg / kg male groups, respectively. Tubular expansion was 1, 3, 5 and 4 cases respectively in 4, 3 and 1 cases, 50, 150 and 500 mg / kg group in males, control group in females, 50, 150 and 500 mg / kg group, respectively Crust was found in 150 mg / kg group of male and female, respectively 3 cases, gangrene of the tail was 1 in case of female in control group, 1 case in female with mucosa, ankle and liver whitening in 500 mg / kg group and skin Crust was observed in one case in a 500 mg / kg group males of F1. Therefore, No adverse effect level(NOAEL) was considered to be 5.1 mg/kg for males and 5.7 mg/kg for females when when FDRL male and female rats were treated with Beta-Naphthyl ethyl ether orally in feed for 90 days. No adverse effect level(NOAEL) was considered to be 500 mg/kg for P and F1 generation when when Crj:CD(SD)IGS male and female rats were treated with Cyclohexene orally by gavage for 48 consecutive days in male and 53 days in female respectively.

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