Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
tom
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
SHORT-TERM TOXICITY STUDY IN RATS DOSED WITH MENTHONE
Author:
C. MADSEN, G. WURTZEN and J. CARSTENSEN
Year:
1986
Bibliographic source:
Toxicology Letters, 32 (1986) 147- 152

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
28 days repeated dose oral toxicity test was performed on rats using menthone.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: 2-isopropyl-5-methylcyclohexanone
- Molecular formula: C10 H18 O
- Molecular weight: 154.2512g/mol
- Smiles notation: C1([C@@H](CC[C@@H](C1)C)C(C)C)=O
- InChl: 1S/C10H18O/c1-7(2)9-5-4-8(3)6-10(9)11/h7-9H,4-6H2,1-3H3
- Substance type: Organic
- Physical state: liquid
Specific details on test material used for the study:
- Name of test material: Menthone racemic
- IUPAC name: 2-isopropyl-5-methylcyclohexanone
- Molecular formula: C10H18O
- Molecular weight: 154.2512 g/mol
- Substance type: Organic
- Physical state: Solid
- Impurities (identity and concentrations): 97% pure

Test animals

Species:
rat
Strain:
other: SPF wistar
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mollegaards Breeding Centre Ltd., Ejby
- Age at study initiation: 4-week-old
- Weight at study initiation: No data
- Fasting period before study: No data available
- Housing: Housed 2/cage in stainless steel wire cages
- Diet (e.g. ad libitum): pelleted diet (Chow 101) ad lib.
- Water (e.g. ad libitum): acidified water (citric acid, pH 3.5) ad lib.
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23°C + 1° C
- Humidity (%): 60% ± 5%
- Air changes (per hr): 6-8 times/h
- Photoperiod (hrs dark / hrs light): Electric light from 21.00-09.00 h.

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
other: Soy bean oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Menthone was dissolved in soy bean oil to give four dosing concentrations 0, 200, 400 and 800 mg menthone/kg b.w. /day respectively.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Pelleted diet (Chow 101)
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Soy bean oil
- Concentration in vehicle: Actual dose: 0, 200, 400 and 800 mg menthone/kg b.w. /day
Males: 0, 200, 400 and 800 mg menthone/kg b.w. /day
Females: 0, 200, 400 and 671 mg menthone/kg b.w. /day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: Food grade
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Actual dose: 0, 200, 400 and 800 mg menthone/kg b.w. /day
Males: 0, 200, 400 and 800 mg menthone/kg b.w. /day
Females: 0, 200, 400 and 671 mg menthone/kg b.w. /day
No. of animals per sex per dose:
Total: 80
0 mg/Kg bw/day: 10 males and 10 females
200 mg/Kg bw/day: 10 males and 10 females
400 mg/Kg bw/day: 10 males and 10 females
800 (males) or 671 (females): mg/Kg bw/day: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. Mortality of rats was observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No data available

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 available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): yes
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: last day of the dosing period
- Anesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. Reticulocyte in whole blood was observed.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: last day of the dosing period
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. Concentration of bilirubin and alkaline phosphatase activity was determined in plasma.

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

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

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, The rats were killed after 4 weeks of dosing by exsanguination in CO2-anaesthesia. A thorough autopsy was performed and the following organs were excised and weighed: kidneys, adrenals, spleen, heart, liver and brain.

HISTOPATHOLOGY: Yes, kidneys, adrenals, spleen, heart, liver and brain lung, aorta, mesenterial lymph node, thymus, stomach, jejunum, colon, thyroid, parathyroid, pancreas, testis, ovary, uterus, spinal cord and ischiatic nerve were fixed in 10 % buffered formalin, prepared for light microscopy and stained with haematoxylin-eosin (all), PerI (liver) and PAS (liver). Frozen sections were prepared from the liver and stained with Oil Red O.
Other examinations:
No data
Statistics:
Body weights, food and water consumption were compared by means of Student’s t-test. Analysis of variance was performed on haematoiogical parameters,
clinicochemical parameters and absolute and relative organ weights. The mean values were compared by the method of Tukey.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality
Clinical signs: 400 mg/Kg bw/day dosed female rats showed pale mucous membranes and showed signs of pain after 19 days of dosing

Mortality: 4 animals died during the study due to accidental intratracheal dosing.

Body weight and weight gain: The reduced food consumption was accompanied by a decrease in body weight gain. The terminal body weights in both males and females were decreased. The decrease was dose-dependent and statistically significant

Food consumption and compound intake: Food consumption was reduced in male rats in 800 mg/Kg bw/day group for entire period while in female rats food consumption was reduced in first 2 week in all dosed animals and for 400 mg/Kg bw/day in 3rd week.

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: Dose-dependent decrease of creatinine concentration and a dose dependent increase of both alkaline phosphatase activity and bilirubin content in blood plasma was observed.

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: A statistically significant dose-related increase of the relative weight of the kidneys, spleen, liver and brain of the female groups and of the spleen, liver and brain of the male groups was observed

Gross pathology: No data available

Histopathology: Histological examination revealed dose-related alterations in the brain. Cyst-like spaces appeared scattered in the white matter of the cerebellum in the two highest dosed groups (males: 400 and 800 mg/Kg bw/day and females: 400 or 671 mg/Kg bw/day)

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
< 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Significant changes in liver weight and bilirubin content of plasma at 200 mg/Kg bw/day
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Significant changes in liver weight and bilirubin content of plasma at 200 mg/Kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Body Weight and Relative Organ Weights of Rats Dosed w ith 0, 200, 400 and 800aMg Menthone/Kg bw/day for 28 Days

 

 Dose (mg/kg bw/day)

 

0

200

400

800a

Number of animals

 

 

 

 

Female

10

9

8

10

Male

10

10

9

10

Body weight (g)

 

 

 

 

Female

187±18

176±13

168±9

166±11**

Male

286±29

258±21

243±25

234±26**

Kidneys (mg/g)

 

 

 

 

Female

7.08±0.44

7.58±0.52

7.68±0.48

7.16±0.42*

Spleen (mg/g)

 

 

 

 

Female

2.73±0.36

3.15±0.42

3.39±0.79

3.53±0.48*

Male

2.36±0.29

2.47±0.27

3.13±0.52

3.10±0.52***

Liver (mg/g)

 

 

 

 

Female

31.14±1.10

34.37±2.01

37.53±1.51

40.21±2.14***

Male

31.46±2.18

34.28±2.61

37.19±1.67

42.40±2.42***

Brain (mg/g)

 

 

 

 

Female

9.84±0.74

10.44±0.84

10.73±0.59

10.91±0.92*

Male

6.84±0.46

7.14±0.91

7.93±0.62

8.35±0.56***

Means±SD (* P<0.05, ** P< 0.01; *** P< 0.001).

aDose reduced to 400 mg/kg in the female group on day 19.

Table 2:Clinical Chemical Parameters in Rats Dosed with Menthone (N = 8/Sex/Group)

 

 Dose (mg/kg bw/day)

 

0

200

400

800a

Creatininea(µmol/L)

 

 

 

 

Female

68.8±7.6

62.9±1.3

59.4±5.5

50.5±7.6 ***

Male

64.2±6.3

66.1±6.8

59.6±6.5

49.2±5.5 ***

Alkaline phosphataseb(U/L)

 

 

 

 

Female

159±38

225±68

211±64

346±149**

Male

260±55

363±178

440±120e

451±144**

Bilirubinb(mg/100 mL)c

 

 

 

 

Female

0.19±0.06

0.23±0.06

0.30±0.19

0.35±0.14***

Male

0.13±0.06

0.20±0.06

0.26±0.05

0.44±0.19***

Results are mean±SD. (** P < 0.01; *** P< 0.001).

aAfter 21 days of dosing.

bAfter 28 days of dosing.

cDose reduced to 400 mg/kg in the female group on day 19.

d5 animals.

e7 animals.

 

Table: 3 Number of Animals with Histological Changes in the Cerebellum After Administration of Menthone for 28 Days

 

 Dose (mg/kg bw/day)

 

0

200

400

800a

Female

0 (10)

0 (9)

1 (8)

7 (10)

Male

0 (10)

0 (10)

3 (9)

5 (10)

aIn the female group the dose was reduced to 400 mg/kg after 19 days of dosing.

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for 2-isopropyl-5-methylcyclohexanone (menthone) is found to be < 200 mg/Kg bw/day using SPF Wistar male and female rats and the low Observed adverse effect level (LOAEL) is found to be 200 mg/Kg bw/day.
Executive summary:

Subacute repeated dose oral toxicity test was performed for the test chemical menthone ( IUPAC name: 2-isopropyl-5-methylcyclohexanone) on SPF Wistar rats at dose concentration 0, 200, 400, and 800 mg/kg/day for 28 days. 10 male and 10 female were grouped in each dose level. Chemical was given orally in form of gavage dissolved in soy bean oil. As the female rats in the highest dose group showed serious weakening, pale mucous membranes and signs of pain and hence after 19 days of dosing the dose was reduced to 400 mg/kg. The average doses for the female groups were 0, 200, 400 and 671 mg/kg b.w./day.

 

A significant reduction of food consumption and body weight was noted in 800 mg/Kg/day males and all dose group female animals and was considered a result of a general toxic effect of the substance. The dose-related increase of the relative brain weight was due to growth depression. The increase of relative kidney (female), spleen and liver weight reflected a true increase as a function of dosing and indicated a toxic effect of the substance at the highest dose (800 mg/Kg/day for males and 671 mg/Kg/day for females). Marked dose-related increase of bilirubin and alkaline phosphatase concentration was noted resulting in an adverse effect on the liver. No histopatholical signs were observed in liver but may express subcellular changes resulting in intrahepatic cholestasis. A decrease in blood creatinine was observed at the highest dose (800 mg/Kg/day for males and 671 mg/Kg/day for females) and might be related to biochemical changes in the liver.

The encephalopathy induced in the rats dosed with menthone at 400 or 800 mg/kg b.w./day was confined to the white matter of the cerebellum. The lesions were cyst like spaces, which caused no cellular reaction in the adjacent tissue. The spaces were not surrounded by a membrane and did not seem to occur intracellularly. The pathomorphological picture indicated that the observed cyst-like spaces were present between unbroken myelin sheets. The encephalopathy produced no clinical symptoms that could be observed by a general clinical inspection. The increase in liver weight and bilirubin content of plasma was statistically significant even at 200 mg/Kg bw/day.

 

The No Observed Adverse Effect Level (NOAEL) is considered to be < 200 mg/Kg bw/day and the low Observed adverse effect level (LOAEL) is found to be 200 mg/Kg bw/day for menthone when exposed to SPF Wistar rats by the oral route of exposure.