3-(2,2-dimethyl-3-hydroxypropyl)toluene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-(2,2-dimethyl-3-hydroxypropyl)toluene

EC Number:

403-140-4

EC Name:

3-(2,2-dimethyl-3-hydroxypropyl)toluene

Cas Number:

103694-68-4

Molecular formula:

C12H18O

IUPAC Name:

2,2-dimethyl-3-(3-methylphenyl)propan-1-ol

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: adult, 3 months old
- Weight at study initiation: males: 28 - 34 g; females: 26 - 32 g
- Assigned to test groups randomly: yes, under following basis: by lot
- Fasting period before study: no
- Housing: collective caging
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2°C
- Humidity: 50 - 80 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: isotonic saline
- Justification for choice of solvent/vehicle: recommended by guideline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test solution was prepared by diluting appropriate amounts with isotonic saline.

Duration of treatment / exposure:

single intraperitoneal application

Frequency of treatment:

1

Post exposure period:

24, 48, 72 hours, respectively

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 48 hours
Male: 1000 mg/kg bw; No. of animals: 5; Sacrifice time: 72 hours

Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 48 hours
Female: 1000 mg/kg bw; No. of animals: 5; Sacrifice times: 72 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: recommended by the guideline
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/ kg bw
- Positive control animals were evaluated 24 h after application

Examinations

Tissues and cell types examined:

Erythrocytes from femura bone marrow.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Preparations were dried, fixed in absolute (99 %) methanol for 5 min. and then allowed to air dry. The slides were stained using a May-Grünwald and Giemsa solution.

METHOD OF ANALYSIS: From each animal 2 preparations were made. Prior to analysis all the slides were randomized and coded (blind evaluation). The cells were examined at a magnification of 1000 x.
A total of 1000 polychromatic erythrocytes was scored from each slide and the number of micronucleated cells in each sample was recorded. The ratio of polychromatic erythrocytes to normochromatic (mature) erythrocytes was calculated on the base of 1000 cells.

Evaluation criteria:

Cell counts are based on a total of 1000 cells per anirnal. Due to own laboratory historical data an incidence of (up to) 0.8 %, respectively of micronucleated polychromatic erythrocytes is considered to be within normal limits.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of toxicity were seen at 1000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No lethal effects, no cytytoxicity (PCE/NCE).

Any other information on results incl. tables

Dose-Range-Finding

A preliminary dose-range-finding study was performed applying single doses of 5000, 2500 and 1000 mg/kg body weight, respectively by intraperitoneal injection in 2 mice each. Animals treated at 5000 mg/kg body weight survived, but exhibited signs like marked sedation, ventral position, ataxia, cyanosis and writhing reflex up to six hours p.a. A dose level of 2500 mg/kg body weight produced similar signs less severe and 1000 mg/kg produced only slight, individually barely perceptible reactions. This dose level was therefore considered to be a suitable dosage, representing the area of the MTD (maximally tolerated dosage).

 

Number of polychromatic erythrocytes with micronuclei

The number of polychromtic erythrocytes with micronuclei was significantly increased 24 h post injection in the positive control animals. This result confirms the sensitivity of the used animal strain. There was no significant difference to control values at any time point in the males of the test compound treated group. All group mean values lay within normal ranges.

 

Number of polychromatic erythrocvtes

The number of polychromatic erythrocytes was not significantly different in the test item treated animals. It decreased, however, significantly in the positive control group (sum of both sexes).

 

Number of normochromatic erythrocytes

There was an analogue increase in the number of normochromatic erythrocytes in the positive control group, but none in the test item treated mice.

 

Ratio polychromatic normochromatic ervthrocytes

Due to the previous results, the ratio dropped significantly only in the positive control group attaining statistical significance in the sum of both sexes.

 

Applicant's summary and conclusion

Conclusions:

The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.

Executive summary:

The test item was administered once intraperitoneally at one high dose level of 1000 mg/kg body weight to mice of the NMRI-strain. Samples of the bone marrow were taken after 24 hours, after 48 hours and finally after 72 hours from 5 males and 5 females each. A concurrent control group (C) received the vehicle only and a positive control group (C-pos.) was treated with Cyclophosphamide (EndoxanR) at a dose level of 40 mg/kg body weight. Results from the data analysis show that there was no significant difference in the number of micronucleated polychromatic erythrocytes compared to the control values at any time point. The number of polychromatic and normochromatic erythrocytes and the ratio polychromatic / normochromatic erythrocytes was also not significantly different to the controls in animals treated with the test item at any time point. The positive control group, treated with 40 mg Cyclophosphamide /kg bw revealed a distinct, significant increase in the incidence of micronuclei (62.3‰, p < 0.001). In this group the ratio of polychromatic erythrocytes to normochromatic erythrocytes was significantly decreased in the sum of both sexes in the positive control group, thus indicating signs of toxicity to erythropoiesis. The decrease in the ratio was due to a significant dropping of polychromatic erythrocytes and a significant increase in the number of normochromatic erythrocytes.

The intraperitoneal administration of the test item at a dose level of 1000 mg/kg bw to male and female mice did not produce significant increases in the frequency of micronuclei in the polychromatic erythrocyte stem cells. All mean values lay completely within laboratory own normal ranges. Therefore, under the experimental conditions of this study, the test item is considered to be non-mutagenic at any tested time point in this test system.