Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 OCT 1994 to 2 AUG 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Version of Guideline not specified
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Principles of method if other than guideline:
Micronucleus cytogenetic assay in mice with thymol (Nr. 259, CAS 89-83-8)
GLP compliance:
yes
Remarks:
with the following exceptions: please refer to 'Any other information on material and methods'
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Thymol
EC Number:
201-944-8
EC Name:
Thymol
Cas Number:
89-83-8
Molecular formula:
C10H14O
IUPAC Name:
5-methyl-2-(propan-2-yl)phenol
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; Protected from exposure to light and moisture

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Males 32.9 to 41.0 g; Females 26.6 to 32.8 g
- Assigned to test groups randomly: yes, under following basis: according to a computer-generated program which is based on distribution according to body weight.
- Housing: AAALAC-accredited facility. Mice of the same sex were housed up to five per cage in plastic autoclavable cages with filter tops. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ≥ 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 27
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil (obtained from Sigma)
- Justification for choice of solvent/vehicle: Thymol was reported to be soluble at 1 g/1.6 mL olive oil at 25°C.
- Concentration of test material in vehicle: 13.8, 27.5 and 55.0 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Individual stock solutions were prepared at 13.8, 27.5 and 55.0 mg/mL for dosing the micronucleus assay.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
single treatment
Post exposure period:
Bone marrow cells were collected 24 hours after treatment for all dose levels and the vehicle and positive control and additionally at 16 and 48 hours after treatment with 1100 mg/kg bw.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
nominal
Dose / conc.:
275 mg/kg bw (total dose)
Remarks:
nominal
Dose / conc.:
550 mg/kg bw (total dose)
Remarks:
nominal
Dose / conc.:
1 100 mg/kg bw (total dose)
Remarks:
nominal
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP, CAS 6055-19-2), obtained from Sigma Chemical Company
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw; 2 mg/mL in distilled water

Examinations

Tissues and cell types examined:
bone marrow cells were examined for micronucleated polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high dose for the micronucleus assay was set to 1100 mg/kg bw which was judged to be the maximum tolerated dose based on toxicity studies.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

number of animals sampled:

16h 24h 48h
Vehicle control 5
275 mg/kg bw 5
550 mg/kg bw 5
1100 mg/kg bw 5 5 5
Positive control (40 mg/kg bw) 5

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded per 1000 erythrocytes.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p <= 0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay will be recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
For the pilot study, thymol was administered by oral gavage to male ICR mice at 10, 100, 1000, and 2500 mg test article/kg body weight and to male and female ICR mice at 5000 mg/kg which was administered in a total volume of 20 mL test article-vehicle mixture/kg body weight. The test article vehicle was distilled water. Mortality occurred in 5/5 males and 5/5 females treated with 5000 mg/kg. Clinical signs, which were noted within two hours after dose administration, included lethargy in male mice treated with 1000 and 2500 mg/kg and piloerection in male mice treated with 1000 mg/kg. All other animals appeared normal throughout the observation period.
For the initial toxicity study, thymol was administered by oral gavage to male and female ICR mice at seven treatment levels: 2500, 3000, 3500, 3800, 4200, 4500, or 4800 mg test article/kg body weight which was administered in a total volume of 20 mL test article-vehicle mixture/kg body weight. The test article vehicle was distilled water. Mortality occurred as follows: 1/3 males and 2/3 females at 3500 mg/kg, 0/3 males and 2/3 females at 3800 mg/kg, 0/3 males and 1/3 females at 4200 mg/kg, and 0/3 males and 1/3 females at 4500 mg/kg. No deaths occurred at 2500, 3000 or 4800 mg/kg. Clinical signs included lethargy and piloerection in mice at all dose levels. In addition, one male mouse treated with 2500 mg/kg exhibited irregular breathing. The test article formed an oily emulsion in the vehicle that required vortexing prior to and between each dosing to assure homogeneity. Based on the difficulty in maintaining a homogeneous dosing stock and the poor relationship between mortality and test article dose, other vehicles were investigated. Thymol was reported to be soluble at 1 gram/1.6 mL olive oil at 25 °C and was judged by the Study Director to be an acceptable vehicle for the test article and compatible with the assay system.
For the repeat toxicity study using olive oil as the vehicle, thymol was administered by oral gavage to male and female ICR mice at six treatment levels: 500, 1000, 1800, 2300, 3000, or 5000 mg test article/kg body weight which was administered in a total volume of 20 mL test article-vehicle mixture/kg body weight. The test article formed solutions at all stock concentrations. Mortality occurred within one day of dose administration as follows: 0/3 males and ,1/3 females at 1000 mg/kg bw, 1/3 males and 2/3 females at 1800 mg/kg, and 3/3 males and 3/3 females at 2300, 3000, and 5000 mg/kg bw. Clinical signs included lethargy in mice at 500, 1000, and 1800 mg/kg bw and prostration and irregular breathing at 1800, 2300, 3000, and 5000 mg/kg bw. The high dose for the micronucleus test was set to 1100 mg/kg bw, which was judged to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was not statistically increased relative to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p ≥ 0.05, Kastenbaum-Bowman Tables) (please refer to 'Any other information on results').
- Ratio of PCE/NCE (for Micronucleus assay): Slight reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observed relative to the vehicle control in high dose male and female mice. These reductions suggest bioavailability of the test article to the bone marrow target (please refer to 'Any other information on results').
- Appropriateness of dose levels and route: The highest dose level induced toxicity. Mortality was observed in 2/20 male and 3/20 female mice receiving 1100 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 275 and 550 mg/kg; lethargy and prostration in male and female mice at 1100 mg/kg. Thus, the test was performed up to toxic dose levels. The route is apprpriate as a route relevant for human exposure was chosen (in accordance with the current version of the OECD TG 474 (2016)).
- Statistical evaluation: please refer to 'Any other information on results'

Any other information on results incl. tables

TABLE 1: SUMMARY OF BONE MARROW MICRONUCLEUS STUDY WITH THYMOLIN ICR MICE

 

MICRONUCLEATEDPOLYCHROMATIC ERYTHROCYTES

TREATMENT

SEX

TIME(h)

NUMBER OF MICE

PCE/TOTAL ERYTHROCYTES

NUMBER PER 1000 PCE'S (MEAN±S.D.)

NUMBER PER PCE'S SCORED1

Olive Oil (vehicle control)

20 mL/kg

M

24

5

0.55

0.0±0.00

0 / 5000

F

24

5

0.60

0.0±0.00

0 / 5000

Thymol

275 mg/kg

M

24

5

0.59

0.4 ± 0.89

2 / 5000

F

24

5

0.51

0.2±0.45

1/ 5000

550 mg/kg

M

24

5

0.53

0.0 ± 0.00

0 / 5000

F

24

5

0.57

0.2 ± 0.45

1/ 5000

1100 mg/kg

M

16

5

0.51

0.4 ± 0.89

2 / 5000

24

5

0.47

0.4 ± 0.89

2 / 5000

48

5

0.47

0.2±0.45

1/ 5000

F

16

5

0.62

0.0±0.00

0 / 5000

24

5

0.57

0.2 ± 0.45

1 / 5000

48

5

0.41

0.2 ± 0.45

1 / 5000

Cyclophosphamide(positive control)

40 mg/kg bw

M

24

5

0.63

6.2±5.22

31/ 5000*

F

24

5

0.57

4.4±2.88

22 / 5000*

1*p0.05 (Kastenbaum-Bowman Tables)

TABLE 2: INDUCTION OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES IN BONE MARROW CELLS COLLECTED 16 HOURS AFTER A SINGLE DOSE OF THYMOL

TREATMENT

SEX

ANIMAL NUMBER

PCE/TOTAL ERYTHROCYTES

MICRONUCLEATED PCE (NUMBER/PCE SCORED)

 

Thymol

 

1100 mg/kg

M

31

0.47

2 /1000

 

32

0.44

0 /1000

 

33

0.64

0 /1000

 

34

0.63

0 /1000

 

35

0.37

0 /1000

 

F

36

0.62

0 /1000

 

37

0.47

0 /1000

38

0.73

0 /1000

 

39

0.63

0 /1000

 

40

0.65

0 /1000

 

TABLE 3: INDUCTION OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES IN BONE MARROW CELLS COLLECTED 24 HOURS AFTER A SINGLE DOSE OF THYMOL

TREATMENT

SEX

ANIMAL NUMBER

PCE/TOTAL ERYTHROCYTES

MICRONUCLEATED PCE (NUMBER/PCE SCORED)

Olive Oil (vehicle control)

20 mL/kg

M

51

0.60

0 /1000

52

0.65

0 /1000

53

0.46

0 /1000

54

0.56

0 /1000

55

0.49

0 /1000

F

56

0.51

0 /1000

57

0.42

0 /1000

58

0.72

0 /1000

59

0.64

0 /1000

60

0.72

0 /1000

Thymol

275 mg/kg

M

61

0.50

0 /1000

62

0.70

0 /1000

63

0.57

0 /1000

64

0.59

2/1000

65

0.58

0 /1000

F

66

0.54

0 /1000

67

0.53

0 /1000

68

0.48

0 /1000

69

0.43

0 /1000

70

0.56

1/1000

550 mg/kg

M

71

0.56

0 /1000

72

0.66

0 /1000

73

0.41

0 /1000

74

0.62

0 /1000

75

0.43

0 /1000

F

76

0.49

0 /1000

77

0.51

0 /1000

78

0.55

1/1000

79

0.66

0 /1000

80

0.63

0 /1000

1100 mg/kg

M

81

0.63

2/1000

82

0.39

0 /1000

83

0.43

0 /1000

84

0.38

0 /1000

85

0.51

0 /1000

F

86

0.51

0 /1000

87

0.59

0 /1000

88

0.52

0 /1000

89

0.64

1/1000

90

0.62

0 /1000

Cyclophosphamide(positive control)

40mg/kg

M

41

0.78

4/1000

42

0.63

10/1000

43

0.58

13 /1000

44

0.64

4/1000

45

0.52

0/1000

F

46

0.48

6/1000

47

0.60

8/1000

48

0.55

1/1000

49

0.53

5/1000

50

0.68

2/1000

TABLE 4: INDUCTION OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES IN BONE MARROW CELLS COLLECTED 48 HOURS AFTER A SINGLE DOSE OF THYMOL

 

TREATMENT

SEX

ANIMAL NUMBER

PCE/TOTAL ERYTHROCYTES

MICRONUCLEATED PCE (NUMBER/PCE SCORED)

 

Thymol

 

1100 mg/kg

M

121

0.60

0 / 1000

 

122

0.40

0 / 1000

 

123

0.37

0 / 1000

 

124

0.48

1/ 1000

 

125

0.48

0 / 1000

 

F

126

0.40

0 / 1000

 

127

0.34

0 / 1000

 

128

0.52

0 / 1000

 

129

0.40

1/ 1000

 

130

0.39

0 / 1000

Applicant's summary and conclusion

Conclusions:
The results of the assay indicate that under the conditions described, thymol did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female ICR mice and was concluded to be negative in the mouse micronucleus assay.
Executive summary:

Male and female ICR mice were exposed to 275, 550 or 1100 mg/kg body weight of thymol which was administered in a total volume of 20 mL/kg as a single oral gavage. The high dose level was calculated to be approximately the maximum tolerated dose. Olive oil was used as vehicle. Mortality was observed in 2/20 male and 3/20 female mice receiving 1100 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 275 and 550 mg/kg; lethargy and prostration in male and female mice at 1100 mg/kg. Bone marrow cells were examined for micronucleated polychromatic erythrocytes.

Slight reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observed relative to the vehicle control in high dose male and female mice. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was not statistically increased relative to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p > 0.05).

The results of the assay indicate that under the conditions described in this report, thymol did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female ICR mice and was concluded to be negative in the mouse micronucleus assay.