Thymol

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Version of Guideline not specified

Deviations:

not specified

Test guideline 2

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

nominal

Doses / concentrations 2

Dose / conc.:

275 mg/kg bw (total dose)

Remarks:

nominal

Doses / concentrations 3

Dose / conc.:

550 mg/kg bw (total dose)

Remarks:

nominal

Doses / concentrations 4

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 SCORED^1
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*p≤0.05 (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.