Thymol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 JAN 1989 to 19 OCT 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

according Ames et al. Mut Res 31, 347-364 (1975), thymol was investigated in the Salmonella/microsome test in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

solid

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: partly deficient in lipopolysaccharide side chains in their cell walls and reduced UV repairability

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days before sacrifice. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C. The livers were washed with cold (4°C), 0.15 M KCl solution (approximately 1 mL KCl per 1 g liver) , and then homogenized in fresh, cold (4°C), 0.15 M KCl (approximately 3 mL KCl per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions.
- method of preparation of S9 mix: The S9 portions were slowly thawed before using. The S9 mix was freshly prepared (Ames et al., 1973a) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix permanently cold. 70 mL of cofactor solution are composed as follows:
MgCl2 x 6 H2O: 162.6 mg
KCl: 246.0 mg
glucose-6-phosphate, disodium salt: 179.1 mg
NADP, disodium salt: 315.0 mg
phosphate buffer: 100.0 mM
S9 mix consists of this cofactor solution and the corresponding volume of S9 fraction. In all tests, the S9 mix comprised 30% (v/v) S9 fraction.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix in final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metabolizing capacity by using 2-aminoanthracene; appropriate activity was demonstrated.

Test concentrations with justification for top dose:

first test: 0, 8, 40, 200, 1000, 5000 µg per plate
repeat test 1: 0, 6, 12, 24, 48, 96, 192 µg per plate (highest dose selected based on the substance's toxicity in the first test, which showed thymol to produce bacteriotoxic effects at 40 µg per plate and above)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, ethanol, acetone, DMSO, DMF, and Ethylene glycol dimethylether (EGDE) according to information given by the internal sponsor in a questionnaire.

Controls

Untreated negative controls:

no

Remarks:

No untreated control was set up for ethanol, since sufficient evidence was available in literature and from our own experience indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: -S9: nitrofurantoin (TA 100), 4-nitro-1,2-phenylene diamine (TA 98, TA 1537), +S9: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : 4 plates/concentration
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; other: marked and dose-dependent reduction in the mutant count per plate compared to the negative controls, titer (titers were determined under the same conditions as mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
-count of mutant colonies

Rationale for test conditions:

The Salmonella/microsome test is a screening method which detects point mutation caused by chemical agents in vitro. Auxotrophic mutants of Salmonella typhimurium are used to demonstrate this effect. For this purpose, the rate of reversion to prototrophy is evaluated in negative control and treated groups. A mutagenic effect is assumed if this rate increases sufficiently in the treated groups.

Evaluation criteria:

A reproducible and dose-related increase in mutant count of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of the negative controls, whereas for TA 1537 at least a threefold increase should be reached. Otherwise the result is evaluated as negative.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
A pre-test was performed at the doses of 0, 8, 40, 200, 1000, 5000 µg per plate. No genotoxicity was observed. Due to the substance's toxicity in the 40 and 200 µg/plate dose groups, doses ranging from 6 µg to 192 µg per plate were chosen for the repeat tests.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The concurrent vehicle controls are in the range of the historical control data. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene, with one exception, increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. The exception was given in Table 3. No effect was noted for 2-aminoanthracene on TA 1537. However, this lack could not be accounted for to a lack of sensitivity of the tester strains, since the strain-specific positive control 4-nitro-1,2-phenylene diamine demonstrated this sensitivity. But there was also no insufficient activating capacity of S9 mix, since the parallel experiments with TA 1535, TA 100 and TA 98 demonstrated the metabolizing capacity of S9 mix. Therefore the lack of activity of 2-aminoanthracene was accounted for to a technical failure, probably to a lack of compound.

Ames test:
- Signs of toxicity : The substance showed bacteriotoxicity at doses above 24 µg/plate as determined by titer.
- Individual plate counts : Please refer to attachment
- Mean number of revertant colonies per plate and standard deviation : please refer to table 1 and 2 under 'Any other information on results'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: please refer to table 3 under 'Any other information on results'
- Negative (solvent/vehicle) historical control data: please refer to table 3 under 'Any other information on results'

Any other information on results incl. tables

Table 1: Summary of Mean Values Without S9 Mix (Mix From Tables 1 - 8)
Results table and dose group [µg/plate]	Strain
	TA 1535	TA 100	TA 1537	TA 98
1-4
0	15	80	6	17
8	13	72	9	21
40	14	87	7	22
200	20	95	7	21
1000	0	--	-	--
5000	0	0	0	0
Na-azid	1089
NF		515
4-NPDA			99	151
5-8
0	8	89	9	22
6	8	97	6	20
12	7	115	6	20
24	5	97	10	22
48	8	69	6	20
96	10	76	7	21
192	5	67	6	21
Na-azid	896
NF		532
4-NPDA			115	197

Table 2: Summary of Mean Values With S9 (Mix From Tables 1-8)
Results table and dose group [µg/plate]	Strain
	TA 1535	TA100	TA 1537	TA 98
1-4
30%S9
0	20	119	15	37
8	14	108	10	27
40	13	103	11	34
200	18	99	7	23
1000	--	---	--	--
5000	0	0	0	0
2-AA	149	480	25	244
5-8
30% S9
0	17	103	8	39
6	13	117	9	34
12	11	88	7	39
24	11	102	8	31
48	10	97	10	31
96	11	87	12	30
192	16	95	9	29
2-AA	153	764	102	475

Table 3: Summary of historical negative and positive controls of experiments performed from July to December 1986 using mean values presented as medians (Z) and semi-Q range (QR)
Compound and S9 Mix		Strain
		TA 1535		TA 100		TA 1537		TA 98
		Z	QR	Z	QR	Z	QR	Z	QR
water	-	17	4	102	20	7	1	17	3
DMSO	-	15	3	91	10	7	1	16	2
DMF	-	18	1	68		7	1	19
Na-azid	-	1062	312
NF	-			354	42
4-NPDA	-					37	22	97	17
30%
water	+	13	2	129	47	9	1	27	3
DMSO	+	14	1	129	13	9	3	28	3
DMF	+	13	2	100		10	1	29
2-AA	+	314	105	1309	328	59	9	549	215
10%
DMSO	+	12	2	124	9	6	2	32	6
2-AA	+	338	94	2045	515	175	51	1426	406

Applicant's summary and conclusion

Conclusions:

In this Ames test none of the four strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied to both, the tests with and without S-9 mix and was confirmed by the results of the repeat tests.

Executive summary:

In a bacterial reverse mutation assay similar to OECD TG 471 according Ames et al. Mut Res 31, 347-364 (1975), thymol was investigated in test doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants ( S. typhimurium TA 1535, TA 1537, TA 98 and TA 100). Doses up to and including 24 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used up to 200 µg per plate for assessment purposes. Evidence of mutagenic activity of thymol was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.