Thymol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, Ames test, GLP, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, up to 5000 µg/plate, negative with and without metabolic activation (Herbold 1989)

Key Ames test, OECD TG 471, 472, GLP, S. typhimurium TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA up to 5000 µg/plate, negative with and without metabolic activation (MHW Japan, 1996)

Key, HPRT test, OECD TG 476, GLP, Chinese hamster V79 cells, up to 10 mM, negative with and without metabolic activation (Harlan 2010)

Key, CA test, OECD TG 473, GLP, Chinese hamster lung (CHL/IU) cells, negative without metabolic activation, ambiguous with metabolic activation (MHW Japan, 1996)

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

histidine

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.

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.

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

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

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

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

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'

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : 7 week aged Sprague Dawley rats were intraperitoneally treated with a combined administration of phenobarbital and 5,6 -benzoflavone
- method of preparation of S9 mix : 0.1 mL S9, 8 µmol MgCl2, 33 µmol KCl, 5 µmol Glucose-6-phosphoric acid, 4 µmol NADH, 4 µmol NADPH, 100 µmol phosphate buffer (pH 7.4)
- concentration or volume of S9 mix and S9 in the final culture medium

Test concentrations with justification for top dose:

pre-test:
+/-S9 mix, 0, 50, 150, 500, 1500, 5000 µg/plate

main test:
-S9 mix, 0, 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate in all five strains
+S9 mix, 0, 62.5, 125, 250, 500, 1000 and 2000 µg/plate (TA 100, WP2, TA98), 0, 31.3, 62.5, 125, 250, 500, 1000 µg/plate (TA1535, TA1537)

The doses for the main test were set based on cytotoxicity observed in the pre-test (growth inhibition of the bacteria).

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: -S9 (TA98, TA100, WP2): 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; + S9 (all strains): 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : single (pre-test), triplicate (main test)
- Number of independent experiments : 2

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

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of mutant colonies generated was scored either macroscopically or under a stereomicroscope

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

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

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

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

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

The chemical did not induce gene mutations in the S.typhimurium and  E.coli strains.

RANGE-FINDING/SCREENING STUDIES (if applicable): In a range-finding test the substance was tested at concentrations of 0, 50, 150, 500, 1500, 5000 µg/plate. No genotoxicity was observed. Cytotoxicity was observed at concentrations of >= 500 µg/plate (please refer to table 1)

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity : Toxicity was observed at 250 µg/plate (TA1537) and 500  µg/plate (TA100, TA1535, WP2, TA98) without S9 mix, and at 500 µg/plate  (TA1535, TA1537) and 1000 µg/plate (TA100, WP2, TA98) with S9 mix. It was determined based on growth inhibition.
- Individual plate counts: please refer to tables 1, 2 and 3
- Mean number of revertant colonies per plate and standard deviation : please refer to tables 1, 2 and 3

Table1: Results of preliminary cytotoxicity test in reverse mutation test of thymol** on bacteria
With (+) or without (-) S9 mix	Test substance dose(µg/plate)	Number of revertants (number of colonies / plate, Mean± S.D.)
		Base - pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9mix (-)	0	92 / 88 / 83 (88 ± 4.5)	14 / 10 / 13 (12 ± 2.1)	31 / 25 / 24 (27 ± 3.8)	17 / 20 / 27 (21 ± 5.1)	8 / 3 / 8(6±2.9)
	50	85	15	26	20	7
	150	81	21	32	21	9
	500	0 *	0 *	9 *	13 *	4 *
	1500	0 *	0 *	0*	0 *	0*
	5000	0 *	0 *	0 *	0 *	0 *
S9mix (+)	0	85 / 85 / 110(93±14.4)	16 / 15 /16 (16±0.6)	23 / 23 / 23 (23±0.0)	26 / 34 / 31 (30 ±4.0 )	8 / 7 / 7 (7 ±0.6)
	50	111	9	31	26	10
	150	89	9	28	40	9
	500	93	5 *	24	40	14 *
	1500	0 *	0 *	0 *	0 *	0 *
	5000	0 *	0 *	0 *	0 *	0 *
Positive control S9 mix (-)	Chemical	AF2	SA	AF2	AF2	9AA
	Dose (µg/plate)	0.01	0.5	0.01	0.1	80
	Number of colonies / plate	451 / 557 / 477 (495 ± 55.2 )	308 / 292 / 250 (283 ± 30.0 )	146 / 123 / 118 (129 ± 14.9)	686 / 670 / 745 (700 ±39.5 )	835 / 1053 / 1077 (988 ±133.3)
Positive control S9 mix (+)	Chemical	2AA	2AA	2AA	2AA	2AA
	Dose (µg/plate)	1	2	10	0.5	2
	Number of colonies/plate	1144 / 1310 / 1219 (1224±83.1 )	333 / 240 / 272 (282±47.2 )	1467 / 1476 / 1419 (1454 ±30.6)	512 / 480 / 517 (503±20.1)	244 / 228 / 208 ( 227 ± 18.0)
AF2: 2-(2-FuryI)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene *: Inhibition was observed against growth of the bacteria. **: Purity was above 98% and fixed compounds and other phenols were contained as impurity.

Table 2. Results of reverse mutation test (I) of thymol ** on bacteria
With (+) or without (-) S9 mix	Test substance dose (µg/plate)	Number of revertants (number of colonies / plate, Mean ± S.D.)
		Base - pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9mix (-)	0	137 / 132 / 121 (130 ± 8.2)	11 / 19 / 24 (18 ± 6.6)	21 / 23 / 26 (23 ± 2.5)	28 / 35 / 36 (33 ± 4.4)	13 / 9 / 8 (10 ± 2.6)
	15.6	140 / 132 / 128 (133 ± 6.1 )	18 / 11 / 15 (15 ± 3.5)	21 / 24 / 12 (19 ± 6.2)	32 / 41 / 30 (34 ± 5.9)	6 / 8 / 9 (8 ± 1.5)
	31.3	151 / 124 / 126 (134 ± 15.0)	22 / 16 / 18 (19 ± 3.1 )	26 / 21 / 14 (20 ± 6.0)	30 / 30 / 35 (32 ± 2.9)	11 / 15 / 7 (11 ± 4.0)
	62.5	125 / 129 / 125 (126 ± 2.3)	18 / 15 / 15 (16 ± 1.7)	19 / 12 / 11 (14± 4.4)	23 / 22 / 39 (28 ± 9.5)	6 / 10 / 10 (9 ± 2.3)
	125	100 / 113 / 121 (111 ± 10.6)	17 / 16 / 19 (17 ± 1.5)	11 / 22 / 12 (15 ± 6.1)	29 / 26 / 27 (27 ± 1.5)	7 / 6 / 14 (9 ± 4.4)
	250	106 / 100 / 118 (108 ± 9.2)	7 / 15 / 8 (10 ± 4.4)	16 / 9 / 7 (11 ± 4.7 )	32 / 26 / 31 (30 ± 3.2)	5* / 8* / 5* (6 ± 1.7)
	500	93* / 79* / 86* (86 ± 7.0)	4* / 0* / 2* (2 ± 2.0)	4* / 12* / 13* (10 ± 4.9)	14* / 18* / 16* (16 ± 2.0)	0* / 0* / 0* (0 ± 0.0)
S9mix (+)	0	111 / 134 / 144 (130 ± 16.9)	11 / 19 / 13 (14 ± 4.2)	30 / 18 / 23 (24 ± 6.0)	39 / 34 / 37 (37 ± 2.5)	11 / 7 / 17 (12 ± 5.0)
	31.3	ND	17 / 19 / 16 (17 ± 1.5)	ND	ND	5 / 14 / 9 (9 ± 4.5 )
	62.5	147 / 124 / 122 (131 ± 13.9)	13 / 25 / 20 (19 ± 6.0)	30 / 30 / 29 (30 ± 0.6)	36 / 43 / 48 (42 ± 6.0)	14 / 13 / 11 (13 ± 1.5)
	125	133 / 128 / 122 (128 ± 5.5)	24 / 15 / 12 (17 ± 6.2)	16 / 27 / 31 (25 ± 7.8)	39 / 38 / 38 (38 ± 0.6)	12 / 9 / 5 (9 ± 3.5)
	250	147 / 137 / 155 (146 ± 9.0)	15 / 10 / 7 (11 ± 4.0)	16 / 24 / 19 (20± 4.0)	42 / 45 / 39 (42 ± 3.0)	10 / 8 / 9 (9 ± 1.0)
	500	117 / 110 / 127 (118 ± 8.5)	11* / 8* / 9* (9 ± 1.5)	31 / 18 / 14 (21 ± 8.9)	32 / 31 / 34 (32 ± 1.5)	11* / 8* / 8* (9 ± 1.7)
	1000	0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)	14* / 1* / 7* (7 ± 6.5)	0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)
	2000	0* / 0* / 0* (0 ± 0.0)		0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)
Positive control S9 mix (-)	Chemical	AF2	SA	AF2	AF2	9AA
	Dose (µg /plate)	0.01	0.5	0.01	0.1	80
	Number of colonies / plate	602 / 611 / 596 (603 ± 7.5)	147 / 120 / 145 (137 ± 15.0 )	108 / 121 / 101 (110 ± 10.1 )	800 / 744 / 777 (774 ± 28.1 )	814 / 760 / 635 (736 ± 91.8 )
Positive control S9 mix (+)	Chemical	2AA	2AA	2AA	2AA	2AA
	Dose (µg /plate)	1	2	10	0.5	2
	Number of colonies / plate	1234 / 1347 / 1501 (1361 ± 134.0)	278 / 249 / 236 (254 ± 21.5 )	1166 / 1335 / 1277 (1259 ± 85.9)	456 / 476 / 462 (465 ± 10.3)	184 / 264 / 259 (236 ± 44.8)
AF2: 2-(2-Fuiyl)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene *: Inhibition was observed against growth of the bacteria. **: Purity was above 98% and fixed compounds and other phenols were contained as impurity. ND: Not done

Table 3: Results of reverse mutation test (II) of thymol ** on bacteria
With (+) or without (-) S9 mix	Test substance dose (µg/plate)	Number of revertants (number of colonies / plate, Mean ± S.D.)
		Base - pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9mix (-)	0	127 / 135 / 160 (141 ± 17.2)	13 / 14 / 8 (12 ± 3.2)	20 / 24 / 17 (20 ± 3.5)	27 / 13 / 30 (23 ± 9.1)	9 / 16 / 10 (12 ± 3.8)
	15.6	110 / 108 / 114 (111 ± 3.1)	5 / 10 / 11 (9 ± 3.2)	23 / 29 / 20 (24 ± 4.6)	26 / 26 / 30 (27 ± 2.3)	5 / 11 / 11 (9 ± 3.5)
	31.3	120 / 112 / 120 (117 ± 4.6)	15 / 10 / 11 (12 ± 2.6 )	18 / 19 / 22 (20 ± 2.1)	26 / 22 / 21 (23 ± 2.6)	14 / 7 / 7 (9 ± 4.0)
	62.5	125 / 125 / 112 (121 ± 7.5)	8 / 12 / 14 (11 ± 3.1)	25 / 19 / 15 (20 ± 5.0)	30 / 27 / 27 (28 ± 1.7)	13 / 5 / 9 (9 ± 4.0)
	125	142 / 137 / 105 (128 ± 20.1)	13 / 13 / 17 (14 ± 2.3)	15 / 27 / 10 (17 ± 8.7)	22 / 21 / 19 (21 ± 1.5)	8 / 7 / 9 (8 ± 1.0)
	250	106 / 106 / 100 (104 ± 3.5 )	10 / 11 / 13 (11 ± 1.5)	15 / 15 / 18 (16 ± 1.7)	17 / 33 / 22 (24 ± 8.2)	5 / 4 / 5 (5 ± 0.6)
	500	0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)	4* / 0* / 0* (1 ± 2.3 )	14* / 0* / 4* (6 ± 7.2)	0* / 0* / 0* (0 ± 0.0)
S9mix (+)	0	131 / 150 / 134 (138 ± 10.2)	16 / 11 / 13 (13 ± 2.5)	23 / 22 / 20 (22 ± 1.5)	31 / 29 / 31 (30 ± 1.2)	13 / 13 / 13 (13 ± 0.0)
	31.3	ND	12 / 20 / 16 (16 ± 4.0)	ND	ND	18 / 18 / 20 (19 ± 1.2)
	62.5	148 / 138 / 167 (151 ± 14.7)	18 / 23 / 15 (19 ± 4.0)	19 / 27 / 24 (23 ± 4.0)	36 / 37 / 42 (38 ± 3.2)	15 / 13 / 14 (14 ± 1.0)
	125	147 / 118 / 115 (127 ± 17.7)	8 / 19 / 19 (15 ± 6.4)	28 / 26 / 22 (25 ± 3.1)	31 / 40 / 56 (42 ± 12.7)	13 / 18 / 15 (15 ± 2.5)
	250	156 / 140 / 126 (141 ± 15.0)	13 / 11 / 16 (13 ± 2.5)	28 / 23 / 17 (23 ± 5.5)	41 / 54 / 48 (48 ± 6.5)	18 / 15 / 8 (14 ± 5.1)
	500	84 / 114 / 135 (111 ± 25.6)	0* / 0* / 0* (0 ± 0.0)	29 / 21 / 23 (24 ± 4.2)	11 / 20 / 16 (16 ± 4.5)	0* / 6* / 0* (2 ± 3.5)
	1000	0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)	16* / 13* / 12* (14 ± 2.1)	0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)
	2000	0* / 0* / 0* (0 ± 0.0)		0* / 0* / 0* (0 ± 0.0)	0* / 0* / 0* (0 ± 0.0)
Positive control S9 mix (-)	Chemical	AF2	SA	AF2	AF2	9AA
	Dose (µg/plate)	0.01	0.5	0.01	0.1	80
	Number of colonies / plate	665 / 691 / 678 (678 ± 13.0)	294 / 315 / 320 (310 ± 13.8)	153 / 146 / 142 (147 ± 5.6 )	878 / 865 / 935 (893 ± 37.2)	1270 / 1518 / 1580 (1456 ± 164.0)
Positive control S9 mix (+)	Chemical	2AA	2AA	2AA	2AA	2AA
	Dose (µg/plate)	1	2	10	0.5	2
	Number of colonies / plate	916 / 943 / 1176 (1012 ± 143.0)	265 / 268 / 313 (282 ± 26.9 )	1423 / 1378 / 1280 (1360 ± 73.1 )	270 / 324 / 303 (299 ± 27.2)	241 / 265 / 253 (253 ± 12.0)
AF2: 2-(2-FuryI)-3-(5-nitro-2-furyl)acrylamide, SA: Sodium azide, 9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene *: Inhibition was observed against growth of the bacteria. **: Purity was above 98% and fixed compounds and other phenols were contained as impurity.

Conclusions:

The chemical did not induce gene mutations in the Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Eschrichia coli WP2 uvrA strains. Toxicity was observed at 250 µg/plate (TA1537) and 500 µg/plate (TA100, TA1535, WP2, TA98) without S9 mix, and at 500 µg/plate (TA1535, TA1537) and 1000 µg/plate (TA100, WP2, TA98) with S9 mix.

Executive summary:

In a plate incorporation method according OECD TG 471 and 472 (plate incorporation method) with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Eschrichia coli WP2 uvrA thymol was negative with and without metabolic activation. The substance was tested up to cytotoxic concentrations. Toxicity was observed at 250 µg/plate (TA1537) and 500 µg/plate (TA100, TA1535, WP2, TA98) without S9 mix, and at 500 µg/plate (TA1535, TA1537) and 1000 µg/plate (TA100, WP2, TA98) with S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 JUN 2010 - 19 OCT 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, ländl. Raum und Verbraucherschutz (30 MAR 2009)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus in V79 cells of the Chinese hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line (supplied by Laboratory for Mutagenicity Testing, Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 -16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

For cell lines:
- Absence of Mycoplasma contamination: yes, checked for each batch
- Methods for maintenance in cell culture: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT- medium.
- Cell cycle length, doubling time or proliferation index: 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, each batch
- Periodically ‘cleansed’ of spontaneous mutants: yes, each batch

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM (minimal essential medium) supplemented with 10 % fetal calf serum (FCS) and 1 % neomycin. The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 is prepared from 8 - 12 weeks old male Wistar HsdCpb:WU rats (Harlan Laboratories GmbH, 33178 Borchen, Germany), weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at - 80° C. Small numbers of the ampoules were kept at -20°C for up to one week. The protein concentration in the S9 preparation was 35.0 mg/mL (Lot No.: 160410) in the pre-experiment and in experiment I. In experiment II the protein concentration was 33.7 mg/mL (Lot No.: 210510).
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCl
33 mM KCI
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During, the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. 1977.
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures

Test concentrations with justification for top dose:

The cell cultures were evaluated at the following concentrations:
Experiment I:
without S9 mix: 2.8; 5.6; 11.3; 22.5; and 45.0 µg/mL
with S9 mix: 11.3; 22.5; 22.5; 45.0; 67.5; and 90 µg/mL
Experiment II:
without S9 mix: 5.0; 10.0; 20.0; 40.0; and 50.0 µg/mL
with S9 mix: 40.0; 80.0; 90.0; 100.0; and 110.0 µg/mL

In the range finding pre-experiment test item concentrations between 11.7 and 1500 µg/mL (=10 mM) were used to evaluate toxicity in the presence and absence of metabolic activation. Strong toxic effects were observed at 93.8 µg/mL and above in the presence and absence of metabolic activation. Precipitation occurred at 750 µg/mL and above in the presence of metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. Based on the data generated in the pre-experiment, the maximum concentration of the first experiment was limited to 90 µg/mL with and without metabolic activation by cytotoxicity. The concentration range of the second experiment was adjusted to cover the rather steep toxic gradient of the first experiment more closely. The individual concentrations were spaced by a factor of 2 at lower concentrations. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

- Justification for percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium was 0.5 % v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Approximately 1.5x10E6 (single culture) and 5x10E2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times): Three days (experiment I) and four days (experiment II) after treatment 1.5x10E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 d
- Selection time (if incubation with a selective agent): 8 d
- Fixation time (start of exposure up to fixation or harvest of cells): 15 d
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: medium containing 11 µg/mL Thioguanin for 8 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 3 - 5 x 10E5 cells

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity:
cloning efficiency, absolute: mean number of colonies per flask divided by the number of cells seeded x 100
cloning efficiency, relative: (mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding control) x 100


METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, 40407 Düsseldorf, Germany).

Rationale for test conditions:

This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. Gene mutations are discussed as an initial step in the carcinogenic process. The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated original population are monitored for the loss of functional HPRT enzyme. HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6TG to the culture medium. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival. In order to establish a concentration response effect of the test item at least four concentration levels are tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival. To demonstrate the sensitivity of the test system reference mutagens are tested in parallel to the test item.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
Howeyer, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.3 - 33.2 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the cultures treated with the test item were compared to the solvent controls. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relevant toxic effects indicated by a relative cloning efficiency I below 50% occurred in the first experiment at 90.0 µg/mL with and at 67.5 µg/mL and above without metabolic activation. In the second main experiments cytotoxic effects as described were

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the test item in the solvent were comparable to the solvent control even at the maximum concentration of the test item.
- Data on osmolality: The osmolality of the test item in the solvent were comparable to the solvent control even at the maximum concentration of the test item.
- Precipitation and time of the determination: No precipitation was observed up to the maximum concentration with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable): In the range finding pre-experiment test item concentrations between 11.7 and 1500 µg/mL (=10 mM) were used to evaluate toxicity in the presence and absence of metabolic activation. Strong toxic effects were observed at 93.8 µg/mL and above in the presence and absence of metabolic activation. Precipitation occurred at 750 µg/mL and above in the presence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table 1

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any : please refer to table 2

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Relevant toxic effects indicated by a relative cloning efficiency I below 50% occurred in the first experiment at 90.0 µg/mL with metabolic activation and at 67.5 µg/mL and above without metabolic activation. In the second main experiments cytotoxic effects as described were noted at 80.0 µg/mL and above with metabolic activation and at 40.0 µg/mL and above without metabolic activation. The recommended cytotoxic range of approximately 10-20% of survival was covered with and without metabolic activation.

- Genotoxicity results: please refer to table 1

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: 69.5 - 1386.4 per 10E6 cells (without metabolic activation); 135.5 - 2574.2 per 10E6 cells (with metabolic activation)
- Negative (solvent/vehicle) historical control data: 3.4 - 31.7 per 10E6 cells (without metabolic activation); 3.3 - 33.2 per 10E6 cells (with metabolic activation)

Table 1: Summary of results
	conc. [µg/mL]	S9 mix	relative clonning efficiency I [%]	relative clonning efficiency II [%]	relative cell density [%]	mutant colonies/10E6 cells	induction factor	relative clonning efficiency I [%]	relative clonning efficiency II [%]	relative cell density [%]	mutant colonies/10E6 cells	induction factor
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4h treatment			culture I					culture II
Solvent control*		-	100.0	100.0	100.0	12.5	1.0	100.0	100.0	100.0	21.9	1.0
Positive control**	150.0	-	58.0	84.7	108.4	109.5	8.7	151.6	104.6	138.7	126.5	5.8
Test item	2.8	-	224.2	90.7	134.6	17.1	1.4	185.7	91.1	108.0	12.5	0.6
Test item	5.6	-	210.0	75.0	116.5	21.9	1.8	164.0	82.2	163.4	25.1	1.1
Test item	11.3	-	186.8	76.7	132.5	20.6	1.6	191.9	126.7	101.0	23.8	1.1
Test item	22.5	-	168.0	65.1	116.9	28.5	2.3	158.9	116.1	120.8	25.1	1.1
Test item	45.0	-	116.0	72.4	23.3	26.1	2.1	156.6	101.0	93.0	20.5	0.9
Test item	67.5	-	0.0	culture was not continued #				0.0	culture was not continued #
Test item	90.0	-	0.0	culture was not continued #				0.0	culture was not continued #
Solvent control*		+	100.0	100.0	100.0	14.9	1.0	100.0	100.0	100.0	18.1	1.0
Positive control***	1.1	+	49.4	61.8	66.5	829.4	55.7	56.6	98.7	66.9	716.4	39.7
Test item	2.8	+	94.0	culture was not continued ##				108.3	culture was not continued ##
Test item	5.6	+	85.7	culture was not continued ##				98.0	culture was not continued ##
Test item	11.3	+	88.2	61.4	103.2	27.8	1.9	86.5	97.3	109.7	17.1	0.9
Test item	22.5	+	82.3	60.4	105.1	23.0	1.5	89.7	90.6	107.1	19.3	1.1
Test item	45.0	+	86.0	57.4	116.1	18.6	1.2	93.9	75.8	108.4	18.6	1.0
Test item	67.5	+	85.6	69.3	97.7	10.0	0.7	91.7	91.7	99.4	20.6	1.1
Test item	90.0	+	40.6	67.0	46.6	19.2	1.3	31.2	94.5	40.3	18.2	1.0
Experiment II / 4 h treatment			culture I					culture II
Solvent control*		-	100.0	100.0	100.0	26.5	1.0	100.0	100.0	100.0	30.4	1.0
Positive control**	150.0	-	111.4	116.2	81.9	144.3	5.5	105.0	111.7	117.9	151.1	5.0
Test item	5.0	-	79.6	99.5	86.3	23.2	0.9	103.0	99.6	120.1	24.7	0.8
Test item	10.0	-	88.8	93.3	108.0	35.3	1.3	87.5	101.3	112.7	42.1	1.4
Test item	20.0	-	84.7	72.7	91.7	44.8	1.7	103.4	84.5	114.5	34.1	1.1
Test item	40.0	-	19.9	68.7	14.5	32.1	1.2	56.5	88.0	105.2	33.1	1.1
Test item	50.0	-	0.0	#	1.8	#		17.7	83.6	18.7	50.8	1.7
Test item	60.0	-	0.0	culture was not continued #				0.0	culture was not continued #
Test item	70.0	-	0.0	culture was not continued #				0.0	culture was not continued #
Solvent control*		+	100.0	100.0	100.0	21.0	1.0	100.0	100.0	100.0	31.6	1.0
Positive control***	1.1	+	53.0	83.7	67.6	931.5	44.4	58.0	91.8	73.9	766.3	24.3
Test item	20.0	+	88.4	##	99.0	##	##	88.9	##	109.1	##	##
Test item	40.0	+	92.8	104.5	93.2	32.0	1.5	86.2	106.6	97.2	21.6	0.7
Test item	80.0	+	19.6	95.1	87.7	53.4	2.5	87.4	103.4	98.6	21.7	. 0.7
Test item	90.0	+	16.6	109.3	91.7	41.3	2.0	40.4	89.3	92.6	37.3	1.2
Test item	100.0	+	0.0	81.4	69.0	47.6	2.3	9.1	113.8	56.0	39.8 .	1.3
Test item	110.0	+	0.0	102.1	56.9	17.2	0.8	0.0	80.2	4.7	10.7	0.3
Test item	120.0	+	0.0	culture was not continued #				0.0	culture was not continued #
* DMSO ** EMS *** DMBA # culture was not continued due to strong toxic effects ## culture was not continued since a minimum of only four analysable concentrations is required

Table 2: p-values from a linear regression (least squares) for the increase of mutant frequencies. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

experimental part	p-value
experiment I, culture I without S9 mix	0.077
experiment I, culture II without S9 mix	0.784
experiment I, culture I with S9 mix	0.468
experiment I, culture II with S9 mix	0.413
experiment II, culture I without S9 mix	0.487
experiment II, culture II without S9 mix	0.159
experiment II, culture I with S9 mix	0.504
experiment II, culture II with S9 mix	0.823

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, thymol is considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP-compliant study according to OECD TG 476 was performed to investigate the potential of thymol to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. Both experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration (1500 µg/mL) applied in the pre-experiment was equal to a molar concentration of approximately 10 mM. The concentration range of the main experiments was limited by cytotoxic effects of the test item.

The evaluated concentrations and the results are:

Experiment I:

without S9 mix: 2.8; 5.6; 11.3; 22.5; and 45.0 µg/mL with S9 mix: 11.3; 22.5; 22.5; 45.0; 67.5; and 90 µg/mL

Experiment II:

without S9 mix: 5.0; 10.0; 20.0; 40.0; and 50.0 µg/mL with S9 mix: 40.0; 80.0; 90.0; 100.0; and 110.0 µg/mL

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, thymol is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Research Resource Bank (JCRB), obtained in February 1988
- Suitability of cells: This CHL / IU cell line is commonly used because it is generally highly sensitive to chemical substances for detecting chromosomal abnormalities.

For cell lines:
- Number of passages if applicable: when obtained 4th generation, at test 12th generation

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's MEM culture medium supplemented with 10% fetal bovine serum

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver (Sprague-Dawley), induced with phenobarbital and 5,6-benzoflavone
- method of preparation of S9 mix: 20 mM HEPES (pH 7.2), 50 mM MgCl2, 330 mM KCl, 50 mM Glucose-6-Phosphate, 40 mM NADP
- concentration or volume of S9 mix and S9 in the final culture medium : 5% S9 (v/v)

Test concentrations with justification for top dose:

-S9 mix (continuous treatment): 0, 0.020, 0.040, 0.080 mg/mL
-S9 mix (short-term treatment): 0, 0.020, 0.040, 0.080 mg/mL
+S9 mix (short-term treatment): 0, 0.020, 0.040, 0.080 mg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2 x 10E4
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 72 h
- Exposure duration/duration of treatment: 6 h, 24 h and 48 h
- Harvest time after the end of treatment (sampling/recovery times): 18 h in case of the 6 h exposure duration

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Before harvesting, the cells were treated with 0.1µg/mL of colcemid for 2 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Chromosome specimens were stained with 3% Giemsa solution for 8 min.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The number of cells with chromatid- and chromosome-type breaks and exchanges were scored per 200 cells at each dose. Polyploid cells were also scored per 800 cells at each dose.
- Determination of polyploidy: A polyploid cell was defined as a metaphase with more than 37 chromosomes, i.e. theoretical “hypertriploid cell”, containing approximately three times the number of chromosomes than half the modal chromosome number in CHL/IU cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cell confluence or cell number to solvent control was measured with a MonocellaterTM (Olympus Optics, Tokyo, Japan)

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

other: cannot be evaluated

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table 4 and 5

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value if any : please refer to table 4 and 5

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: The growth inhibition of CHL/IU cells treated with thymol (TM) for 6 h with S9 mix does not show a dose-response relationship (please refer to tables 1 - 3). Thus, it cannot reliably be evaluated how cytotoxic the concentration of 0.08 mg/mL was. Especially, as the positive response was only observed at the highest concentration tested, it is crucial to know the exact extent of cytotoxicity. In fact, for the test without metabolic activation, the dose of 0.08 mg/mL was excluded, because of high cytotoxicity.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : please refer to table 4 and 5
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : please refer to table 4 and 5

Table 1: Growth inhibition of CHL/IU cells continuously treated with thymol (TM) for 48 h without S9 mix
Concentration of TM (mg/mL)	Cell growth (% of control)
			Average
0	100	100	100.0
0.0031	92	91	91.5
0.0063	87	83	85.0
0.013	82	81	81.5
0.025	98	96	97.0
0.050	70	89	79.5
0.10	0	0	0.0
Cell growth was measured by Monocellater™ (OLYMPUS)

Table 2: Growth inhibition of CHL/IU cells treated with thymol (TM) for 6 h with S9 mix
Concentration of TM (mg/mL)	Cell growth (% of control)
			Average
0	100	100	100.0
0.047	85	69	77.0
0.094	0	3	1.5
0.19	19	10	14.5
0.38	30	30	30.0
0.75	66	97	81.5
1.5	64	71	67.5
Cell growth was measured by Monocellater™ (OLYMPUS)

Table 3: Growth inhibition of CHL/IU cells treated with thymol (TM) for 6 h without S9 mix
Concentration of TM	Cell growth (% of control)
			Average
0	100	100	100.0
0.047	64	69	66.5
0.094	0	0	0.0
0.19	0	0	0.0
0.38	0	0	0.0
0.75	7	7	7.0
1.5	9	7	8.0
TM Cell growth was measured by Monocellater (OLYMPUS)

Table 4: Chromosome analysis of Chinese hamster cells (CHL/IU) continuously treated with thymol (TM)* without S9 mix
Group	Concentration (mg/mL)	Time of exposure (h)	No. of cells analysed	No. of structural aberrations							Others^3)	No. of cells with aberrations		Polyploid (%)	Trend test
				gap	ctb	cte	csb	cse	mul^2)	total
												TAG (%)	TA (%)		SA	NA
Control			200	0	0	1	0	0	0	1	0	1 (0.5)	1 (0.5)	0.50
Solvent^1)	0	24	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.13
TM	0.020	24	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)	0.38
TM	0.040	24	200	2	0	1	0	0	0	3	0	3 (1.5)	1 (0.5)	0.00	NT	NT
TM	0.080	24	15^T	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.00^6)T
MC	0.00005	24	200	3	29	61	0	4	0	97	0	72 (36.0)	70 (35.0)	0.00
Solvent^1)	0	48	200	0	0	0	0	1	0	1	0	1 (0.5)	1 (0.5)	0.38
TM	0.020	48	200	0	1	0	0	0	0	1	0	1 (0.5)	1 (0.5)	0.13
TM	0.040	48	200	0	0	0	0	0	0	0	1	0 (0.0)	0 (0.0)	0.63	NT	NT
TM	0.080	48	0^T											T
MC	0.00005	48	200	4	31	59	0	1	20	115	5	60 (30.0)	57 (28.5)	0.75
Abbreviations : gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse chromosome exchange (dicentric and ring etc.), mul: multiple aberrations, TAG: total no.of cells with aberrations, TA: total no. of cells , with aberrations except gap, SA: structural aberration, NA: numerical aberration, MC: mitomycin C, NT: not tested, T: Toxic; this group was excluded from judgement in case of less than one hundred cells for structural aberration analysed or less than four hundred cells for polyploid cells analysed. 1) Dimethyl sulfoxide was used as solvent.  2) More than ten aberrations in a cell were scored as 10.   3) Others, such as attenuation and premature chromosome condensation, were excluded from the no. of structural aberrations.  4) Eight hundred cells were analysed in each group.  5) Cochran • Armitage's trend test was done at p<0.05 when the incidence of TAG or polyploid in the treatment groups was significantly different from historical solvent control at p<0.05 by Fisher's exact test. 6) Forty cells were analysed. * : Purity was more than 98.0 %. Unvolatile materials (<=0.05 %) and other kinds of phenol (<=1.95 %) were contained as impurities.

Table 5: Chromosome analysis of Chinese hamster cells (CHL/IU) treated with thymol (TM)** with and without S9 mix
Group	Concentration (mg/mL)	S 9 mix	Time of exposure (h)	No. of cells analysed	No. of structural aberrations							Others^3)	No. of cells with aberrations			Polyploid^4)(%)	Trend test^5)
					gap	ctb	cte	csb	cse	mul^2)	total
													TAG (%)	TA (%)			SA	NA
Control				200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)		0.00
Solvent^1)	0	-	6-(18)	200	1	0	0	0	1	0	2	0	2 (1.0)	1 (0.5)		0.13
TM	0.020	-	6-(18)	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)		0.38
TM	0.040	-	6-(18)	200	1	0	0	0	0	10	11	0	2 (1.0)	1 (0.5)		0.00	NT	NT
TM	0.080	-	6-(18)	77^T	1	1	2	0	0	0	4	0	4 (5.2)	3 (3.9)		0.58^6)T
CPA	0.005	-	6-(18)	200	0	0	1	0	0	0	1	0	1 (0.5)	1 (0.5)		0.13
Solvent^1)	0	+	6-(18)	200	1	1	0	1	0	0	3	0	3 (1.5)	2 (1.0)		0.25
TM	0.020	+	6-(18)	200	1	1	0	0	0	0	2	1	2 (1.0)	1 (0.5)		0.13
TM	0.040	+	6-(18)	200	0	0	1	0	0	0	1	0	1 (0.5)	1 (0.5)		0.13	+	NT
TM	0.080	+	6-(18)	200	1	5	7	0	2	0	15	0	11* (5.5)	10 (5.0)		0.75
CPA	0.005	+	6-(18)	200	10	193	272	1	1	360	837	1	176 (88.0)	175 (87.5)		0.00
Abbreviations: gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.), mul: multiple aberrations, TAG: total no.of cells with aberrations, TA: total no. of cells with aberrations except gap, SA: structural aberration, NA: numerical aberration, CPA: cyclophosphamide, NT: not tested, T: Toxic; this group was excluded from judgement in case of less than one hundred cells for structural aberration analysed or less than four hundred cells for polyploid cells analysed. 1) Dimethyl sulfoxide was used as solvent.   2) More than ten aberrations in a cell were scored as 10.   3) Others, such as attenuation and premature chromosome condensation, were excluded from the no. of structural aberrations. 4) Eight hundred cells were analysed in each group.   5) Cochran • Armitage's trend test was done at p<0.05. 6) One hundred and seventy-three cells were analysed. * : Significantly different from historical solvent control data with respect to TAG and polyploid at p < 0.05 by Fisher's exact test using a Bonferroni correction for multiple comparisons. ** : Purity was more than 98.0 %. Unvolatile materials (<= 0.05 %) and other kinds of phenol (<= 1.95 %) were contained as impurities.

Conclusions:

At the highest concentration (0.080 mg/mL) in the short-term treatment (6 h exposure) in the presence of an exogenous metabolic activation system, structural chromosomal aberrations (5.5%) were induced. However, the growth inhibition of CHL/IU cells treated with thymol for 6 h with S9 mix does not show a dose-response relationship. Thus, it cannot reliably be evaluated how cytotoxic the concentration of 0.08 mg/mL was. Especially, as the positive response was only observed at the highest concentration tested, it is crucial to know the exact extent of cytotoxicity. In fact, for the test without metabolic activation, the dose of 0.08 mg/mL was excluded, because of high cytotoxicity.

Executive summary:

A chromosomal aberration test of thymol according to OECD TG 473 under GLP conditions was conducted using Chinese hamster lung (CHL/IU) cells. Cytogenetic effects were seen as follows: At the high (0.08 mg/mL) concentration with short-term treatment in the presence of an exogenous metabolic activation system, structural chromosomal aberration (5.5%) were induced. Thymol did not induce polyploidy under the condition tested. However, the growth inhibition of CHL/IU cells treated with thymol for 6 h with S9 mix does not show a dose-response relationship. Thus, it cannot reliably be evaluated how cytotoxic the concentration of 0.08 mg/mL was. Especially, as the positive response was only observed at the highest concentration tested, it is crucial to know the exact extent of cytotoxicity. In fact, for the test without metabolic activation, the dose of 0.08 mg/mL was excluded, because of high cytotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key, MNA, OECD 474, GLP, mouse, gavage, up to 1100 mg/kg bw (MTD), negative (MHW Japan 1996)

Key, MNA, OECD 474, GLP, mouse, gavage, negative (BG Chemie 1995)

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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

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.

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'

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

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Wako Pure Chemical Industries, Ltd.,
- Lot No. CAN1119

Species:

mouse

Strain:

other: Crj:BDF1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +- 1
- Humidity (%): 55 +- 5

Route of administration:

oral: gavage

Vehicle:

olive oil

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

24 hours

Dose / conc.:

0 mg/kg bw (total dose)

Dose / conc.:

312.5 mg/kg bw (total dose)

Dose / conc.:

625 mg/kg bw (total dose)

Dose / conc.:

1 250 mg/kg bw (total dose)

No. of animals per sex per dose:

5 male and 5 female mice

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide 50 mg/kg

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

procedure: bone marrow/acridine orange staining

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1250 mg/kg lowest dose producing toxicity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In order to determine the dose of thymol used in the micronucleus test, 7 groups consisting of 5 animals in each group were set up for both males and females, and the doses were 500, 750, 1000, 1250, 1500, 1750 and 2000 mg/kg bw, respectively. The general condition was observed every day for 4 days and the presence or absence of death was examined. As a result, immediately after administration, a decrease in locomotor activity was observed in all administration groups, and with increasing dose, toxic signs such as staggering, abdominal recumbence, and respiratory urgency appeared. Based on this study the maximum tolerated dose was 2000 mg/kg bw in males and 1250 mg/kg bw in females.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The frequency of micronucleated immature erythrocytes was not significantly increased in males and females up to the dose of 1250 mg/kg oral gavage.
- Ratio of PCE/NCE (for Micronucleus assay): Inhibition of bone marrow cell proliferation was not observed under the test conditions.
- Appropriateness of dose levels and route: The dose was selected based on a reliable range-finding study as the maximum tolerated dose. The oral route is recommended in the OECD TG 474. Thus, the route and the dose are appropriate.

Table1. Results of micronucleus test in BDF1 male mice after single administration of thymol by gavage Group Animal No. MNPCE / PCE ^a) PCE / ERY ^b) Solvent control Olive oil 10 mL/kg 1 2 / 2000 261/ 500 2 3 / 2000 261/ 500 3 4 / 2000 174 / 500 4 0 / 2000 232 / 500 5 3 / 2000 286 / 500 Total 12 /10000 1214 / 2500 (0.12 ± 0.08 ) (48.6 ± 8.6) TM 312.5 mg/kg 11 5 / 2000 238 / 500 12 3 / 2000 293 / 500 13 1/ 2000 272 / 500 14 5 / 2000 311/ 500 15 6 / 2000 279 / 500 Total 20 /10000 1393 / 2500 % (Mean ± SD) (0.20 ± 0.10) (55.7 ± 5.4 ) TM 625 mg/kg 16 3 / 2000 152 / 500 17 - 1/ 2000 263 / 500 18 1/ 2000 207 / 500 19 8 / 2000 306. / 500 20 6 / 2000 276 / 500 Total 19 /10000 1204 / 2500 % (Mean ± S.D.) (0.19 ±0.16 ) (48.2 ± 12.3 ) TM 1250 mg/kg 21 1/ 2000 228 / 500 22 3 / 2000 198 / 500 23 3 / 2000 308 / 500 24 1/ 2000 319 / 500 25 7 / 2000 287 / 500 Total 15 /10000 1340 / 2500 % (Mean ± S.D.) (0.15 ± 0.12 ) (53.6 ± 10.5 ) Positive control CPA 50 mg/kg 26 27 / 2000 179 / 500 27 17 / 2000 164 / 500 28 22 / 2000 201/ 500 29 51/ 2000 298 / 500 30 40 / 2000 299 / 500 Total 157 /10000 1141/2500 % (Mean ± S.D.) (1.57 ±0.70)*** (45.6 ± 13.1) a) Number of micronucleated polychromatic erythrocytes/total number of polychromatic erythrocytes observed b) Number of polychromatic erythrocytes/total number of erythrocytes observed  TM: thymol CPA: Cyclophosphamide ***: Data significantly different from the solvent control at 0.1% level

Table 2 Results of micronucleus test BDF1 female mice after single administration of thymol by gavage
Group	Animal
	No	MNPCE/PCEa)	PCE/ERYb)
Solvent control Olive oil 10 mL/kg	51	7 / 2000	293 / 500
	52	4 / 2000	297 / 500
	53	0 / 2000	347 / 500
	54	5 / 2000	337 / 500
	55	1 / 2000	322 / 500
	Total	17 /10000	1596 / 2500
	% (Mean ± S.D.)	(0.17 ± 0.14 )	(63.8 ± 4.8 )
TM 312.5 mg/kg	61	5 / 2000	242 / 500
	62	2 / 2000	302 / 500
	63	3 / 2000	302 / 500
	64	3 / 2000	352 / 500
	65	1 / 2000	318 / 500
	Total	14 / 10000	1516 / 2500
	%(Mean ± SD)	(0.14 ± 0.07 )	(60.6 ± 8.0)
TM 625 mg/kg	66	3 / 2000	341 / 500
	67	1/ 2000	304 / 500
	68	3 / 2000	306 / 500
	69	2 / 2000	335 / 500
	70	6 / 2000	283 / 500
	Total	15 /10000	1569 / 2500
	%(Mean ± SD)	(0.15 ± 0.09 )	(62.8 ±4,8 )
TM 1250 mg/kg	71	2 / 2000	347 / 500
	72	3 / 2000	355 / 500
	73	1 / 2000	304 / 500
	74	3 / 2000	342 / 500
	75	2 / 2000	256 / 500
	Total	11 / 10000	1604 / 2500
	%(Mean ± S.D.)	(0.11 ± 0.04 )	(64.2 ± 8.2 )
Positive control CPA 50 mg/kg	76	28 / 2000	306 / 500
	77	31/ 2000	272 / 500
	78	31/ 2000	225 / 500
	79	17 / 2000	275 / 500
	80	36 / 2000	294 / 500
	Total	143 /10000	1372 / 2500
	%(Mean ± S.D.)	(1.43 ± 0.35)***	(54.9 ± 6.2)
a) Number of micronucleated polychromatic erythrocytes/total number of polychromatic erythrocytes observed
b) Number of polychromatic erythrocytes/total number of erythrocytes observed
TM: thymol
CPA: Cyclophosphamide
*** Data significantly different from the solvent control at 0.1% level

Conclusions:

Thmyol was negative in the in vivo micronucleus test.

Executive summary:

In an in vivo micronucleus test according OECD 474 the frequency of micronucleated erythrocytes was not significantly increased in male and female mice up to the dose of 1250 mg/kg bw. The test item was administered by oral gavage in a single treatment. Animals were killed 24 hours after treatment. Inhibition of bone marrow cell proliferation was not observed under the test conditions. As a result, thmyol was negative in the in vivo micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All Ames tests were negative. The chromosomal aberration tests had ambiguos results; this is also the case for SCE and comet assays. A gene mutation assay in chinese hamster V79 cells in vitro (V79/HPRT) with thymol was negative with and without metabolic activation.

The results of one chromosomal aberration assay (Azirak, 2008) could not be assessed because after critical evaluation of the test procedure revealed some drawbacks and deviations to the actual guideline. First of all the documentation did not comply with the current standards. Additional the number of test animals was lower as required by the actual guideline. Effects were examined at an early stage, the type of aberration was not described and individual animals data were missing. Therefore the test has essential limitations and is not regarded as reliable.

In 2 additional NMTs (mammalian erythrocyte micronucleus tests) which fully comply with a reliable study a negative test result was found. The result is supported by a SLRL test (sex-linked recessive lethal test for mutagenesis in Drosophila melanogaster) which was also negative.

 

Overall, no clear evidence for a mutagenic potential of thymol was found.

 

Justification for classification or non-classification

Based on the available studies, it is concluded that the substance does not need to be classified for genetic toxicity according to Regulation (EC) No 1272/2008.