Sodium mentholate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

first strain (TA 1535) missing

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-menthol

EC Number:

218-690-9

EC Name:

L-menthol

Cas Number:

2216-51-5

Molecular formula:

C10H20O

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Test material form:

solid

Specific details on test material used for the study:

purchased from Sigma Chemical St. Louis, MO, USA

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox Molecular Toxicology, Annapolis, USA..
- method of preparation of S9 mix: The S9 mixture [21 mL] was prepared for use as follows: 7.0 ml of distilled water; 10.5 ml of 100 mM phosphate buffer pH 7.4; 0.84 ml of 100 mM NADP; 0.105 ml of 1 M glucose-6-phosphate; 0.420 ml of 1.65 M KCl + 0.4
M MgCl2 salt solution and 2.1 ml of lyophilized S9 fraction reconstituted with distilled water.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): enzymatic activity was confirmed by the specific positive control substances

Test concentrations with justification for top dose:

0, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700 µg/plate; doses up to 600–700 µg/plate(TA97a, TA98 and TA100) and 200–400 µg/plate (TA102)
Toxicity to S. typhimurium was investigated in a preliminary test carried out with TA100 strain without and with addition of S9 mixture. Toxicity was observed at doses of 600 µg/plate and higher in the preliminary assay.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

mitomycin C

other: nitro-o-phenilene-diamine (NPD), TA98, -S9; 2-Aminofluorene (2AF), TA97a, +S9 and 2-aminoanthracene (2AA), TA100, +S9

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.2 x 10E9 bacteria/mL
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Results and discussion

Test results

Test results 1

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 2

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 3

Species / strain:

S. typhimurium TA 97

Remarks:

TA97a

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 102

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:

RANGE-FINDING/SCREENING STUDIES (if applicable): Toxicity to S. typhimurium was investigated in a preliminary test carried out with TA100 strain without and with addition of S9 mixture. In all subsequent assays, the upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. Toxicity was apparent either as a reduction in the number of revertants, and or as an alteration of the auxotrophic background growth i.e., background lawn. Please refer to the results in Table 1.

Ames test:
- Mean number of revertant colonies per plate and standard deviation : please refer to Table 2

Any other information on results incl. tables

Table 1: Toxicity of (-)-menthol to S. typhimurium TA100 strain

Dose (µg/plate)	(-)-Menthol
	- S9	+ S9
3000
2750
2500
2000
1500
1250
1000
900		101 * /0/0^+
800	93 ±39*	167* /0/0^+
700	134±29*	77±6*
600	159±14	120±10*
500	165±12	166±17
400
300
200
0	174±15^a	184±26^b
PC	924±41^a	591±69^b
(*) Toxicity apparent as an alteration of the background lawn.  (a,b) Toxicity assays carried out concomitantly shared the same solvent- and positive controls.  (-) Dose not tested. (/ + ) Mutant counts of individual plates. Data are shown as mutant counts (mean±SD) of three plates.

Table 2: Mutagenicity testing of (-)-menthol (5-methyl-2-(1-methyl-ethyl)cyclohexanol) in the Salmonella/microsome assay [TA100, TA98, TA97a and TA102 tester strains]

NUMBER OF REVERTANTS (Mean ±S.D.)
(-) - MENTHOL	DOSE (µg/plate)	TA100		TA98		TA97a		TA102
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
	800	-	-	-	43/0/0^+	-	93±4*	-	-
	700	-	168 ± 15	-	48±6	86±54*	154±8	-	-
	600	161 ± 7	170 ± 10	41±4	50±10	132 ± 11	181 ± 8	-	-
	500	161±10	179 ± 11	46±1	64±3	137±6	186±11	-	552±114*
	400	183 ± 3	182 ± 18	40 ± 8	57 ± 12	148±11	198 ±13	312±135	826±21
	300	199 ± 17	182±13	39 ± 8	57 ± 9	155 ± 6	209±14	409±152	754±33
	200	205 ± 11	183 ± 7	35±4	55 + 10	169±13	209 ± 16	643±62	897±18
	100	211 ± 12	-	42 ± 2	-	149±2	-	665±34	873±66
	50	-	-	-	-	-	-	686±35	738±19
	25	-	-	-	-	-	-	574±50	-
	10	-	-	-	-	-	-	648±32	-
	5	-	-	-	-	-	-	708±34	-
	0	219 ± 21	196 ± 18	47±3	63 ± 1	160±22	172±6	719±25	832±67
	PC	860 ± 1	1003 ± 66	159±16	343 ± 57	998±52	844±18	5967±1198	1589±157

Dose 0 — Negative Control: 100 µL ethanol PA; PC — Positive Control: TA100/-S9, SA (0.5 µg/plate); TA100/+S9, 2AA (1 µg/plate); TA98/-S9, NPD (1 µg/plate); TA98/+S9, 2AA (0.5 µg/plate); TA97a/-S9, 4-NQNO (1 µg/plate); TA97a/+S9, 2AF (10 µg/plate); TA102/+S9, MC (0.5 µg/plate); TA102/+S9, B-[a]-P (50 µg/plate)

(-) Dose not tested.

(*) Toxicity apparent as an alteration of the background lawn.

(/+) mutant counts of individual plates.

Values are the means ± SD of three plates of one (out of two) representative experiment.

Applicant's summary and conclusion

Conclusions:

In a bacterial reverse mutation assay d-menthol was negative in S. typhimurium strains TA 97a, TA98, TA100 and TA102 with and without metabolic activation.

Executive summary:

In this study similar to OECD Test Guideline 471 d-menthol was tested in S. typhimurium strains TA 97a, TA98, TA100 and TA102 with and without rat liver S9 fraction. As a result, d-menthol was negative in all test strains with and without metabolic activation.