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Additional information

Testing data on genetic toxicity of L-Menthol is not available. The evaluation of L-Menthol is based on read across data and/or published data. We used all available information for a weight of evidence approach to assess the genetic toxicity of L-Menthol.

Justification for Read-across:

Based on the identical profiles of the different Menthols as laid out in Read-Across Justification for Menthols (see file MentholsReadAcrossFinal.pdf in section 13) we use the different stereoisomers of Menthol for read across studies. These isomers are L-Menthol (CAS 2216-51-5), D-Menthol (CAS 15356-60-2) and DL-Menthol (CAS 89-78-1). Moreover, a comparative physico-chemical profile of these isomers reinforces this similarity. As structural isomers, the members of the Menthol category share the same molecular weight. Of particular importance to environmental effects and human effects are the values for partition coefficient (log Kow around 3), vapour pressure (from 17 Pa at 25°C for the DL-Menthol to 21 Pa at 25°C for the natural L Menthol) and water solubility (moderately soluble from 410 mg/l at 25°C for the natural L-Menthol to 470 mg/l at 25°C for the DL-Menthol).

In addition investigations on toxicokinetics showed that D-, L-, DL- and the unspecified Menthol were well absorbed via the oral route. For all isomers, elimination is rapid and mainly as glucuronic acid conjugates via urine, minor amounts via faces. Significant differences in toxicokinetic properties of Menthol isomers were not found (please refer to the IUCLID section 7.1). The common test substance DL-Menthol is a racemic mixture of the D- and L- isomers and contains both isomers. Data gaps for L-Menthol can therefore be filled by the respective results with the racemic mixture. The read across is consistent based on these physico-chemical, environmental and (eco-) toxicological parameters.

In a OECD SIDS, L-Menthol (CAS 2216-51-5), D-Menthol (CAS 15356-60-2) and DL-Menthol (CAS 89-78-1) were recognized as a category group.

The available toxicity data indicate very similar toxicity profiles for D-, L-, DL Menthol and a unspecified Menthol isomer mixture. In mammalian species the low toxicity is manifested by the LD50 values, generally tested to be greater than 2000 mg/kg bw in acute studies, limited toxicity in repeated dose studies, and no effects in the teratology evaluations. Irritation to skin and eyes was slight to moderate. DL Menthol is a racemic mixture of the D- and L- isomers and contains both isomers in equal proportion. Data gaps for D- or L Menthol and the unspecified isomer mixture can therefore be filled by the respective results with the racemic mixture and the doses for each isomer should be equivalent to half of the total tested DL-dose.


Due to above discussion, to this endpoint, genotoxic properties of L-Menthol can be considered identical to the properties of the other isomers or racemic mixture.


Detail on the genetic toxicity study endpoint:

Despite the absence of experimental data on L-Menthol we conclude by read across from the genetic toxicity information of the other isomers and on the basis of a weight of evidence approach that this endpoint is sufficiently covered.


Bacteria reverse test:

Menthol (DL-Menthols and the isomeric mixture) was not mutagenic in Ames tests using the tested strains S. typhimurium TA92, TA 97a, TA 98, TA 100, TA 102, TA 1535, TA 1537, FU 100, and TA 1538 with and without metabolic activation (M. T. King, 1992, Andersen and Jensen, 1984, M. Ishidate Jr, et al., 1984, Judith E. Miller, et al., 2005, Errol Zeiger, et al., 1988, Gomes -Carneiro, et al., 1998).

We conclude that the L-Menthol is not mutagenic to the Salmonella typhimurium strains mentioned above.


In vitro mammaliam cells tests:

·        Chromosomal aberration tests performed with DL Menthol showed mainly negative results.

·        Tests conducted with CHO cells in concentrations of 100, 150 and 200 µg/ml without metabolic activation and in concentrations of 50, 124 and 200 µg/ml with metabolic activation by Ivett et al. (1989) were negative.

·        A cytogenetic assay with CHL cells performed by Ishidate et al. (1984) showed a negative result without metabolic activation. The concentrations tested were 100, 150 and 200µg/ml.

·        A study of Hilliard et al. (1998) showed ambiguous results. Weak but statistically significant increases in chromosomal aberrations were observed in CHO cells and TK6 human lymphocytes after treatment with DL Menthol in concentrations of 250 to 281 µg/ml (cell viability 47-33% of controls) and 128 to 187 µg/ml (cell viability at 187 µg/ml 20% of controls), respectively, without metabolic activation. Due to the cytotoxic doses tested the observed weak chromosomal aberration is interpreted as false positive result, being generated by secondary effects of toxicity.

·        A further chromosome aberration test with CHO cells was positive, showing maximal 7% aberrant metaphases (Galloway et al., 1998). Again Menthol was tested at cytotoxic concentrations. The authors conclude that “the data, however, support the concept that when there is good evidence that a compound and its metabolites do not react with DNA (or topoisomerases), aberrations at toxic doses that inhibit DNA synthesis may be induced indirectly, and may not constitute a risk of mutagenicity at low exposure levels associated with a good safety margin.

·        Experimental data from the testing of 31 chemicals (including DL-Menthol) for mutagenicity at the TK locus in L5178Y mouse lymphoma cells are presented and evaluated. If mutagenic activity was not obtained for the chemical added to suspension cultures for 4 hr, then the testing was repeated in the presence of hepatic S9 mix prepared from Aroclor 1254 induced male Fischer 344 rats. Multiple trials were performed for each chemical, and mutagenic treatments were analyzed for the induction of small and large mutant colony populations. A negative result was obtained for DL-Menthol with and without metabolic activation (Myhr and Caspary, 1991). The concentration range tested was 12.5 to 200 µg/ml; the lethal dose was 200 µg/ml.

·        The genotoxic potential of Menthol was investigated by analyzing the frequencies of sister chromatid exchange (SCE) and chromosomal aberrations in cultured human lymphocytes exposed to Menthol. Phytohaemagglutinin-stimulated human lymphocyte cultures grown in the presence of Menthol at final concentrations of 0.1, 1 or 10mM, with or without S-9, had polyploid cell and structural chromosomal aberration frequencies similar to those seen in the solvent controls. Furthermore, Menthol, either in the presence or absence of S-9, did not alter the SCE frequency in the tested human chromosomes. The results suggest that Menthol does not have a chromosomal-damaging effect in human lymphocytes (Murthy et al., 1991).

·        An alkaline elution assay to detect DNA damage in primary rat hepatocytes – testing concentrations of 0.1, 0.3, 0.7, 1.0, 1.3mM up to cytotoxic concentrations - was negative (Storer et al., 1996).

·        A sister chromatid exchange test with chinese hamster ovary cells was concluded negative with and without metabolic activation (Ivett et al., 1989)

·        The chromatids damage induced by the Menthol was investigated by in vitro method to human embryonic lung cells, at concentrations of 0.1, 1.0 and 10.0mg/ml in 0.85 % saline. Cells were incubated at 37 °C and examined twice daily. Cells were harvested by shaking when sufficient mitoses were observed, usually 24-48 hours after planting, and fixed in absolute methanol: glacial acetic acid (3:1) for 30 minutes. Microscopic inspection was conducted to count aberrations (bridges pseudochiasmata, multipolar cells, acentric fragments, etc.) in treated- and control groups. Outcome was that the test substance produced no significant aberration in the anaphase of human tissue culture chromosomes under test conditions (Litton Bionetics, Inc., 1975).

Test results in detail are given in following table.

Test system







Exp. [µg/ml]

Cytotox.[µg/ml](% cell viability) 

+ MA

- MA



Exposure time: 8 hrs(-); 2 hrs(+) Harvest time: 10.50 (-),12.50 (+) hrs

100, 150, 200 (- MA), 50, 124, 250 (+ MA)




Ivett, et al.,


Exposure time: 24, 48 hrs

100, 150, 200

200 = 50% cell-
growth inhibition



Ishidate, et al., 1984


Exposure time: 3 hrs
Harvest time: 20 hrs

203, 219, 234

234 (45 %)


Galloway, et al., 1998


Exposure time: 3 hrs
Harvest time: 20 hrs


281 (33%)



Hilliard, et al. , 1998

TK6 human lymphocytes

Exposure time: 3 hrs
Harvest time: 17-35 hrs


187 (20%) 



Hilliard, et al. , 1998

 L5178Y mouse lymphoma cells  

Exposure time:

4 hrs
Harvest time: 2 days

 15 - 200  Lethal at 200µg/ml in the absence or presence of S9 mix, and 150µg/ml caused average RTG values that ranged from 24% to 27% without S9 and 52% to 94% with S9.  

Myhr and Caspary, 1991

 Human lymphocytes chromosome aberration assay  no data  0.1, 1, and 10 nM  At concentrations higher than 10 mM, it significantly affected the growth of human lymphocytes in phytohaemagglutinin-stimulated cultures  -  -  

Murthy et al., 1991

  Human lymphocytes SCE assay

no data

   no data  At concentrations higher than 10 mM, it significantly affected the growth of human lymphocytes in phytohaemagglutinin-stimulated cultures   -  -   

Murthy et al., 1991

 alkaline elution assay

Exposure time: 3 hr

0.1, 0.3, 0.7, 1.0, 1.3 mM

 negative  n.d.  n.d.  

Storer et al., 1996

Human embryonic lung culture (WI-38)  24 -48hours  0.1, 1.0 and 10.0 mg/ml  negative without metabolic activation  n.d  - Litton Bionetics, Inc., 1975 

Genotoxicity in vivo:

·        In a micronucleus assay in bone marrow cells of B6C3F1 mice, the test organisms received daily intraperitoneal injections of 0, 250, 500 or 1000 mg/kg bw/day DL-Menthol (CAS No. 15356-70-4) for 3 days. No increase in micronuclei in bone marrow polychromatic erythrocytes was observed. The data indicated that cytotoxic effects on bone marrow cells could not be expected under the test conditions.

·        In a host mediated assay in mice effects of oral administered Menthol on genmutation and recombination was investigated in Salmonella typhimurium (his G-46 and TA-1530) and saccharomyces cerevisiae (the diploid strain D-3) injected into the mice peritoneum.Menthol caused no significant increases in mutant or recombinant frequencies when tested against Salmonella G-46 at all dose levels, Salmonella TA-1530 subacute dose levels and Saccharomyces D3 acute dose levels, respectively. Test against TA-1530 acute levels showed increasing mutant frequencies with increasing dose levels with the high dose being weakly positive but significant reaction. The subacute levels with Saccheromyces D3 showed increased recombinant frequencies with no dose response (Litton Bionetics, Inc., 1975).

·        In the further study of same report (Litton Bionetics, Inc., 1975), the in vivo chromosomal aberration test in bone marrow cells of rat was conducted for Menthol. Test substance was suspended in 0.85 % saline and administered to male rats by intubation. The dose levels used were 145 mg/kg, 14.5 mg/kg and 1.45 mg/kg in the first test both by acute method (single dose/ after 6, 24 and 48 hours animals were killed) and subacute method (one dose per day for 5 days, animals were killed 6 hours after last administration.). In test II the used concentrations were 500 and 3000 mg/kg for the acute treatment, and 1150 mg/kg for the subacute treatment. After exposure the bone marrow from one femur was isolated. Polychromatic erythrocytes were stained by Giemsa. Polychromatic erythrocytes were microscopically inspected to count the chromosomal aberrations. The results showed that under test conditions the substance can not induce chromosomal aberration in rat bone marrow polychromatic erythrocytes.

·        Additionally, the in vivo Rodent Dominant Lethal Test in rat was conducted with Menthol. Test substance was suspended in 0.85 % saline and administered to male rats by intubation. The dose levels used were 145 mg/kg, 14.5 mg/kg and 1.45 mg/kg in the first test both by acute method (single dose) and subacute method (one dose per day for 5 days). In test II the 500 and 3000 mg/kg were used for the acute test, and 1150 mg/kg for the subacute test. Treated males were sequentially mated to 2 females per week for 8 weeks and 7 weeks for acute and subacute test repectively. Two virgin female rats were housed with male for 5 days (Monday to Friday). These two females were removed and housed in a cage until killed. The males were rested on Saturday and Sunday and two new females introduced to cages on Monday. Females were killed using CO2 at day 14 after separating from males, and at necropsy the uterus was examined for total number of implantations, total number of corpora lutea, pre-implantation losses, and dead implants. The results showed that under test conditions the substance can not induce a dominant lethal event after exposure to the test substance, which indicates that the substance has not affected the germinal tissue of rat (Litton Bionetics, Inc., 1975).

Overall we can conclude that Menthol and its isomers do not possess a genotoxic and/or carcinogenic potential. Subsequently additional carcinogenicity test can be waived based on the weight of evidence approach.

Justification for selection of genetic toxicity endpoint
Putting all available information of Menthol together we conclude that L Menthol does not possess a genotoxic potential.

Short description of key information:
In most available information about the testing of Methanol's genotoxic potential, no activity over background was found. In those chromosome aberration tests in which Menthols were due to cytotoxic doses weak positive tested the chromosomal aberration is interpreted as false positive result, being generated by secondary effects of toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Weighing all available information dealing with the genetic toxicity of L Menthol we assume that L Menthol does not need to be classified for this endpoint.