Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available for (±)-neomenthol. Reliable data are available from L-menthol and menthol.

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.

L-menthol and menthol and thus (±)-neomenthol is not genotoxic in vitro as found from a weight of evidence approach.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to IUCLID section 13 for a detailed justification of the category approach.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not specified
Genotoxicity:
ambiguous
Cytotoxicity:
yes
Remarks:
at 1.5 mM of menthol the number of cells in culture was only 45% of control
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results from study from 1998a
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity:
yes
Remarks:
33% of controls surviving cells
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results from study from 1998b
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
20% of control surviving cells
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results from study from 1998b
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study from 1989
Species / strain:
other: Chinese Hamster Lung Fibroblast (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
at the highest non cytotoxic dose
Cytotoxicity:
no
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
not specified
Remarks on result:
other: results from study from 1984
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study from 1991
Species / strain:
other: human embryonic lung cells (WI-38)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: results from study from 1975
Conclusions:
In four of six cytogencity tests menthol did not show chromosome aberration potential. In two studies postive or ambigous results were observed at cytotoxic concentrations therefore these were interpreted as false positive results, being generated by secondary effects of toxicity.
In conclusion no cytogenicity is considered for menthol.
Executive summary:

Cytogenicity tests are available from structural analogue menthol (CAS 89-78-1). It is concluded that (±)-neomenthol does not induce cytogenicity in mammalian cells. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to IUCLID section 13 for a detailed justification of the category approach.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study from 1996
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study from 1991
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study from 1989
Conclusions:
Methanol did not show DNA damaging potential in 3 different tests.
Executive summary:

DNA damaging tests are available from structural analogue menthol (CAS 89-78-1). It is concluded that (±)-neomenthol does not induce DNA damage in mammalian cells. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to IUCLID section 13 for a detailed justification of the category approach.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Evaluation criteria:
no data
Species / strain:
other: TA98, 100, 97a, 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at highest concentration with and without S9 mix for TA97a and 102
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: reults from Ames 1998 (L-menthol)
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: results from Ames 1992 (menthol)
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 92, TA 94
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
at the highest non-cytotoxic dose
Cytotoxicity:
no
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results from Ames 1984a (menthol)
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at the highest concentration with and without S9 mix
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: results from Ames 1984b (menthol)
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: results from Ames 1988 (menthol)
Species / strain:
other: FU 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: LD50 without S9 >16 µg/mL; LD50 with S9 >9 µg/mL
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: results from study fluorouracil resistance (2005)
Conclusions:
Based on 5 gene mutation studies in bacteria menthol showed no genotoxic potential in these in vitro tests. One Ames test performed with L-menthol showed also negative results.
Executive summary:

Ames tests are available from structural analogue L-menthol (CAS 2216-51-5) and menthol (CAS 89-78-1). It is concluded that (±)-neomenthol does not induce gene mutation in bacteria. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to IUCLID section 13 for a detailed justification of the category approach.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
DL Menthol was usually lethal at concentration of 200 µg/ml in the absence or presence of S9 mix, and 150 µg /ml caused average RTG values that ranged from 24% to 27% without S9 and 52% to 94% with S9 respectively.
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
No evidence for mutagenicity was obtained. In nonactivation experiment Trial 2, a highly toxic (10% RTG) treatment of one culture with 200 pg/mL caused no change in the mutant frequency (MF).
Conclusions:
Under the conditions tested, menthol showed in C5178Y Mouse Lymphoma Cells no gene mutation effects.
Executive summary:

A mouse lymphoma test is available from structural analogue menthol (CAS 89 -78 -1). It is concluded that (±)-neomenthol does not induce gene mutation in mammalian cells. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data is available for (±)-neomenthol. Reliable data are available from menthol.

All in vivo tests showed no genotoxic potential.

Menthol and thus (±)-neomenthol is not genotoxic in vivo as found from a weight of evidence approach.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
micronucleus, dominant lethal assay, bone marrow chromosome aberration, host mediated assay
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to IUCLID section 13 for a detailed justification of the category approach.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Control animals:
yes, concurrent vehicle
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
only for the highest dose (1000 mg/kg)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: results from mouse micronucleus assay (1993)
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: results from dominant lethal assay (1975)
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: results from bone marrow chromosome aberration test (1975)
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: results from host mediated assay (1975)
Conclusions:
Menthol did not show genetic toxicity in mouse micronucleus test, dominant lethal test or bone marrow chromosme aberreation test. In a host mediated assay ambigous results were found. Since this is an outdated assay and all other assays were negative it can be concluded that menthol does not possess a genotoxic potential.
Executive summary:

Four different in vivo genotoxicity tests are available from structural analogue menthol (CAS 89-78-1 and CAS 15356-70-4). It is concluded that (±)-neomenthol does not possess genotoxic potential in vivo. As explained in the justification for type of information, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Testing data on genetic toxicity of ±-neomenthol is not available. The evaluation of ±-neomenthol is based on read across data and/or published data. All available information were used for a weight of evidence approach to assess the genetic toxicity of ±-neomenthol.

Read Across Justification:

Based on the identical profiles of the different menthols and supported by the Read-Across Justification for menthols (IUCLID chapter 13) all studies on stereoisomers of (±)-neomenthol are used for read across. These isomers are L-menthol (CAS 2216-51-5), (+)-menthol (CAS 15356-60-2), D/L-menthol (CAS 1490 -04 -6) and 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 Pow between 3.12 and 3.45 at 25°C), vapour pressure (from 3.6 to 21 Pa at 25°C) and water solubility (moderately soluble from 231 to 456 mg/L at 25°C). The read across is consistent based on these physico-chemical parameters.

  

Detail on the genetic toxicity study endpoint:

Despite the absence of experimental data on ±-neomenthol 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 (1992, 1984 (1), 1984, 2005, 1988, 1998).

It is concluded that the menthols and thus (±)-neomenthol is not mutagenic to the Salmonella typhimurium strains mentioned above.

 

In vitro mammaliam cells tests:

·        Chromosomal aberration tests performed with 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 (1989) were negative.

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

·        A study of (1998 (1)) 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 (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 hours, 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 (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 (1991).

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

·        A sister chromatid exchange test with chinese hamster ovary cells was concluded negative with and without metabolic activation (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 (1975).

Test results in detail are given in following table.

Test system

Protocol

Concentrations

Results

Reference

 

 

Exp. [µg/mL]

Cytotox.[µg/ml](% cell viability) 

+ MA

- MA

 

CHO

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

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

200

-

-

1989

CHL 

Exposure time: 24, 48 hrs

100, 150, 200

200 = 50% cell-
growth inhibition

n.d. 

-

1984

CHO

Exposure time: 3 hrs
Harvest time: 20 hrs

203, 219, 234

234 (45 %)

+

1998

CHO

Exposure time: 3 hrs
Harvest time: 20 hrs

46-297

(47%),
266
(39%),
281 (33%)
 

+

+

1998 (1)

TK6 human lymphocytes

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

128-187

187 (20%) 

n.d. 

+

1998 (1)

 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.  

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  -  -  

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   -  -   

1991

 alkaline elution assay

Exposure time: 3 hr

0.1, 0.3, 0.7, 1.0, 1.3 mM

 negative  n.d.  n.d.  

1996

Human embryonic lung culture (WI-38)  24 -48hours  0.1, 1.0 and 10.0 mg/mL  negative without metabolic activation  n.d  - 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 menthol (CAS No. 15356-70-4) for 3 days (1993). 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 (1975).

·        In the further study of same report (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 does 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 does 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 (1975).

Overall it can be conclude that menthol and its isomers do not possess a genotoxic potential.

Justification for classification or non-classification

(±)-neomenthol does not meet the criteria for classification and labelling for genetic toxicity as set out in Regulation (EC) No 1272/2008.