D,L- Menthol / D,L-Isomenthol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD471: Salmonella typhimurium and Escherichia coli reverse mutation assay. The study was performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. Reverse mutation assays determine the frequency at which an agent abolishes or suppresses the effect of the forward mutation.

The reversion of bacteria from growth-dependence on a particular amino acid to grow in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker.

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The Salmonella typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.

According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted. To validate the test, reference mutagens were tested in parallel to the test item.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2018 to 21 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

S9 Liver homogenates from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I (plate incorporation test):
All strains with and without S9: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation test):
All strains without S9: 3, 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Strains TA 1535, TA 1537, and TA 100 with S9 mix: 3; 10; 33; 100; 333; and 1000 μg/plate
Strain TA 98 and WP2 uvrA with S9 mix: 10; 33; 100; 333; 1000; and 2500 μg/plate

Top dose concentrations based on results of the initial toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (-S9 at 10ug/plate in TA98 and 50 ug/plate in TA1537); methyl methane sulfonate (-S9 at 2.0uL/plate in WP2 uvrA); 2-aminoanthracene (+S9 at 2.5 ug/plate in all strains)

Remarks:

The positive control 2-aminoanthracene was only tested with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding: 108-109 cells/mL

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable



NUMBER OF REPLICATIONS: 3 replicate plates were kept in the dark for 48 hours at 37 ºC


DETERMINATION OF CYTOTOXICITY
- Method: manually counting cells and observing the reduction in the number of revertants.
- Any supplementary information relevant to cytotoxicity: Not specified

OTHER EXAMINATIONS: Precipitation of the test item was observed in the incubated overlay agar plate in both experiments. The undissolved particles had no influence on the results.

Rationale for test conditions:

Following standard guidelines

Evaluation criteria:

The number of revertant colonies on the plates, with and without the test compound, were compared to evaluate mutagenicity. A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of cytotoxicity.

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not specified
- Effects of osmolality: Not specified
- Evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation: The test item showed signs of precipitation in experiment I (with metabolising system) and II (with metabolising system). The undissolved particulates had no effect on the recorded data.
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: Not specified


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The data set without S9 are: TA 1535 (334-1816); TA 1537 (43-157); TA 98 (211-627); TA 100 (498-2767); WP2 uvrA. The data set with S9 are: TA 1535 (176-668); TA 1537 (83-434); TA 98 (360-6586); TA 100 (536-6076); WP2 uvrA (167-1265).

- Negative (solvent/vehicle) historical control data: The data set without S9 are: TA 1535 (6-25); TA 1537 (6-19); TA 98 (13-43); TA 100 (78-209); WP2 uvrA (27-63). The data set with S9 are: TA 1535 (7-26); TA 1537 (7-30); TA 98 (15-58); TA 100 (73-208); WP2 uvrA (28-72).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Revertant colonies

Table 1- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment I)

Strain	Dose level per plate (µg)	Mean revertants per plate	Standard deviation	Ratio treated/solvent	Individual revertant colony counts
TA 1535	3	9.7	2.5	0.9	7,10, 12
	10	10.0	3.6	0.9	14,9,7
	33	14.7	1.2	1.4	14,16,14
	100	9.0	4.4	0.8	6,7,14
	333	4.7	0.6	0.4	5,4,5
	1000	8.3	1.2	0.8	7 R, 9 R, 9 R
	2500	8.7	1.5	0.8	7 M R,9 M R, 10 MR
	5000	6.7	1.2	0.6	6 M R, 6 M R, 8 M R
	Solvent control	10.7	1.5		9,12,11
	Negative control	8.3	1.2		9,9,7
TA 1537	3	7.7	1.2	0.9	9,9,7
	10	10.7	0.6	1.2	11,10,11
	33	5.3	0.6	0.6	6,5,5
	100	8.3	2.1	0.9	6,9,10
	333	6.7	2.1	0.7	6,5,9
	1000	3.7	1.5	0.4	5 R, 2 R, 4 R
	2500	6.0	1.0	0.7	5 R, 7 R, 6R
	5000	4.3	2.1	0.5	6 R, 5 R, 2 R
	Solvent control	9.0	0.0		9, 9, 9
	Negative control	7.3	2.3		6, 6, 10
TA 98	3	19.3	4.7	0.9	14, 23, 21
	10	20.3	5.5	0.9	26, 15, 20
	33	19.3	4.0	0.9	15, 20, 23
	100	24.7	7.5	1.1	25, 17, 32
	333	18.7	3.5	0.9	22, 15, 19
	1000	21.0	6.2	1.0	14 R, 26 R, 23 R
	2500	15.0	5.3	0.7	17 R, 19 R, 9 R
	5000	10.0	1.0	0.5	11 R, 10 R, 9 R
	Solvent control	21.7	5.8		25, 25, 15
	Negative control	29.3	7.1		37, 28, 23
TA 100	3	136.7	22.9	0.9	163, 121, 126
	10	148.3	15.6	1.0	150, 163, 132
	33	137.3	15.5	0.9	150, 142, 120
	100	154.7	8.7	1.0	145, 157, 162
	333	107.0	5.2	0.7	104, 104, 113
	1000	32.3	10.0	0.2	40 R, 36 R, 21 R
	2500	34.3	8.0	0.2	42 R, 26 R, 35 R
	5000	26.7	4.7	0.2	32 R, 25 R, 23 R
	Solvent control	153.7	28.3		146, 130, 185
	Negative control	181.7	3.2		183, 178, 184
WP2 uvrA	3	22.0	3.0	0.7	19, 22, 25
	10	25.0	4.4	0.8	23, 22, 30
	33	30.0	5.3	1.0	26, 28, 36
	100	31.0	4.0	1.0	27,31,35
	333	27.0	1.0	0.9	26,27,28
	1000	23.7	8.3	0.8	21,33,17
	2500	22.0	1.0	0.7	21, 33, 22
	5000	14.0	3.5	0.5	16, 10, 16
	Solvent control	29.7	3.5		33, 26, 30
	Negative control	37.0	6.1		41, 40, 30

M= Manuel count

R= Reduced background growth

P= Precipitate

Table 2- Induction of revertants inSalmonella typhimuriumandEscherichia coliby the test item in the presence of a metabolising system (Experiment I)

Strain	Dose level per plate (µg)	Mean revertants per plate	Standard deviation	Ratio treated/solvent	Individual revertant colony counts
TA 1535	3	11.3	4.0	1.0	7,10, 15
	10	12.3	2.9	1.1	14,14,9
	33	9.0	3.0	0.8	9,12,6
	100	11.7	2.5	1.0	12,9,14
	333	10.7	5.5	0.9	16,5,11
	1000	3.3	1.5	0.3	3 M R, 5 M R, 2 M R
	2500	0.0	0.0	0.0	0 R,0 R, 0 R
	5000	0.0	0.0	0.0	0 R P, 0 R P, 0 R P
	Solvent control	11.3	5.1		7,17,10
	Negative control	10.0	1.0		10,9,11
TA 1537	3	14.0	1.7	1.2	15,15,12
	10	8.7	1.5	0.7	7,10,9
	33	10.0	1.0	0.9	9,10,11
	100	9.3	3.8	0.8	5,12,11
	333	12.0	2.0	1.0	12,14,10
	1000	2.3	0.6	0.2	3 M R, 2 M R, 2 M R
	2500	0.7	1.2	0.1	0 R, 2 R, 0 R
	5000	0.0	0.0	0.0	0 R P, 0 R P, 0 R P
	Solvent control	11.7	2.5		9, 14, 12
	Negative control	9.7	3.2		12,11,6
TA 98	3	33.0	8.9	1.3	36, 40,23
	10	30.0	2.0	1.2	28,30,32
	33	34.3	6.4	1.3	38,38,27
	100	26.7	4.7	1.0	23,32,25
	333	28.0	11.5	1.1	40,27,17
	1000	11.7	2.5	0.5	12 M R, 9 M R, 14 M R
	2500	2.3	1.2	0.1	1 M R, 3 M R, 3 M R
	5000	0.0	0.0	0.0	0 R P, 0 R P, 0 R P
	Solvent control	25.7	5.5		31,20,26
	Negative control	32.0	4.0		36,28,32
TA 100	3	142.3	16.7	1.0	161, 137, 129
	10	116.0	4.4	0.8	121, 113, 114
	33	123.3	10.1	0.9	122, 134, 114
	100	135.3	7.1	1.0	143, 129, 134
	333	100.3	21.0	0.7	79, 121, 101
	1000	27.0	8.2	0.2	36 M R, 25 M R, 20 M R
	2500	0.7	0.6	0.0	1 R, 1 R, 0 R
	5000	0.0	0.0	0.0	0 R P, 0 R P, 0 R P
	Solvent control	137.7	8.6		136, 147, 130
	Negative control	157.7	14.2		173, 155, 145
WP2 uvrA	3	33.0	2.0	1.1	35,33,31
	10	30.7	2.3	1.0	28,32,32
	33	39.7	8.1	1.3	47,31,41
	100	33.7	9.5	1.1	41,23,37
	333	27.3	8.1	0.9	36,26,20
	1000	23.0	3.6	0.7	22 R, 27 R, 20 R
	2500	6.7	0.6	0.2	7 M R, 7 M R, 6 M R
	5000	7.3	0.6	0.2	8 M R P, 7 M R P, 7 M R P
	Solvent control	31.3	4.6		26,40,28
	Negative control	40.3	3.8		43, 42, 36

M= Manuel count

R= Reduced background growth

P= Precipitate

Table 3- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment II)

Strain	Dose level per plate (µg)	Mean revertants per plate	Standard deviation	Ratio treated/solvent	Individual revertant colony counts
TA 1535	3	9.3	4.5	0.8	9,5,14
	10	8.7	3.2	0.8	5,10,11
	33	10.7	0.6	1.0	10,11,11
	100	9.3	2.1	0.8	10,11,7
	333	4.0	3.0	0.4	7 R,4 R,1 R
	1000	0.3	0.6	0.0	1 R, 0 R, 0 R
	2500	0.0	0.0	0.0	0 R, 0 R, 0 R
	5000	0.0	0.0	0.0	0 R, 0 R, 0 R
	Solvent control	11.0	1.0		12,10,11
	Negative control	12.0	4.4		10,17,9
TA 1537	3	8.7	3.5	1.0	9,12,5
	10	7.3	1.5	0.8	9,6,7
	33	8.3	3.1	0.9	11,5,9
	100	8.7	3.5	1.0	9,5,12
	333	5.7	2.9	0.6	9,5,4
	1000	0.3	0.6	0.0	0 R, 1 R, 0 R
	2500	0.0	0.0	0.0	0 R, 0 R, 0 R
	5000	0.0	0.0	0.0	0 R, 0 R ,0 R
	Solvent control	9.0	1.7		10,10,7
	Negative control	10.0	3.6		7,14,9
TA 98	3	20.7	1.2	0.8	20,20,22
	10	29.0	3.6	1.2	28,33,26
	33	24.3	6.4	1.0	28,17,28
	100	26.7	2.9	1.1	25,30,25
	333	14.7	4.6	0.6	20,12,12
	1000	0.7	1.2	0.0	2 R, 0 R, 0 R
	2500	0.0	0.0	0.0	0 R, 0 R, 0 R
	5000	0.0	0.0	0.0	0 R, 0 R, 0 R
	Solvent control	24.3	3.2		28,22,23
	Negative control	28.0	5.3		32,22,30
TA 100	3	120.0	5.2	0.9	126,117,117
	10	134.7	16.8	1.0	120,131,153
	33	137.0	1.7	1.0	135,138,138
	100	120.0	9.5	0.9	111,130,119
	333	47.3	4.5	0.3	52,47,43
	1000	9.3	2.5	0.1	12 R, 9 R, 7 R
	2500	0.3	0.6	0.0	1 R, 0 R, 0 R
	5000	0.0	0.0	0.0	0 R,0 R, 0 R
	Solvent control	137.3	8.0		129,138,145
	Negative control	203.7	35.0		169,239,203
WP2 uvrA	3	24.0	3.5	0.9	28,22,22
	10	24.3	4.0	0.9	25,20,28
	33	29.0	2.6	1.1	31,26,30
	100	29.3	9.3	1.1	19,32,37
	333	18.3	4.2	0.7	15,17,23
	1000	0.7	0.6	0.0	0 R, 1 R, 1 R
	2500	0.0	0.0	0.0	0 R, 0 R, 0 R
	5000	0.0	0.0	0.0	0 R, 0 R, 0 R
	Solvent control	27.3	0.6		27,27,28
	Negative control	31.0	1.0		31,32,30

 R= Reduced background growth

Table 4- Induction of revertants in Salmonella typhimurium and Escherichia coli by the test item in the absence of a metabolising system (Experiment II)

Strain	Dose level per plate (µg)	Mean revertants per plate	Standard deviation	Ratio treated/solvent	Individual revertant colony counts
TA 1535	3	10.3	1.5	1.0	12,9,10
	10	9.7	3.2	1.0	11,6,12
	33	11.3	5.5	1.1	15,5,14
	100	11.7	2.9	1.2	15,10,10
	333	10.3	4.7	1.0	14,5,12
	1000	1.0	1.0	0.1	0 R, 2 R, 1 R
	Solvent control	10.0	1.0		10,9,11
	Negative control	9.7	3.2		11,12,6
TA 1537	3	14.0	2.0	0.9	14,16,12
	10	16.0	2.6	1.1	15,19,14
	33	11.7	0.6	0.8	11,12,12
	100	13.0	2.6	0.9	12,11,16
	333	7.0	2.0	0.5	9,7,5
	1000	0.0	0.0	0.0	0 R, 0 R, 0 R
	Solvent control	15.0	2.6		12,17,16
	Negative control	16.3	4.6		19,11,19
TA 98	10	40.7	5.1	1.1	42,35,45
	33	42.3	4.0	1.2	47,40,40
	100	36.0	7.9	1.0	30,33,45
	333	13.3	2.9	0.4	10,15,15
	1000	1.3	0.6	0.0	1 R, 1R, 2 R
	2500	0.3	0.6	0.0	0 R, 0 R, 1 R
	Solvent control	36.7	3.5		40, 33, 37
	Negative control	46.3	3.8		48, 49, 42
TA 100	3	112.0	12.1	0.9	105, 126, 105
	10	125.3	18.6	1.0	146, 120, 110
	33	127.7	7.1	1.0	129, 134, 120
	100	96.3	14.5	1.7	96, 111, 82
	333	17.0	3.6	0.1	14, 16, 21
	1000	0.3	0.6	0.0	1 R, 0 R, 0 R
	Solvent control	130.7	6.1		136, 132, 124
	Negative control	189.0	1.0		190, 188, 189
WP2 uvrA	10	31.7	9.0	0.8	42,27,26
	33	36.3	11.5	0.9	36,48,25
	100	36.7	5.0	0.9	36,32,42
	333	38.3	11.0	0.9	31,33,51
	1000	18.0	3.6	0.4	14,21,19
	2500	0.0	0.0	0.0	0 R, 0 R, 0 R
	Solvent control	41.0	7.0		36,38,49
	Negative control	46.7	1.5		45,48,47

R= Reduced background growth

 

 

Table 5- Concentrations where the test item showed reduced background growth

Strain	Experiment 1		Experiment 2
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 1535	1000-5000	1000-5000	333-5000	1000
TA 1537	1000-5000	1000-5000	1000-5000	1000
TA 98	1000-5000	1000-5000	1000-5000	1000-2500
TA 100	1000-5000	1000-5000	1000-5000	1000
EP2 uvrA	/	1000-5000	1000-5000	2500

 

Table 6- Concentrations where the test item showed toxic effects based on the reduction in the number of revertants 

Strain	Experiment 1		Experiment 2
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 1535	333	1000-5000	333-5000	1000
TA 1537	1000	1000-5000	1000-5000	1000
TA 98	/	2500-5000	1000-5000	333-2500
TA 100	1000-5000	1000-5000	333-5000	333-1000
EP2 uvrA	/	2500-5000	1000-5000	1000-2500

 

Conclusions:

Under the reported experimental conditions, no biological relevant increase in the number of revertant colonies were observed. Therefore, the test item is considered to not induce mutagenic effects via the processes of base pair changes or frameshifts within the genome of the tested amino stains used up to the highest concentration (5000 µg/plate). 

Executive summary:

The potential mutagenicity of the test item was investigated in an Ames test with the Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) and Escherichia coli strain (WP2uvrA) in the presence and absence of a metabolising system in the concentration range of 3-5000 µg/plate. Each assay was observed for precipitation, normal background lawn and for the number of revertant colonies. Precipitation was observed in both experiments.

Under the reported experimental conditions, no biological relevant increase in the number of revertant colonies were observed. Therefore, the test item is considered to not induce mutagenic effects via the processes of base pair changes or frameshifts within the genome of the tested amino stains used up to the highest concentration (5000 µg/plate). 

This study (2018) is GLP-compliant and follows the OECD Test Guideline 471 and is therefore reliable without restrictions (Klimisch 1).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 μg/plate with S9 mix and in experiment II from 2500 to 5000 μg/plate without S9 mix respectively at 2500 μg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate with S9 mix. In experiment II no precipitation of the test item was observed on the incubated agar plates up to the highest investigated dose. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Justification for classification or non-classification

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is, therefore, considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.