3-methyl-2-butenyl salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

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:

January - April 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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine, tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Strain TA 100: 0.3, 1, 3,10, 33, 100, 333, and 1000 μg/plate
all others: 33, 100, 333, 1000, 2500, and 5000 μg/plate
Based on the observed toxicity in the pretest 1000 μg/plate were chosen as maximal concentration for strain TA 100, 5000 μg/plate for the remaining strains.

Vehicle / solvent:

DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537, TA 98 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA (With metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in Experiment I and pre-incubation in Experiment II

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h at 37 °C

SELECTION AGENT: L-Histidin for S. typhimurium, Tryptophan for E. coli

NUMBER OF REPLICATIONS: 3

Titer of the incubated culture: 10^8 - 10^9 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn

ACCEPTANCE CRITERIA:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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:

cytotoxicity

Remarks:

at 100 μg/plate

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:

cytotoxicity

Remarks:

at 333 μg/plate

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:

cytotoxicity

Remarks:

at 100 μg/plate

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since toxic effects were observed six concentrations were tested in experiment II. Based on the observed toxicity 1000 μg/plate were chosen as maximal concentration for strain TA 100, 5000 μg/plate for the remaining strains.

HISTORICAL CONTROL DATA
see table at "Any other information"
In experiment I the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain WP2 uvrA with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduced background lawn
- TA 1537: with metabolic activation at 333 μg/plate and without metabolic activation at 100 μg/plate cytotoxic effects
- TA 98: with metabolic activation at 1000 μg/plate and without metabolic activation at 333 μg/plate cytotoxic effects
- TA 100: with metabolic activation at 1000 μg/plate and without metabolic activation at 100 μg/plate cytotoxic effects

Mean number of revertants with S9 mix

Test substance	Dose/Plate (µg)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
DMSO	-	11	18	28	135	35
Test substance	0.3				150
	1				124
	3	13	16	37	161
	10	14	15	32	141
	33	12	15	28	137	40
	100	10	8	35	134	39
	333	9	8R	25	69	40
	1000	1	0R	14R	5R	40
	2500	0	0R	1R		24
	5000	0	0R	0R		27
Positive control	10					369
	2.5	425	157	3959	5070

Positive Control: 2 –Aminoanthracene (2 -AA)

R: reduced background growth

Mean number of revertants without S9 mix

Test substance	Dose/Plate (µg)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
DMSO	-	15	13	26	156	30
Test substance	0.3				140
	1				187
	3	11	10	27	145
	10	11	10	24	154
	33	8	8	20	142	32
	100	7	5R	17	48	30
	333	8	7R	20R	48	31
	1000	6	7R	15R	43	32
	2500	3	4R	14R		30
	5000	1	2R	7R		28
NaN3	10	1151	-	-	2171	-
4-NOPD	10	-	-	317	-	-
4-NOPD	50	-	68	-	-	-
MMS	2.0	-	-	-	-	843

R: reduced background growth

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Historical control data

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
TA1537	Solvent control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent control	25	4.4	13	43	34	6.2	15	58
	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent control	156	26.0	78	209	148	32.3	73	208
	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
WP2 uvr A	Solvent control	41	5.6	27	63	50	6.8	28	72
	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Conclusions:

The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Strain TA 100: 0.3; 1; 3;10; 33; 100; 333; and 1000 µg/plate

Strain WP2 uvrA: 33; 100; 333; 1000; 2500 and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with S9 mix in both experiments. 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. The undissolved particles had no influence on the data recording. In experiment II no precipitation of the test item was observed in the overlay agar on the incubated agar plates. In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix up to 5000 µg/plate. In experiment II reduced background growth was observed in strains TA 1537, TA98, and TA 100 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. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Strain TA 100: 0.3; 1; 3;10; 33; 100; 333; and 1000 µg/plate

Strain WP2 uvrA: 33; 100; 333; 1000; 2500 and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with S9 mix in both experiments. 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. The undissolved particles had no influence on the data recording. In experiment II no precipitation of the test item was observed in the overlay agar on the incubated agar plates. In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix up to 5000 µg/plate. In experiment II reduced background growth was observed in strains TA 1537, TA98, and TA 100 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. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled as mutagen according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.