Ethyl salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is one in vitro genotoxicity study available. The test item did not induce an increase in mutation frequency in the Ames test, and therefore, it can be concluded that the test substance does not exert gentoxic effects.

The test was performed according to OECD TG 471 and in compliance to GLP.

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:

15.-30.09.2016

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:

July 21, 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

- histidine for Salmonella typhimurium
- tryptophan for Escherichia coli

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:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

5000 μg/plate were chosen as maximal concentration.

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

- Experiment II:
-- Strains TA 1535 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
-- Strains TA 1537, TA 98, and WP2 uvrA : 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

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

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Positive controls:

yes

Positive control substance:

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

Remarks:

Without metabolic activation

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

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
- Exposure duration: 48h at 37°C

NUMBER OF REPLICATIONS: 3

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.

Rationale for test conditions:

these are according to the OECD guideline 471

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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

solvent

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

solvent

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

Vehicle controls validity:

valid

Remarks:

solvent

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

solvent

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

Remarks:

solvent

Positive controls validity:

valid

Additional information on results:

- The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in the first experiment and from 2500 to 5000 μg/plate in the second experiment. In the overlay agar on the incubated agar plates no precipitation of the test item was observed.
- 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), were observed in all strains.

Remarks on result:

other: not mutagenic

Experiment I 

Metabolic	Test	Dose Level	Revertant Colony Counts (Mean ±SD)
Activation	Group	(per plate)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		13 ± 4	12 ± 4	31 ± 12	150 ± 10	45 ± 3
	Untreated		12 ± 3	11 ± 3	27 ± 5	190 ± 12	39 ± 6
	ETHYL	3 µg	12 ± 1	13 ± 4	28 ± 6	172 ± 8	47 ± 3
	SALICYLATE	10 µg	9 ± 3	8 ± 2	22 ± 3	173 ± 11	40 ± 3
		33 µg	11 ± 4	9 ± 3	23 ± 3	169 ± 12	42 ± 8
		100 µg	14 ± 4	8 ± 1	23 ± 8	168 ± 5	35 ± 13
		333 µg	15 ± 1	8 ± 2	25 ± 3	121 ± 8	31 ± 1
		1000 µg	12 ± 3^R	10 ± 3^R	18 ± 3	67 ± 6^R	27 ± 6
		2500 µg	11 ± 4^R	8 ± 3^R	29 ± 4^R	63 ± 6^R	31 ± 6
		5000 µg	10 ± 1^R	4 ± 1^M R	23 ± 7^R	57 ± 4^R	37 ± 3
	NaN3	10 µg	1343 ± 27			1842 ± 401
	4-NOPD	10 µg			526 ± 36
	4-NOPD	50 µg		75 ± 13
	MMS	2.0 µL					791 ± 40
With Activation	DMSO		15 ± 5	10 ± 3	36 ± 13	110 ± 5	69 ± 9
	Untreated		14 ± 3	12 ± 2	42 ± 10	189 ± 6	62 ± 16
	ETHYL	3 µg	12 ± 3	12 ± 2	44 ± 6	123 ± 8	42 ± 4
	SALICYLATE	10 µg	13 ± 5	10 ± 3	35 ± 10	158 ± 24	60 ± 6
		33 µg	9 ± 5	12 ± 3	30 ± 3	149 ± 19	53 ± 12
		100 µg	14 ± 3	6 ± 1	38 ± 8	164 ± 2	50 ± 3
		333 µg	11 ± 4	10 ± 1	34 ± 8	132 ± 13	43 ± 4
		1000 µg	7 ± 2^R	7 ± 3^R	27 ± 7^R	41 ± 8^R	48 ± 9
		2500 µg	2 ± 0^M R	2 ± 1^R M	6 ± 1^M R	7 ± 2^M R	34 ± 5^R
		5000 µg	2 ± 1^M R	0 ± 0^R	1 ± 1^R	0 ± 0^R	17 ± 4^M R
	2-AA	2.5 µg	349 ± 10	361 ± 23	4292 ± 624	2005 ± 255
	2-AA	10.0 µg					400 ± 19

Experiment II

Metabolic	Test	Dose Level	Revertant Colony Counts (Mean ±SD)
Activation	Group	(per plate)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 3	15 ± 3	32 ± 3	171 ± 16	51 ± 7
	Untreated		10 ± 4	19 ± 2	31 ± 11	202 ± 5	42 ± 2
	ETHYL	3 µg	8 ± 2			181 ± 11
	SALICYLATE	10 µg	11 ± 2	15 ± 6	23 ± 4	158 ± 3	46 ± 2
		33 µg	13 ± 4	15 ± 1	28 ± 2	171 ± 21	36 ± 14
		100 µg	11 ± 1	14 ± 4	30 ± 6	160 ± 6	39 ± 7
		333 µg	14 ± 2	8 ± 3	23 ± 1	52 ± 3^R	23 ± 4
		1000 µg	7 ± 2^R	5 ± 1^M R	11 ± 1^M R	62 ± 7^R	15 ± 3^M R
		2500 µg	10 ± 5^R	5 ± 1^M R	7 ± 2^M R	48 ± 13^R	3 ± 1^R M
		5000 µg	11 ± 3^R	5 ± 1^M R	4 ± 1^M R	1 ± 0^R	3 ± 1^M R
	NaN3	10 µg	1333 ± 40			2108 ± 156
	4-NOPD	10 µg			548 ± 54
	4-NOPD	50 µg		84 ± 6
	MMS	2.0 µL					841 ± 127
With Activation	DMSO		16 ± 4	15 ± 4	34 ± 3	165 ± 23	56 ± 5
	Untreated		14 ± 2	19 ± 7	46 ± 8	206 ± 4	56 ± 7
	ETHYL	3 µg	14 ± 6			172 ± 6
	SALICYLATE	10 µg	13 ± 2	15 ± 1	37 ± 8	151 ± 12	54 ± 10
		33 µg	14 ± 2	12 ± 3	30 ± 2	152 ± 9	63 ± 4
		100 µg	16 ± 4	10 ± 6	40 ± 9	141 ± 9	53 ± 5
		333 µg	11 ± 3	8 ± 3	27 ± 3	51 ± 5^R	58 ± 2
		1000 µg	3 ± 1^M R	1 ± 0^M R	9 ± 3^M R	23 ± 2^R	21 ± 5^M R
		2500 µg	0 ± 0^M R	1 ± 0^M R	0 ± 0^M R	1 ± 0^R	15 ± 4^M R
		5000 µg	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^R	0 ± 0^M R
	2-AA	2.5 µg	365 ± 11	193 ± 9	3421 ± 397	4468 ± 178
	2-AA	10.0 µg					394 ± 16

NaN3 = sodium azide

2-AA = 2-aminoanthracene

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

R = Reduced background growth

M = Manual count

Conclusions:

The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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 test was performed according to OECD TG 471 and in compliance to GLP.

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 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Strains TA 1537, TA 98, and WP2 uvrA : 10; 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 in the first experiment and from 2500 to 5000 μg/plate in the second experiment. In the overlay agar on the incubated agar plates no precipitation of the test item was observed.

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), were observed in all strains.

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. 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.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is one in vitro study available that assessed the possible genotoxic potential of the test substance. It was performed according to GLP and internationally accepted guidelines.

Ames assay:

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 test was performed according to OECD TG 471 and in compliance to GLP. 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 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Strains TA 1537, TA 98, and WP2 uvrA : 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

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.

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. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

The key study is well documented and according to GLP and internationally accepted guidelines.

As no genotoxic effects is observed in the available study addressing genetic toxicity, classification for genetic toxicity under EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) is not required.