Isobutyl salicylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.09.-17.10.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Test guideline 2

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:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Strasse 80, 65189 Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl salicylate

EC Number:

201-729-9

EC Name:

Isobutyl salicylate

Cas Number:

87-19-4

Molecular formula:

C11H14O3

IUPAC Name:

2-methylpropyl 2-hydroxybenzoate

Method

Target gene:

histidine, tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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:

- in the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate
- toxic effects were observed in experiment I
- Pre-Experiment/Experiment I & Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Experiment II:
Strains TA 1537 and TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
The remaining strains: 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.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Controls 2

Positive controls:

yes

Remarks:

10 μg/plate

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 3

Positive controls:

yes

Remarks:

10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537

Positive control substance:

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

Remarks:

Without metabolic activation

Controls 4

Positive controls:

yes

Remarks:

2.0 μL/plate

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 5

Positive controls:

yes

Remarks:

2.5 μg/plate (10.0 μg/plate in WP2 uvrA)

Positive control substance:

other: 2-aminoanthracene, 2-AA

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.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: 2500 – 5000 with S9; Exp. II: 2500 – 5000 with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: 1000 – 5000 without S9, 2500 – 5000 with S9; Exp. II: 333 – 2500 without S9, 1000 – 2500 with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: 2500 – 5000 with S9, 5000 without S9; Exp. II: 5000 without S9, 1000 – 5000 with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: 333 – 5000 without S9; 333, 5000 with S9; Exp. II: 100 – 2500 without S6, 333 – 2500 with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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:

The assay was performed in three independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Any other information on results incl. tables

Summary of experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100
Without	DMSO		12 ± 4	15 ± 2	32 ± 4	146 ± 13
Activation	Untreated		12 ± 5	12 ± 2	29 ± 4	165 ± 6
	Test item	3 μg	13 ± 6	14 ± 2	24 ± 3	153 ± 14
		10 μg	11 ± 5	10 ± 1	36 ± 6	141 ± 13
		33 μg	15 ± 5	17 ± 3	25 ± 8	158 ± 21
		100 μg	15 ± 7	13 ± 2	30 ± 2	84 ± 12
		333 μg	9 ± 1	10 ± 5	20 ± 5	40 ± 5
		1000 μg	9 ± 3	5 ± 2 M R	23 ± 3	17 ± 3 M R
		2500 μg	9 ± 4	2 ± 1 M R	13 ± 3	22 ± 6 M R
		5000 μg	11 ± 3	2 ± 1 M R	10 ± 2	18 ± 4 M R
	NaN3	10 μg	1520 ± 36			2311 ± 58
	4-NOPD	10 μg			415 ± 38
	4-NOPD	50 μg		87 ± 8
	MMS	2.0 μL
With	DMSO		15 ± 3	17 ± 5	40 ± 11	122 ± 7
Activation	Untreated		20 ± 5	20 ± 1	43 ± 4	143 ± 15
	Test item	3 μg	15 ± 5	17 ± 4	40 ± 4	115 ± 13
		10 μg	10 ± 1	23 ± 4	37 ± 5	108 ± 9
		33 μg	16 ± 6	15 ± 6	44 ± 7	139 ± 2
		100 μg	13 ± 6	17 ± 1	39 ± 3	118 ± 5
		333 μg	10 ± 2	16 ± 1	41 ± 2	43 ± 9
		1000 μg	8 ± 4	9 ± 5	25 ± 2	57 ± 7
		2500 μg	1 ± 1	3 ± 3 M R	21 ± 1 R	64 ± 16
		5000 μg	0 ± 0	1 ± 1 M R	8 ± 1 M R	3 ± 1
	2-AA	2.5 μg	366 ± 30	359 ± 22	3655 ± 326	3766 ± 228
	2-AA	10.0 μg

Summary of experiment Ia

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			WP2 uvrA
Without	DMSO		39 ± 6
Activation	Untreated		43 ± 7
	ISOBUTYL	3 μg	37 ± 12
	SALICYLATE	10 μg	47 ± 3
		33 μg	50 ± 8
		100 μg	49 ± 5
		333 μg	34 ± 4
		1000 μg	32 ± 3
		2500 μg	41 ± 1
		5000 μg	36 ± 8
	MMS	2.0 μL	1135 ± 75
With	DMSO		44 ± 7
Activation	Untreated		44 ± 12
	ISOBUTYL	3 μg	42 ± 6
	SALICYLATE	10 μg	42 ± 5
		33 μg	44 ± 11
		100 μg	43 ± 14
		333 μg	47 ± 9
		1000 μg	51 ± 4
		2500 μg	39 ± 9
		5000 μg	33 ± 8
	2-AA	10.0 μg	431 ± 55

Summary of experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without	DMSO		15 ± 5	9 ± 2	22 ± 2	153 ± 9	39 ± 6
Activation	Untreated		17 ± 3	9 ± 3	27 ± 10	187 ± 7	43 ± 2
	ISOBUTYL	0.3 μg		9 ± 1		150 ± 11
	SALICYLATE	1 μg		11 ± 2		144 ± 14
		3 μg		12 ± 1		147 ± 19
		10 μg	13 ± 3	13 ± 2	26 ± 9	157 ± 8	42 ± 6
		33 μg	14 ± 3	9 ± 3	22 ± 1	105 ± 7	52 ± 5
		100 μg	9 ± 2	10 ± 2 R	22 ± 7	58 ± 18 R	40 ± 4
		333 μg	11 ± 1	3 ± 1 M R	23 ± 10 R	57 ± 10 M R	39 ± 4
		1000 μg	10 ± 3 R	4 ± 2 M R	11 ± 3 M R	41 ± 8 M R	45 ± 11
		2500 μg	13 ± 3 R	1 ± 1 M R	13 ± 1 M R	21 ± 3 M R	36 ± 2
		5000 μg	17 ± 2 R		9 ± 1 M R		37 ± 8
	NaN3	10 μg	1268 ±			2344 ± 86
			57
	4-NOPD	10 μg			406 ± 21
	4-NOPD	50 μg		69 ± 11
	MMS	2.0 μL					762 ± 9
With	DMSO		12 ± 4	9 ± 2	37 ± 9	118 ± 8	56 ± 7
Activation	Untreated		10 ± 3	10 ± 3	36 ± 4	186 ± 14	61 ± 7
	ISOBUTYL	0.3 μg		12 ± 2		113 ± 11
	SALICYLATE	1 μg		8 ± 2		130 ± 2
		3 μg		10 ± 1		122 ± 16
		10 μg	12 ± 5	7 ± 2	29 ± 3	113 ± 3	50 ± 8
		33 μg	11 ± 3	12 ± 2	27 ± 5	101 ± 2	55 ± 3
		100 μg	10 ± 4	7 ± 3	28 ± 3	114 ± 9 R	59 ± 10
		333 μg	10 ± 5	9 ± 2 R	35 ± 10 R	51 ± 9 R	56 ± 8
		1000 μg	13 ± 2 R	1 ± 1 M R	15 ± 4 M R	13 ± 3 M R	49 ± 11
		2500 μg	3 ± 1 R	1 ± 0 M R	7 ± 2 M R	14 ± 2 M R	45 ± 11
		5000 μg	0 ± 0 R		2 ± 1 M R		46 ± 1
	2-AA	2.5 μg		83 ± 1	3843 ± 77	4598 ± 151
	2-AA	10.0 μg	367 ± 32				333 ± 17

Applicant's summary and conclusion

Conclusions:

In 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.

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 Ia) 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 performet according to OECD TG 471 and in compliance to GLP.

The assay was performed in three 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 & Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:

Strains TA 1537 and TA 100: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

The remaining strains: 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. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.

The plates incubated with the test item showed reduced background growth in all strains with and without S9, except of strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains, except of strain WP2 uvrA.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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. They showed a distinct in-crease in 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.