Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria:Key study. Test method according to OECD 471, GLP study. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 μg/plate. Based on the available data, the test item is not mutagenic.

In vitro cytogenicity chromosome aberration study in mammalian cells:Weight of evidence. The genotoxic potential of the test substance methyl salicylate (99% purity) was studied under non-GLP conditions in an in vitro mammalian chromosomal aberration assay using Chinese hamster lung fibroblast cells in accordance with the publication by Ishidate and Odashima 1977). All tested doses were negative and did not produce a significant increase in the number of chromosomal aberrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 29th, 2018 to June 6th, 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his D (S. typhimurium TA 98); his C (S. typhimurium TA 1537); his G (S. typhimurium TA 100 and TA1535); tryp E (E. coli WP2 uvrA pKM101)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: ΔuvrB and rfa mutated (TA 98 and TA 100: pKM 101)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA, pKM 101 mutated

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of liver rats

Test concentrations with justification for top dose:

Initial mutation test (plate incorporation) / Confirmatory mutation test (pre-incubation): 50, 150, 500, 1500 and 5000 μg/plate. The maximum test concentration was 5000 μg test item/plate since in the preliminary test the numbers of revertant colonies were mostly in the normal range, no cytotoxicity was observed and there was no precipitation up to the highest dose tested (TA100 without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was found to be soluble in Dimethyl sulfoxide (DMSO) at 100 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine (1, 2 μg/plate; S. typhimurium strains, + S9), /cis-Platinum (II) Diammine D ichloride (1 μg/plate; E.coli, - S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
1.Plate incorporation (initial mutation test): A stock solution of the test item was prepared at 100 mg / mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9E09 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled at 45ºC containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains, or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain. In the assay with metabolic activation, either a standard plate incorporation method where the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates. After a 48-72 hour incubation period at 37 ºC, revertant colonies are counted in each plate.
2.Pre-incubation (confirmatory mutation test): Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes were gently mixed and incubated for 30 min at 37ºC in a shaking incubator. After the incubation period, molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37ºC for 48 hours.

DURATION
- Preincubation period: 30 minutes (confirmatory mutation test)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): The lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3 (test item, negative and positive controls).

DETERMINATION OF CYTOTOXICITY
- Method: other: relative total growth.
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9E 03 bacteria /mL) and 0.1 mL of the stock solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 h at 37 ºC, and the colonies counted. A negative control containing the blank alone is run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

- OTHER:
- Sterility test: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.
- Preparation of the metabolic activation system (S9 fraction): Obtention of S9 fraction: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3805 validated on 5.2017 - expiry date: 25.05.2019). Preparation of S9-mix 10% (v/v): The final concentration of cofactors and salts is as follows: S9 fraction 10%; MgCl2-6H2O 8 mM; KCl 33 mM; Glucose-6-Phosphate Na2 5 mM; NADP Na2 4mM; Phosphate buffer pH 7.4 0.1 M.

Rationale for test conditions:

Results of sterility controls show the absence of any bacterial growth in the presence of test item S9-mix. Results of the bacteriostatic activity control show no toxicity. Values and frequency are within the laboratory's historical control ranges.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation. The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537. All results must be confirmed in an independent experiment.

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:

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:

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
- Precipitation: None observed

RANGE-FINDING/SCREENING STUDIES: No precipitate and no inhibitory, cytotoxic effect of the test item was observed up to the highest dose tested (5000 μg/plate).


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (- S9): values for spontaneous revertants (revertants/plate) in the period of 2008 - 2017 were as follows:
Salmonella typhimurium TA98: 496.3 ± 219.9, TA100: 991.8 ± 331.2, TA1535: 731.5 ± 220.2, TA1537: 876.5 ± 448.3, Escherichia coli WP2 uvrA: 484.6 ± 168.2.
- Positive historical control data (+ S9): values with metabolic activation in the period of 2008 - 2017 were:
Salmonella typhimurium TA98: 572.9 ± 222.1, TA100: 846.8 ± 359.5, TA1535: 109.2 ± 56.0, TA1537: 55.1 ± 24.7, Escherichia coli WP2 uvrA: 686.5 ± 253.3.
- Negative (solvent/vehicle) historical control data (- S9): values for untreated control sample without metabolic activation in the period of 2008 - 2017 were as follows:
Salmonella typhimurium TA98: 16.0 ± 3.9, TA100: 59.8 ± 11.8, TA1535: 11.0 ± 3.6, TA1537: 6.0 ± 2.5, Escherichia coli WP2 uvrA: 48.8 ± 7.8.
- Negative (solvent/vehicle) historical control data (+ S9): values with metabolic activation in the period of 2008 - 2017 were:
Salmonella typhimurium TA98: 23.2 ± 5.0, TA100: 96.2 ± 22.3, TA1535: 12.2 ± 4.1, TA1537: 8.1 ± 3.5, Escherichia coli WP2 uvrA: 156.8 ± 34.6.
- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-- No toxic effects of the test item were observed in any of the five tested strains used up to the highest dose (with and without metabolic activation) in assays 1 and 2.

Table 3. Sterility control

Serie	Doses	Colony number/plate
Control n° 1		1	2	3
Solution of  Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 2		1	2	3
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate BATCH: L4284945   (LEMI code : 18/0147-110618-S1)	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

Table 4. Bacteriostatic activity control nº1

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	2500 µg	5000 µg
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	N1	849	900	789	856	630	405	445	221
	N2	844	750	825	823	758	569	420	235
	N3	674	715	819	869	789	589	439	132
	N	789 ± 100	788 ± 98	811 ± 19	849 ± 24	726 ± 84	521 ± 101	435 ± 13	196 ± 56
	%	-	100%	103%	108%	92%	66%	55%	25%

Table 5. Bacteriostatic activity control n° 2

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	5000 µg
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	N1	420	406	429	408	389	309	89
	N2	396	386	435	389	401	245	121
	N3	384	365	418	409	425	274	92
	N	400 ± 18	386 ± 21	427 ± 9	402 ± 11	405 ± 18	276 ± 32	101 ± 18
	%	-	96%	107%	101%	101%	69%	25%

Table 6. Assays of metabolic activity without metabolic activation

TA 1535 Assay n°1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	14	9	6	9.67	4.04	-
Positive control solvent	5 µL	13	9	7	9.67	3.06	-
Positive control : Sodium azide	5 µg in 5 µL	878	948	980	935.33	52.37	96.76
Vehicle	50 µL	15	9	11	11.67	3.06	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg* 1500 µg 500 µg 150 µg 50 µg	10 4 16 8 7	4 10 9 14 13	6 7 9 4 7	6.67 7.00 11.33 8.67 9.00	3.06 3.00 4.04 5.03 3.46	0.57 0.60 0.97 0.74 0.77

 

TA 1535 Assay n°2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	11	13	18	14.00	3.61	-
Positive control solvent	5 µL	12	14	10	12.00	2.00	-
Positive control : Sodium azide	5 µg in 5 µL	844	814	883	847.00	34.60	70.58
Vehicle	50 µL	13	15	13	13.67	1.15	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg* 1500 µg 500 µg 150 µg 50 µg	9 3 7 14 6	5 9 10 7 15	7 8 6 17 14	7.00 6.67 7.67 12.67 11.67	2.00 3.21 2.08 5.13 4.93	0.51 0.49 0.56 0.93 0.85

TA 1537 Assay n°1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	8	4	7	6.33	2.08	-
Positive control solvent	20 µL	12	4	10	8.67	4.16	-
Positive control : 9-Aminoacridine	50 µg in 20 µL	1609	1978	1739	1775.33	187.16	204.85
Vehicle	50 µL	9	10	18	12.33	4.93	-
	5000 µg*	10	5	7	7.33	2.52	0.59
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	10 9	13 7	4 10	9.00 8.67	4.58 1.53	0.73 0.70
	150 µg	8	10	7	8.33	1.53	0.68
	50 µg	10	9	9	9.33	0.58	0.76

TA 1537 Assay n°2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	6	5	6.00	1.00	-
Positive control solvent	20 µL	8	4	9	7.00	2.65	-
Positive control : 9-Aminoacridine	50 µg in 20 µL	1209	869	841	873.00	204.86	139.00
Vehicle	50 µL	5	7	8	6.67	1.53	-
	5000 µg*	8	6	6	6.67	1.15	1.00
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	9 4	6 10	7 7	7.33 7.00	1.53 3.00	1.10 1.05
	150 µg	10	9	9	9.33	0.58	1.40
	50 µg	9	9	9	9.00	0.00	1.35

TA 98 Assay n°1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	17	20	16.33	4.04	-
Positive control solvent	20 µL	19	23	17	19.67	3.06	-
Positive control : 2-Nitrofluorene	2 µg in 20 µL	559	648	598	601.67	44.61	30.59
Vehicle	50 µL	17	11	19	15.67	4.16	-
	5000 µg*	15	17	12	14.67	2.52	0.94
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	23 19	20 14	19 18	20.67 17.00	2.08 2.65	1.32 1.09
	150 µg	29	17	19	21.67	6.43	1.38
	50 µg	19	26	21	22.00	3.61	1.40

TA 98 Assay n°2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	21	23	20	21.33	1.53	_
Positive control solvent	20 µL	25	27	25	25.67	1.15	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	494	689	563	582.00	98.88	22.68
Vehicle	50 µL	23	20	22	21.67	1.53	_
	5000 µg*	13	11	14	12.67	1.53	0.58
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	12 17	15 13	15 15	14.00 15.00	1.73 2.00	0.65 0.69
	150 µg	17	19	17	17.67	1.15	0.82
	50 µg	20	22	23	21.67	1.53	1.00

TA 100 Assay n°1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	51	63	52	55.33	6.66	-
Positive control solvent	20 µL	67	68	52	62.33	8.96	-
Positive control : Sodium azide	20 µg in 20 µL	1346	1287	1293	1308.67	32.47	20.99
Vehicle	50 µL	59	56	68	61.00	6.24	-
	5000 µg**	24	24	28	25.33	2.31	0.42
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	62 58	63 69	59 60	61.33 62.33	2.08 5.86	1.01 1.02
	150 µg	55	64	57	58.67	4.73	0.96
	50 µg	48	58	58	54.67	5.77	0.90

TA 100 Assay n°2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	75	46	54	58.33	14.98	-
Positive control solvent	20 µL	48	54	52	51.33	3.06	-
Positive control : Sodium azide	20 µg in 20 µL	1512	1351	1441	1434.67	80.69	27.95
Vehicle	50 µL	55	69	62	62.00	7.00	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg** 1500 µg 500 µg 150 µg 50 µg	32 50 54 51 51	38 53 52 55 50	37 52 48 53 54	35.67 51.67 51.33 53.00 51.67	3.21 1.53 3.06 2.00 2.08	0.58 0.83 0.83 0.85 0.83

E. coli Assay n°1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	119	121	110	116.67	5.86	-
Positive control solvent	10 µL	113	124	126	121.00	7.00	-
Positive control : cis-Platinum (II)	1 µg in 10 µL	453	511	477	480.33	29.14	3.97
Vehicle	50 µL	107	104	132	114.33	15.37	-
	5000 µg**	61	108	99	89.33	24.95	0.78
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	99 114	109 106	107 116	105.00 112.00	5.29 5.29	0.92 0.98
	150 µg	104	102	105	103.67	1.53	0.91
	50 µg	101	105	109	105.00	4.00	0.92

E. coli Assay n°2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	177	167	174	172.67	5.13	-
Positive control solvent	10 µL	172	177	181	176.67	4.51	-
Positive control : cis-Platinum (II)	1 µg in 10 µL	577	610	554	580.33	28.15	3.28
Vehicle	50 µL	153	178	169	166.67	12.66	-
	5000 µg **	137	169	172	159.33	19.40	0.96
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	147 151	160 157	146 144	151.00 150.67	7.81 6.51	0.91 0.90
	150 µg	152	181	178	170.33	15.95	1.02
	50 µg	162	167	176	168.33	7.09	1.01

Table 7. Assays with metabolic activation

TA 1535 -Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	6	9	7.33	1.53	-
Positive control solvent	20 µL	9	8	9	8.67	0.58	-
Positive control : 2-Anthramine	2 µg in 20 µL	113	203	133	149.67	47.26	17.27
Vehicle	50 µL	7	10	16	11.00	4.58	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg** 1500 µg 500 µg 150 µg 50 µg	1 4 5 10 11	1 6 5 6 7	1 4 6 6 8	1.00 4.67 5.33 7.33 8.67	0.00 1.15 0.58 2.31 2.08	0.09 0.42 0.48 0.67 0.79

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

 TA 1535 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	9	14	15	12.67	3.21	-
Positive control solvent	10 µL	13	10	12	11.67	1.53	-
Positive control : 2-Anthramine	1 µg in 10 µL	88	116	93	99.00	14.93	8.49
Vehicle	50 µL	7	14	16	12.33	4.73	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg** 1500 µg* 500 µg 150 µg 50 µg	0 1 4 6 12	0 3 4 9 13	0 2 5 10 9	0.00 2.00 4.33 8.33 11.33	0.00 1.00 0.58 2.08 2.08	0.00 0.16 0.35 0.68 0.92

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

TA 1537 -Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	9	5	19	11.00	7.21	-
Positive control solvent	20 µL	10	19	17	15.33	4.73	-
Positive control : 2-Anthramine	2 µg in 20 µL	45	54	51	50.00	4.58	3.26
Vehicle	50 µL	18	18	10	15.33	4.62	-
	5000 µg**	0	0	0	0.00	0.00	0.00
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1 500 µg** 500 µg	1 5	6 3	6 6	4.33 4.67	2.89 1.53	0.28 0.30
	150 µg	11	13	10	11.33	1.53	0.74
	50 µg	11	9	14	11.33	2.52	0.74

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

 TA 1537 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	13	10	5	9.33	4.04	-
Positive control solvent	10 µL	5	16	4	8.33	6.66	-
Positive control : 2-Anthramine	1 µg in 10 µL	62	53	55	56.67	4.73	6.80
Vehicle	50 µL	8	13	11	10.67	2.52	-
	5000 µg**	0	0	0	0.00	0.00	0.00
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg** 500 µg	5 11	1 14	2 8	2.67 11.00	2.08 3.00	0.25 1.03
	150 µg	7	10	8	8.33	1.53	0.78
	50 µg	5	10	7	7.33	2.52	0.69

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

TA 98 -Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	27	31	26	28.00	2.65	-
Positive control solvent	20 µL	19	20	26	21.67	3.79	-
Positive control : 2-Anthramine	2 µg in 20 µL	807	645	907	786.33	132.22	36.29
Vehicle	50 µL	25	23	27	25.00	2.00	-
	5000 µg**	5	7	16	9.33	5.86	0.37
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg* 500 µg	17 8	18 16	14 18	16.33 14.00	2.08 5.29	0.65 0.56
	150 µg	23	20	23	22.00	1.73	0.88
	50 µg	32	24	26	27.33	4.16	1.09

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

 TA 98 Assay n°2 –with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	22	25	23	23.33	1.53	-
Positive control solvent	10 µL	26	29	23	26.00	3.00	-
Positive control : 2-Anthramine	1 µg in 10 µL	622	739	603	654.67	73.65	25.18
Vehicle	50 µL	23	26	30	26.33	3.51	-
	5000 µg**	6	2	4	4.00	2.00	0.15
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg** 500 µg	1 23	7 21	6 15	4.67 19.67	3.21 4.16	0.18 0.75
	150 µg	20	21	25	22.00	2.65	0.84
	50 µg	26	27	29	27.33	1.53	1.04

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

TA 100 -Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	64	76	65	68.33	6.66	-
Positive control solvent	20 µL	63	67	65	65.00	2.00	-
Positive control : 2-Anthramine	2 µg in 20 µL	675	693	769	712.33	49.89	10.96
Vehicle	50 µL	50	59	65	58.00	7.55	-
	5000 µg**	53	46	33	44.00	10.15	0.76
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg 500 µg	58 71	60 66	54 55	57.33 64.00	3.06 8.19	0.99 1.10
	150 µg	51	70	69	63.33	10.69	1.09
	50 µg	65	69	71	68.33	3.06	1.18

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

 

TA 100 Assay n°2 –with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	67	70	65	67.33	2.52	-
Positive control solvent	10 µL	74	84	87	81.67	6.81	-
Positive control : 2-Anthramine	1 µg in 10 µL	724	760	655	713.00	53.36	8.73
Vehicle	50 µL	76	58	69	67.67	9.07	-
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	5000 µg** 1500 µg* 500 µg 150 µg 50 µg	10 20 21 70 69	10 10 65 58 72	20 30 28 68 75	13.33 20.00 38.00 65.33 72.00	5.77 10.00 23.64 6.43 3.00	0.20 0.30 0.56 0.97 1.06

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

E.coli -Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	167	192	176	178.33	12.66	-
Positive control solvent	5 µL	212	207	184	201.00	14.93	-
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	709	690	740	713.00	25.24	3.55
Vehicle	50 µL	159	166	166	163.67	4.04	-
	5000 µg**	113	136	76	108.33	30.27	0.66
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg* 500 µg	122 152	121 139	146 158	129.67 149.67	14.15 9.71	0.79 0.91
	150 µg	161	133	144	146.00	14.11	0.89
	50 µg	146	152	155	151.00	4.58	0.92

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

E.coli Assay n°2 –with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	222	197	218	212.33	13.43	-
Positive control solvent	5 µL	218	220	205	214.33	8.14	-
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	724	829	659	737.33	85.78	3.44
Vehicle	50 µL	181	177	197	185.00	10.58	-
	5000 µg**	160	47	18	75.00	75.03	0.41
Solution of Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate	1500 µg* 500 µg	106 223	97 166	111 176	104.67 188.33	7.09 30.44	0.57 1.02
	150 µg	213	201	192	202.00	10.54	1.09
	50 µg	208	213	183	201.33	16.07	1.09

*light thinning of the bacterial lawn

**thinning of the bacterial lawn

Conclusions:

The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 μg/plate. Based on the available data, the test item is not mutagenic.

Executive summary:

A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471, under GLP conditions. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were exposed to five concentrations of the test substance ranging from 50 to 5000 μg/plate in DMSO, with and without metabolic activation, according to preliminary assays. The metabolic activation system (S9 fraction) prepared from Sprague Dawley rat liver homogenate was provided by MOLTOX. Two independent assays were performed: an initial mutation test (plate incorporation method) and a confirmatory mutation test (pre-incubation method) were carried out. Untreated, solvent controls and strain specific positive controls were included in the assays and were within the historical control range in all strains. All validity criteria were fulfilled. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 μg/plate. Based on the available data, the test item is not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Tests were performed according to Ishidate and Odashima (1977)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung fibroblast cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium supplemented with 10% calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

without

Test concentrations with justification for top dose:

Three different doses up to 0.25 mg/mL were tested

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: no justification provided

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa solution (1.5% at pH 6.8)

NUMBER OF REPLICATIONS: one for each exposure duration

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases were observed

DETERMINATION OF CYTOTOXICITY
- Method: was determined in a pre-test in which the dose leading to 50% cell-growth inhibition was determined (this dose was used as maximum dose in the test)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The results were considered to be negative if the incidence of chromosome aberrations weas less than 4.9%.

Statistics:

Not reported

Species / strain:

other: Chinese hamster lung fibroblast cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: the maximum dose was established in a pre-test and defined as the dose leading to 50% growth inhibition

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

The incidence of polyploid cells was 4% and the incidence of structural aberrations was 1% after 48 hours of exposure.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The substance (Methyl salicylate) was negative in an in vitro mammalian chromosomal aberration assay using Chinese hamster lung fibroblast cells in the absence of metabolic activation.

Executive summary:

The genotoxic potential of the test substance methyl salicylate (99% purity) was studied under non-GLP conditions in an in vitro mammalian chromosomal aberration assay using Chinese hamster lung fibroblast cells in accordance with the publication by Ishidate and Odashima 1977). The cells were obtained from a cell line established from the lung of a newborn female at the Cancer Research Institute at Tokyo and was maintained by 4 -day passage in Minimum Essential Medium supplemented with 10% calf serum. The modal chromosome number was 25 and the doubling time was approximately 15 hours. Cells were exposed to the substance for 24 or 48 hours in the absence of metabolic activation. The substance was dissolved in DMSO and tested at three doses, whereby the highest dose was defined as the dose leading to 50% inhibition of cell growth (as determined in a preliminary test). Chromosome preparations were made by adding colcemid (at a concentration of 0.2 µg/mL) to the medium 2 hours before cell harvest. Cells were then trypsinised and suspended in a hypotonic KCl solution (0.075 M) for 13 minutes at room temperature, centrifuged and fixed with acetic acid-methanol (1:3, v:v) and spread on glass slides. After air-drying the slides were stained with Giemsa solution (1.5% at pH 6.8) for 12 to 15 minutes. A hundred well-spread metaphases were observed under the microscope. The incidence of polyploid cells and cells with structural chromosomal aberrations was recorded. Untreated cells and solvent controls served as negative controls. All tested doses were negative and did not produce a significant increase in the number of chromosomal aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mouse micronucleus test (Micronucleus):

Under the conditions of this study, the test substance did not show any evidence of mutagenic potential when administered orally.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CF-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male mice were individually housed and females were housed 4/cage and given free access to rodent diet and drinking water except for 16 hours prior and 3 hours post dosing of the high-dose group and 4-6 prior to 3 hours post dosing of the other test groups. The animal room had a light photoperiod of 12 hours.

Route of administration:

oral: gavage

Vehicle:

Peanut Oil

Details on exposure:

8-week-old CFW-1 (Winkelmann) albino mice (7/sex/group) weighing 20-30 g were gavaged with 3000 mg test substance/kg body weight in peanut oil at a dose volume of 10 ml/kg body weight and euthanized at 24, 48, or 72 hours post dosing.

Duration of treatment / exposure:

dose volume of 10 ml/kg body weight and euthanized at 24, 48, or 72 hours post dosing.

Frequency of treatment:

Once

Post exposure period:

24, 48, or 72 hours

Remarks:

Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

7/sex/group

Positive control(s):

Positive control (20 mg/kg body weight of Endoxan)

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The smears were fixed in methanol, rinsed with distilled water 2x, stained in 10% Giemsa, rinsed with distilled water, and air-dried. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes.

Evaluation criteria:

The proportion of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes and the number of micronucleated normochromatic erythrocytes was recorded.

Statistics:

Statistical analyses were conducted using the tables of Kastenbaum and Bowman.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Dose: 300 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.

Dose: 1500 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.

Dose: 3000 mg/kg-Effects:no effects. Results:No mortalities; no statistically significant change in number of micronucleated polychromatic erythrocytes or micronucleated normochromatic erythrocytes at any of the 3 sampling times; no statistically significant change in the ratio of polychromatic to normochromatic erythrocytes compared to vehicle control.

Conclusions:

Under the conditions of this study, the test substance did not show any evidence of mutagenic potential when administered orally.

Executive summary:

Under the conditions of this study, the test substance did not show any evidence of mutagenic potential when administered orally.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Short description of key information:
Ames test: non-mutagenic
In vitro cytogenicity (chromosome aberration on Methyl salicylate): non-mutagenic with and without S9
In vitro gene mutation in mammalian cells: Data waived as reliable in vivo data on structural analogue is available.
In vivo Mouse micronucleus (Cyclo hexyl salicylate): did not show any evidence of mutagenic potential when administered orally.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

For classification and labelling purposes this indicates that the Source Substances are suitable structural analogues and based on the response seen for the above end points the Target substance is considered not to be mutagenic. Classification and labelling is therefore not required.