3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

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:

From July 23rd, 2018 to August 9th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

EC Number:

600-896-9

Cas Number:

109089-77-2

Molecular formula:

C17H18O3

IUPAC Name:

3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

Test material form:

solid: particulate/powder

Method

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

Species / strain 1

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

Remarks:

(TA 98 and TA 100: pKM 101)

Species / strain 2

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/plate test item 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.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO), acetone, NaCl 0.15M

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (1, 2 μg/plate; S. typhimurium strains, + S9), cis-Platinum (II) Diamine Dichloride (1 μg/plate; E.coli, - S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
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 1-9 E09 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 37ºC, 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 (1-9 E03 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 37ºC, 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 retined 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-11101-5-3919 validated on 28 06.2018 – expiry date: 07.02.2020). Preparation of S9-mix 10% (v/v): The final concentration of co-factors and salts is as follows: S9 fraction 10%; MgCl2-6H2O 8 mM; KCl 33 mM; Glucose-6-Phophate 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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

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:

TEST-SPECIFIC CONFOUNDING FACTORS : 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.

Any other information on results incl. tables

Table 3. Sterility control.

Serie	Doses	Colony number/plate
Control n° 1		1	2	3
Solution of   test item   LEMI code : 18/0187-010818-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
Control n° 2		1	2	3
Solution of   test item     LEMI code :  18/0187-060818-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 controls.

					Doses (/plate)
		0 (neg. Ctrl.)	DMSO	50 µg	150 µg	500 µg	1 500 µg	2 500 µg	5 000 µg
Control nº1	N1 N2 N3 N %	531 545 556 544 ± 13 -	564 532 543 546 ± 16 100%	557 568 573 566 ± 8 104%	560 579 544 561 ± 18 103%	529 556 545 543 ± 14 100%	571 578 531 560 ± 25 103%	526 567 532 542 ± 22 100%	502 434 455 464 ± 35 85%
Control Nº2	N1 N2 N3 N %	558 586 542 562 ± 22 -	578 551 564 564 ± 14 100%	585 580 565 577 ± 10 103%	584 571 553 569 ± 16 101%	583 565 526 558 ± 29 99%	555 530 501 529 ± 27 94%		464 488 483 478 ± 13 85%

Table 5. Result tables.

Table

TA 1535 – Assay n°1 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	10	12	9.33	3.06	_
Positive control solvent	5 µL	6	9	8	7.67	1.53	_
Positive control : Sodium azide	5 µg in 5 µL	697	710	757	721.33	31.56	94.09
Vehicle	50 µL	12	11	14	12.33	1.53	_
Test item  18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	7 9 10 8 10	10 9 12 17 10	11 8 13 7 7	9.33 8.67 11.67 10.67 9.00	2.08 0.58 1.53 5.51 1.73	0.76 0.70 0.95 0.86 0.73

  

TA 1535 – Assay n°1 (+10 % S9-mix; without pre-incubation)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	14	16	12.33	4.73	_
Positive control solvent	20 µL	15	20	14	16.33	3.21	_
Positive control: 2-Anthramine	2 µg in 20 µL	45	54	49	49.33	4.51	3.02
Vehicle	50 µL	17	16	11	14.67	3.21	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	14 14 20 16 18	16 18 16 17 19	15 19 13 14 15	15.00 17.00 16.33 15.67 17.33	1.00 2.65 3.51 1.53 2.08	1.02 1.16 1.11 1.07 1.18

 

TA 1535 – Assay n°2 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
n° 1	n° 2	n° 3
Negative control	100 µL	11	9	4	8.00	3.61	_
Positive control solvent	5 µL	8	5	12	8.33	3.51	_
Positive control : Sodium azide	5 µg in 5 µL	864	987	841	897.33	78.50	107.68
Vehicle	50 µL	12	6	10	9.33	3.06	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	9 5 11 7 8	5 8 10 14 7	9 7 7 12 12	7.67 6.67 9.33 11.00 9.00	2.31 1.53 2.08 3.61 2.65	0.82 0.71 1.00 1.18 0.96

  

TA 1535 – Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	6	10	9.33	3.06	_
Positive control solvent	10 µL	10	14	11	11.67	2.08	_
Positive control : 2-Anthramine	1 µg in 10 µL	50	62	58	56.67	6.11	4.86
Vehicle	50 µL	9	9	12	10.00	1.73	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	8 8 9 11 9	12 9 15 9 15	11 10 8 13 10	10.33 9.00 10.67 11.00 11.33	2.08 1.00 3.79 2.00 3.21	1.03 0.90 1.07 1.10 1.13

   

TA 1537 – Assay n°1 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	11	5	9	8.33	3.06	_
Positive control solvent	20 µL	9	6	7	7.33	1.53	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	891	568	748	735.67	161.85	100.32
Vehicle	50 µL	3	3	6	4.00	1.73	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	10 2 6 5 4	1 6 5 4 1	1 6 2 7 6	4.00 4.67 4.33 5.33 3.67	5.20 2.31 2.08 1.53 2.52	1.00 1.17 1.08 1.33 0.92

 

TA 1537 – Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	3	12	5	6.67	4.73	_
Positive control solvent	20 µL	4	5	3	4.00	1.00	_
Positive control : 2-Anthramine	2 µg in 20 µL	38	45	61	48.00	11.79	12.00
Vehicle	50 µL	4	3	13	6.67	5.51	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	1 3 3 6 4	7 3 3 7 5	11 12 4 4 5	6.33 6.00 3.33 5.67 4.67	5.03 5.20 0.58 1.53 0.58	0.95 0.90 0.50 0.85 0.70

 

TA 1537 – Assay n°2 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	8	5	6	6.33	1.53	_
Positive control solvent	20 µL	4	8	3	5.00	2.65	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	854	748	855	819.00	61.49	163.80
Vehicle	50 µL	7	10	9	8.67	1.53	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	4 11 9 5 6	5 4 4 8 5	4 5 6 8 6	4.33 6.67 6.33 7.00 5.67	0.58 3.79 2.52 1.73 0.58	0.50 0.77 0.73 0.81 0.65

  

TA 1537 – Assay n°2 – with metabolic activation (10% S9-mix) – with pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	11	3	8	7.33	4.04	_
Positive control solvent	10 µL	5	6	8	6.33	1.53	_
Positive control : 2-Anthramine	1 µg in 10 µL	20	36	48	34.67	14.05	5.47
Vehicle	50 µL	10	10	6	8.67	2.31	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	6 9 8 3 13	6 8 9 5 6	8 10 11 13 8	6.67 9.00 9.33 7.00 9.00	1.15 1.00 1.53 5.29 3.61	0.77 1.04 1.08 0.81 1.04

  

TA 98 –Assay n°1 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	9	11	8	9.33	1.53	_
Positive control solvent	20 µL	9	13	8	10.00	2.65	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	601	541	721	621.00	91.65	62.10
Vehicle	50 µL	10	12	6	9.33	3.06	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	6 9 9 9 13	8 6 7 11 8	9 9 8 7 9	7.67 8.00 8.00 9.00 10.00	1.53 1.73 1.00 2.00 2.65	0.82 0.86 0.86 0.96 1.07

   

TA 98 –Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	12	14	12.67	1.15	_
Positive control solvent	20 µL	17	11	13	13.67	3.06	_
Positive control : 2-Anthramine	2 µg in 20 µL	951	850	1057	952.67	103.51	69.71
Vehicle	50 µL	11	13	13	12.33	1.15	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	13 13 13 15 9	7 11 11 11 13	12 9 11 8 9	10.67 11.00 11.67 11.33 10.33	3.21 2.00 1.15 3.51 2.31	0.86 0.89 0.95 0.92 0.84

 

TA 98 –Assay n°2 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	11	12	15	12.67	2.08	_
Positive control solvent	20 µL	26	17	14	19.00	6.24	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	684	578	577	613.00	61.49	32.26
Vehicle	50 µL	17	12	13	14.00	2.65	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	9 12 15 14 24	15 19 10 13 19	15 21 18 12 14	13.00 17.33 14.33 13.00 19.00	3.46 4.73 4.04 1.00 5.00	0.93 1.24 1.02 0.93 1.36

  

TA 98 – Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	18	19	16	17.67	1.53	_
Positive control solvent	10 µL	16	20	14	16.67	3.06	_
Positive control : 2-Anthramine	1 µg in 10 µL	758	801	964	841.00	108.67	50.46
Vehicle	50 µL	14	21	10	15.00	5.57	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	19 23 19 30 16	21 34 20 36 12	19 30 15 22 23	19.67 29.00 18.00 29.33 17.00	1.15 5.57 2.65 7.02 5.57	1.31 1.93 1.20 1.96 1.13

  

TA 100 – Assay n°1 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	65	74	56	65.00	9.00	_
Positive control solvent	20 µL	73	58	67	66.00	7.55	_
Positive control : Sodium azide	20 µg in 20 µL	1078	949	921	982.67	83.74	14.89
Vehicle	50 µL	41	62	59	54.00	11.36	_
Test item 18/0187-010818-S1	5000 µg* 1500 µg 500 µg 150 µg 50 µg	41 59 60 47 48	47 58 72 73 74	46 75 64 54 68	44.67 64.00 65.33 58.00 63.33	3.21 9.54 6.11 13.45 13.61	0.83 1.19 1.21 1.07 1.17

   

TA 100 – Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	130	119	128	125.67	5.86	_
Positive control solvent	20 µL	131	124	115	123.33	8.02	_
Positive control : 2-Anthramine	2 µg in 20 µL	1655	1710	1627	1664.00	42.23	13.49
Vehicle	50 µL	143	132	126	133.67	8.62	_
Test item 18/0187-010818-S1	5000 µg* 1500 µg 500 µg 150 µg 50 µg	96 141 131 113 130	101 122 135 124 127	115 127 135 134 127	104.00 130.00 133.67 123.67 128.00	9.85 9.85 2.31 10.50 1.73	0.78 0.97 1.00 0.93 0.96

 

TA 100 – Assay n°2 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	80	83	71	78.00	6.24	_
Positive control solvent	20 µL	57	71	73	67.00	8.72	_
Positive control : Sodium azide	20 µg in 20 µL	1678	1291	1660	1543.00	218.42	23.03
Vehicle	50 µL	74	55	61	63.33	9.71	_
Test item 18/0187-010818-S1	5000 µg* 1500 µg 500 µg 150 µg 50 µg	42 76 81 80 81	58 64 68 70 69	73 60 70 56 73	57.67 66.67 73.00 68.67 74.33	15.50 8.33 7.00 12.06 6.11	0.91 1.05 1.15 1.08 1.17

     

TA 100 – Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	60	56	79	65.00	12.29	_
Positive control solvent	10 µL	71	68	64	67.67	3.51	_
Positive control : 2-Anthramine	1 µg in 10 µL	1205	1089	1011	1101.67	97.62	16.28
Vehicle	50 µL	86	81	61	76.00	13.23	_
Test item 18/0187-010818-S1   * Light thinning of the bacterial lawn	5000 µg* 1500 µg 500 µg 150 µg 50 µg	75 83 77 65 66	62 70 69 78 88	58 68 87 67 68	65.00 73.67 77.67 70.00 74.00	8.89 8.14 9.02 7.00 12.17	0.86 0.97 1.02 0.92 0.97

   

E. COLI – Assay n°1 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	84	74	81	79.67	5.13	_
Positive control solvent	10 µL	83	79	84	82.00	2.65	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	315	410	317	347.33	54.28	4.24
Vehicle	50 µL	91	88	74	84.33	9.07	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	89 81 92 84 88	76 85 83 90 89	75 86 87 81 92	80.00 84.00 87.33 85.00 89.67	7.81 2.65 4.51 4.58 2.08	0.95 1.00 1.04 1.01 1.06

 

E. COLI – Assay n°1– with metabolic activation (10 % S9-mix) – without pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	113	129	121	121.00	8.00	_
Positive control solvent	5 µL	126	131	128	128.33	2.52	_
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	524	621	489	544.67	68.38	4.24
Vehicle	50 µL	133	152	136	140.33	10.21	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	120 144 153 132 128	144 125 136 141 150	109 127 138 140 149	124.33 132.00 142.33 137.67 142.33	17.90 10.44 9.29 4.93 12.42	0.89 0.94 1.01 0.98 1.01

       

E. COLI Assay n°2 – without metabolic activation (-S9-mix)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	144	131	130	135.00	7.81	_
Positive control solvent	10 µL	148	118	135	133.67	15.04	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	291	395	354	346.67	52.39	2.59
Vehicle	50 µL	124	157	122	134.33	19.66	_
Test item 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	142 136 148 142 131	113 124 137 139 118	119 129 162 162 118	124.67 129.67 149.00 147.67 122.33	15.31 6.03 12.53 12.50 7.51	0.93 0.97 1.11 1.10 0.91

      

E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	211	221	201	211.00	10.00	_
Positive control solvent	5 µL	212	199	234	215.00	17.69	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	710	840	654	734.67	95.42	3.42
Vehicle	50 µL	216	183	209	202.67	17.39	_
Test item LEMI code : 18/0187-010818-S1	5000 µg 1500 µg 500 µg 150 µg 50 µg	218 225 213 231 203	236 220 198 237 218	205 212 229 228 220	219.67 219.00 213.33 232.00 213.67	15.57 6.56 15.50 4.58 9.29	1.08 1.08 1.05 1.14 1.05

* Light thinning of the bacterial lawn

Applicant's summary and conclusion

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 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 (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.