Dibenzoyl-L-tartaric acid monohydrate

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:

15/10/2018 - 31/10/2018

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:

2019

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:

Dibenzoyl-L-tartaric acid monohydrate

EC Number:

220-374-0

EC Name:

Dibenzoyl-L-tartaric acid monohydrate

Cas Number:

2743-38-6

Molecular formula:

C18H14O8

IUPAC Name:

(2R,3R)-2,3-bis(benzoyloxy)butanedioic 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 test item/plate since in the preliminary test the numbers of revertant colonies were mostly in the normal range, no citotoxicity 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) Diammine 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 1-9E09 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 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 (1-9E 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 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-11101-5-3805 validated on 7.2017 - expiry date: 25.05.2019). 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 : 18/0260-221018-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 :  18/0260-291018-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 %	508 549 504 520 ± 25 -	510 489 515 505 ± 14 97%	504 523 515 514 ± 10 99%	509 511 520 513 ± 6 99%	522 516 508 515 ± 7 99%	489 503 517 503 ± 14 97%	499 515 516 510 ± 10 98%	526 489 420 478 ± 54 92%
Control Nº2	N1 N2 N3 N %	495 464 486 482 ± 16 -	475 463 487 475 ± 12 99%	474 485 502 487 ± 14 101%	519 466 491 492 ± 27 102%	484 497 469 483 ± 14 100%	505 495 477 492 ± 14 102%		492 491 457 480 ± 20 100%

Table 5. Mutagenic activity tests: assay 1 (plate incorporation).

Assay 1 – TA 1535 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	21	16	22	19.67	3.21	_
Positive control solvent	5 µL	16	16	11	14.33	2.89	_
Positive control : Sodium azide	5 µg in 5 µL	728	903	750	793.67	95.32	55.37
Vehicle	50µL	19	16	9	14.67	5.13	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	17 10 21 12 17	9 16 19 14 19	12 8 17 20 15	12.67 11.33 19.00 15.33 17.00	4.04 4.16 2.00 4.16 2.00	0.86 0.77 1.30 1.05 1.16

Assay 1 – TA 1535 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	11	11	13.67	4.62	_
Positive control solvent	20 µL	13	13	15	13.67	1.15	_
Positive control : 2-Anthramine	2 µg in 20 µL	203	187	214	201.33	13.58	14.73
Vehicle	50µL	16	21	19	18.67	2.52	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	12 12 13 19 10	17 14 23 15 24	13 14 12 16 19	14.00 13.33 16.00 16.67 17.67	2.65 1.15 6.08 2.08 7.09	0.75 0.71 0.86 0.89 0.95

Assay 1 – TA 1537 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	10	5	5	6.67	2.89	_
Positive control solvent	20 µL	8	8	12	9.33	2.31	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	1817	1962	1901	1893.33	72.80	202.86
Vehicle	50µL	4	9	4	5.67	2.89	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	6 5 3 4 9	12 4 5 7 7	8 8 4 4 10	8.67 5.67 4.00 5.00 8.67	3.06 2.08 1.00 1.73 1.53	1.53 1.00 0.71 0.88 1.53

Assay 1 – TA 1537 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	10	11	6	9.00	2.65	_
Positive control solvent	20 µL	13	11	14	12.67	1.53	_
Positive control : 2-Anthramine	2 µg in 20 µL	48	61	48	52.33	7.51	4.13
Vehicle	50µL	13	5	9	9.00	4.00	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	15 13 14 15 8	6 10 12 12 14	13 11 10 8 12	11.33 11.33 12.00 11.67 11.33	4.73 1.53 2.00 3.51 3.06	1.26 1.26 1.33 1.30 1.26

Assay 1 – TA 98 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	9	12	13.33	5.13	_
Positive control solvent	20 µL	14	9	12	11.67	2.52	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	583	573	551	569.00	16.37	48.77
Vehicle	50µL	13	12	18	14.33	3.21	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	16 23 11 11 16	19 10 14 18 8	7 11 20 9 11	14.00 14.67 15.00 12.67 11.67	6.24 7.23 4.58 4.73 4.04	0.98 1.02 1.05 0.88 0.81

Assay 1 – TA 98 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	29	20	20.33	8.50	_
Positive control solvent	20 µL	22	18	12	17.33	5.03	_
Positive control : 2-Anthramine	2 µg in 20 µL	569	576	489	544.67	48.34	31.42
Vehicle	50µL	14	17	23	18.00	4.58	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	16 31 21 21 21	20 12 28 25 23	15 22 22 25 27	17.00 21.67 23.67 23.67 23.67	2.65 9.50 3.79 2.31 3.06	0.94 1.20 1.31 1.31 1.31

Assay 1 – TA 100 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	93	71	72	78.67	12.42	_
Positive control solvent	20 µL	73	82	69	74.67	6.66	_
Positive control : Sodium azide	20 µg in 20 µL	1563	1498	1561	1540.67	36.96	20.63
Vehicle	50µL	70	73	65	69.33	4.04	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	83 86 78 64 75	79 79 84 86 70	64 70 70 72 88	75.33 78.33 77.33 74.00 77.67	10.02 8.02 7.02 11.14 9.29	1.09 1.13 1.12 1.07 1.12

Assay 1 – TA 100 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	80	79	104	87.67	14.15	_
Positive control solvent	20 µL	108	86	94	96.00	11.14	_
Positive control : 2-Anthramine	2 µg in 20 µL	1068	1153	854	1025.00	154.07	10.68
Vehicle	50µL	90	112	84	95.33	14.74	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	84 88 86 103 98	95 91 110 102 105	79 92 107 118 100	86.00 90.33 101.00 107.67 101.00	8.19 2.08 13.08 8.96 3.61	0.90 0.95 1.06 1.13 1.06

Assay 1 – E. coli (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	116	129	117	120.67	7.23	_
Positive control solvent	10 µL	112	129	102	114.33	13.65	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	629	722	811	720.67	91.01	6.30
Vehicle	50µL	126	116	107	116.33	9.50	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	104 107 105 108 111	108 116 119 113 108	120 124 115 126 107	110.67 115.67 113.00 115.67 108.67	8.33 8.50 7.21 9.29 2.08	0.95 0.99 0.97 0.99 0.93

Assay 1 – E. coli (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	180	181	188	183.00	4.36	_
Positive control solvent	5 µL	178	187	195	186.67	8.50	_
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	1204	1008	1220	1144.00	118.05	6.13
Vehicle	50µL	180	171	219	190.00	25.51	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	184 179 177 207 190	170 182 174 193 181	183 198 179 184 183	179.00 186.33 176.67 194.67 184.67	7.81 10.21 2.52 11.59 4.73	0.94 0.98 0.93 1.02 0.97

Table 6. Mutagenic activity tests: assay 2 (pre-incubation).

Assay 2 – TA 1535 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	15	12	9	12.00	3.00	_
Positive control solvent	5 µL	14	11	10	11.67	2.08	_
Positive control : Sodium azide	5 µg in 5 µL	889	962	848	899.67	57.74	77.11
Vehicle	50µL	11	9	7	9.00	2.00	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	9 7 11 10 7	8 9 11 13 12	12 16 7 14 12	9.67 10.67 9.67 12.33 10.33	2.08 4.73 2.31 2.08 2.89	1.07 1.19 1.07 1.37 1.15

Assay 2 – TA 1535 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	10	13	12	11.67	1.53	_
Positive control solvent	10 µL	13	13	9	11.67	2.31	_
Positive control : 2-Anthramine	1 µg in 10 µL	151	164	152	155.67	7.23	13.34
Vehicle	50µL	11	7	10	9.33	2.08	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	9 16 16 11 17	12 15 13 13 10	9 24 14 16 8	10.00 18.33 14.33 13.33 11.67	1.73 4.93 1.53 2.52 4.73	1.07 1.96 1.54 1.43 1.25

Assay 2 – TA 1537 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	3	4	6	4.33	1.53	_
Positive control solvent	20 µL	5	7	7	6.33	1.15	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	1564	1756	1361	1560.33	197.53	246.37
Vehicle	50µL	1	5	7	4.33	3.06	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	4 7 4 2 4	4 8 3 1 6	7 4 6 5 3	5.00 6.33 4.33 2.67 4.33	1.73 2.08 1.53 2.08 1.53	1.15 1.46 1.00 0.62 1.00

Assay 2 – TA 1537 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	15	1	7.33	7.09	_
Positive control solvent	10 µL	4	7	5	5.33	1.53	_
Positive control : 2-Anthramine	1 µg in 10 µL	51	61	47	53.00	7.21	9.94
Vehicle	50µL	7	4	7	6.00	1.73	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	7 7 6 8 4	4 9 5 5 10	6 4 1 5 4	5.67 6.67 4.00 6.00 6.00	1.53 2.52 2.65 1.73 3.46	0.94 1.11 0.67 1.00 1.00

Assay 2 – TA 98 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	14	21	23	19.33	4.73	_
Positive control solvent	20 µL	21	20	17	19.33	2.08	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	441	508	621	523.33	90.97	27.07
Vehicle	50µL	12	24	17	17.67	6.03	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	22 22 26 21 20	21 16 22 23 27	18 21 20 14 19	20.33 19.67 22.67 19.33 22.00	2.08 3.21 3.06 4.73 4.36	1.15 1.11 1.28 1.09 1.25

Assay 2 – TA 98 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	34	32	32	32.67	1.15	_
Positive control solvent	10 µL	32	30	35	32.33	2.52	_
Positive control : 2-Anthramine	1 µg in 10 µL	328	427	500	418.33	86.33	12.94
Vehicle	50µL	32	38	34	34.67	3.06	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	26 27 31 40 25	33 26 38 27 28	31 29 30 34 37	30.00 27.33 33.00 33.67 30.00	3.61 1.53 4.36 6.51 6.24	0.87 0.79 0.95 0.97 0.87

Assay 2 – TA 100 (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	66	57	65	62.67	4.93	_
Positive control solvent	20 µL	63	60	61	61.33	1.53	_
Positive control : Sodium azide	20 µg in 20 µL	1452	1388	1411	1417.00	32.42	23.10
Vehicle	50µL	60	65	58	61.00	3.61	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	58 61 58 60 63	67 62 61 62 60	69 64 66 67 71	64.67 62.33 61.67 63.00 64.67	5.86 1.53 4.04 3.61 5.69	1.06 1.02 1.01 1.03 1.06

Assay 2 – TA 100 (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	102	95	95	97.33	4.04	_
Positive control solvent	10 µL	87	92	83	87.33	4.51	_
Positive control : 2-Anthramine	1 µg in 10 µL	371	522	419	437.33	77.15	5.01
Vehicle	50µL	71	79	80	76.67	4.93	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	89 96 101 87 91	93 92 82 92 85	90 85 95 81 94	90.67 91.00 92.67 86.67 90.00	2.08 5.57 9.71 5.51 4.58	1.18 1.19 1.21 1.13 1.17

Assay 2 – E. coli (+S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	76	82	83	80.33	3.79	_
Positive control solvent	10 µL	81	85	89	85.00	4.00	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	817	583	756	718.67	121.39	8.45
Vehicle	50µL	67	90	75	77.33	11.68	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	88 87 83 74 76	86 76 79 80 89	77 75 81 87 79	83.67 79.33 81.00 80.33 81.33	5.86 6.66 2.00 6.51 6.81	1.08 1.03 1.05 1.04 1.05

Assay 2 – E. coli (-S9)	Dose/Plate		Plate		Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	140	139	129	136.00	6.08	_
Positive control solvent	5 µL	134	118	135	129.00	9.54	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	1045	992	1156	1064.33	83.69	8.25
Vehicle	50µL	119	108	104	110.33	7.77	_
Test item	5000 µg 1500 µg 500 µg 150 µg 50 µg	122 123 137 109 112	137 115 105 117 124	112 113 110 120 94	123.67 117.00 117.33 115.33 110.00	12.58 5.29 17.21 5.69 15.10	1.12 1.06 1.06 1.05 1.00

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 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.