[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2020

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-11-2020 to 18-12-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

histidine and tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased from recognized supplier (dates within full study report); Post-mitochondrial fraction (S9) Lot No 4269.
- method of preparation of S9 mix: Documented in the full study report. Stored at -80ºC.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including activity is presented in the full study report.

Test concentrations with justification for top dose:

Test 1 (plate incorporation method, +S9 and -S9): 0, 1.6, 5, 16, 50, 160, 500, 1600, 5000 μg/plate.
Test 2 (pre-incubation method, +S9 and -S9): 0, 1.6, 5, 16, 50, 160, 500, 1600, 5000 μg/plate.
Test 3 (pre-incubation method):
Test 3 (+S9): 0, 1.6, 5, 16, 50, 160, 500, 1600, 5000 μg/plate
Test 3 (-S9): 0, 0.05, 0.16, 0.5, 1.6, 5, 16, 50, 160, 500, 1600, 5000 μg/plate
Doses were selected to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on cytotoxicity observed the results of Test 1 and Test 2.

Vehicle / solvent:

- Vehicle/solvent used: dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: A preliminary solubility assessment was conducted. As the test item was found to be soluble in the preferred solvent, DMSO, at 100 mg per mL, solubility was not assessed with any other solvents.
- Justification for percentage of solvent in the final culture medium: Cytotoxicity not occuring in the solvent controls.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: potassium dichromate

Remarks:

Without metabolic activation S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Test 1. in medium; in agar (plate incorporation); Test 2, Test 3. in medium; in agar (pre-incubation).
The choice of application was due to standard OECD Test Guideline 471 protocol. Pre-incubation test was conducted after the negative result from the first test (plate incorporation).

DURATION:
Exposure duration:
Test 1. Frozen aliquots of the bacterial strains (-70°C or lower) were incubated overnight (8 to 10 hours) at 37°C in an orbital incubator (120 rpm) in nutrient broth. For strains TA98, TA100 and uvrA/pKM101, the nutrient broth was supplemented with ampicillin at 25 μg/mL.
Aliquots (100 μL) of bacterial culture were mixed with 100 μL of solvent, positive control or test formulation dilution and 500 μL of sodium phosphate buffer or S9 mix. Finally, 2 mL of molten 0.6% agar maintained at approximately 50°C and supplemented with low concentrations of biotin and histidine (each at 0.05 mmol/L) for the S. typhimurium strains, and tryptophan (0.018 mmol/L) for the E. coli strain, were added. The mixture was poured immediately onto minimal glucose agar plates and incubated for 3 days at 37°C. There were three plates in all solvent control, test item and positive control groups. After incubation at 37ºC for 3 days, all plates were examined both macroscopically and microscopically for evidence of cytotoxicity, precipitates and any other effects relevant to the interpretation of the test.
Test 2, Test 3. The liquid pre-incubation experiment was performed as above with the following differences:
Aliquots (100 μL) of bacterial culture were mixed with 50 μL of solvent, positive control or test item formulation dilution and 500 μL of sodium phosphate buffer or S9 mix and then incubated for 60 minutes at 37°C in an orbital incubator at 120 rpm.
After incubation, 2 mL of molten 0.6% agar containing biotin and histidine/tryptophan (as above) was added to the mixture and poured onto minimal glucose agar plates and incubated for a further 3 days at 37°C. There were three plates in all solvent control, test item and positive control groups. After incubation at 37ºC for 3 days, all plates were examined both macroscopically and microscopically for evidence of cytotoxicity, precipitates and any other effects relevant to the interpretation of the test.

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: relative total growth

Rationale for test conditions:

In accordance with OECD TG 471 (adpoted 1997, corrected 2020) guidelines.

Evaluation criteria:

The test item is considered mutagenic using the following criteria:
1. A concentration-related increase in the number of revertant colonies per plate in at least one strain with or without S9
2. Reproducible increase at one or more test item concentrations in the number of revertant colonies per plate in at least one strain with or without S9
3. At least one test item concentration with an increase outside the historical control database of the laboratory
4. Expert judgment
5. If criteria not met, the test item is non-mutagenic

Statistics:

Mean +/- Standard Deviation (SD)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: See Tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test 1 (+S9) Plate incorporation

Dose per plate		Number of revertant colonies  per plate
		S. typhimurium  LT2								E. coli  WP2
mg	mmol	TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		14	12	11	12	30	34	150	147	206	195
		12	2.0	11	1.7	35	3.2	153	7.9	199	14.0
		10		14		36		138		179
1.6	0.00768	11	11	12	11	32	35	157	156	189	189
		10	1.0	11	0.6	30	6.4	159	3.1	193	3.5
		12	0.9	11	0.9	42	1.0	153	1.1	186	1.0
5	0.024	11	12	11	11	25	28	139	147	216	205
		11	2.3	12	1.0	27	3.1	147	8.0	194	11.0
		15	1.0	10	0.9	31	0.8	155	1.0	205	1.1
16	0.0768	12	11	10	12	34	34	150	157	190	189
		10	1.2	11	2.1	30	3.5	157	7.0	191	2.6
		10	0.9	14	1.0	37	1.0	164	1.1	186	1.0
50	0.24	13	12	13	11	30	34	154	160	224	213
		14	3.2	10	1.5	37	3.6	159	6.6	212	10.1
		8	1.0	11	0.9	35	1.0	167	1.1	204	1.1
160	0.768	12	11	10	11	29	31	152	138	198	196
		11	1.0	10	1.2	34	2.5	133	11.9	201	6.8
		10	0.9	12	0.9	31	0.9	130	0.9	188	1.0
500	2.4	11	12	11	11	27	30	133	147	162	168
		13	1.2	10	1.0	32	2.9	145	15.6	181	11.6
		13	1.0	12	0.9	32	0.9	164	1.0	160	0.9
1600	7.68	10 r	8	12 r	10	22 r	26	86 r	87	157	144
		7 r	1.5	9 r	1.5	30 r	4.0	87 r	1.0	152	18.4
		8 r	0.7	10 r	0.8	27 r	0.8	88 r	0.6	123	0.7
5000	24	3 vp	3	5 vp	8	27  vp	23	73  vp	76	133  rp	97
		4 vp	1.5	8 vp	3.0	21  vp	3.8	86  vp	9.3	71 rp	32.3
		1 vp	0.3	11  vp	0.7	20  vp	0.7	68  vp	0.5	86 rp	0.5
Positive  control		133	146	153	167	1215	1402	1825	1927	1935	2053
		138	18.9	169	13.6	1429	175.5	2043	109.6	2125	103.2
		168	12.2	180	13.9	1563	41.2	1914	13.1	2100	10.5

 

Test 1 (-S9) Plate incorporation

Dose per plate		Number of revertant colonies  per plate
		S. typhimurium  LT2								E. coli  WP2
mg	mmol	TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		13	14	11	12	27	29	119	121	175	171
		14	1.5	11	1.7	31	2.1	124	2.5	163	6.7
		16		14		30		121		174
1.6	0.00768	15	13	10	10	30	32	122	126	183	176
		12	2.1	9	1.0	31	2.1	127	3.2	175	6.1
		11	0.9	11	0.8	34	1.1	128	1.0	171	1.0
5	0.024	13	14	10	11	27	24	124	116	193	177
		13	1.7	12	1.0	25	3.6	109	7.6	168	14.2
		16	1.0	11	0.9	20	0.8	114	1.0	169	1.0
16	0.0768	13	12	11	12	32	32	119	120	160	157
		12	1.5	12	1.5	31	0.6	117	3.6	158	4.2
		10	0.9	14	1.0	32	1.1	124	1.0	152	0.9
50	0.24	11	12	10	9	29	27	118	119	165	171
		13	1.0	8	1.2	29	4.0	118	2.3	168	7.4
		12	0.9	8	0.8	22	0.9	122	1.0	179	1.0
160	0.768	13	13	10	10	24	26	119	114	154	149
		12	0.6	10	0.0	26	2.5	118	8.4	137	10.1
		13	0.9	10	0.8	29	0.9	104	0.9	155	0.9
500	2.4	12	11	10	10	25	28	114	110	171	171
		11	1.5	8	2.0	34	5.2	118	10.6	165	5.5
		9	0.8	12	0.8	25	1.0	98	0.9	176	1.0
1600	7.68	10 r	9	7 r	6	29 r	26	91 r	99	140	140
		8 r	1.0	8 r	2.1	27 r	3.6	98 r	8.5	148	8.0
		9 r	0.6	4 r	0.5	22 r	0.9	108 r	0.8	132	0.8
5000	24	7 vp	8	7 vp	7	21  vp	20	103  vp	100	117  rp	120
		7 vp	1.2	5 vp	1.5	20  vp	1.0	98  vp	2.6	128  rp	6.7
		9 vp	0.6	8 vp	0.6	19  vp	0.7	99  vp	0.8	116  rp	0.7
Positive  control		476	435	158	185	307	345	613	579	995	976
		394	41.0	260	65.8	319	55.2	568	30.4	954	20.6
		436	31.1	137	15.4	408	11.9	555	4.8	978	5.7

 

Test 2 (+S9) Pre-incubation

Dose per plate		Number of revertant colonies  per plate
		S. typhimurium  LT2								E. coli  WP2
mg	mmol	TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		11	13	9	11	40	33	145	137	216	211
		12	2.1	11	1.5	29	5.9	139	8.6	201	8.4
		15		12		31		128		215
1.6	0.00768	15	13	10	11	37	32	126	136	207	212
		12	1.5	11	0.6	36	7.2	134	11.1	215	4.2
		13	1.0	11	1.0	24	1.0	148	1.0	213	1.0
5	0.024	13	12	10	11	32	34	132	135	211	197
		12	1.5	12	1.2	34	1.5	128	8.9	192	12.7
		10	0.9	10	1.0	35	1.0	145	1.0	187	0.9
16	0.0768	12	9	13	12	31	35	126	127	212	214
		7	2.5	11	1.2	38	3.8	121	7.1	205	10.1
		9	0.7	11	1.1	37	1.1	135	0.9	225	1.0
50	0.24	14	11	12	9	29	30	104	115	205	193
		9	2.9	8	2.3	25	5.6	143	24.1	188	10.8
		9	0.8	8	0.8	36	0.9	99	0.8	185	0.9
160	0.768	10	11	11	10	16	20	114	126	195	188
		9	3.2	6	3.2	30	8.4	127	11.5	177	9.9
		15	0.8	12	0.9	15	0.6	137	0.9	193	0.9
500	2.4	6 rp	8	6 rp	6	27 rp	21	111  rp	111	161  p	172
		9 rp	1.5	7 rp	1.5	15 rp	6.0	109  rp	1.5	179  p	9.9
		8 rp	0.6	4 rp	0.5	20 rp	0.6	112  rp	0.8	177  p	0.8
1600	7.68	4 vp	2	7 vp	6	22  vp	20	101  vp	75	149  sp	155
		1 vp	1.7	6 vp	1.5	26  vp	7.2	57  vp	23.1	154  sp	6.6
		1 vp	0.2	4 vp	0.5	12  vp	0.6	67  vp	0.5	162  sp	0.7
5000	24	2 vp	2	6 vp	5	19  vp	16	75  vp	73	147  rp	147
		2 vp	0.6	6 vp	1.2	15  vp	2.3	71  vp	2.0	153  rp	5.5
		1 vp	0.2	4 vp	0.5	15  vp	0.5	73  vp	0.5	142  rp	0.7
Positive  control		147	147	137	144	1338	1403	0 k	0	2001	1933
		150	3.0	156	10.2	1509	92.8	0 k	0.0	1948	76.6
		144	11.3	140	13.1	1361	42.5	0 k	0.0	1850	9.2

 

Test 2 (-S9) Pre-incubation

Dose per plate		Number of revertant colonies  per plate
		S. typhimurium  LT2								E. coli  WP2
mg           mmol		TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		13	14	8	9	32	28	121	121	153	152
		14	0.6	10	1.0	27	3.2	118	3.0	147	4.2
		14		9		26		124		155
1.6	0.00768	12	14	11	9	17	14	123	122	159	148
		14	1.5	8	1.5	10	3.6	117	4.6	142	9.3
		15	1.0	9	1.0	15	0.5	126	1.0	144	1.0
5	0.024	13	15	11	9	16 s	13	121	123	148	150
		16	1.5	7	2.1	11 s	2.5	130	6.2	147	3.8
		15	1.1	10	1.0	13 s	0.5	118	1.0	154	1.0
16	0.0768	8 v	9	3 v	5	10 s	13	119 s	108	149	153
		7 v	2.6	5 v	2.5	12 s	3.1	113 s	14.7	156	3.6
		12 v	0.6	8 v	0.6	16 s	0.5	91 s	0.9	154	1.0
50	0.24	0 f	0	0 f	0	10 v	11	102  v	103	150	150
		0 f	0.0	0 f	0.0	11 v	1.5	112  v	8.1	157	7.5
		0 f	0.0	0 f	0.0	13 v	0.4	96 v	0.9	142	1.0
160	0.768	0 f	0	0 f	0	8 v	8	108  v	80	154	145
		0 f	0.0	0 f	0.0	7 v	1.5	68 v	24.0	134	10.1
		0 f	0.0	0 f	0.0	10 v	0.3	65 v	0.7	146	1.0
500	2.4	0 fp	0	0  np	0	8 vp	9	41  vp	46	134  p	138
		0 fp	0.0	0  np	0.0	8 vp	2.3	48  vp	4.7	137  p	4.0
		0 fp	0.0	0  np	0.0	12  vp	0.3	50  vp	0.4	142  p	0.9
1600	7.68	0 fp	0	0  np	0	11  vp	10	35  vp	36	123  sp	126
		0 fp 0 fp	0.0 0.0	0  np 0  np	0.0 0.0	7 vp 12  vp	2.6 0.4	30  vp 43  vp	6.6 0.3	127  sp 129  sp	3.1 0.8
5000	24	0 fp	0	0  np	0	4 vp	6	34  vp	29	121  rp	118
		0 fp 0 fp	0.0 0.0	0  np 0  np	0.0 0.0	8 vp 6 vp	2.0 0.2	25  vp 29  vp	4.5 0.2	113  rp 119  rp	4.2 0.8
Positive  control		431	449	195	200	399	407	0 k	0	969	904
		440	24.4	211	9.9	406	8.0	0 k	0.0	926	77.8
		477	32.1	193	22.2	415	14.5	0 k	0.0	818	5.9

 

Test 3 (+S9) Pre-incubation

Dose per plate	Number of revertant colonies per plate
	S. typhimurium LT2
mg           mmol	TA100
Solvent control	154	156
	158                2.1 157
1.6         0.00768	148	146
	140                5.3 150                0.9
5             0.024	153	148
	140                6.8 150                0.9
16           0.0768	137	137
	139                2.0 135                0.9
50             0.24	159	154
	156                6.2 147                1.0
160           0.768	139	139
	127              11.5 150                0.9
500             2.4	111 rp	116
	107 rp         11.7 129 rp          0.7
1600           7.68	88 vp	85
	83 vp             2.6 84 vp             0.5
5000             24	84 vp	78
	78 vp             5.5 73 vp             0.5
Positive  control	1393	1445
	1468              45.1 1474                9.3

 

Test 3 (-S9) Pre-incubation

Dose per plate		Number of revertant colonies per plate
		S. typhimurium  LT2								E. coli WP2
mg	mmol	TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		13	13	9	10	30	28	117	118	150	148
		12	1.5	10	1.0	27	1.7	117	1.2	142	5.3
		15		11		27		119		152
0.05	0.00024	12	12	11	11	29	29	126	128	137	137
		13	0.6	11	0.6	26	2.5	121	7.6	135	1.5
		12	0.9	10	1.1	31	1.0	136	1.1	138	0.9
0.16	0.00077	11	14	11	9	30	29	119	118	138	147
		13	3.1	9	1.5	29	0.6	117	1.0	158	10.1
		17	1.1	8	0.9	29	1.0	118	1.0	145	1.0
0.5	0.0024	12	13	11	9	29	29	116	120	142	139
		13	1.0	9	1.5	30	0.6	118	5.3	138	2.3
		14	1.0	8	0.9	29	1.0	126	1.0	138	0.9
1.6	0.00768	12	11	10	10	30	29	116	122	149	145
		9	1.7	11	0.6	25	3.6	123	5.6	148	5.5
		12	0.8	10	1.0	32	1.0	127	1.0	139	1.0
5	0.024	14	12	9	9	27	28	120	117	150	143
		10	2.1	10	1.0	25	3.1	113	3.5	140	5.8
		13	0.9	8	0.9	31	1.0	117	1.0	140	1.0
16	0.0768	6 r	7	7 r	6	25 r	27	116 r	112	140	137
		10 r	2.3	8 r	2.6	25 r	3.5	109 r	3.6	149	13.7
		6 r	0.5	3 r	0.6	31 r	1.0	111 r	0.9	122	0.9
50	0.24	9 v	8	5 v	5	22 r	17	106 r	106	155	146
		8 v	0.6	3 v	2.5	15 r	4.4	111 r	5.0	140	7.9
		8 v	0.6	8 v	0.5	14 r	0.6	101 r	0.9	143	1.0
160	0.768	0 f	0	0 f	0	19 v	20	87 v	95	154	150
		0 f	0.0	0 f	0.0	20 v	1.0	101 v	7.2	149	3.6
		0 f	0.0	0 f	0.0	21 v	0.7	97 v	0.8	147	1.0
500	2.4	0 f	0	0 n	0	16 v	16	72 v	68	142	134
		0 f	0.0	0 n	0.0	15 v	0.6	63 v	4.6	139	11.4
		0 f	0.0	0 n	0.0	16 v	0.6	69 v	0.6	121	0.9
1600	7.68	0 fp	0	0 np	0	24 vp	20	66 vp	63	124 p	125
		0 fp	0.0	0 np	0.0	12 vp	6.9	62 vp	3.1	118 p	7.0
		0 fp	0.0	0 np	0.0	24 vp	0.7	60 vp	0.5	132 p	0.8
5000	24	0 fp	0	0 np	0	16 vp	17	37 vp	50	112 rp	108
		0 fp	0.0	0 np	0.0	16 vp	2.3	60 vp	11.8	112 rp	7.5
		0 fp	0.0	0 np	0.0	20 vp	0.6	53 vp	0.4	99 rp	0.7
Positive control		406	387	149	168	1793	1775	576	574	1002	968
		358	25.3	134	47.1	1763	15.9	570	3.5	995	53.5
		396	29.8	222	16.8	1769	63.4	576	4.9	906	6.5

 

 

Dose per plate		Number of revertant colonies  per plate
		S. typhimurium  LT2								E. coli  WP2
mg	mmol	TA1535		TA1537		TA98		TA100		uvrA /pKM101
Solvent control		14	12	11	12	30	34	150	147	206	195
		12	2.0	11	1.7	35	3.2	153	7.9	199	14.0
		10		14		36		138		179
1.6	0.00768	11	11	12	11	32	35	157	156	189	189
		10	1.0	11	0.6	30	6.4	159	3.1	193	3.5
		12	0.9	11	0.9	42	1.0	153	1.1	186	1.0
5	0.024	11	12	11	11	25	28	139	147	216	205
		11	2.3	12	1.0	27	3.1	147	8.0	194	11.0
		15	1.0	10	0.9	31	0.8	155	1.0	205	1.1
16	0.0768	12	11	10	12	34	34	150	157	190	189
		10	1.2	11	2.1	30	3.5	157	7.0	191	2.6
		10	0.9	14	1.0	37	1.0	164	1.1	186	1.0
50	0.24	13	12	13	11	30	34	154	160	224	213
		14	3.2	10	1.5	37	3.6	159	6.6	212	10.1
		8	1.0	11	0.9	35	1.0	167	1.1	204	1.1
160	0.768	12	11	10	11	29	31	152	138	198	196
		11	1.0	10	1.2	34	2.5	133	11.9	201	6.8
		10	0.9	12	0.9	31	0.9	130	0.9	188	1.0
500	2.4	11	12	11	11	27	30	133	147	162	168
		13	1.2	10	1.0	32	2.9	145	15.6	181	11.6
		13	1.0	12	0.9	32	0.9	164	1.0	160	0.9
1600	7.68	10 r	8	12 r	10	22 r	26	86 r	87	157	144
		7 r	1.5	9 r	1.5	30 r	4.0	87 r	1.0	152	18.4
		8 r	0.7	10 r	0.8	27 r	0.8	88 r	0.6	123	0.7
5000	24	3 vp	3	5 vp	8	27  vp	23	73  vp	76	133  rp	97
		4 vp	1.5	8 vp	3.0	21  vp	3.8	86  vp	9.3	71 rp	32.3
		1 vp	0.3	11  vp	0.7	20  vp	0.7	68  vp	0.5	86 rp	0.5
Positive  control		133	146	153	167	1215	1402	1825	1927	1935	2053
		138	18.9	169	13.6	1429	175.5	2043	109.6	2125	103.2
		168	12.2	180	13.9	1563	41.2	1914	13.1	2100	10.5

Conclusions:

Interpretation of results: negative.
Under the conditions of this study, the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

Test item was tested for mutagenic activity using genetically modified Salmonella typhimurium LT2 bacteria of strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 as indicator organisms, according to the methods of Maron and Ames, 1983, Venitt et al, 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000, and in compliance with OECD Test Guideline 471 (2020).

A preliminary solubility assessment was conducted. As solubility was satisfactory in dimethyl sulphoxide, it was used throughout this study as the solvent for the test item.

Test 1

A mutagenicity test was conducted for test item using the plate incorporation method (Test 1) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The dose range used was 1.6 to 5000 μg (0.00768 to 24 μmol) per plate.

Test 2

As the result from Test 1 was clearly negative, a confirmatory test was carried out using the liquid pre-incubation method (Test 2) with a dose range of 1.6 to 5000 μg (0.00768 to 24 μmol) per plate in the presence and absence of S9 mix.

Test 3

Test 3 consisted of a repeat mutagenicity test using the liquid pre-incubation method for S. typhimurium strain TA100 in the presence of S9 mix with the same dose range as Test 2, i.e. 1.6 to 5000 μg (0.00768 to 24 μmol) per plate and a retest using the liquid pre-incubation method for all five strains in the absence of S9 mix with a modified dose range of 0.05 to 5000 μg (0.00024 to 24 μmol) per plate .

The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 5 μg (0.024 μmol) per plate. The maximum dose level scored for revertant colonies was 5000 μg (24 μmol) per plate. The minimum dose at which precipitate was seen was on the test plates was 500 μg (2.4 μmol) per plate.

No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

It was concluded that the test item was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key study: Test item was tested for mutagenic activity using genetically modified Salmonella typhimurium LT2 bacteria of strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 as indicator organisms, according to the methods of Maron and Ames, 1983, Venitt et al, 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000, and in compliance with OECD Test Guideline 471 (2020).

A preliminary solubility assessment was conducted. As solubility was satisfactory in dimethyl sulphoxide, it was used throughout this study as the solvent for the test item.

Test 1

A mutagenicity test was conducted for test item using the plate incorporation method (Test 1) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The dose range used was 1.6 to 5000 μg (0.00768 to 24 μmol) per plate.

Test 2

As the result from Test 1 was clearly negative, a confirmatory test was carried out using the liquid pre-incubation method (Test 2) with a dose range of 1.6 to 5000 μg (0.00768 to 24 μmol) per plate in the presence and absence of S9 mix.

Test 3

Test 3 consisted of a repeat mutagenicity test using the liquid pre-incubation method for S. typhimurium strain TA100 in the presence of S9 mix with the same dose range as Test 2, i.e. 1.6 to 5000 μg (0.00768 to 24 μmol) per plate and a retest using the liquid pre-incubation method for all five strains in the absence of S9 mix with a modified dose range of 0.05 to 5000 μg (0.00024 to 24 μmol) per plate .

The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 5 μg (0.024 μmol) per plate. The maximum dose level scored for revertant colonies was 5000 μg (24 μmol) per plate. The minimum dose at which precipitate was seen was on the test plates was 500 μg (2.4 μmol) per plate.

No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

It was concluded that the test item was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity.