[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

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:

02 - 23 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2:
1, 3, 10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation for TA 98 and TA 100
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 1535, TA 1537 and WP2 uvr A

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviations were calculated.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9: starting at 2500 µg/plate; +S9: starting at 1000 µg/plate Experiment II: -S9: -; +S9: at 5000 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9 and +S9: starting at 2500 µg/plate Experiment II: -S9: starting at 1000 µg/plate; +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9 and +S9: starting at 333 µg/plate Experiment II: -S9 and +S9: at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9 and +S9: starting at 333 µg/plate Experiment II: -S9: at 33µg/plate; +S9: at 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9: - ;+S9: starting at 2500 µg/plate Experiment II: -S9 and +S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 up to 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.


RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): Please refer to table 1 in "Any other information on results incl. tables".

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test substance showed reduced background growth in all tester strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all tester strains used.

 Table 1: Historical control data

Strain			without S9 mix			with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
TA1537	Solvent control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent control	25	4.4	13	43	34	6.2	15	58
	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent control	156	26	78	209	148	32.3	73	208
	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
WP2uvrA	Solvent control	41	5.6	27	63	50	6.8	28	72
	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Mean = mean value of revertants/plate

SD = standard deviation

Min = minimal value/Max = maximal value                             

Data from November 2014 until November 2016 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 350 experiments).

Table 2: Test results of Experiment I (plate incorporation).

	Number of revertant colonies per plate
S9-Mix	Without
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent control (DMSO)	18 ± 3	13 ± 1	25 ± 4	134 ± 12	36 ± 8
Negative control (untreated)	15 ± 1	11 ± 6	26 ± 8	162 ± 15	38 ± 11
3	16 ± 5	9 ± 5	26 ± 7	125 ± 10	38 ± 4
10	17 ± 3	12 ± 2	24 ± 8	104 ± 6	41 ± 2
33	17 ± 2	12 ± 2	30 ± 6	83 ± 4	29 ± 7
100	22 ± 10	17 ± 3	29 ± 3	62 ± 6 r	40 ± 2
333	14 ± 3 rm	13 ± 2	7 ± 2 rm	2 ± 1 r	30 ± 2
1000	9 ± 1 rm	7 ± 2 rm	0 ± 1 rm	0 ± 0 r	30 ± 5
2500	8 ± 2 rm	2 ± 1 rm	0 ± 0 rm	1 ± 1 r	32 ±1 r
5000	4 ± 2 rm	0 ± 0 rm	0 ± 0 rm	0 ± 1 r	21 ± 6 r
Positive Control	NaN3	4-NOPD	4-NOPD	NaN3	MMS
Dose (µg/plate)	10	50	10	10	2
Number of revertant colonies/plate	1607 ± 89	100 ± 9	426 ± 35	2404 ± 194	1013 ± 144
S9-Mix	With
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent control (DMSO)	16 ± 3	15 ± 6	35 ± 6	105 ± 8	53 ± 10
Negative control (untreated)	11 ± 5	15 ± 3	43 ± 6	136 ± 11	48 ± 1
3	17 ± 5	18 ± 8	43 ± 1	94 ± 13	50 ± 8
10	16 ± 2	21 ± 4	30 ± 3	107 ± 8	51 ± 3
33	16 ± 6	12 ± 3	29 ± 2	83 ± 18	44 ± 16
100	14 ± 2	16 ± 6	32 ± 7	60 ± 7	41 ± 14
333	11 ± 5	17 ± 2	15 ± 5 rm	15 ± 3 rm	37 ± 8
1000	7 ± 2 rm	9 ± 2 rm	1 ± 1 rm	1 ± 1 r	25 ± 5
2500	7 ± 2 rm	5 ± 1 rm	0 ± 0 rm	0 ± 0 r	18 ± 4 r
5000	4 ± 2 rm	4 ± 1 rm	0 ± 0 rm	0 ± 0 r	6 ± 1r
Positive Control	2-AA	2-AA	2-AA	2-AA	2-AA
Dose (µg/plate)	2.5	2.5	2.5	2.5	10
Number of revertant colonies/plate	396 ± 51	121 ± 5	4563 ± 298	2777 ± 315	439 ± 51
NaN3: sodium acide; 2-AA: 2-Aminoanthracene; 4-NOPD: 4-nitro-o-phenylene-diamine; MMS: methyl methane sulfonate r: reduced background growth m: manual count

 

 Table 2: Results of Experiment II (pre-incubation)

	Number of revertant colonies per plate
S9-Mix	Without
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent control (DMSO)	10 ± 2	11 ± 5	31 ± 5	169 ± 12	41 ± 2
Negative control (untreated)	11 ± 1	16 ± 5	29 ± 3	203 ± 11	42 ± 6
1			27 ± 7	157± 20
3			24 ± 8	168 ± 6
10	9 ± 2	11 ± 4	36 ± 5	169 ± 15	37 ± 6
33	10 ± 1	8 ± 2	19 ±2	70 ± 10	38 ± 10
100	11 ± 4	8 ± 2	25 ± 3	69 ± 13	35 ± 3
333	7 ± 3	11 ± 2	16 ± 2	43 ± 6 r	22 ± 3
1000	5 ± 1 rm	3 ± 1 rm	2 ± 1	1 ± 1 rm	26 ± 5
2500	6 ± 2 rm	1 ± 1 rm			16 ± 4 rm
5000	5 ± 1 rm	0 ± 1 rm			10 ± 2 rm
Positive Control	NaN3	4-NOPD	4-NOPD	NaN3	MMS
Dose (µg/plate)	10	50	10	10	2
Number of revertant colonies/plate	1761 ± 16	93 ± 6	548 ± 32	2534 ± 43	1052 ± 96
S9-Mix	With
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent control (DMSO)	13 ± 6	13 ± 3	33 ± 6	156 ± 11	52 ± 8
Negative control (untreated)	13 ± 1	16 ± 4	47 ± 8	206 ± 6	54 ± 11
1			33 ± 13	167± 1
3			38 ± 6	149 ± 10
10	10 ± 4	14 ± 4	39 ± 10	161 ± 4	49 ± 10
33	14 ± 3	15 ± 4	44 ± 8	137 ± 18	48 ± 6
100	10 ± 4	17 ± 2	39 ± 9	85 ± 4	52 ± 10
333	9 ± 2	18 ± 2	25 ± 7 r	28 ± 7 rm	47 ± 9
1000	11 ± 1	18 ± 4 r	1 ± 1 rm	0 ± 1 rm	28 ± 3
2500	7 ± 1 rm	4 ± 1 rm			17 ± 5 rm
5000	6 ± 1 rm	2 ± 1 rm			11 ± 2 rm
Positive Control	2-AA	2-AA	2-AA	2-AA	2-AA
Dose (µg/plate)	2.5	2.5	2.5	2.5	10
Number of revertant colonies/plate	453 ± 31	109 ± 2	5475 ± 288	4801 ± 140	385 ± 10
NaN3: sodium acide; 2AA: 2-Aminoanthracene; 4-NOPD: 4-nitro-o-phenylene-diamine; MMS: methyl methane sulfonate r: reduced background growth m: manual count

 

 

 

 

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 98 and TA 100: 1; 3; 10; 33; 100; 333; and 1000 µg/plate

Strains TA 1535, TA 1537, and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

No Precipitation of the test item was observed in the overlay agar on the incubated agar plates. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2017). In this study the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate or cytotoxic concentrations.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008.