T-A06

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria: Key study. Method according to OECD 471, GLP study. The test item does not have mutagenic activity under the test conditions, with or 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:

21 September 2020 to 15. October 2020

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, his C, his G, tryp E

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 Sprague Dawley rat liver homogenate by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox11101-5-4244 validated on 21.08.2020 – expiry date: 28.04.2022)
- method of preparation of S9 mix:
S9 fraction 10 %
MgCL2-6H2O 8 mM
KCl 33 mM
Glucose-6-Phosphate Na2 5 mM
NADP Na2 4 mM
Phosphate buffer pH 7.4 0.1 M
- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9-mix/plate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): sterility test

Test concentrations with justification for top dose:

Preliminary bacteriostatic activity controls with TA100: 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg.The highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. No bacterostatic activity could be detected.
Main Assay with 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
data concerning the solubility, the visual homogeneity and the stability of the test item
in the vehicle are available.

- Justification for percentage of solvent in the final culture medium:
No control of the concentration of the test item in the vehicle by means of an analytical method has been performed

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine & cis-Platinum (II) Diammine Dichloride

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2 assays, 5 bacteria , each with and without metabolic activation + sterility test and bacteriostatic activity control

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): (1-9 x 103 bacteria/mL
- Test substance added in suspension from stock solution 100mg/mL in EtOH

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Assay 2 with metabolic activation preincubation 30min @ 37C
- Exposure duration/duration of treatment: 48 to 72 hours
- Harvest time after the end of treatment (sampling/recovery times): after 48 to 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.:relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The determination of mutagenic activity of the test item is its capacity to induce revertant colonies in mutated Salmonella typhimurium and Escherichia coli strains. Revertant colonies grow in absence of the amino-acid of which they are originally dependant.

Evaluation criteria:

The following validity criteria were checked to validate each experiment:
• the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
• the spontaneous reversion rate of the absolute negative control shall comply with the historical
values of the laboratory,
• The spontaneous reversion rate of the blank control shall not be statistically different from
absolute negative control.
• the mean number of revertant colonies obtained for each strain and the corresponding positive
control, with and/or without metabolic activation shall comply with the historical values of the
laboratory.
• Negative and positive values should not show significant difference with the historical values of
the laboratory (± 2 standard deviations)

Statistics:

n/a

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

no cytotoxicity

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

no cytotoxicity

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

no cytotoxicity

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Remarks:

5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary cytotoxicity testing (strain TA100)


Ames test:
- Signs of toxicity Non observed
- Individual plate counts 3 plates per stain per dilution with and without s9
- Mean number of revertant colonies per plate and standard deviation See tables in 'any other information on results incl.tables'



HISTORICAL CONTROL DATA not speciefied. Historical data without S9, with S9 and without preincubation and with s9 and with preincubation mean and min max values available.

Table TA1538

Strain		TA 1535
Assay		1						2
Metabolic activation		Without			With			Without			With
		Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R
Negative Control		12.67	2.31	_	14.33	5.69	_	9.67	3.51	_	15	6.08	_
Positive Control solvent		8.33	2.08	_	16.33	2.08	_	12	4.36	_	13.33	2.52	_
Positive Control		736	47.84	88.32	175.67	7.77	10.8	673.67	99.49	56.14	59.67	7.37	4.48
Vehicle		13.33	1.53	_	11	2.65	_	7	2.65	_	11	2.65	_
Test item:	5000 µg*	13.67	2.08	1.03	18.33	2.08	1.67	11	5.2	1.57	12.33	2.52	1.12
	1500 µg	10.67	1.53	0.8	18	1.73	1.64	9.33	2.52	1.33	10.33	4.04	0.94
	500 µg	12	1.73	0.9	12	4.36	1.09	7.67	2.08	1.1	6.67	2.08	0.61
	150 µg	14	3	1.05	16.67	1.15	1.52	6.67	2.08	0.95	10.33	3.21	0.94
	50 µg	14.33	0.58	1.08	14.33	5.86	1.3	7.33	1.53	1.05	11.33	1.53	1.03

Table TA1537

Strain		TA 1537
Assay		1						2
Metabolic activation		Without			With			Without			With
		Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R
Negative Control		6.67	3.06	_	6	1	_	3.33	1.53	_	5.33	1.53	_
Positive Control solvent		6.67	2.52	_	9.67	3.21	_	5.33	3.06	_	5	1.73	_
Positive Control		1007.33	123.33	151.1	48.67	2.52	5.03	649.33	134.17	121.8	48.33	6.66	9.67
Vehicle		5.33	1.15	_	7	4.36	_	5.33	1.15	_	5	2.65	_
Test item:	5000 µg*	8.33	3.21	1.56	8.33	4.16	1.19	5.67	2.08	1.06	5.33	0.58	1.07
	1500 µg	7	1	1.31	8.33	3.21	1.19	4.33	1.15	0.81	3.67	0.58	0.73
	500 µg	8.33	1.53	1.56	10.67	4.62	1.52	3	1	0.56	4.67	3.21	0.93
	150 µg	6.33	2.08	1.19	7.33	2.08	1.05	5.67	0.58	1.06	4.67	0.58	0.93
	50 µg	5.33	1.53	1	8.67	1.15	1.24	5	2	0.94	5.67	2.08	1.13

Table TA98

Strain		TA 98
Assay		1						2
Metabolic activation		Without			With			Without			With
		Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R
Negative Control		17.33	6.66	_	30	2.65	_	19.67	0.58	_	31.33	1.53	_
Positive Control solvent		17.67	0.58	_	32.67	4.62	_	25.33	2.08	_	33	1	_
Positive Control		283.33	56.05	16.04	658.33	57.1	20.2	420	58.85	16.58	282.67	23.16	8.57
Vehicle		22.67	5.03	_	30.33	6.03	_	24	1.73	_	35	1	_
Test item:	5000 µg*	19.33	2.08	0.85	32.67	5.86	1.08	20	5.57	0.83	31.67	1.53	0.9
	1500 µg	16.33	4.04	0.72	26.67	1.15	0.88	18.33	3.51	0.76	40.67	2.52	1.16
	500 µg	24.33	3.21	1.07	29	2	0.96	23	1.73	0.96	32.67	2.52	0.93
	150 µg	27.67	2.52	1.22	26.67	6.03	0.88	23	1.73	0.96	37.67	2.08	1.08
	50 µg	17.33	4.04	0.76	29	4.36	0.96	27.33	2.08	1.14	36.33	4.04	1.04

Table TA100

Strain		TA 100
Assay		1						2
Metabolic activation		Without			With			Without			With
		Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R
Negative Control		86	3.46	_	88	2	_	68	6.08	_	79.67	9.29	_
Positive Control solvent		85.67	6.11	_	90.33	7.57	_	70	7	_	81.67	2.52	_
Positive Control		1240	105.48	14.47	962.67	99.89	10.7	1075.7	49.03	15.37	513	11.53	6.28
Vehicle		68.67	17.01	_	72.33	10.07	_	70.67	7.09	_	82	8.54	_
Test item:	5000 µg*	70.33	2.52	1.02	78.33	10.69	1.08	68.67	6.03	0.97	79.67	5.51	0.97
	1500 µg	67.33	14.64	0.98	79.67	2.08	1.1	58.67	7.09	0.83	81.67	7.23	1
	500 µg	68.67	7.64	1	90.33	6.03	1.25	53	6.08	0.75	76	7	0.93
	150 µg	65.67	7.09	0.96	76.33	8.74	1.06	57	6.56	0.81	71.67	6.51	0.87
	50 µg	59	2	0.86	75.33	11.68	1.04	56.67	3.51	0.8	67.67	2.52	0.83

Table Ecoli

Strain		E Coli
Assay		1						2
Metabolic activation		Without			With			Without			With
		Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R	Mean	Standard deviation	R
Negative Control		62.33	4.16	_	100	6.56	_	63	4	_	93	10.39	_
Positive Control solvent		60	2	_	96.67	5.13	_	80.67	0.58	_	93.33	12.5	_
Positive Control		306	7.94	5.1	398	24.64	4.12	326	15.52	4.04	307.33	16.44	3.29
Vehicle		57.33	4.51	_	89.33	8.02	_	62	5.2	_	87	7.94	_
Test item:	5000 µg*	54.33	3.79	0.95	68.67	7.09	0.77	67	5.57	1.08	90.67	9.87	1.04
	1500 µg	60	3	1.05	84	9.54	0.94	74.67	7.37	1.2	88.33	17.39	1.02
	500 µg	57.33	4.51	1	68.33	3.06	0.76	57	6.56	0.92	83.67	10.5	0.96
	150 µg	53	2.65	0.92	70.67	7.02	0.79	67.67	9.29	1.09	91.67	10.07	1.05
	50 µg	57.33	5.13	1	68	3	0.76	70	2.65	1.13	84.33	7.51	0.97

Conclusions:

The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

Executive summary:

The mutagenic activity of the test item was determined according to OECD 471 under GLP conditions. Four different histidine dependent Salmonella typhimurium strains (TA1535, 1537, 98 & 100) and a tryptophan dependent Escherichia coli WP2 uvrA strain were exposed to the test subtance in EtOH solution ranging from 50 to 5000 μg/plate in triplicate, including negative, positive and positive-solvent controls with and without metabolic activation. The plates were screened for revertant colonies that gained the ability to be independent of the former dependent histidine or tryptophane.  No increase in revertant colonies could be observed. The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro gene mutation in bacteria:

The mutagenic activity of the test item was determined according to OECD 471 under GLP conditions. Four different histidine dependent Salmonella typhimurium strains (TA1535, 1537, 98 & 100) and a tryptophan dependent Escherichia coli WP2 uvrA strain were exposed to the test subtance in EtOH solution ranging from 50 to 5000 μg/plate in triplicate, including negative, positive and positive-solvent controls with and without metabolic activation. The plates were screened for revertant colonies that gained the ability to be independent of the former dependent histidine or tryptophane.  No increase in revertant colonies could be observed. The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

Justification for classification or non-classification

Based on the available information, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.