Adenine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a GLP-compliant bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

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:

2018-07-23 to 2018-08-02

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:

1997

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Sofuni, 1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA 98, TA 100, TA 1535, and TA 1537: histidine operon; His G 46, His D 3052, His C 3076
Escherichia coli WP2 uvrA: trp-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats

Test concentrations with justification for top dose:

1st serie: 1.58, 5.0, 15.8, 50.0, 158, 500, 1500 µg/plate
2nd serie: 1.58, 5.0, 15.8, 50.0, 158, 500, 1500 µg/plate
Top dose: Due to the limited solubility of the test item, 1000 µg/plate was chosen as the appropriate maximum concentration.

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

Untreated negative controls:

no

Remarks:

covered by solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (CAS no 67-68-5, purity > 99.9%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene, daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants (scored electronically with the validated „Ames Study Manager", Instem, Stone, Staffordshire, UK).

Rationale for test conditions:

Salmonella typhimurium and Escherichia coli strains were tested in accordance with the plate incorporation method (Ames, 1975) and the OECD, EEC and Japanese guidelines for this test system.

Evaluation criteria:

A test material was to be defined as negative or non-mutagenic in this assay if
- The assay was to be considered valid, and
- "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
- a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
- "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

vehicle control = negative control

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Remarks:

vehicle control = negative control

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Summary 1st Series

Metabolic Activation	Test  Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without activation	DMSO		31 ± 9	100 ± 13	20 + 6	9 ± 3	32 ± 7
	Test item	1.58	30 ± 8	96 ± 20	21 ± 4	5 ± 1	29 ± 3
		5.00	22 ± 6	96 ± 1	20 ± 1	12 ± 1	37 ± 2
		15.80	26 ± 6	100 ± 2	28 ± 5	9 ± 5	35 ± 6
		50.00	32 ± 10	103 ± 2	19± 7	7 ±2	35 ± 2
		158.00	39 ± 14	94 ± 10	23 ± 7	10 ±7	41 ± 13
		500.00	29 ± 5	98 ± 19	20 ± 7	13 ± 4	35 ± 6
		1000.00	40 ± 8	111+ 9	21 ± 6	10 ± 2	41 ± 6
	NaN3	2.00		2147 ± 144	1055 ± 24
	NQO	2.00					1967 ± 48
	DAUN	2.00	295 ± 43
	9-AA	50.00				1608 ± 956
With activation	DMSO		37 ±13	126 ± 8	18 ± 3	9 ± 4	46 ± 8
	Test item	1.58	28 ± 7	126 ± 28	23 ± 5	13 ± 1	45 ± 8
		5.00	27 ± 6	113 + 7	18 ± 5	16 ± 4	38 ±6
		15.80	37 ±13	122 ± 3	22 ± 3	9 ± 4	38 ± 10
		50.00	37 ± 4	114 ± 17	18± 2	11 ± 6	44 ± 13
		158.00	35 ± 2	110 ± 25	18 ± 6	12 ± 2	44 ± 11
		500.00	34 ± 13	110 ± 17	21 ±7	22 ± 4	53 + 8
		1000.00	31 ± 2	120 ± 5	18 ± 5	20 ± 5	45 ± 4
	2-AA	2.00	612 ± 64	2450 ± 64
	2-AA	10.00					477 ± 40
	2-AA	5.00			117 ± 12	394 ± 46

NQO      4-Nitroquinoline-N-oxide

2-AA       2-Aminoanthracene

9-AA       9-Aminoacridine

DAUN    Daunomycin

NaN3     Sodium azide

Table 2: Summary 2nd Series

Metabolic Activation	Test  Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without activation	DMSO		26 ± 6	91 ± 13	16 ± 2	9 ± 3	29 ± 6
	Test item	15.80	33 ± 0	114 ± 6	17 ± 9	10 ± 4	39 ± 4
		50.00	24 ± 8	115 + 16	15 ± 9	15 ± 2	34 ± 2
		158.00	32 ± 3	96 ± 13	17 ±4	9 ± 4	33 ± 5
		500.00	32 ± 8	98 ± 14	22 ± 6	12 ± 6	34 ± 6
		1000.00	29 ± 2	94 ± 6	19 ± 4	11 ± 1	42 ± 7
	NaN3	2.00		1991 ± 50	836 ± 77
	NQO	2.00					2303 ± 192
	DAUN	2.00	498 ± 37
	9-AA	50.00				1540 ± 294
With activation	DMSO		29 ± 10	129 ± 14	15 ± 2	10 ± 4	47 ± 9
	Test item	15.80	35 ± 8	124 ± 21	14 ± 5	11 ± 5	42 ± 17
		50.00	27 ± 4	118 ± 5	16 ± 6	14 ± 1	45 ± 9
		158.00	33 ± 6	114 ± 6	15 ± 1	13 ± 6	50 ± 11
		500.00	34 ± 4	103 ± 22	21 ± 10	20 ± 4	52 ± 7
		1000.00	33 ± 14	124 ± 6	14 ± 6	23 ± 4	57 ± 14
	2-AA	10.00					297 ± 33
	2-AA	2.00	208 ± 78	785 ±128
	2-AA	5.00			148 ± 27	168 ± 40

NQO     4-Nitroquinoline-N-oxide

2-AA      2-Aminoanthracene

9-AA      9-Aminoacridine

DAUN   Daunomycin

NaN3    Sodium azide

Conclusions:

In a GLP-compliant bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix).

Executive summary:

The mutagenic potential of the test item was assessed in a GLP-compliant bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 in the absence and presence of a rat liver metabolizing system (S9 mix) from rats pretreated with beta-Naphthoflavone/Phenobarbital. As tester strains Salmonella typhimarium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA were used. Two experimental series were performed: The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. Due to the limited solubility of the test item, 1000 µg/plate was chosen as the appropriate maximum concentration (1.58, 5.0, 15.8, 50.0, 158, 500, 1000 µg test item/plate in both series). Solvent and positive control treatments were included for all strains.  3 replicates for test item concentrations and positive controls as well as 6 replicates for solvent controls were used. The numbers of revertants were scored electronically with the validated „Ames Study Manager" (Instem, Stone, Staffordshire, UK). The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Under the experimental conditions reported, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic potential of the test item was assessed in a GLP-compliant bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 in the absence and presence of a rat liver metabolizing system (S9 mix) from rats pretreated with p-Naphthoflavone/Phenobarbital. As tester strains Salmonella typhimarium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA were used. Two experimental series were performed: The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. Due to the limited solubility of the test item, 1000 µg/plate was chosen as the appropriate maximum concentration (1.58, 5.0, 15.8, 50.0, 158, 500, 1500 µg test item/plate in both series). Solvent and positive control treatments were included for all strains. 3 replicates for test item concentrations and positive controls as well as 6 replicates for solvent controls were used. The numbers of revertants were scored electronically with the validated "Ames Study Manager" (Instem, Stone, Staffordshire, UK). The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Under the experimental conditions reported, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the eleventh time in Regulation (EU) No 2018/669.