Asparagine

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:

2016-10-0 to 2017-01-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st, July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30th, 2008

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:

Asparagine

EC Number:

200-735-9

EC Name:

Asparagine

Cas Number:

70-47-3

Molecular formula:

C4H8N2O3

IUPAC Name:

asparagine

Test material form:

solid: particulate/powder

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-diamine (without metabolic activation), 2-AA; 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation) and Experiment II: preincubation
- Cell density at seeding (if applicable): 100 µL of Bacteria suspension containing approximately 1E09 cells/mL for both experimental settings

DURATION
- Preincubation period: 60 min in Experiment II
- Exposure duration: 48 h in both experimental settings

NUMBER OF REPLICATIONS: 3 replicates for each strain and dose level, including the controls


DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.

Rationale for test conditions:

The OECD Guideline for Testing of Chemicals, Section 4, No. 471 — Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 98, TA, 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000,2500 and 5000 µg/plate.

Any other information on results incl. tables

Table 3:Number of revertants per plate (mean of 3 plates); incorporation method

	[strain 1535]		[strain 1537]		[strain 102]		[strain 98]		[strain 100]
Conc. [µg/plate]	— MA	+ MA	— MA	+ MA	— MA	+ MA	— MA	+ MA	— MA	+ MA
0 A dest.	29	15	5	6	270	370	19	28	115	117
0 *	28	14	11	12	261	339	27	28	120	110
31.6	29	17	11	10	282	383	18	22	114	113
100	22	12	11	7	275	342	18	17	128	127
316	24	11	10	9	268	307	21	24	116	129
1000	25	11	11	7	269	313	19	29	112	126
2500	25	15	9	9	319	308	23	24	98	112
5000	27	19	9	7	343	401	25	22	110	112
Positive control [-MA]	976	------	68	-------	1470	------	330	---------	757	-------
Positive control [+MA]	-------	276	-------	356	--------	925	-------	2405	------	2142

*solvent control with DMSO

Table 4:Number of revertants per plate (mean of 3 plates); preincubation method

	[strain 1535]		[strain 1537]		[strain 102]		[strain 98]		[strain 100]
Conc. [µg/plate]	— MA	+ MA	— MA	+ MA	— MA	+ MA	— MA	+ MA	— MA	+ MA
0 A dest.	7	16	6	9	236	303	19	22	85	108
0 *	8	12	7	7	206	316	22	17	73	95
31.6	5	14	5	9	214	295	21	14	69	93
100	8	13	5	6	219	358	20	20	85	93
316	5	13	4	5	247	307	24	19	91	110
1000	9	11	4	5	267	316	21	16	70	102
2500	3	16	6	7	252	305	21	25	84	96
5000	5	13	11	6	256	368	24	22	84	94
Positive control [-MA]	473	------	81	-------	1470	------	321	-------	573	-------
Positive control [+MA]	-------	164	-------	226	--------	677	-------	1751	------	1238

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:

The present study was conducted according to OECD Test Guideline 471 in order to determine the mutagenicity of L-asparagine. There was no increase in the amount of revertants for each incubation scheme and strain. Therefore, L-asparagine is considered to be non-mutagenic in the bacterial reverse mutation assay.