Sodium hydrogen L-aspartate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay according to OECD Guideline 471, the test substance was considered to be non mutagenic (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:

2017-01-19 to 2017-03-03

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:

other: Japanese Guidelines for this test system (Sofuni, 1993)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine in Salmonella typhimurium
tryptophane in Escherichia coli

Species / strain / cell type:

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

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolizing system (S9 mix) from beta-Naphthoflavone/Phenobarbital treated male Wistar rats

Test concentrations with justification for top dose:

A maximum concentration of 5000 µg/plate was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD TG 471, 1997).
1st series: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate, 10 % S9 mix
2nd series: 50, 158, 500, 1580, 5000 µg/plate, 20 % S9 mix

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent: Ultra-pure water showed best performance as solvent and was thus used for this experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin (DAUN)

Remarks:

1.0 µg/plate, TA98, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2.0 µg/plate, TA100, TA1535, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate, TA1537, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2.0 µg/plate, E. coli, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

2.0, 5.0, 10.0 µg/plate, all strains, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 to 3 days

NUMBER OF REPLICATIONS: 3 plates per concentration, 6 plates per control

DETERMINATION OF CYTOTOXICITY
- Method: colony number

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous re-vertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. Based upon the historical controls of the laboratory and statistical considerations the criteria were established for “No increase” and “Clear increase”. All results between “No” and “Clear” were considered as “weak increase”.
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.
In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item soluble in water.
- Precipitation: No precipitation of the test material on the agar plates occurred.

HISTORICAL CONTROL DATA
- Positive historical control data: Without S9: TA98 352 ± 146.3, TA100 1337 ± 316.8, TA1535 900 ± 193.4, TA1537 1175 ± 564.2, WP2 uvrA 1613 ± 438.7; With S9: TA98 529 ± 296.1, TA100 1262 ± 435.4, TA1535 200 ± 64.2, TA1537 418 ± 201.3, WP2 uvrA 347 ± 192.4
- Negative (solvent/vehicle) historical control data: Without S9: TA98 34 ± 5.2, TA100 110 ± 11.7, TA1535 27 ± 3.2, TA1537 28 ± 3.5, WP2 uvrA 30 ± 4.9; With S9: TA98 40 ± 7.0, TA100 126 ± 11.9, TA1535 26 ± 3.9, TA1537 30 ± 4.4, WP2 uvrA 36 ± 5.9

Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Results without S9
Water	39 ± 5	130 ± 14	27 ± 5	6 ± 2	39 ± 9
5	44 ± 2	117 ± 25	26 ± 10	9 ± 2	37 ± 4
15.8	48 ± 3	117 ± 15	27 ± 4	3 ± 2	30 ± 6
50	41 ± 5	119 ± 13	27 ± 3	9 ± 1	30 ± 11
158	43 ± 8	132 ± 12	25 ± 4	6 ± 3	30 ± 2
500	46 ± 11	120 ± 6	28 ± 5	7 ± 2	27 ± 5
1580	43 ± 4	129 ± 15	35 ± 6	6 ± 2	35 ± 40
5000	39 ± 4	131 ± 9	24 ± 3	7 ± 3	33 ± 5
DAUN (1)	203 ± 40
NaN3 (2)		1649 ± 43	924 ± 12
9-AA (50)				556 ± 150
NQO (2)					2058 ± 65
	Results with S9
Water	47 ± 10	132 ± 9	25 ± 6	11 ± 5	40 ± 4
5	47 ± 4	115 ± 6	22 ± 7	8 ± 3	40 ± 9
15.8	44 ± 8	125 ± 14	30 ± 2	9 ± 1	37 ± 2
50	53 ± 12	134 ± 11	23 ± 6	16 ± 9	46 ± 11
158	54 ± 13	130 ± 5	27 ± 4	5 ± 3	45 ± 3
500	55 ± 13	134 ± 13	23 ± 8	11 ± 4	40 ± 5
1580	44 ± 7	134 ± 13	23 ± 3	11 ± 4	34 ± 8
5000	40 ± 12	117 ± 4	24 ± 4	10 ± 5	34 ± 10
2-AA (2)	958 ± 96	1677 ± 280
2-AA (5)			181 ± 21	555 ± 157
2-AA (10.0)					471 ± 4

 

Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Results without S9
Water	36 ± 5	125 ± 24	24 ± 4	9 ± 4	22 ± 4
50	50 ± 6	129 ± 12	22 ± 2	5 ± 4	26 ± 4
158	43 ± 5	137 ± 8	25 ± 4	7 ± 4	28 ± 11
500	39 ± 2	121 ± 7	26 ± 8	7 ± 5	20 ± 6
1580	33 ± 3	121 ± 10	24 ± 6	7 ± 1	25 ± 6
5000	38 ± 8	127 ± 8	24 ± 7	10 ± 2	26 ± 1
DAUN (1)	281 ± 36
NaN3 (2)		1584 ± 58	860 ± 47
9-AA (50)				1119 ± 170
NQO (2)					1751 ± 17
	Results with S9
Water	50 ± 8	142 ± 11	25 ± 7	11 ± 3	35 ± 3
50	45 ± 2	156 ± 19	26 ± 7	12 ± 5	36 ± 10
158	45 ± 3	152 ± 11	26 ± 3	15 ± 3	44 ± 6
500	43 ± 9	150 ± 14	24 ± 2	12 ± 6	38 ± 8
1580	44 ± 3	144 ± 5	29 ± 8	8 ± 2	41 ± 3
5000	52 ± 9	158 ± 8	26 ± 6	9 ± 4	44 ± 6
2-AA (2)	450 ± 64	754 ± 27
2-AA (5)			185 ± 15	201 ± 22
2-AA (10.0)					284 ± 36

Conclusions:

Under the experimental conditions the test substance was considered non mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to investigate the test substance for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix) according to OECD Guideline 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from β-Naphthoflavone/Phenobarbital -pretreated male Wistar rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10 % and 20 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for solvent control treatments, and 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 test item treatments of all the tester strains in the absence and presence of S9 mix, no relevant increase in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test substance was not mutagenic under the experimental conditions described.

Furthermore, it was assumed, that the anhydrate form has the same mutagenic potential to the skin and thus the result of the test item is also applicable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix) according to OECD 471 (reference 7.6.1 -1). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from β-Naphthoflavone/Phenobarbital -pretreated male Wistar rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10 % and 20 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for solvent control treatments, and 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 test item treatments of all the tester strains in the absence and presence of S9 mix, no relevant increase in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test substance was not mutagenic under the experimental conditions described.

Furthermore, it was assumed, that the anhydrate form has the same mutagenic potential and thus the result of the test item is also applicable.

Justification for classification or non-classification

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

Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.