Oxalacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria (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:

23 October 2016 - 25 January 2017

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd series: 50, 281, 500, 1580 and 5000 µg/plate
top dose: in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD 471, 1997)

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water
- Justification for choice of solvent/vehicle: based on the available information from the preliminary solubility test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: daunomycin, 2-aminoanthracene

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 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

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 TA 1535

Metabolic activation:

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:

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:

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
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Table 2: Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2uvrA
Without Activation	H2O		43 ± 8	133 ± 10	25 ± 6	29 ± 4	36 ± 10
	Test item	5.00	41 ± 9	154 ± 10	33 ± 10	31 ± 2	36 ± 2
		15.8	44 ± 6	135 ± 17	27 ± 4	31 ± 3	39 ± 3
		50.0	36 ± 6	135 ± 18	27 ± 6	32 ± 5	34 ± 10
		158	41 ± 6	145 ± 7	25 ± 10	28 ± 7	37 ± 2
		500	40 ± 5	127 ± 5	28 ± 7	33 ± 7	34 ± 4
		1580	41 ± 4	125 ± 11	30 ± 2	37 ± 6	32 ± 2
		5000	44 ± 9	115 ± 8	28 ± 5	31 ± 6	41 ± 6
	DAUN	1.00	213 ± 29
	NaN3	2.00		1621 ± 31	983 ± 48
	9-AA	50.0				623 ± 97
	NQO	2.00					1548 ± 38
With Activation	H2O		56 ± 18	143 ± 13	26 ± 2	32 ± 6	41 ± 9
	Test item	5.00	43 ± 5	137 ± 13	30 ± 3	35 ± 2	50 ± 10
		15.8	57 ± 4	151 ± 17	30 ± 1	29 ± 5	39 ± 4
		50.0	46 ± 3	152 ± 21	30 ± 2	35 ± 14	43 ± 8
		158	58 ± 2	148 ± 12	30 ± 5	33 ± 2	39 ± 2
		500	63 ± 3	146 ± 12	33 ± 8	38 ± 10	42 ± 3
		1580	51 ± 12	142 ± 8	25 ± 3	38 ± 13	40 ± 6
		5000	54 ± 5	116 ± 8	30 ± 4	31 ± 4	36 ± 6
	2-AA	2.00	986 ± 30	1575 ± 61
	2-AA	5.00			229 ± 26	559 ± 9
	2-AA	10.0					277 ± 8

Key to Positive Control: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

Table 3: Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2uvrA
Without Activation	H2O		45 ± 4	138 ± 18	27 ± 4	31 ± 4	38 ± 8
	Test item	50.0	37 ± 6	137 ± 13	32 ± 3	29 ± 8	39 ± 10
		281	34 ± 6	122 ± 12	27 ± 10	30 ± 7	34 ± 9
		500	39 ± 8	123 ± 7	31 ± 3	39 ± 6	29 ± 9
		1580	42 ± 11	135 ± 3	31 ± 3	34 ± 6	33 ± 5
		5000	39 ± 11	118 ± 10	27 ± 2	32 ± 5	33 ± 5
	DAUN	1.00	424 ± 117
	NaN3	2.00		1329 ± 45	790 ± 36
	9-AA	50.0				1156 ± 534
	NQO	2.00					1618 ± 51
With Activation	H2O		48 ± 7	135 ± 14	21 ± 3	34 ± 4	36 ± 9
	Test item	50.0	50 ± 7	131 ± 7	24 ± 4	41 ± 5	45 ± 3
		281	52 ± 9	137 ± 5	24 ± 3	36 ± 10	38 ± 3
		500	50 ± 1	140 ± 5	27 ± 4	38 ± 8	35 ± 11
		1580	56 ± 11	130 ± 8	26 ± 2	43 ± 8	42 ± 8
		5000	52 ± 7	106 ± 17	17 ± 6	40 ± 9	38 ± 9
	2-AA	2.00	479 ± 39	708 ± 65
	2-AA	5.00			96 ± 2	207 ± 24
	2-AA	10.0					149 ± 32

Key to Positive Control: NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-Nitroquinoline-N-oxide

 

Table 4: Historical data - Negative Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	380	379	388	400	180	179	176	176	304	304
Number of Values	89	89	91	94	39	39	38	38	70	70
Minimum	28	31	89	95	23	19	15	13	23	26
Maximum	49	54	147	166	54	39	29	33	49	51
Mean	37	42	111	120	31	28	24	25	33	38
Standard Deviation	4.3	5.6	12.2	11.3	6.9	4.4	3.4	4.7	5.3	5.3

The historical data have been obtained in experiments between 01/2015 and 12/2015

 

Table 5: Historical data - Positive Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3	2-AA	NaN3	2-AA	9-AA	2-AA	NQO	2-AA
Total Plates	190	190	190	200	90	90	88	88	152	152
Number of Values	89	89	89	94	39	39	38	38	70	70
Minimum	89	201	195	450	546	138	120	106	222	126
Maximum	1197	1544	2289	2229	1562	352	2313	588	2613	737
Mean	362	769	1360	1365	867	262	977	378	1606	394
Standard Deviation	201.9	257.4	305.0	314.9	172.3	51.8	429.9	142.3	488.8	142.2

The historical data have been obtained in experiments between 01/2015 and 12/2015

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

The present 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).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537,and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from ß-Naphthoflavone/Phenobarbital-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 20% S9 in the 2nd series, respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle 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.

No relevant increases in revertant numbers were observed after treatment in any of the tester strains in the absence and presence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Test

The present 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).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537,and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from ß-Naphthoflavone/Phenobarbital-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 20% S9 in the 2nd series, respectively.

Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle 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.

No relevant increases in revertant numbers were observed after treatment in any of the tester strains in the absence and presence of S9 mix.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.