L-cystine dihydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of a bacterial mutagenicity assay (Ames test), the test item is considered to be non-mutagenic in the absence and presence of metabolic activation (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:

06 September 2016 - 14 November 2016

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 (incl. QA statement)

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:

Rat liver S9 (pre-treated with Aroclor 1254)

Test concentrations with justification for top dose:

Top dose: 5000 µg/plate (maximum recommended concentration)
1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 158, 500, 1580, 2810, 5000 µg/plate (with and without S9 mix)
3rd series: 158, 500, 1580, 2810, 5000 µg/plate (strain TA 1537, with and without S9 mix)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria

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 1 µg/plate (TA 98, + S9), 2-aminoanthracene 2-10 µg/plate (all strains, -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: exposure 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls
Three experimental series were performed.

DETERMINATION OF CYTOTOXICITY:
- Method: counting numbers of revertants

OTHER:
- S9 concentration: 10% (1st series) and 30% S9 (2nd and 3rd series)

Evaluation criteria:

Classification as negative or non-mutagenic 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)

Classification as 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.

Statistics:

Not performed as not mandatory for this test system.

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:

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 precipitation occurred.

In the second series there was a high spontaneous rate for TA 1537 observed, that exceeded the tolerance interval of the historical control values. Therefore, it was decided to perform a third series with this strain under the same conditions. In this third series the spontaneous rate was within the tolerance interval of the historical controls thus confirming the validity of the assay.

Table 2: Summary 1^st series

Metabolic Activation	Test material	Concentr. (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		37 ± 6	93 ± 13	31 ± 6	36 ± 4	25 ± 6
	Test item	5	34± 7	98±3	20±5	30±2	26±5
		15.8	38±4	107±9	24±6	29±9	26±4
		50	34±5	117±15	24±4	27±3	30±6
		158	39±7	98±6	27±4	34±6	28±13
		500	48±6	117±17	23±1	31±3	28±1
		1580	39±7	111±9	23±8	27±8	33±13
		5000	41±4	116± 12	27±7	32±9	32±10
	DAUN	1	364±93
	NaN3	2		1197±253	781±40
	9-AA	50				1361±441
	NQO	2					1499± 224
With Activation	DMSO		41±5	122±13	30±2	33±6	31±8
	Test item	5	37± 2	138±7	24±5	31±1	37±4
		15.8	37±6	123±15	22±1	26±2	34±2
		50	37±7	107±15	28±1	39±5	26±11
		158	39±7	112±11	21±7	40±8	32±6
		500	46±7	131±16	22±7	32±2	45±10
		1580	39±11	113± 14	27±3	34±6	31±3
		5000	31±2	122±16	21±3	27±10	40±2
	2-AA	2	557± 56	1660±111
	2-AA	5			266±7	649±12
	2-AA	10					769±38

 

Table 3: Summary 2^nd 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		30 ± 8	107 ± 11	28 ± 4	36 ± 8	30 ± 5
	Test item	158	30± 11	103± 3	34±8	34± 5	32± 4
		500	33± 9	102± 5	26± 2	37± 7	27± 4
		1580	31± 6	106± 5	23± 5	37± 9	29± 2
		2810	33± 12	110± 16	17± 8	35±1	37± 2
		5000	30± 5	107± 6	26± 3	43± 7	34± 10
	DAUN	1	255± 32
	NaN3	2		977± 91	876± 13
	9-AA	50				1141± 292
	NQO	2					1309± 31
With Activation	DMSO		31± 7	125± 16	29± 6	43± 5	35± 8
	Test item	158	31± 4	135± 8	28± 8	30± 6	39± 11
		500	33±11	126± 13	34± 4	45± 15	37± 9
		1580	26±4	126± 17	30± 8	41± 9	38± 10
		2810	33± 5	124± 21	24± 6	40± 4	29± 1
		5000	34± 3	118± 7	22± 2	29±6	28± 1
	2-AA	2	197± 40
	2-AA	5		1875± 53
	2-AA	10			221± 22	484± 25	171±24

 

Table 4: Summary 3^rd series

Metabolic Activation	Test material	Concentr. (µg/plate)	Revertants per plate (Mean ± SD)
			TA 1537
Without Activation	DMSO		33± 8
	Test item	158	27± 9
		500	28± 3
		1580	35± 4
		2810	30± 7
		5000	36± 5
	9-AA	50	1994± 562
With Activation	DMSO		35± 8
	Test item	158	31± 12
		500	36± 6
		1580	39± 13
		2810	35± 2
		5000	29± 2
	2-AA	10	257± 40

Key to Positive Controls

NaN₃                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

Conclusions:

The test item is considered non-mutagenic under the test conditions described.

Executive summary:

The test item was examined for its mutagenic activity in three series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pre-treated rats was used. In this study, three experimental series were performed. In the experiments with S9 mix, 10 % (1^st series) and 30% S9 (2^nd and 3^rd series) were used.

Treatments of all tester strains were performed using test item formulations prepared in anhydrous analytical grade dimethyl sulfoxide (DMSO) in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the acceptance criteria have been met in total and the study is considered valid.

Following test item treatments, no precipitation of the test material on the agar plates occurred. There was no toxicity to the bacteria observed.

In the second series there was a high spontaneous rate for TA 1537 observed, that exceeded the tolerance interval of the historical control values. Therefore, it was decided to perform a third series with this strain under the same conditions. In this third series the spontaneous rate was within the tolerance interval of the historical controls thus confirming the validity of the assay.

Under the conditions described, there were no relevant increases in revertant numbers after test item exposure observed in the absence and presence of S9 mix.

According to the criteria for negative and positive results, the test item was not mutagenic under the described experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471, Bacterial Reverse Mutation Assay

The test item was examined for its mutagenic activity in three series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pre-treated rats was used. In this study, three experimental series were performed. In the experiments with S9 mix, 10 % (1^stseries) and 30% S9 (2^ndand 3^rdseries) were used.

Treatments of all tester strains were performed using test item formulations prepared in anhydrous analytical grade dimethyl sulfoxide (DMSO) in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the acceptance criteria have been met in total and the study is considered valid. Following test item treatments, no precipitation of the test material on the agar plates occurred. There was no toxicity to the bacteria observed.

In the second series there was a high spontaneous rate for TA 1537 observed, that exceeded the tolerance interval of the historical control values. Therefore, it was decided to perform a third series with this strain under the same conditions. In this third series the spontaneous rate was within the tolerance interval of the historical controls thus confirming the validity of the assay. Under the conditions described, there were no relevant increases in revertant numbers after test item exposure observed in the absence andpresence of S9 mix. According to the criteria for negative and positive results, the test item was not mutagenic under the described experimental conditions.

Justification for classification or non-classification

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

The available experimental data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity (UN GHS: no category) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.