N-hydroxysuccinimide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Key study: Similar to OECD 471. The test item did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation up to 5000 µg/plate.

In vitro gene mutation study in yeast: Supporting study: Under the test conditions, the test item showed neither mitotic conversion nor death in Saccharomyces cerevisiae D4-RD up to 5 mg/ml.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Chemical Dynamics (South Plainfield, NJ).

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Bacterial strains were received from B.N. Ames.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 100, 500, 1000 and 5000 µg/plate.
No toxicity was observed at the highest dose (5000 µg/plate).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test item is completely soluble in the vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: N-methyl-N'-nitro-N-nitrosoguanidine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1E08 cells/ml
The plate incorporation assay was conducted accoding to Ames et al. (1975). To 2 ml of molten top agar at 45ºC were added 0.1 ml bacterial culture containing approximately 1E08 cells/ml, 0.1 ml test solution or solvent and 0.5 ml S) mix or buffer. After mixing, the soft agar was poured onto selective minimal agar plates.

DURATION
- Exposure duration: 2 days at 37 ºC

SELECTION AGENT (mutation assays): lack of histidine in the media

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was stimated by examining background bacterial lawns using a stereoscope.

- OTHER:
Preparation of the metabolic activation system: S9 was prepared from aroclor 1254-induced male Sprague-Dawley rat livers.

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:

valid

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:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Table 1.Number of revertant colonies after exposure to N-hydroxisuccinimide or positive control with metabolic activation.

 

Strain	Positive control	Amount tested (µg/plate)	Mean number of colonies per plate
			N-hydroxysuccinimide	Positive control
TA 1535	-	5000	11	-
	-	1000	11	-
	-	500	14	-
	-	100	10	-
	-	0	13	-
	2-AA	10	-	183
TA 1537	-	5000	9	-
	-	1000	5	-
	-	500	7	-
	-	100	9	-
	-	0	11	-
	2-AA	50	-	175
TA 98	-	5000	30	-
	-	1000	28	-
	-	500	26	-
	-	100	28	-
	-	0	35	-
	2-AA	20	-	1137
TA 100	-	5000	168	-
	-	1000	184	-
	-	500	176	-
	-	100	176	-
	-	0	211	-
	2-AA	10	-	1535

2-AA: 2-aminoanthracene

 

Table 2.Number of revertant colonies after exposure to N-hydroxisuccinimide or positive control without metabolic activation.

 

Strain	Positive control	Amount tested (µg/plate)	Mean number of colonies per plate
			N-hydroxysuccinimide	Positive control
TA 1535	-	5000	15	-
	-	1000	10	-
	-	500	15	-
	-	100	12	-
	-	0	13	-
	MNNG	5	-	1190
TA 1537	-	5000	4	-
	-	1000	7	-
	-	500	7	-
	-	100	5	-
	-	0	7	-
	9-AA	5	-	322
TA 98	-	5000	19	-
	-	1000	22	-
	-	500	15	-
	-	100	23	-
	-	0	15	-
	2-NF	5	-	959
TA 100	-	5000	160	-
	-	1000	163	-
	-	500	157	-
	-	100	174	-
	-	0	172	-
	MNNG	5	-	1055

MNNG: N-methyl-N’-nitro-N-nitrosoguanidine

9-AA: 9-aminoacrinide

2-NF: 2-nitrofluorene

Conclusions:

The test item did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation up to 5000 µg/plate.

Executive summary:

A study to determine the ability of the test item to induce mutation was assessed by the bacterial reverse mutation test, performed according to the method described by Ames, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA 1535, TA1537, TA 98 and TA 100) were exposed to 0, 100, 500, 1000 and 5000 µg/plate of the test item in presence and absence of S9 rat liver metabolic activation, by the plate incorporation method. Under the experimental conditions used, the test item did no induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Reference 2

Endpoint:

genetic toxicity in vitro, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1971

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: To evaluate the mitotic gene conversion in Saccharomyces cerevisiae of a substance.
- Short description of test conditions: Determination of mutation frequencies of the diploid strain D4-RD of Saccharomyces cerervisiae after the cehmical exposure by the liquid-holding system. The diploid Saccaromyces cerevisiae strain D4-RD cells only grow after addion of anenine and tryptophan to the medium, in addition to non-fermentable carbon sources.
- Parameters analysed / observed: Determination of the spontaneous convertion frequency.

GLP compliance:

no

Type of assay:

mitotic recombination assay with Saccharomyces cerevisiae

Target gene:

Adenine 2-locus and tryptophan 5-locus.

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

D4-RD

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Maximum dose: 5 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: No data.

Details on test system and experimental conditions:

METHOD OF APPLICATION.The assay was carried out according to the method described by Maquart and Zimmermann (1970) [see attached background material].
- Cell density at seeding (if applicable): Each tube was inoculated with about 300 cells.

Cells were grown in 5 ml liquid YEP (1% Difco yeast extract, 2% Difco bactopeptone, 2% glucose). The test item was dissolved and diluted to 1:100 in the cell suspension.

DURATION
- Exposure duration: 4 days at 25ºC.

SELECTION AGENT (mutation assays): glucose free solution.

Rationale for test conditions:

The saccharomyces D4-RD strain is much more sensitive to genetically active substances at 25ºC.

Key result

Species / strain:

Saccharomyces cerevisiae

Remarks:

D4-RD

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Under the test conditions, the test item showed neither mitotic conversion nor death in Saccharomyces cerevisiae D4-RD up to 5 mg/ml.

Executive summary:

A mitotic gene conversion test in Saccharomyces cerevisiae was performed with the test item. The test was carried out with the diploid strain D4 -RD of Saccharomyces cerevisiae in adenine 2 -locus and tryptophan 5 -locus, which additionally has a respiratory defect. This strain is much more sensitive to genetically active substances at 25ºC. A suspension of about 300 cells was exposed to the test item in a glucose-free 5 ml of liquid-holding solution for 4 days at 25ºC. Under the test conditions, the test item showed neither mitotic conversion nor death in Saccharomyces cerevisiae D4-RD up to 5 mg/ml.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria:

Key study: A study to determine the ability of the test item to induce mutation was assessed by the bacterial reverse mutation test, performed according to the method described by Ames, similar to OECD 471. Four histidine dependent strains of Salmonella typhimurium (TA 1535, TA1537, TA 98 and TA 100) were exposed to 0, 100, 500, 1000 and 5000 µg/plate of the test item in presence and absence of S9 rat liver metabolic activation, by the plate incorporation method. Under the experimental conditions used, the test item did no induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

In vitro gene mutation study in yeast:

Supporting study: A mitotic gene conversion test in Saccharomyces cerevisiae was performed with the test item. The test was carried out with the diploid strain D4 -RD of Saccharomyces cerevisiae in adenine 2 -locus and tryptophan 5 -locus, which additionally has a respiratory defect. This strain is much more sensitive to genetically active substances at 25ºC. A suspension of about 300 cells was exposed to the test item in a glucose-free 5 ml of liquid-holding solution for 4 days at 25ºC. Under the test conditions, the test item showed neither mitotic conversion nor death in Saccharomyces cerevisiae D4-RD up to 5 mg/ml.

Justification for classification or non-classification

Based on the available information, the test item is not classified as mutagen according to CLP Regulation (EC) no. 1272/2008.