1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-08-15 - 2001-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Remarks:

UK Department of Health

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

EC Number:

245-910-0

EC Name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Cas Number:

23847-08-7

Molecular formula:

C12H20N2O2S2

IUPAC Name:

1-[(2-oxoazepan-1-yl)disulfanyl]azepan-2-one

Constituent 2

Reference substance name:

CLDS (Caprolactam disulfide)

IUPAC Name:

CLDS (Caprolactam disulfide)

Details on test material:

- Name of test material (as cited in study report): CLDS (Caprolactam disulfide)
- Substance type: organic
- Physical state: solid (off-white powder)
- Lot/batch No.: 0110079/007
- Storage condition of test material: room temperature, in the dark
- Other: data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor.

Method

Target gene:

histidin- (S. typhimurium) / tryptophan- (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The Salmonella strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors and the spontaneous reversion rate. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Mutation experiment 1: 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation experiment 2: 50 to 5000 μg/plate for Escherichia coli strain WP2uvrA- and 15 to 5000 μg/plate for the Salmonella tester strains (use of fresh cultures of the bacterial strains and fresh test material formulations)
An additional dose (15 μg/plate) was used to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: low water solubility

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 3 μg/plate for TA100, 5 μg/plate for TA1535 and 2 μg/plate for WP2uvrA-

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 μg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 μg/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

other:

Positive control substance:

other: 2-Aminoanthracene, 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA-

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

other:

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 μg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): no histidine or tryptophan supplemented, top agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Test substance and controls:
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 15 minutes at 40°C on the day of each experiment.
Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10E-4 microns.
Vehicle and positive controls were used in parallel with the test material.
In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix.

S9-Mix:
S9 was prepared in-house on 28 April 2001 and 04 August 2001 from the livers of male Sprague-Dawley rats weighing ~ 250g. These had each orally received three consecutive daily doses of phenobarbitone/b-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolise the indirect mutagens 2-Aminoanthracene and Benzo(a)pyrene. The S9 was stored at -196ºC.
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test. The S9-mix used in both experiments was shown to be sterile.
- S9: 5.0 ml
- 1.65 M KCl/0.4 M MgCl2: 1.0 mL
- 0.1 M Glucose-6-phosphate: 2.5 mL
- 0.1 M NADPH: 2.0 mL
- 0.1 M NADH: 2.0 mL
- 0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
- Sterile distilled water: 12.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from Fred Baker Scientific.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E9 bacteria per mL.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

no data on statistical methods employed was available.

Results and discussion

Test results

Test results 1

Species / strain:

other: TA 100 and WP2urvA-

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material was toxic at and above 1500 and 5000 μg/plate to the strains of bacteria used, TA100 and WP2uvrA-, respectively.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9-mix, at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9-mix, at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
The dose range was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose (15 μg/plate) was used to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out. The test material was toxic at and above 1500 and 5000 μg/plate to the strains of bacteria used, TA100 and WP2uvrA-, respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes and results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9-mix, at 5000 μg/plate. No toxicity was observed to Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Remarks on result:

other: other: Preliminary Toxicity Study

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 and Table 4 for Experiment 1 and Table 5 and Table 6 for Experiment 2.

A history profile of vehicle and positive control values for 1999 and 2000 is presented in Table 7.

Table 3: Test Results: Experiment 1 - Without Metabolic Activation
TEST PERIOD		FROM: 07 SEPTEMBER 2001					TO:10SEPTEMBER2001
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	67	(72) 7.0#	13	(13) 1.0	31	(23) 6.7	35	(26) 7.9	12	(12) 1.0
		69		14		20		20		11
		80		12		19		23		13
-	15	81	(75) 6.0	10	(11) 1.0	22	(20) 1.7	12	(18) 6.0	6	(8) 2.0
		74		11		19		24		8
		69		12		19		19		10
-	50	111	(85) 24.2	14	(14) 3.0	19	(21) 1.5	21	(20) 1.2	14	(11) 3.1
		63		17		21		21		8
		81		11		22		19		12
-	150	80	(82) 7.6	13	(13) 1.5	19	(16) 3.8	21	(19) 4.0	8	(10) 3.8
		90		15		12		21		14
		75		12		18		14		7
-	500	62	(70) 11.9	11	(11) 0.0	19	(18) 1.7	16	(18) 2.9	16	(10) 6.0
		65		11		16		16		11
		84		11		19		21		4
-	1500	91	(78) 15.9	13	(13) 3.0	20	(17) 4.6	22	(23) 1.7	9	(7) 2.1
		82		16		20		22		6
		60		10		12		25		5
-	5000	34S	(28) 6.7	8S	(8) 1.5	19	(17) 3.5	17S	(17) 2.5	0V	(0) 0.0
		21S		6S		13		15S		0V
		30S		9S		19		20S		0V
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
S9-Mix
	No. colonies per plate	555	(564) 19.2	676	(527) 230.3	733	(745) 12.5	149	(144) 5.6	1456	(1467) 170.3
-		586		262		745		138		1303
		551		644		758		145		1643
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S        Sparse bacterial background lawn
V        Very weak bacterial background lawn
#         Standard deviation

Table 4: Test Results: Experiment 1 - With Metabolic Activation
TEST PERIOD		FROM: 07 SEPTEMBER 2001					TO:10SEPTEMBER2001
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	74	(71) 10.3#	12	(12) 2.0	15	(19) 5.1	23	(30) 7.5	20	(11) 8.2
		60		10		18		38		9
		80		14		25		29		4
+	15	77	(75) 2.1	13	(12) 1.0	20	(20) 0.6	35	(28) 7.0	10	(6) 3.5
		74		11		21		28		4
		73		12		20		21		4
+	50	82	(84) 8.2	14	(14) 2.5	18	(19) 4.0	27	(30) 3.0	9	(9) 1.5
		77		16		23		33		7
		93		11		15		30		10
+	150	78	(83) 4.6	13	(15) 1.5	10	(20) 8.7	24	(32) 7.4	12	(11) 2.3
		87		15		26		35		12
		84		16		24		38		8
+	500	86	(80) 14.9	18	(14) 3.8	29	(24) 5.0	27	(29) 4.0	10	(9) 3.1
		91		11		19		34		6
		63		12		23		27		12
+	1500	86	(83) 2.9	11	(11) 1.0	19	(18) 1.0	34	(34) 3.5	9	(6) 2.5
		81		12		18		38		4
		81		10		17		31		6
+	5000	0V	(0) 0.0	8S	(7) 1.0	26	(25) 3.1	26	(28) 2.9	0V	(0) 0.0
		0V		6S		28		26		0V
		0V		7S		22		31		0V
Positive controls	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
S9-Mix
	No. colonies per plate	1908	(1681)201.3	512	(496) 14.2	762	(787) 21.8	264	(285) 21.5	243	(266) 20.0
+		1525		491		802		285		281
		1609		485		797		307		273
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
S        Sparse bacterial background lawn
V        Very weak bacterial background lawn
#         Standard deviation

Table 5: Test Results: Experiment 2 - Without Metabolic Activation
TEST PERIOD		FROM:18SEPTEMBER2001					TO:21SEPTEMBER2001
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	123	(135) 11.5#	11	(13) 1.5	18	(18) 3.5	14	(16) 2.8	12	(11) 1.7
		135		13		15		C		9
		146		14		22		18		12
-	15	138	(134) 4.6	15	(13) 4.0			24	(24) 3.0	8	(7) 1.7
		129		15		N/T		27		5
		135		8				21		8
-	50	108	(117) 7.8	14	(12) 2.1	19	(18) 1.0	13	(16) 5.8	10	(11) 2.6
		121		10		18		13		14
		122		11		17		23		9
-	150	121	(133) 10.4	9	(11) 2.5	14	(17) 4.6	15	(18) 3.1	9	(9) 1.5
		141		14		22		21		7
		136		11		14		17		10
-	500	125	(131) 15.3	14	(16) 8.2	15	(17) 5.7	C	(18) 1.4	5	(9) 3.2
		148		9		12		19		10
		119		25		23		17		11
-	1500	120	(131) 11.5	10	(8) 1.5	11	(14) 4.4	10	(13) 3.1	7	(9) 2.1
		143		7		12		16		11
		130		8		19		12		10
-	5000	58S	(37) 19.8	2S	(4) 1.5	18	(21) 2.5	14	(21) 7.5	0V	(0) 0.0
		33S		5S		23		20		0V
		19S		4S		21		29		0V
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Concentration (µg/plate)	3		5		2		0.2		80
S9-Mix
	No. colonies per plate	659	(681) 33.0	189	(228) 39.0	570	(691) 113.9	125	(134) 12.1	1217	(1372) 248.0
-		665		228		708		130		1241
		719		267		796		148		1658
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
C Contaminated
N/T     Not tested at this dose level
S        Sparse bacterial background lawn
V        Very weak bacterial background lawn
#         Standard deviation

Table 6: Test Results: Experiment 2 - With Metabolic Activation
TEST PERIOD		FROM: 18 SEPTEMBER 2001					TO:21SEPTEMBER2001
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	155	(157) 11.7#	12	(10) 2.9	20	(21) 1.2	32	(29) 7.0	12	(13) 2.1
		147		12		22		21		15
		170		7		22		34		11
+	15	150	(148) 4.9	9	(12) 3.6			18	(24) 6.6	19	(17) 3.2
		142		11		N/T		23		18
		151		16				31		13
+	50	147	(137) 13.8	11	(9) 2.1	15	(20) 6.2	28	(27) 1.5	16	(18) 2.5
		142		7		27		27		21
		121		10		18		25		18
+	150	132	(137) 14.2	10	(11) 3.2	28	(25) 2.9	32	(28) 5.3	25	(18) 6.5
		126		15		23		22		12
		153		9		23		30		18
+	500	145	(139) 9.0	9	(8) 1.2	26	(25) 3.6	27	(26) 3.2	9	(11) 2.5
		129		9		28		28		11
		144		7		21		22		14
+	1500	110	(115) 5.0	9	(7) 1.5	15	(18) 3.8	15	(19) 5.5	10	(13) 2.5
		114		6		16		16		13
		120		7		22		25		15
+	5000	0V	(0) 0.0	0V	(0) 0.0	22	(22) 5.5	22	(15) 6.4	0T	(0) 0.0
		0V		0V		17		11		0T
		0V		0V		28		11		0T
Positive controls	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
S9-Mix
	No. colonies per plate	1454	(1440) 33.9	471	(460) 9.6	1070	(1203) 137.8	266	(312) 41.5	273	(297) 32.5
+		1401		453		1193		347		334
		1464		456		1345		322		284
2AA 2-Aminoanthracene
BP Benzo(a)pyrene

N/T     Not tested at this dose level

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#         Standard deviation

The test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9-mix, at 5000 μg/plate. No toxicity was observed to Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 7: history profile of vehicle and positive control values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 1999
Strain	TA100			TA98			TA1535		TA1538		TA1537			WP2uvrA^-		TA102
S9-Mix	-S9	+S9		-S9		+S9	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9		+S9
Mean	102	107		24		31	22	16	13	19	11	17		23	27	263		298
SD	19	20		6		7	6	4	5	5	3	4		5	6	42		32
Min	57	63		10		12	10	7	5	8	3	5		10	11	188		222
Max	179	186		46		56	40	35	32	31	25	26		52	46	352		363
Values	751	592		646		510	645	488	57	31	634	481		641	495		89	37
POSITIVE CONTROL VALUES1999
Strain	TA100			TA98			TA1535		TA1538		TA1537			WP2uvrA^-		TA102
S9-Mix	-S9	+S9		-S9		+S9	-S9	+S9	-S9	+S9	-S9	+S9		-S9	+S9	-S9		+S9
Mean	549	1019		146		312	368	245	327	446	1017	345		787	598	882		737
SD	176	378		32		129	237	60	157	184	307	152		251	232	186		117
Min	238	406		95		113	104	123	113	215	350	112		200	208	583		509
Max	1170	2060		290		717	1229	467	653	837	2031	870		1760	1294	1673		1261
Values	161	160		162		160	158	157	27	27	159	158		153	152	52		51
COMBINED VEHICLE AND UNTREATED CONTROL VALUES2000
Strain S9-Mix	TA100		TA1535			WP2uvrA^-		TA102	TA98		TA1537		TA1538		WP2uvrA^-p1im101		TA97a
	-S9	+S9	-S9		+S9	-S9	+S9	-S9 +S	9-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	115	118	22		16	22	24	284 31;	> 23	32	12	16	14	31	222	198	102	125
SD	22	22	5		4	5	6	41 35	6	6	4	4	6	3	99	42	14	4
Min	63	63	8		9	8	10	193 22/	I11	13	4	8	8	29	113	168	82	122
Max	198	181	40		31	45	47	38137^-	J55	63	25	27	21	33	377	228	123	127
Values	801	621	761		588	706	540	149 81	764	594	749	575	6	2	5	2	6	2
POSITIVE CONTROL VALUES 2000
Strain S9-Mix	TA100		TA1535			WP2i	vrA^-	TA102	TA	98	TA15	37	TA	1538	WP2u p1m	vrA^-01	TA	97a
	-S9	+S9	-S9		+S9	-S9	+S9	-S9 +S	9-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	488	1620	382		278	663	664	877 79	4 138	259	1071	466	225	607	2089	1389	261	685
SD	137	490	260		89	243	212	137 12	3 34	70	327	121	28	40	175	967	1	141
Min	234	443	125		128	203	185	522 38	8 89	129	290	191	193	568	1965	705	260	585
Max	1190	3079	1843		630	1520	1534	1161 109	0 262	506	2160	768	245	648	2213	2073	261	785
Values	172	172	172		170	166	166	69 7	0 173	172	171	170	3	3	2	2	2	2
SD=Standard deviation
Min =Minimal value
Max=Maximum value

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The study was performed according to the OECD Guideline 471, EU Method B13/14 and EPA OPPTS 870.5100 without deviations and considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The Salmonella/microsome test, employing doses up to 5000 µg per plate, showed Caprolactam disulphide to produce no bacteriotoxic effects in Escherichia coli strain WP2uvrA-. A Cytotoxic effect was seen in the majority of the Salmonella strains at 5000 µg/plate. The test material did not induce significant increases in the frequency of revertant colonies in the bacterial strains used. No indications of mutagenic effects of Caprolactam disulphide could be found at assessable doses up to 5000 µg per plate in any of the strains tested. The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Caprolactam disulfide was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants (TA 1535, TA 1537, TA 100 and TA 98 ) and Escherichia coli strain WP2uvrA-. The study was performed according to the OECD Guideline 471, EU Method B13/14 and EPA OPPTS 870.5100 without deviations and considered to be of the highest quality (reliability Klimisch 1). The bacteria were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose (15 μg/plate) was used to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. The positive controls N-ethyl-N'-nitro-N-nitrosoguanidine(ENNG, 9-Aminoacridine (9AA) and 4-Nitroquinoline-1-oxide (4NQO) had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. So all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9 -mix, at 5000 μg/plate. No toxicity was observed to Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.