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Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 / strainopen allclose all
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 / 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
Controlsopen allclose all
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-
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
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
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-
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 resultsopen allclose all
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
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
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
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:
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.