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EC number: 695-735-2 | CAS number: 68489-14-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The key study according to OECD TG 471 (2017) was performed to evaluate WS-5 (CAS 68489-14-5) for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of following strains of bacteria:Salmonella typhimurium: TA1537, TA98, TA1535, TA100 and Escherichia coli WP2uvrA strain.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) in both first and second experiments.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Also, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in both first and second experiments.
Based on these results, WS-5 (CAS 68489-14-5) was considered to be non-mutagenic under the conditions of this test and OECD TG 471.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 May 2017 - 15 June 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Qualifier:
- according to guideline
- Guideline:
- other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Test item: WS-5
- EC Number: 695-735-2
- CAS Number: 68489-14-5
- Source and lbatch No.of test material: 83100003
- Expiration date of the batch: 12 September 2018
- Purity test date: 99.86%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
TEST ITEM PREPARATION
WS-5 was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 05 minutes at 40 °C on
the day of each experiment. No correction was required for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. - Target gene:
- Strain Genotype
TA1537 his C 3076; rfa-; uvrB-:
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-:
TA100 his G 46; rfa-; uvrB-;R-factor
WP2uvrA trp-; uvrA-: - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- The test item was tested using the following method according to the OECD Guidelines for Testing of Chemicals No. 471 (1997) “Bacterial Reverse Mutation Test”. The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: In solubility checks performed in-house, the test item was noted as insoluble in sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- (untreated) controls
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 4-Nitroquinoline-1-oxide (4NQO); 2-Aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Top agar was prepared using 0.6% Bacto agar (lot number 6147883 03/21) 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 SGL Ltd (lot number 44572 07/17).
Prior to use, the master strains (all bacterial strains) were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
DURATION
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method was performed with and without metabolic activation.
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain.
All of the plates (with and without of metabolic activation) were incubated at 37 ± 3 °C for approximately 48 hours.
The scoring for the presence of revertant colonies was performed using an automated colony counting system. A number of manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.
Test for Mutagenicity: Experiment 2 – Pre-Incubation Method was performed using the pre-incubation method in the presence and absence of metabolic activation
The dose range was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
DETERMINATION OF CYTOTOXICITY in Experiment 1 and 2 all plates were viewed microscopically for evidence of thinning (toxicity). - Rationale for test conditions:
- The purpose of the study was to evaluate WS-5 (CAS 68489-14-5) for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria according to the OECD Guidelines for Testing of Chemicals No. 471 (1997) “Bacterial Reverse Mutation Test”.
- Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- 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
- Conclusions:
- Based on the results, WS-5 (CAS 68489-14-5) was considered to be non-mutagenic under the conditions of this AMES test.
WS-5 is not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. - Executive summary:
The purpose of the study performed according to OECD TG 471 was to evaluate WS-5 (CAS 68489-14-5) for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of following strains of bacteria:Salmonella typhimurium: TA1537, TA98, TA1535, TA100 and Escherichia coli WP2uvrA strain.
The maximum dose level of the test item, WS-5 in the first experiment (Plate Incorporation Method) was selected as the maximum recommended dose level of 5000 µg/plate according to OECD TG 471. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed.
There was no visible reduction in the growth of the bacterial background lawn at any dose level tested, either in the presence or absence of metabolic activation (S9-mix). There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), as well.
As the result of the first Experiment was deemed negative, second Experiment was performed using the pre-incubation method in the presence and absence of metabolic activation. Six test item dose levels per bacterial strain were selected in the second mutation test (Pre-Incubation Method):15, 50, 150, 500, 1500 and 5000 µg/plate in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Similarly, to the first experiment, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) in the second experiment.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
Also, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in the second Experiment .
Conclusions:
Based on these results, WS-5 (CAS 68489-14-5) was considered to be non-mutagenic under the conditions of this test. WS-5 is not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Reference
Table 1. Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 02 June 2017 |
To: 05 June 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
101 67 74 |
(81) 18.0# |
12 17 10 |
(13) 3.6 |
30 39 29 |
(33) 5.5 |
32 24 29 |
(28) 4.0 |
13 27 16 |
(19) 7.4 |
||
1.5 µg |
101 105 63 |
(90) 23.2 |
18 13 15 |
(15) 2.5 |
19 36 32 |
(29) 8.9 |
19 29 20 |
(23) 5.5 |
17 15 9 |
(14) 4.2 |
||
5 µg |
64 71 62 |
(66) 4.7 |
10 16 14 |
(13) 3.1 |
34 34 29 |
(32) 2.9 |
14 12 9 |
(12) 2.5 |
13 13 16 |
(14) 1.7 |
||
15 µg |
63 73 68 |
(68) 5.0 |
12 13 8 |
(11) 2.6 |
26 18 39 |
(28) 10.6 |
18 19 16 |
(18) 1.5 |
19 16 11 |
(15) 4.0 |
||
50 µg |
84 67 73 |
(75) 8.6 |
12 12 11 |
(12) 0.6 |
27 32 21 |
(27) 5.5 |
16 15 9 |
(13) 3.8 |
10 9 15 |
(11) 3.2 |
||
150 µg |
75 61 82 |
(73) 10.7 |
12 12 9 |
(11) 1.7 |
45 32 31 |
(36) 7.8 |
21 19 23 |
(21) 2.0 |
16 12 14 |
(14) 2.0 |
||
500 µg |
62 71 67 |
(67) 4.5 |
15 14 10 |
(13) 2.6 |
31 27 42 |
(33) 7.8 |
24 10 19 |
(18) 7.1 |
10 6 8 |
(8) 2.0 |
||
1500 µg |
65 72 68 |
(68) 3.5 |
10 14 8 |
(11) 3.1 |
22 29 22 |
(24) 4.0 |
16 8 15 |
(13) 4.4 |
10 9 11 |
(10) 1.0 |
||
5000 µg |
85 65 62 |
(71) 12.5 |
11 8 12 |
(10) 2.1 |
41 36 35 |
(37) 3.2 |
11 20 11 |
(14) 5.2 |
11 19 18 |
(16) 4.4 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
299 338 326 |
(321) 20.0 |
279 385 360 |
(341) 55.4 |
347 290 346 |
(328) 32.6 |
202 191 209 |
(201) 9.1 |
144 240 224 |
(203) 51.4 |
|||
Table 2. Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 02 June 2017 |
To: 05 June 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
74 90 75 |
(80) 9.0# |
17 13 16 |
(15) 2.1 |
37 26 42 |
(35) 8.2 |
28 13 29 |
(23) 9.0 |
7 11 13 |
(10) 3.1 |
||
1.5 µg |
83 65 63 |
(70) 11.0 |
16 13 12 |
(14) 2.1 |
44 34 35 |
(38) 5.5 |
29 26 31 |
(29) 2.5 |
12 19 12 |
(14) 4.0 |
||
5 µg |
72 86 67 |
(75) 9.8 |
12 13 8 |
(11) 2.6 |
31 41 39 |
(37) 5.3 |
20 21 27 |
(23) 3.8 |
12 10 6 |
(9) 3.1 |
||
15 µg |
72 65 64 |
(67) 4.4 |
18 16 21 |
(18) 2.5 |
42 43 23 |
(36) 11.3 |
23 32 23 |
(26) 5.2 |
11 13 4 |
(9) 4.7 |
||
50 µg |
63 67 65 |
(65) 2.0 |
10 10 17 |
(12) 4.0 |
29 32 30 |
(30) 1.5 |
22 21 13 |
(19) 4.9 |
11 5 6 |
(7) 3.2 |
||
150 µg |
72 86 87 |
(82) 8.4 |
12 13 12 |
(12) 0.6 |
30 32 31 |
(31) 1.0 |
21 17 21 |
(20) 2.3 |
5 6 10 |
(7) 2.6 |
||
500 µg |
64 90 74 |
(76) 13.1 |
10 11 11 |
(11) 0.6 |
28 20 41 |
(30) 10.6 |
15 12 13 |
(13) 1.5 |
3 11 8 |
(7) 4.0 |
||
1500 µg |
82 76 76 |
(78) 3.5 |
11 22 10 |
(14) 6.7 |
39 26 36 |
(34) 6.8 |
10 17 21 |
(16) 5.6 |
6 7 8 |
(7) 1.0 |
||
5000 µg |
102 86 98 |
(95) 8.3 |
16 16 19 |
(17) 1.7 |
31 33 34 |
(33) 1.5 |
29 19 19 |
(22) 5.8 |
14 7 4 |
(8) 5.1 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1195 929 1077 |
(1067) 133.3 |
361 345 370 |
(359) 12.7 |
309 311 342 |
(321) 18.5 |
240 231 229 |
(233) 5.9 |
312 351 359 |
(341) 25.1 |
|||
Table 3. Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation).
Test Period |
From: 12 June 2017 |
To: 15 June 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
80 73 68 |
(74) 6.0# |
11 11 7 |
(10) 2.3 |
34 36 36 |
(35) 1.2 |
16 13 16 |
(15) 1.7 |
7 10 7 |
(8) 1.7 |
||
15 µg |
82 81 78 |
(80) 2.1 |
7 9 11 |
(9) 2.0 |
33 45 26 |
(35) 9.6 |
10 11 11 |
(11) 0.6 |
8 7 5 |
(7) 1.5 |
||
50 µg |
73 77 68 |
(73) 4.5 |
7 11 12 |
(10) 2.6 |
26 31 32 |
(30) 3.2 |
14 13 13 |
(13) 0.6 |
5 11 6 |
(7) 3.2 |
||
150 µg |
81 63 80 |
(75) 10.1 |
11 8 11 |
(10) 1.7 |
39 46 35 |
(40) 5.6 |
15 16 18 |
(16) 1.5 |
11 12 4 |
(9) 4.4 |
||
500 µg |
83 78 67 |
(76) 8.2 |
14 11 11 |
(12) 1.7 |
34 28 28 |
(30) 3.5 |
14 14 11 |
(13) 1.7 |
11 4 11 |
(9) 4.0 |
||
1500 µg |
87 84 88 |
(86) 2.1 |
10 9 12 |
(10) 1.5 |
32 42 40 |
(38) 5.3 |
15 21 10 |
(15) 5.5 |
11 10 3 |
(8) 4.4 |
||
5000 µg |
83 68 80 |
(77) 7.9 |
14 11 11 |
(12) 1.7 |
42 37 36 |
(38) 3.2 |
7 18 10 |
(12) 5.7 |
10 11 4 |
(8) 3.8 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
1061 966 1193 |
(1073) 114.0 |
1372 1469 1359 |
(1400) 60.1 |
827 783 627 |
(746) 105.1 |
244 331 293 |
(289) 43.6 |
158 117 239 |
(171) 62.1 |
|||
Table 4. Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation).
Test Period |
From: 12 June 2017 |
To: 15 June 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
75 83 80 |
(79) 4.0# |
14 8 14 |
(12) 3.5 |
47 46 44 |
(46) 1.5 |
29 18 14 |
(20) 7.8 |
6 11 16 |
(11) 5.0 |
||
15 µg |
76 81 65 |
(74) 8.2 |
14 15 7 |
(12) 4.4 |
39 51 48 |
(46) 6.2 |
26 23 17 |
(22) 4.6 |
13 7 13 |
(11) 3.5 |
||
50 µg |
66 83 72 |
(74) 8.6 |
12 8 10 |
(10) 2.0 |
43 46 49 |
(46) 3.0 |
19 16 11 |
(15) 4.0 |
4 14 12 |
(10) 5.3 |
||
150 µg |
67 79 71 |
(72) 6.1 |
7 10 10 |
(9) 1.7 |
45 36 52 |
(44) 8.0 |
15 15 20 |
(17) 2.9 |
11 10 14 |
(12) 2.1 |
||
500 µg |
80 66 64 |
(70) 8.7 |
12 14 8 |
(11) 3.1 |
44 49 52 |
(48) 4.0 |
20 15 22 |
(19) 3.6 |
9 9 7 |
(8) 1.2 |
||
1500 µg |
76 63 63 |
(67) 7.5 |
8 8 8 |
(8) 0.0 |
49 42 44 |
(45) 3.6 |
15 18 12 |
(15) 3.0 |
12 5 9 |
(9) 3.5 |
||
5000 µg |
78 73 82 |
(78) 4.5 |
11 8 13 |
(11) 2.5 |
58 40 47 |
(48) 9.1 |
17 12 12 |
(14) 2.9 |
11 11 5 |
(9) 3.5 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1145 1166 1315 |
(1209) 92.7 |
194 227 239 |
(220) 23.3 |
223 234 259 |
(239) 18.4 |
120 135 155 |
(137) 17.6 |
233 251 256 |
(247) 12.1 |
|||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
The available data on AMES test performed accroding to OECD TG 471, indicated that classification for genotoxicity is not warranted.
WS-5 is not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
