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Registration Dossier
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EC number: 946-413-8 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).
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:
- 26 July - 10 September 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD Guideline 471 without any deviation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- dated 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- dated August 1998
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1003398942
- Expiration date of the lot/batch: 18 May 2018
- Purity test date: 18 July 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark (closed containers)
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions were prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 10 minutes at approximately 40°C. The test item was confirmed as a UVCB product and, therefore, no purity correction was required. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period. - Target gene:
- histidine locus for Salmonella strains and tryptophan for E. coli strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- 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:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: In solubility checks performed in house, the test item was noted as insoluble in dimethyl sulphoxide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL but fully soluble in dimethyl formamide at 50 mg/mL. Dimethyl formamide was selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system. - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours for both Experiments
CONTROLS:
- Vehicle/solvent control: DMF
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
NUMBER OF REPLICATIONS: Triplicate
- OTHER: 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. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count. - Rationale for test conditions:
- The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (i.e. maximum recommended dose level). The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item dose levels per bacterial strain were selected in the second mutation test 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.
- Evaluation criteria:
- Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it will be considered to exhibit mutagenic activity in this test system. (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- None
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (small, flake-like in appearance) was noted in both experiments with and without S9-mix from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
MUTAGENICITY
- The vehicle (dimethyl formamide (DMF)) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).
- No significant 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 Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6
OTHERS:
- Prior to use, the master 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.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. - Conclusions:
- Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (dimethyl formamide (DMF)) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).
A test item precipitate (small, flake-like in appearance) was noted in both experiments with and without S9-mix from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
No significant 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 Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
Reference
Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
Experiment 1 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
75 |
|
22 |
|
19 |
|
34 |
|
10 |
|
72 |
(80) |
19 |
(21) |
15 |
(18) |
40 |
(37) |
9 |
(9) |
94 |
|
22 |
|
20 |
|
36 |
|
8 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
89 |
|
18 |
|
19 |
|
33 |
|
13 |
|
101 |
(96) |
20 |
(20) |
20 |
(18) |
39 |
(34) |
13 |
(13) |
99 |
|
23 |
|
15 |
|
31 |
|
12 |
|
Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 29 August 2017 |
To: 01 September 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 (DMF) |
111 94 110 |
(105) 9.5# |
16 21 13 |
(17) 4.0 |
22 24 16 |
(21) 4.2 |
21 33 33 |
(29) 6.9 |
9 11 10 |
(10) 1.0 |
||
1.5 µg |
90 90 68 |
(83) 12.7 |
14 25 12 |
(17) 7.0 |
17 28 17 |
(21) 6.4 |
23 29 32 |
(28) 4.6 |
5 15 4 |
(8) 6.1 |
||
5 µg |
89 81 81 |
(84) 4.6 |
16 14 17 |
(16) 1.5 |
19 21 20 |
(20) 1.0 |
23 27 28 |
(26) 2.6 |
14 8 10 |
(11) 3.1 |
||
15 µg |
121 101 116 |
(113) 10.4 |
14 11 14 |
(13) 1.7 |
11 22 25 |
(19) 7.4 |
19 34 28 |
(27) 7.5 |
9 8 7 |
(8) 1.0 |
||
50 µg |
96 95 113 |
(101) 10.1 |
16 19 10 |
(15) 4.6 |
22 19 20 |
(20) 1.5 |
30 31 32 |
(31) 1.0 |
7 8 10 |
(8) 1.5 |
||
150 µg |
115 94 124 |
(111) 15.4 |
20 18 14 |
(17) 3.1 |
19 25 16 |
(20) 4.6 |
28 21 26 |
(25) 3.6 |
9 14 7 |
(10) 3.6 |
||
500 µg |
90 111 114 |
(105) 13.1 |
17 21 18 |
(19) 2.1 |
15 19 24 |
(19) 4.5 |
34 28 24 |
(29) 5.0 |
8 11 9 |
(9) 1.5 |
||
1500 µg |
89 P 94 P 107 P |
(97) 9.3 |
10 P 19 P 17 P |
(15) 4.7 |
21 P 20 P 18 P |
(20) 1.5 |
21 P 29 P 27 P |
(26) 4.2 |
12 P 9 P 8 P |
(10) 2.1 |
||
5000 µg |
117 P 86 P 96 P |
(100) 15.8 |
14 P 19 P 18 P |
(17) 2.6 |
19 P 17 P 24 P |
(20) 3.6 |
25 P 23 P 31 P |
(26) 4.2 |
10 P 9 P 11 P |
(10) 1.0 |
||
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 |
||||||||
517 413 507 |
(479) 57.4 |
583 747 701 |
(677) 84.6 |
991 917 1015 |
(974) 51.1 |
200 259 249 |
(236) 31.6 |
184 205 508 |
(299) 181.3 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Precipitate
#: Standard deviation
Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 29 August 2017 |
To: 01 September 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 (DMF) |
99 78 101 |
(93) 12.7# |
29 23 15 |
(22) 7.0 |
23 24 23 |
(23) 0.6 |
37 32 25 |
(31) 6.0 |
11 9 9 |
(10) 1.2 |
||
1.5 µg |
97 76 75 |
(83) 12.4 |
19 28 18 |
(22) 5.5 |
30 29 24 |
(28) 3.2 |
28 41 35 |
(35) 6.5 |
7 10 9 |
(9) 1.5 |
||
5 µg |
88 77 92 |
(86) 7.8 |
24 25 19 |
(23) 3.2 |
23 26 22 |
(24) 2.1 |
27 40 26 |
(31) 7.8 |
6 11 10 |
(9) 2.6 |
||
15 µg |
95 85 81 |
(87) 7.2 |
25 18 22 |
(22) 3.5 |
29 27 30 |
(29) 1.5 |
33 26 31 |
(30) 3.6 |
7 15 8 |
(10) 4.4 |
||
50 µg |
91 102 95 |
(96) 5.6 |
20 21 17 |
(19) 2.1 |
22 25 24 |
(24) 1.5 |
33 24 24 |
(27) 5.2 |
7 13 9 |
(10) 3.1 |
||
150 µg |
116 103 94 |
(104) 11.1 |
20 14 20 |
(18) 3.5 |
31 16 36 |
(28) 10.4 |
44 33 31 |
(36) 7.0 |
10 11 12 |
(11) 1.0 |
||
500 µg |
106 98 89 |
(98) 8.5 |
24 25 19 |
(23) 3.2 |
23 18 24 |
(22) 3.2 |
31 39 37 |
(36) 4.2 |
12 8 9 |
(10) 2.1 |
||
1500 µg |
91 P 86 P 100 P |
(92) 7.1 |
20 P 29 P 21 P |
(23) 4.9 |
26 P 21 P 19 P |
(22) 3.6 |
41 P 36 P 37 P |
(38) 2.6 |
7 P 10 P 9 P |
(9) 1.5 |
||
5000 µg |
79 P 90 P 98 P |
(89) 9.5 |
19 P 16 P 18 P |
(18) 1.5 |
28 P 30 P 17 P |
(25) 7.0 |
23 P 38 P 27 P |
(29) 7.8 |
12 P 7 P 11 P |
(10) 2.6 |
||
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 |
||||||||
1112 1058 1385 |
(1185) 175.3 |
225 248 220 |
(231) 14.9 |
380 380 401 |
(387) 12.1 |
207 197 199 |
(201) 5.3 |
451 499 533 |
(494) 41.2 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Precipitate
#: Standard deviation
Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 07 September 2017 |
To: 10 September 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 (DMF) |
72 67 75 |
(71) 4.0# |
16 26 17 |
(20) 5.5 |
17 17 22 |
(19) 2.9 |
39 36 23 |
(33) 8.5 |
22 16 16 |
(18) 3.5 |
||
15 µg |
63 68 74 |
(68) 5.5 |
20 28 22 |
(23) 4.2 |
24 15 11 |
(17) 6.7 |
22 21 36 |
(26) 8.4 |
19 10 12 |
(14) 4.7 |
||
50 µg |
80 71 62 |
(71) 9.0 |
25 18 21 |
(21) 3.5 |
24 26 18 |
(23) 4.2 |
26 23 27 |
(25) 2.1 |
20 11 16 |
(16) 4.5 |
||
150 µg |
70 64 78 |
(71) 7.0 |
18 20 21 |
(20) 1.5 |
27 18 22 |
(22) 4.5 |
26 24 28 |
(26) 2.0 |
13 15 14 |
(14) 1.0 |
||
500 µg |
73 70 74 |
(72) 2.1 |
19 19 11 |
(16) 4.6 |
19 25 19 |
(21) 3.5 |
31 33 28 |
(31) 2.5 |
17 21 27 |
(22) 5.0 |
||
1500 µg |
65 P 69 P 67 P |
(67) 2.0 |
15 P 14 P 18 P |
(16) 2.1 |
22 P 20 P 23 P |
(22) 1.5 |
28 P 22 P 31 P |
(27) 4.6 |
12 P 12 P 19 P |
(14) 4.0 |
||
5000 µg |
62 P 59 P 69 P |
(63) 5.1 |
16 P 24 P 18 P |
(19) 4.2 |
17 P 15 P 21 P |
(18) 3.1 |
27 P 24 P 25 P |
(25) 1.5 |
19 P 20 P 12 P |
(17) 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 |
||||||||
925 1037 1652 |
(1205) 391.4 |
2016 1867 2120 |
(2001) 127.2 |
724 763 664 |
(717) 49.9 |
318 345 335 |
(333) 13.7 |
118 118 120 |
(119) 1.2 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Precipitate
#: Standard deviation
Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 07 September 2017 |
To: 10 September 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 (DMF) |
68 70 69 |
(69) 1.0# |
23 26 28 |
(26) 2.5 |
15 19 20 |
(18) 2.6 |
38 38 32 |
(36) 3.5 |
16 13 20 |
(16) 3.5 |
||
15 µg |
69 69 52 |
(63) 9.8 |
25 14 19 |
(19) 5.5 |
21 28 31 |
(27) 5.1 |
31 29 30 |
(30) 1.0 |
13 14 14 |
(14) 0.6 |
||
50 µg |
56 71 71 |
(66) 8.7 |
20 18 17 |
(18) 1.5 |
24 19 20 |
(21) 2.6 |
30 40 36 |
(35) 5.0 |
9 20 19 |
(16) 6.1 |
||
150 µg |
69 69 65 |
(68) 2.3 |
14 14 20 |
(16) 3.5 |
25 24 25 |
(25) 0.6 |
34 41 32 |
(36) 4.7 |
7 12 12 |
(10) 2.9 |
||
500 µg |
73 78 80 |
(77) 3.6 |
22 16 17 |
(18) 3.2 |
34 30 22 |
(29) 6.1 |
32 33 34 |
(33) 1.0 |
13 9 15 |
(12) 3.1 |
||
1500 µg |
73 P 63 P 66 P |
(67) 5.1 |
22 P 20 P 22 P |
(21) 1.2 |
17 P 26 P 33 P |
(25) 8.0 |
32 P 23 P 36 P |
(30) 6.7 |
10 P 19 P 12 P |
(14) 4.7 |
||
5000 µg |
61 P 65 P 60 P |
(62) 2.6 |
18 P 25 P 27 P |
(23) 4.7 |
27 P 22 P 20 P |
(23) 3.6 |
24 P 39 P 35 P |
(33) 7.8 |
18 P 15 P 11 P |
(15) 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 |
||||||||
1178 1179 1197 |
(1185) 10.7 |
268 284 273 |
(275) 8.2 |
174 173 189 |
(179) 9.0 |
316 318 313 |
(316) 2.5 |
272 269 268 |
(270) 2.1 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
P: Precipitate
#: Standard deviation
Table 7.6.1/6:History Profile of Vehicle and Positive Control Values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values† |
274 |
278 |
504 |
285 |
26 |
13 |
461 |
229 |
526 |
299 |
506 |
282 |
42 |
51 |
39 |
49 |
|||||||||||||||||
Min |
60 |
61 |
7 |
7 |
222 |
278 |
10 |
12 |
11 |
10 |
4 |
6 |
87 |
98 |
89 |
93 |
|||||||||||||||||
Max |
166 |
175 |
31 |
29 |
376 |
388 |
58 |
43 |
45 |
46 |
27 |
27 |
237 |
254 |
174 |
177 |
|||||||||||||||||
Mean |
91 |
95 |
16 |
14 |
286 |
333 |
24 |
27 |
21 |
24 |
12 |
13 |
156 |
164 |
123 |
137 |
|||||||||||||||||
SD |
19.3 |
19.1 |
4.5 |
4.0 |
48.7 |
37.6 |
5.6 |
5.9 |
6.2 |
6.1 |
3.8 |
3.4 |
42.2 |
35.6 |
23.1 |
21.2 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2015 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
276 |
280 |
252 |
264 |
13 |
13 |
231 |
227 |
262 |
276 |
253 |
261 |
20 |
35 |
20 |
35 |
|
||||||||||||||||
Min |
222 |
250 |
79 |
118 |
953 |
673 |
116 |
103 |
100 |
78 |
164 |
97 |
430 |
494 |
745 |
325 |
|
||||||||||||||||
Max |
2266 |
2402 |
2779 |
457 |
3140 |
1655 |
2769 |
550 |
502 |
705 |
2318 |
823 |
1696 |
2264 |
3662 |
1174 |
|
||||||||||||||||
Mean |
614 |
927 |
472 |
246 |
2303 |
1093 |
792 |
266 |
222 |
218 |
911 |
336 |
761 |
1461 |
2257 |
569 |
|
||||||||||||||||
SD |
260.6 |
452.5 |
434.8 |
55.7 |
815.2 |
376.5 |
342.1 |
97.7 |
70.2 |
107.6 |
412.4 |
135.7 |
350.0 |
382.0 |
790.7 |
220.3 |
|
||||||||||||||||
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values |
399 |
401 |
758 |
393 |
60 |
30 |
690 |
345 |
788 |
415 |
762 |
398 |
32 |
32 |
16 |
24 |
|||||||||||||||||
Min |
63 |
66 |
8 |
8 |
216 |
221 |
10 |
13 |
8 |
12 |
3 |
4 |
97 |
104 |
78 |
52 |
|||||||||||||||||
Max |
154 |
156 |
34 |
39 |
340 |
375 |
53 |
53 |
49 |
51 |
24 |
23 |
268 |
243 |
148 |
166 |
|||||||||||||||||
Mean |
90 |
93 |
15 |
15 |
268 |
310 |
22 |
27 |
21 |
25 |
12 |
13 |
161 |
159 |
118 |
110 |
|||||||||||||||||
SD |
14.5 |
14.3 |
4.5 |
5.2 |
26.4 |
31.1 |
5.8 |
6.3 |
4.8 |
5.7 |
3.5 |
3.5 |
39.2 |
32.3 |
17.0 |
29.3 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2016 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
409 |
406 |
381 |
386 |
30 |
28 |
341 |
335 |
388 |
385 |
379 |
381 |
14 |
24 |
8 |
16 |
|
||||||||||||||||
Min |
221 |
284 |
84 |
92 |
897 |
629 |
107 |
102 |
100 |
96 |
95 |
101 |
445 |
574 |
1674 |
372 |
|
||||||||||||||||
Max |
2222 |
2863 |
2994 |
879 |
2326 |
2140 |
1611 |
637 |
449 |
4357 |
1413 |
639 |
1117 |
1855 |
2823 |
945 |
|
||||||||||||||||
Mean |
724 |
1264 |
854 |
240 |
1633 |
950 |
718 |
240 |
186 |
188 |
406 |
290 |
743 |
1271 |
2379 |
535 |
|
||||||||||||||||
SD |
320.4 |
562.9 |
664.9 |
62.1 |
564.5 |
382.7 |
338.6 |
98.2 |
49.8 |
230.8 |
227.0 |
92.7 |
214.6 |
326.5 |
426.2 |
143.3 |
|
||||||||||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Table 7.6/1: Summary of genotoxicity test
Test n° |
Test / Guideline Reliability |
Focus |
Strains tested |
Metabolic activation |
Test concentration |
Statement |
1
Thompson, 2017 |
Ames Test (OECD 471) K, rel. 1 |
Gene mutation |
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA- |
-S9 +S9 |
Up to cytotoxic or highest recommended concentration |
-S9 : non mutagenic +S9 : non mutagenic |
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (dimethyl formamide (DMF)) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).
A test item precipitate (small, flake-like in appearance) was noted in both experiments with and without S9-mix from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
No significant 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 Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
Justification for classification or non-classification
Harmonized classification:
The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.
Self-classification:
Based on the available information, no classification is proposed.
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