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EC number: 203-214-4 | CAS number: 104-57-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
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:
- from 2015-11-18 to 2015-11-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- ENVIGO CRS GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: Sodium azide (NaN3; TA 1535, TA 100), 4-nitro-o-phenylene-diamine (4-NOPD; TA 1537, TA 98), methyl methane sulfonate (MMS; WP2 uvrA); +S9: 2-aminoanthracene (2-AA; TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- Experiment I: in agar (plate incorporation)
- Experiment II: preincubation
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Key result
- Species / strain:
- 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:
- cytotoxicity
- Remarks:
- In experiment II toxic effects occured in strains TA 1537, TA 98 and TA 100.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in experiment II with and without S9 mix. The undissolved particles had no influence on the data recording.
COMPARISON WITH HISTORICAL CONTROL DATA: Done
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation in experiment I. In experiment II toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98, and TA 100. - Conclusions:
- Under the experimental conditions reported, the test item is considered negative in the Ames test.
- Executive summary:
The genetic toxicity of the test item was tested according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA (Ames test). The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate
No substantial increase in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Therefore, the test substance is considered to be non-mutagenic in this reverse mutation assay.
Reference
Table 1: Toxic effects, evidence as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate)
Strain |
Experiment I |
Experiment II |
||
|
Without S9 mix |
With S9 mix |
Without S9 mix |
With S9 mix |
TA 1535 |
/ |
/ |
/ |
/ |
TA 1537 |
/ |
/ |
5000 |
5000 |
TA 98 |
/ |
/ |
5000 |
/ |
TA 100 |
/ |
/ |
2500-5000 |
/ |
WP2 uvrA |
/ |
/ |
/ |
/ |
/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor 0.5)
Table 2: Summary of Experiment I (plate incorporation)
Test group |
Dose level (µg/plate) |
Revertant Colony Counts (Mean +/- SD) |
|||||||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
DMSO |
|
11+/- 2 |
16 +/- 3 |
12 +/- 3 |
12 +/- 4 |
23 +/- 6 |
32 +/- 6 |
141 +/- 14 |
146 +/- 4 * |
39 +/- 2 |
46 +/- 3 |
Untreated |
|
8 +/- 3 |
12 +/- 4 |
10 +/- 4 |
17 +/- 3 |
23 +/- 2 |
34 +/- 9 |
154 +/- 9 |
182 +/- 11 * |
42 +/- 9 |
49 +/- 8 |
Test item |
3 |
9 +/- 3 |
16 +/- 4 |
10 +/- 3 |
12 +/- 3 |
25 +/- 5 |
38 +/- 7 |
133 +/- 29 |
134 +/- 16* |
36 +/- 6 |
49 +/- 4 |
10 |
12 +/-3 |
11 +/- 4 |
12 +/- 5 |
16 +/- 6 |
21 +/- 1 |
52 +/- 5 |
122 +/- 8 |
123 +/- 13* |
38 +/- 4 |
53 +/- 10 |
|
33 |
14 +/- 2 |
15 +/- 2 |
9 +/-3 |
11 +/- 3 |
28 +/- 1 |
36 +/- 4 |
124 +/- 9 |
148 +/- 26* |
44 +/- 1 |
45 +/- 8 |
|
100 |
12 +/- 2 |
13 +/- 1 |
10 +/- 1 |
12 +/- 7 |
24 +/- 6 |
39 +/- 3 |
138 +/- 22 |
142 +/- 7* |
42 +/- 5 |
50 +/- 5 |
|
333 |
11 +/- 5 |
11 +/- 2 |
8 +/- 2 |
12 +/- 2 |
21 +/- 1 |
45 +/- 8 |
132 +/- 14 |
143 +/- 15* |
40 +/- 8 |
53 +/- 8 |
|
1000 |
11 +/- 3 |
11 +/- 3 |
11 +/- 3 |
13 +/- 4 |
22 +/- 2 |
32 +/- 5 |
135 +/- 25 |
140 +/- 20* |
43 +/- 10 |
55 +/- 15 |
|
2500 |
9 +/- 1 |
10 +/- 2 |
8 +/- 2 |
13 +/- 4 |
19 +/- 5 |
35 +/- 9 |
84 +/- 17 |
121 +/- 17* |
40 +/- 10 |
48 +/- 4 |
|
5000 |
8 +/- 1 |
12 +/- 6 |
6 +/- 3 |
16 +/- 3 |
24 +/- 3 |
32 +/- 10 |
70 +/- 7 |
78 +/- 6* |
38 +/- 7 |
47 +/- 10 |
|
NaN3 |
10 |
1185+/- 65 |
|
|
|
|
|
2241 +/- 56 |
|
|
|
4-NOPD |
10 |
|
|
|
|
318 +/- 27 |
|
|
|
|
|
4-NOPD |
50 |
|
|
104 +/- 13 |
|
|
|
|
|
|
|
MMS |
2.0 |
|
|
|
|
|
|
|
|
742 +/- 48 |
|
2-AA |
2.5 |
|
359 +/- 23 |
|
185 +/- 13 |
|
4104 +/- 292 |
|
4899 +/- 171* |
|
|
2-AA |
10.0 |
|
|
|
|
|
|
|
|
|
331 +/- 12 |
Table 3: Summary of Experiment II (preincubation)
Test group |
Dose level (µg/plate) |
Revertant Colony Counts (Mean +/- SD) |
|||||||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
DMSO |
|
14 +/- 1 |
16 +/- 5 |
8 +/- 2 |
11 +/- 3 |
26 +/- 4 |
27 +/- 5 |
130 +/- 9 |
111 +/- 8 |
36 +/- 8 |
49 +/- 2 |
Untreated |
|
22 +/- 2 |
15 +/- 4 |
10 +/- 3 |
10 +/- 2 |
30 +/- 9 |
28 +/- 3 |
180 +/- 17 |
180 +/- 34 |
55 +/- 4 |
57 +/- 4 |
Test item |
33 |
11 +/- 4 |
15 +/- 7 |
9 +/- 5 |
9 +/- 4 |
21 +/- 5 |
31 +/- 4 |
122 +/- 8 |
119 +/- 10 |
36 +/- 6 |
53 +/- 3 |
|
100 |
10 +/- 3 |
14 +/- 2 |
9 +/- 0 |
7 +/- 3 |
23 +/- 8 |
28 +/- 5 |
130 +/- 14 |
123 +/- 12 |
42 +/- 1 |
56 +/- 8 |
|
333 |
14 +/- 2 |
15 +/- 6 |
9 +/- 3 |
9 +/- 1 |
28 +/- 9 |
31 +/- 3 |
141 +/- 4 |
128 +/- 17 |
30 +/- 3 |
58 +/- 11 |
|
1000 |
15 +/- 2 |
14 +/- 5 |
8 +/- 2 |
10 +/- 3 |
29 +/- 4 |
34 +/- 2 |
100 +/- 12 |
125 +/- 8 |
25 +/- 2 |
52 +/- 4 |
|
2500 |
10 +/- 2 |
13 +/- 6 |
8 +/- 3 |
8 +/- 1 |
23 +/- 7 |
27 +/- 3 |
34 +/- 7 |
98 +/- 18 |
38 +/- 3 |
53 +/- 3 |
|
5000 |
14 +/- 1 (P) |
8 +/- 2 (P,M) |
0 +/- 1 (P) |
3 +/- 1 (P,M) |
11 +/- 3 (P) |
22 +/- 3 (P) |
37 +/- 6 (P, M) |
69 +/- 22 (P) |
27 +/- 1 (P) |
52 +/- 8 (P) |
NaN3 |
10 |
1195 +/- 37 |
|
|
|
|
|
1787 +/- 163 |
|
|
|
4-NOPD |
10 |
|
|
|
|
319 +/- 5 |
|
|
|
|
|
4-NOPD |
50 |
|
|
81 +/- 12 |
|
|
|
|
|
|
|
MMS |
2.0 |
|
|
|
|
|
|
|
|
509 +/- 50 |
|
2-AA |
2.5 |
|
352 +/- 31 |
|
153 +/- 12 |
|
4087 +/- 458 |
|
4012 +/- 671 |
|
|
2-AA |
10.0 |
|
|
|
|
|
|
|
|
|
310 +/- 25 |
NaN3: sodium azide
2-AA: 2-aminoanthracene
4-NOPD: 4-nitro-o-phenylene-diamine
MMS: methyl methane sulfonate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key study
The genetic toxicity of the
test item was tested according to the plate incorporation test
(experiment I) and the pre-incubation test (experiment II) using
Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the
Escherichia coli strain WP2 uvrA (Ames test). The assay was performed
with and without liver microsomal activation. Each concentration,
including the controls, was tested in triplicate. The test item was
tested at the following concentrations:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000
µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate
No substantial increase in revertant colony numbers of any of the five
tester strains were observed following treatment with the test item at
any dose level, neither in the presence nor absence of metabolic
activation (S9 mix). Therefore, the test substance is considered to be
non-mutagenic in this reverse mutation assay.
Supporting studies:
Yoo, 1986 (J. Osaka City Med. Centre, 34: 267-288)
In a reverse gene mutation assay in bacteria
(Ames test), strain WP2 uvrA of E. coli was exposed to the
concentrations of 0.5 - 4.0 mg/plate. As a result, the test substance
was considered to be non mutagenic under the test conditions chosen.
Ono et al., 2012 (Food and Chemistry Toxicology 50(5): 1538 -1546)
In this study the information on the results of 367 flavor items in
the Ames test were summarised. The test substance was one of the
substances studied and was considered negative in the Ames test.
However, no information about test system and no information about
strains are given.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.
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.
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