Registration Dossier
Registration Dossier
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EC number: 228-667-5 | CAS number: 6320-03-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Key study. Method according to OECD 471, GLP study. Based on the available information, the test item is not mutagenic.
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 December 9th, 2015 to January 14th, 2016.
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2 uvrA.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- other: See Table 1 (in 'Any other information on materials and methods incl. tables').
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix with rat liver homogenate of phenobarbital and B-naphthoflavone induced male rats
- Test concentrations with justification for top dose:
- Preliminary range-finding assay: 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (top dose was the maximum recommended dose for soluble non-cytotoxic substances). Based on the results of the Range Finding Test (no insolubility or toxic effect of the test item were detected, see 'Any other information on materials and methods' for further detail), the concentrations in the main test were as follows:
Initial mutation test: 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate.
Confirmatory mutation test: 1.581, 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: in a preliminary compatibility test, the solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The test item was insoluble at 100 mg/mL concentration using distilled water as vehicle. However, a clear solution was achieved observed at the same concentrations using DMSO and DMF as vehicles. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. - Untreated negative controls:
- yes
- Remarks:
- Untreated.
- Remarks:
- With and without metabolic activation.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Remarks:
- With and without metabolic activation.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Water
- Remarks:
- With and without metabolic activation.
- Positive controls:
- yes
- Remarks:
- Vehicle: distilled water
- Positive control substance:
- sodium azide
- Remarks:
- S. typhimurium TA100 and TA1535 without metabolic activation
- Positive controls:
- yes
- Remarks:
- Vehicle: DMSO
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine (NPD)
- Remarks:
- S. typhimurium TA98 without metabolic activation
- Positive controls:
- yes
- Remarks:
- Vehicle: DMSO
- Positive control substance:
- 9-aminoacridine
- Remarks:
- S. typhimurium TA1537 without metabolic activation
- Positive controls:
- yes
- Remarks:
- Vehicle: distilled water
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- E. coli WP2 uvrA without metabolic activation
- Positive controls:
- yes
- Remarks:
- Vehicle: DMSO
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- All Salmonella typhimurium strains and E. coli with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- Plate incorporation method (initial mutation test): Molten top agar was prepared and kept at 45ºC. The equivalent number of minimal glucose agar plates was properly labelled (3 plates per control or concentration level). The test item and other components were prepared freshly and added to the overlay (45ºC): 2000 µL top agar, 50 µL test item formulation or vehicle or reference control, 100 µL bacterial culture, 500 µL S9 mix or phosphate buffer (pH 7.4). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37ºC for 48 hours.
- Pre-incubation method (confirmatory mutation test): Before the overlaying, 50 µL of the test item formulation (or vehicle/solvent or reference control), 100 µL of bacterial culture and 0.5 mL S9 mix or phosphate buffer (pH 7.4) were added into appropiate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37ºC for 48 hours.
DURATION
- Preincubation period: 20 minutes (confirmatory mutation test)
- Exposure duration: 48 hours
- Other: After the 48 hours of incubation the colony numbers were determined by manual counting.
NUMBER OF REPLICATIONS: Each sample per triplicate in each of the three mutation tests.
DETERMINATION OF CYTOTOXICITY
- Method: other: inhibition of bacterial lawn of auxotrophic cells. - Rationale for test conditions:
- In the Preliminary Range Finding Test performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix), no observations of insolubility of the test item or clear inhibitory or toxic effect of the test item were detected at the concentrations tested (see 'Any other information on materials and methods').
- Evaluation criteria:
- Criteria for Validity: The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response: A test item was considered mutagenic if:
- a dose-related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel(TM) software.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See additional information on results.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- See additional information on results.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was observed on the plates of the initial mutation test in all strains with and without metabolic activation at 5000 µg/plate and in the Confirmatory Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 strains with and/or without metabolic activation and in Escherichia coli WP2 uvrA strain without metabolic activation at 5000 μg/plate concentration.
RANGE-FINDING/SCREENING STUDIES
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in 2 tester strains of S. typhimurium (TA98 and TA100). The numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate was observed in the Preliminary Concentration Range Finding Test. No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
COMPARISON WITH HISTORICAL CONTROL DATA: Some relative high number of colonies were observed with the test substance at the some concentrations but they were not dose-related, were within the historical control range and were below the biologically relevant threshold limit, thus they were considered as biological variability of the test system. Untreated, solvent and positive controls were within the historical range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Inhibitory, cytotoxic effect (slightly reduced background lawn development or lower numbers of revertant colonies) was observed in the initial mutation test in S. typhimurium TA100 and TA1537 strains with metabolic activation at 5000 µg/plate.
- Inhibitory cytotoxic effect (slightly reduced background lawn development or lower numbers of revertant colonies) was observed in the confirmatory mutation test in S. typhimurium TA98 and TA100 strains with and without metabolic activation at 5000 and 1581 µg/plate and in S. typhimurium TA1535 and Escherichia Coli WP2 uvrA strains with and without metabolic activation at 5000 µg/plate and without metabolic activation at 1581 µg/plate and in S. typhimurium TA1537 strain with and without metabolic activation at 5000 and 1581 µg/plate concentrations and with metabolic activation at 500 µg/plate.
- Measurement of cytotoxicity used: background lawn development. - Remarks on result:
- other: strain/cell type: S. typhimurium TA1535, TA1537, TA100, TA98
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under test conditions, the test item did not induce gene mutations in any of the strains tested. Therefore, the test item is not mutagenic.
- Executive summary:
A bacterial reverse mutation test was conducted according to OECD guideline 471 under GLP conditions with the test item. The study consisted of a preliminary compatibility test, a preliminary range finding test, an initial mutation test (plate incorporation method) and a confirmatory test (pre-incubation method). Four histidine requiring strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and tryptophan requiring strain E. coli WP2 uvrA were tested in triplicate at concentrations of 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, with and without metabolic activation (S9 mix, prepared from the livers of phenobarbital / β-naphtoflavone induced rats). Vehicle, untreated and positive controls were run in parallel. The validity criteria were fulfilled. In the main tests, precipitate and inhibitory, cytotoxic effects were observed at the highest dose tested. Under test conditions, the test item did not induce gene mutations in any of the strains tested. Therefore, the test item is not mutagenic.
Reference
Table 5. Summary Table of the Initial Mutation Test (Plate Incorporation Method)
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
24.0 |
25.3 |
85.0 |
84.3 |
13.0 |
14.3 |
8.3 |
7.3 |
32.7 |
28.7 |
|
MF |
1.16 |
1.09 |
1.04 |
0.90 |
0.66 |
0.73 |
1.79 |
0.92 |
1.04 |
1.02 |
||
DMSO control |
Mean |
20.7 |
23.3 |
82.0 |
93.3 |
19.7 |
19.7 |
4.7 |
8.0 |
31.3 |
28.0 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
Distilled water control |
Mean |
-- |
-- |
91.0 |
-- |
17.0 |
-- |
-- |
-- |
30.0 |
-- |
|
MF |
-- |
-- |
1.11 |
-- |
0.86 |
-- |
-- |
-- |
0.96 |
-- |
||
5000 |
Mean |
5.7 |
16.0 |
67.0 |
46.0 |
13.7 |
12.0 |
3.0 |
1.3 |
7.7 |
4.7 |
|
MF |
0.27 |
0.69 |
0.82 |
0.49 |
0.69 |
0.61 |
0.64 |
0.17 |
0.24 |
0.17 |
||
1581 |
Mean |
18.3 |
20.0 |
95.0 |
49.7 |
16.0 |
16.3 |
6.3 |
9.3 |
13.3 |
15.3 |
|
MF |
0.89 |
0.86 |
1.16 |
0.53 |
0.81 |
0.83 |
1.36 |
1.17 |
0.43 |
0.55 |
||
500 |
Mean |
21.0 |
27.3 |
83.3 |
57.3 |
16.3 |
14.7 |
5.7 |
7.3 |
23.7 |
23.0 |
|
MF |
1.02 |
1.17 |
1.02 |
0.61 |
0.83 |
0.75 |
1.21 |
0.92 |
0.76 |
0.82 |
||
158.1 |
Mean |
24.7 |
22.0 |
81.7 |
65.3 |
15.0 |
18.7 |
4.0 |
5.7 |
27.7 |
31.0 |
|
MF |
1.19 |
0.94 |
1.00 |
0.70 |
0.76 |
0.95 |
0.86 |
0.71 |
0.88 |
1.11 |
||
50 |
Mean |
25.7 |
27.0 |
80.7 |
67.3 |
13.3 |
17.0 |
5.7 |
4.3 |
32.3 |
34.3 |
|
MF |
1.24 |
1.16 |
0.98 |
0.72 |
0.68 |
0.86 |
1.21 |
0.54 |
1.03 |
1.23 |
||
15.81 |
Mean |
22.7 |
27.0 |
76.0 |
84.0 |
12.7 |
16.7 |
8.3 |
8.7 |
29.0 |
37.0 |
|
MF |
1.10 |
1.16 |
0.93 |
0.90 |
0.64 |
0.85 |
1.79 |
1.08 |
0.93 |
1.32 |
||
NPD (4mg) |
Mean |
394.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
19.10 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2288.0 |
-- |
2221.3 |
-- |
225.3 |
-- |
213.3 |
-- |
-- |
|
MF |
-- |
98.06 |
-- |
23.80 |
-- |
11.46 |
-- |
26.67 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
149.3 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.33 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1073.3 |
-- |
1184.0 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
11.79 |
-- |
69.65 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
381.7 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
81.79 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
926.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
30.89 |
-- |
||
Table 6.Summary Table of the Confirmatory Mutation Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||
Untreated control |
Mean |
28.3 |
33.7 |
105.7 |
119.7 |
18.3 |
19.7 |
6.7 |
5.0 |
38.0 |
37.0 |
|||
MF |
1.25 |
1.38 |
1.09 |
1.20 |
1.15 |
1.40 |
1.11 |
1.25 |
1.01 |
1.10 |
||||
DMSO control |
Mean |
22.7 |
24.3 |
96.7 |
99.7 |
16.0 |
14.0 |
6.0 |
4.0 |
37.7 |
33.7 |
|||
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||||
Distilled water control |
Mean |
-- |
-- |
109.3 |
-- |
14.0 |
-- |
-- |
-- |
37.3 |
-- |
|||
MF |
-- |
-- |
1.13 |
-- |
0.88 |
-- |
-- |
-- |
0.99 |
-- |
||||
5000 |
Mean |
0.0 |
0.0 |
24.7 |
3.0 |
4.3 |
11.7 |
0.7 |
0.0 |
13.3 |
2.7 |
|||
MF |
0.00 |
0.00 |
0.26 |
0.03 |
0.27 |
0.83 |
0.11 |
0.00 |
0.35 |
0.08 |
||||
1581 |
Mean |
14.3 |
13.7 |
41.3 |
23.7 |
8.3 |
12.7 |
2.3 |
4.7 |
18.3 |
21.7 |
|||
MF |
0.63 |
0.56 |
0.43 |
0.24 |
0.52 |
0.90 |
0.39 |
1.17 |
0.49 |
0.64 |
||||
500 |
Mean |
17.0 |
26.7 |
93.0 |
69.0 |
15.0 |
17.7 |
5.0 |
4.7 |
35.7 |
31.7 |
|||
MF |
0.75 |
1.10 |
0.96 |
0.69 |
0.94 |
1.26 |
0.83 |
1.17 |
0.95 |
0.94 |
||||
158.1 |
Mean |
26.0 |
31.3 |
93.7 |
86.3 |
15.0 |
17.0 |
10.0 |
11.0 |
31.3 |
40.7 |
|||
MF |
1.15 |
1.29 |
0.97 |
0.87 |
0.94 |
1.21 |
1.67 |
2.75 |
0.83 |
1.21 |
||||
50 |
Mean |
20.0 |
29.7 |
96.7 |
94.7 |
18.0 |
17.7 |
10.3 |
8.0 |
32.7 |
40.7 |
|||
MF |
0.88 |
1.22 |
1.00 |
0.95 |
1.13 |
1.26 |
1.72 |
2.00 |
0.87 |
1.21 |
||||
15.81 |
Mean |
25.0 |
28.3 |
94.3 |
93.3 |
20.0 |
21.0 |
4.7 |
4.7 |
30.0 |
40.3 |
|||
MF |
1.10 |
1.16 |
0.98 |
0.94 |
1.25 |
1.50 |
0.78 |
1.17 |
0.80 |
1.20 |
||||
5 |
Mean |
21.7 |
34.7 |
102.7 |
102.3 |
20.0 |
19.3 |
4.7 |
7.3 |
32.0 |
35.3 |
|||
MF |
0.96 |
1.42 |
1.06 |
1.03 |
1.25 |
1.38 |
0.78 |
1.83 |
0.85 |
1.05 |
||||
1.581 |
Mean |
22.3 |
25.7 |
100.0 |
103.0 |
13.0 |
13.0 |
6.7 |
9.0 |
26.7 |
38.7 |
|||
MF |
0.99 |
1.05 |
1.03 |
1.03 |
0.81 |
0.93 |
1.11 |
2.25 |
0.71 |
1.15 |
||||
NPD (4mg) |
Mean |
374.0 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|||
MF |
16.50 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||||
2AA (2mg) |
Mean |
-- |
2234.7 |
-- |
2437.3 |
-- |
213.3 |
-- |
205.3 |
-- |
-- |
|||
MF |
-- |
91.84 |
-- |
24.45 |
-- |
15.24 |
-- |
51.33 |
-- |
-- |
||||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
196.0 |
|||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.82 |
||||
SAZ (2mg) |
Mean |
-- |
-- |
1100.0 |
-- |
1158.7 |
-- |
-- |
-- |
-- |
-- |
|||
MF |
-- |
-- |
10.06 |
-- |
82.76 |
-- |
-- |
-- |
-- |
-- |
||||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
392.0 |
-- |
-- |
-- |
|||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
65.33 |
-- |
-- |
-- |
||||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
997.3 |
-- |
|||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
26.71 |
-- |
||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity in vitro: A bacterial reverse mutation test was conducted according to OECD guideline 471 under GLP conditions with the test item. Four histidine requiring strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and tryptophan requiring strain E. coli WP2 uvrA were tested in triplicate at concentrations of 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, with and without metabolic activation (S9 mix, prepared from the livers of phenobarbital /β-naphtoflavone induced rats), based on the results of a preliminary range-finding test. Vehicle, untreated and positive controls were run in parallel. Under test conditions, the test item did not induce gene mutations in any of the strains tested. Therefore, the test item is not mutagenic.
Justification for classification or non-classification
Based on the available information, the substance is not classified for mutagenicity according to CLP Regulation (EC) No. 1272/2008.
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