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EC number: 629-721-4 | CAS number: 308062-60-4
- Life Cycle description
- Uses advised against
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
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- Endocrine disrupter testing in aquatic vertebrates – in vivo
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 Sep 2021 - 07 Oct 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 June 2020
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 31 May 2008
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N-(C16-C18)alkyl(C16-C18)alkane-1-amine Amines, di-C16-18-alkyl Amines, di-C16-18 (evennumbered) alkyl
- Cas Number:
- 308062-60-4
- IUPAC Name:
- N-(C16-C18)alkyl(C16-C18)alkane-1-amine Amines, di-C16-18-alkyl Amines, di-C16-18 (evennumbered) alkyl
- Test material form:
- solid
- Details on test material:
- Chemical name: Amines, di-C16-18 (even numbered) alkyl
EC no.: 629-721-4
To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.
Constituent 1
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: All contained the following additional mutations in: rfa: deep rough (defective LPS cellcoat); gal: galactose metabolism; chl: nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair (deletion of UV repair B gene)
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: the strain lacks an excision repair system and is sensitive to agents such as UV
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
- volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 : Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively. - Test concentrations with justification for top dose:
- Dose Range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Six concentrations, 10, 25, 50, 100, 250 and 500 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.
First Experiment: Direct plate
The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. Based on the results of the dose-range finding test, the following concentrations were selected for the remaining tester strains:
- TA1535, TA1537 and TA98: 10, 25, 50, 100, 250 and 500 μg/plate (with (5% v/v S9 fraction) and without metabolic activation)
Second Experiment: Direct plate
Based on the results of the first mutation assay, the test item was tested up to the dose level of
400 μg/plate in all tester strains:
- TA1535, TA1537, TA98, TA100 and WP2uvrA: 5, 20, 50, 100, 200, 400 μg/plate (with (10% v/v S9 fraction) and without metabolic activation)
The test item was tested beyond a precipitating dose level in all experiments. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colorless solution THF.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) - Experiment 1and Experiment 2
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h
- Temperature: 37.0 ± 1.0°C (actual range 36.9 – 39.7°C)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with the colony counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. - Rationale for test conditions:
- Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix.
The test item was soluble in the selected vehicle. The number of cultures and the strains selected are in agreement with the OECD TG 471. - Evaluation criteria:
- A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Exp. 1 + 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- but tested beyond a precipitating dose level.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Exp. 1 + 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- but tested beyond a precipitating dose level.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Exp.1 + 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- but tested beyond a precipitating dose level.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Exp. 1 + 2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- but tested beyond a precipitating dose level
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Exp. 1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- but tested beyond a precipitating dose level.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First Experiment (dose-range finding study is reported as part of the first experiment):
In the dose-range finding test, the test item was tested up to concentrations of 500 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 250 μg/plate and upwards.
In the first mutation assay, test item was tested at a concentration range of 10 to 500 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 100 μg/plate and upwards in tester strain TA1535 and at dose levels of 250 μg/plate and upwards in tester strains TA1537 and TA98.
In a follow-up experiment, the test item was tested at a concentration range of 5 to 400 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested beyond a precipitating dose level.
RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 10, 25, 50, 100, 250 and 500 μg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 10, 25, 50, 100, 250 and 500 μg/plate.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- Signs of toxicity
First Experiment (dose-range finding study is reported as part of the first experiment):
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Second Experiment:
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. In strains TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range were observed at 200 μg/plate. However, since no dose-relationship was observed, this reduction was not considered to be caused by toxicity of the test item. It is more likely that this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- Mean number of revertant colonies per plate:
No increase in the number of revertants was observed upon treatment with the test item under all conditions tested. See Table 2 to 4 in section "Any other information on results incl. tables"
HISTORICAL CONTROL DATA
- Negative vehicle historical control data: Valid; Please see table 5 in "Any other information on results incl. tables" section.
- Positive historical control data: Valid; Please see table 6 in "Any other information on results incl. tables" section.
Any other information on results incl. tables
Table 2 - Dose-Range Finding Test: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Dose (µg/plate) | TA100 | WP2uvrA | |||||||
Without S9-mix | |||||||||
Positive control | 988 | ± | 43 |
| 1191 | ± | 32 |
| |
Solvent control | 111 | ± | 11 |
| 21 | ± | 9 |
| |
10 | 105 | ± | 10 |
| 18 | ± | 5 |
| |
25 | 103 | ± | 10 |
| 30 | ± | 11 |
| |
50 | 96 | ± | 24 |
| 18 | ± | 4 |
| |
100 | 91 | ± | 12 | NP | 20 | ± | 9 | NP | |
250 | 80 | ± | 23 | SP | 30 | ± | 4 | SP | |
500 | 81 | ± | 11 | n MP | 22 | ± | 5 | n MP | |
With S9-mix1 | |||||||||
Positive control | 1437 | ± | 192 |
| 395 | ± | 28 |
| |
Solvent control | 86 | ± | 25 |
| 30 | ± | 8 |
| |
10 | 75 | ± | 16 |
| 31 | ± | 6 |
| |
25 | 78 | ± | 7 |
| 29 | ± | 10 |
| |
50 | 82 | ± | 9 |
| 26 | ± | 7 |
| |
100 | 80 | ± | 9 | NP | 28 | ± | 11 | NP | |
250 | 101 | ± | 14 | SP | 29 | ± | 9 | SP | |
500 | 75 | ± | 7 | n MP | 17 | ± | 4 | n MP | |
Mean number of revertant colonies (3 replicate plates) ± SD is reported | |||||||||
1 | Plate incorporation assay (5% S9) | ||||||||
MP | Moderate Precipitate | ||||||||
NP | No precipitate | ||||||||
SP | Slight Precipitate | ||||||||
n | Normal bacterial background lawn |
Table 3 - Experiment 1: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay
Dose (µg/plate) | TA1535 | TA1537 | TA98 | |||||||||||
Without S9-mix | ||||||||||||||
Positive control | 861 | ± | 17 | 694 | ± | 95 |
| 1053 | ± | 78 |
| |||
Solvent control | 16 | ± | 6 |
| 5 | ± | 2 |
| 15 | ± | 8 |
| ||
10 | 10 | ± | 5 |
| 2 | ± | 2 |
| 9 | ± | 4 |
| ||
25 | 7 | ± | 4 |
| 2 | ± | 1 |
| 16 | ± | 6 |
| ||
50 | 14 | ± | 2 | NP | 2 | ± | 3 |
| 19 | ± | 5 |
| ||
100 | 13 | ± | 5 | SP | 2 | ± | 3 | NP | 15 | ± | 3 | NP | ||
250 | 6 | ± | 2 | MP | 8 | ± | 6 | SP | 13 | ± | 4 | SP | ||
500 | 8 | ± | 1 | n MP | 4 | ± | 3 | n SP | 15 | ± | 5 | n SP | ||
With S9-mix1 | ||||||||||||||
Positive control | 286 | ± | 44 | 236 | ± | 44 |
| 578 | ± | 156 |
| |||
Solvent control | 19 | ± | 1 |
| 4 | ± | 3 |
| 12 | ± | 3 |
| ||
10 | 6 | ± | 3 |
| 4 | ± | 3 |
| 13 | ± | 6 |
| ||
25 | 6 | ± | 3 |
| 2 | ± | 2 |
| 15 | ± | 9 |
| ||
50 | 9 | ± | 2 |
| 2 | ± | 2 | n | 22 | ± | 2 |
| ||
100 | 10 | ± | 7 | SP | 5 | ± | 6 | NP | 13 | ± | 3 | NP | ||
250 | 4 | ± | 1 | MP | 5 | ± | 4 | SP | 11 | ± | 4 | SP | ||
500 | 5 | ± | 1 | n MP | 3 | ± | 2 | n SP | 17 | ± | 8 | n SP | ||
Mean number of revertant colonies (3 replicate plates) ± SD is reported | ||||||||||||||
1 | Plate incorporation assay (5% S9) | |||||||||||||
MP | Moderate Precipitate | |||||||||||||
NP | No precipitate | |||||||||||||
SP | Slight Precipitate | |||||||||||||
n | Normal bacterial background lawn |
Table 4 - Experiment 2: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Dose | TA1535 | TA1537 | TA98 | TA100 | WP2uvrA | ||||||||||||||||
(µg/plate) | |||||||||||||||||||||
Without S9-mix | |||||||||||||||||||||
Positive control | 851 | ± | 79 | 1128 | ± | 118 | 928 | ± | 199 | 825 | ± | 79 | 1696 | ± | 52 | ||||||
Solvent control | 11 | ± | 6 | 8 | ± | 4 | 12 | ± | 2 | 107 | ± | 11 | 21 | ± | 8 | ||||||
5 | 12 | ± | 7 | 6 | ± | 2 | 16 | ± | 4 | 108 | ± | 21 | 25 | ± | 7 | ||||||
20 | 12 | ± | 5 | NP | 4 | ± | 2 | NP | 12 | ± | 4 | NP | 118 | ± | 15 | 21 | ± | 4 | NP | ||
50 | 10 | ± | 4 | SP | 3 | ± | 2 | SP | 21 | ± | 5 | SP | 111 | ± | 13 | NP | 20 | ± | 5 | SP | |
100 | 8 | ± | 2 | SP/MP | 6 | ± | 4 | SP | 14 | ± | 4 | SP/MP | 115 | ± | 11 | SP | 23 | ± | 5 | SP | |
200 | 7 | ± | 4 | MP | 3 | ± | 1 | MP | 10 | ± | 4 | MP | 102 | ± | 16 | SP | 22 | ± | 4 | MP | |
400 | 8 | ± | 2 | n MP | 2 | ± | 2 | n MP | 7 | ± | 2 | n MP | 111 | ± | 14 | SP | 15 | ± | 8 | n MP | |
With S9-mix1 | |||||||||||||||||||||
Positive control | 151 | ± | 14 | 255 | ± | 40 | 608 | ± | 66 | 646 | ± | 159 | 281 | ± | 40 | ||||||
Solvent control | 10 | ± | 3 | 3 | ± | 2 | 12 | ± | 3 | 62 | ± | 11 | 25 | ± | 6 | ||||||
5 | 7 | ± | 2 | 5 | ± | 2 | 16 | ± | 3 | 53 | ± | 16 | 24 | ± | 2 | ||||||
20 | 9 | ± | 3 | 6 | ± | 3 | 23 | ± | 1 | 60 | ± | 4 | 20 | ± | 7 | NP | |||||
50 | 10 | ± | 2 | 4 | ± | 1 | 19 | ± | 0 | 64 | ± | 16 | NP | 26 | ± | 7 | |||||
100 | 7 | ± | 7 | NP | 4 | ± | 4 | NP | 22 | ± | 6 | NP | 66 | ± | 9 | SP | 30 | ± | 5 | SP | |
200 | 10 | ± | 6 | SP/MP | 1 | ± | 2 | n MP | 15 | ± | 3 | MP | 65 | ± | 6 | SP | 32 | ± | 13 | SP | |
400 | 6 | ± | 1 | n MP | 3 | ± | 2 | n MP | 10 | ± | 3 | n MP | 75 | ± | 7 | n SP | 39 | ± | 8 | n SP | |
Mean number of revertant colonies (3 replicate plates) ± SD is reported | |||||||||||||||||||||
1 | Plate incorporation assay (10% S9) | ||||||||||||||||||||
MP | Moderate Precipitate | ||||||||||||||||||||
NP | No precipitate | ||||||||||||||||||||
SP | Slight Precipitate | ||||||||||||||||||||
n | Normal bacterial background lawn |
Table 5 - Historical Control Data of the Solvent Control
| TA1535 | TA1537 | TA98 | TA100 | WP2uvrA | |||||
S9-mix | - | + | - | + | - | + | - | + | - | + |
Range | 3 – 26 | 3 – 23 | 2 – 24 | 2 – 20 | 3 – 61 | 5 – 60 | 58 – 188 | 46 – 176 | 9 – 61 | 9 – 68 |
Mean | 9 | 10 | 5 | 5 | 13 | 18 | 106 | 98 | 23 | 26 |
SD | 3 | 3 | 2 | 2 | 5 | 6 | 20 | 23 | 9 | 10 |
Total number of plates | 2215 | 2193 | 2219 | 2220 | 2301 | 2337 | 2365 | 2293 | 2155 | 2146 |
SD = Standard deviation
Historical control data from experiments performed between May 2018 and May 2021.
Table 6 – Historical Control Data of the Positive Control Items
| TA1535 | TA1537 | TA98 | TA100 | WP2uvrA | |||||
S9-mix | - | + | - | + | - | + | - | + | - | + |
Range | 107 – 1425 | 78 – 1481 | 64 – 1475 | 48 – 1843 | 379 – 2118 | 272 – 3369 | 173 – 1852 | 371 – 2666 | 93 – 2027 | 109 – 1968 |
Mean | 924 | 285 | 829 | 282 | 1310 | 933 | 832 | 1403 | 1253 | 415 |
SD | 167 | 119 | 367 | 156 | 299 | 396 | 185 | 409 | 472 | 203 |
Total number of plates | 2073 | 2072 | 1710 | 2094 | 2209 | 2155 | 2178 | 2156 | 2062 | 2032 |
SD = Standard deviation
Historical control data from experiments performed between May 2018 and May 2021.
Applicant's summary and conclusion
- Conclusions:
- The results of an AMES test, performed according to OECD TG 471 and in accordance with GLP principles, showed that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Executive summary:
In an Ames test, performed according to OECD TG 471 and in accordance with GLP principles, the test item was assessed for its potential to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and at the tryptophan locus of one Escherichia coli strain (WP2uvrA).
The test was performed in two independent direct plate experiments. The vehicle of the test item was tertrahydrofuran.
In the dose-range finding test, the test item was tested up to concentrations of 500 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 250 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.
Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 10 to 500 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 100 μg/plate and upwards in tester strain TA1535 and at dose levels of 250 μg/plate and upwards in tester strains TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 5 to 400 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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