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EC number: 209-099-7 | CAS number: 555-45-3
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Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537,TA 1538, TA 97 TA 100 and TA 98 and in E. coli WP2 uvrA pKM
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 31 Jan - 10 Feb 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The test system included 5 S. typhimurium strains, but neither the tester strain TA102 nor an additional E. Coli strain were tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 1992
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- First experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Second experiment: 6.25, 12.5, 25, 50 and 100 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80/bidistilled water (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The suspension medium Tween 80/bidist. water was chosen according to the solubility properties tested preliminary before start of the study. - Untreated negative controls:
- yes
- Remarks:
- untreated fresh cell suspensions
- Negative solvent / vehicle controls:
- yes
- Remarks:
- suspension medium Tween 80/bidist. water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9: sodium azide (2 µg/plate) for TA100 and TA1535; 9-aminoacridine (80 µg/plate) for TA1537; 4-nitro-o-phenylendiamine (40 µg/plate) for TA98 and TA1538; with S9: 2 aminoanthracene (2.5 or 5 µg/plate) for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn - Evaluation criteria:
- A combination of the following criteria was considered as a positive result:
- The plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls.
- The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1).
- As a rule, the positive control showed mutation rates exceeding the control values of TA100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0.
- At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.
Reproducibility
If the test substance induces reverse mutations in only one test, and if this effect cannot be reproduced in one or several repeated tests, the initially positive test data will lose their significance.
The criteria for interpretation specified here do not apply absolutely. Other aspects in connection with this in vitro test system may as well be taken into account for the final assessment of the test substance. - Statistics:
- Mean values and standard deviation were calculated.
- 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:
- at 40 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitations were not noted.
COMPARISON WITH HISTORICAL CONTROL DATA: number of revertants in the negative and vehicle controls (with and without S9) were all within the characteristic spontaneous mutation ranges of the test batches presented in Table 1.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first experiment, the background lawn was reduced or completely cleared in:
TA100 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA1535 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA1537 from 40 µg/plate (-S9 and +S9)
TA1538 from 40 µg/plate (-S9) and from 200 µg/plate (+S9)
TA98 from 200 µg/plate (-S9 and +S9)
- In the second experiment, the background lawn was reduced or completely cleared in:
TA100 from 50 µg/plate (-S9) and from 100 µg/plate (+S9)
TA1535 from 100 µg/plate (-S9 and +S9)
TA1537 from 50 µg/plate (-S9 and +S9)
TA1538 from 50 µg/plate (-S9 and +S9)
TA98 from 100 µg/plate (-S9 and +S9) - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 17 Apr -21 Apr 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- yes
- Remarks:
- only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-Naphthoflavone
- Test concentrations with justification for top dose:
- 5000 µg/plate with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 95% ethanol (100 and 50 µL/plate in the plate incorporation and preincubation tests, respectively).
- Justification for choice of solvent/vehicle: in a preliminary test, the solubility of the test compound was determined in a number of solvents suitable for the Ames test, namely water, dimethylsulfoxide, glycerol, dimethyl formamide, formamide, ethanol, acetone, dioxane, tetrahydrofuran and tetrahydrofurfuryl alcohol. In all these solvents, the test compound could not be dissolved in appreciable amounts. Therefore, a suspension of the test material was prepared on the day of testing by mixing the test compound with 95% ethanol, stirring it on a magnetic stirrer and using samples of this stirred suspension for testing. No stability testing or composition analysis was performed on the dosing suspension. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: 2-nitrofluorene (2-NF; 2.5 µg/plate for TA98); sodium azide (SA; 5 and 2.5 µg/plate for TA100 and TA1535, respectively); 9-aminoacridine (9-AA; 40 µg/plate for TA1537; +S9: 2-aminoanthracene (2-AA; 2.5 µg/plate for all strains)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation
DURATION
- Preincubation period: 30 min (second experiment)
- Exposure duration: 72 h (first and second experiment)
NUMBER OF REPLICATIONS: 3 replications in the first and second experiment, respectively.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth - Evaluation criteria:
- Criteria for determination of a valid test:
The following criteria had to be met for the mutagenicity assay to be considered valid:
- In the solvent control, each tester strain culture exhibited a characteristic mean number of spontaneous revertants.
- To ensure that appropriate numbers of bacteria were plated, overnight culture titers had to be in excess of 1E08 bacteria/mL.
- The mean of each positive control exhibited a significant increase in the number of revertants over the mean value of the respective vehicle control.
- In a standard Ames test, at least four non-toxic dose levels were required to evaluate the assay data. In the current test, due to non-solubility of the test compound, only a limit dose was employed.
Criteria for evaluation of test results:
For a test compound to be considered positive, it had to (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. In a standard Ames test, this increase had to be accompanied by a dose response towards increasing concentrations of the test article. In the current test, a reproducible caused by a treatment with the limit dose would have been sufficient. A test article that did not meet these criteria would be called non-mutagenic in bacteria. Single increases in revertant frequencies, which were not reproducible in two independent tests were considered non-relevant. - Statistics:
- Mean values and standard deviation were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 5000 µg/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance is not soluble in water.
- Precipitation: due to non-solubility of the test susbtance in all solvents used routinely in the Ames test, the tester strains were exposed to a suspension of the test compound at a single limit concentration of 5000 µg/plate. Test compound-treated bacterial colonies were counted by hand, as the precipitate formed by the test compound prevented automatic counting. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 12 May - 22 Jun 1987
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- only 3 S. typhimurium strains tested, lack of details on test material
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium, other: TA97, TA98 and TA100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- Toxicity range-finder experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Mutation experiment: 1.6, 8, 40, 200, 1000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethylene glycol dimethyl ether (EGDME)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- EGDME
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9 mix: 2-nitrofluorene (50 µg/plate, TA98); sodium azide (2 µg/plate, TA100); 9-aminoacridine (50 µg/plate, TA97); with S9 mix: 2-aminoanthracene (5 µg/plate, all strains)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
in medium; in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: thinning of background layer and/or presence of microcolonies - Evaluation criteria:
- At least 2-fold increase of revertant numbers compared to controls and dose-response correlation.
- Statistics:
- Analysis of variance and regression analysis.
- Key result
- Species / strain:
- S. typhimurium, other: TA97, TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: limited solubility at 1000 µg/plate, but at this dose the precipitated test agent did not interfere with the counting of colonies. Thus 1000 µg/plate was chosen as the highest concentration fur mutation testing.
- Precipitation: yes, at 1000 µg/plate
RANGE-FINDING/SCREENING STUDIES:
Stain TA100 was used for testing up to 5000 µg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in TA100 in the absence or presence of S9 mix. At 5000 µg/plate precipitation occurred. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 09 Apr - 24 Apr 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Only low test concentrations used due to growth inhibition in Salmonella, but not in E.coli strains.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)
- Test concentrations with justification for top dose:
- 0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metablic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation);
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition - Evaluation criteria:
- If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.
- Statistics:
- The results at each concentration were demonstrated with the mean and the standard deviation.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Referenceopen allclose all
Table 2. Group mean values of revertant colonies per treatment group (first experiment).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
|
|
TA 100 |
TA1535 |
TA1537 |
TA1538 |
TA98 |
– |
Untreated |
168 |
20 |
13 |
27 |
28 |
– |
Vehicle |
134 |
17 |
14 |
28 |
32 |
– |
8 |
119 |
16 |
9 |
29 |
28 |
– |
40 |
87 |
10 |
4 |
T |
24 |
– |
200 |
T |
T |
T |
T |
20 |
– |
1000 |
T |
T |
T |
T |
T |
– |
5000 |
T |
T |
T |
T |
T |
Positive controls, –S9 |
Name |
Sodium azide |
Sodium azide |
9-AA |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3) |
474 |
455 |
604 |
1675 |
1359 |
|
+ |
Untreated |
152 |
22 |
17 |
23 |
51 |
+ |
Vehicle |
159 |
18 |
13 |
31 |
38 |
+ |
8 |
126 |
18 |
12 |
23 |
32 |
+ |
40 |
132 |
13 |
12 |
25 |
30 |
+ |
200 |
96 |
15 |
5 |
23 |
27 |
+ |
1000 |
45 |
T |
T |
8 |
10 |
+ |
5000 |
20 |
T |
T |
T |
T |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3) |
1562 |
183 |
87 |
1339 |
940 |
T = toxic
9-AA = 9-aminoacridine
4-NOPD = 4-nitro-o-phenylendiamine
2-AA = 2-aminoanthracene
Table 3. Group mean values of revertant colonies per treatment group (second experiment).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
|
|
TA 100 |
TA1535 |
TA1537 |
TA1538 |
TA98 |
– |
Untreated |
121 |
13 |
12 |
28 |
24 |
– |
Vehicle |
141 |
13 |
9 |
24 |
31 |
– |
6.25 |
135 |
20 |
14 |
26 |
26 |
– |
12.5 |
137 |
19 |
11 |
34 |
26 |
– |
25 |
112 |
12 |
7 |
30 |
29 |
– |
50 |
79 |
13 |
8 |
T |
28 |
– |
100 |
T |
T |
T |
T |
20 |
Positive controls, –S9 |
Name |
Sodium azide |
Sodium azide |
9-AA |
4-NOPD |
4-NOPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3) |
612 |
518 |
457 |
1741 |
1449 |
|
+ |
Untreated |
128 |
14 |
12 |
26 |
38 |
+ |
Vehicle |
129 |
16 |
17 |
27 |
26 |
+ |
6.25 |
135 |
14 |
14 |
27 |
42 |
+ |
12.5 |
114 |
13 |
12 |
30 |
27 |
+ |
25 |
129 |
17 |
13 |
28 |
26 |
+ |
50 |
119 |
15 |
8 |
21 |
28 |
+ |
100 |
111 |
15 |
12 |
T |
23 |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3) |
1591 |
166 |
143 |
1499 |
780 |
T = toxic
9-AA = 9-aminoacridine
4-NOPD = 4-nitro-o-phenylendiamine
2-AA = 2-aminoanthracene
Table 1. Test Results of Experiment 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
untreated |
110 |
8 |
31 |
12 |
– |
Ethanol |
105 |
7 |
33 |
16 |
– |
5000 |
125P |
3P |
30P |
20P |
Positive controls, –S9 |
Name |
SA |
SA |
2-NF |
9-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
40 |
|
Mean No. of colonies/plate (average of 3) |
575 |
267 |
123 |
125 |
|
+ |
untreated |
126 |
11 |
25 |
12 |
+ |
Ethanol |
116 |
14 |
33 |
16 |
+ |
5000 |
136P |
8P |
29P |
10P |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
1394 |
123 |
835 |
120 |
SA = sodium azide
2-NF = 2-nitrofluorene
9-AA = 9-aminoacridine
2-AA = 2-Aminoanthracene
P = Precipitate
Table 2. Test Results of Experiment 2 (preincubation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
untreated |
142 |
13 |
30 |
14 |
– |
Ethanol |
126 |
9 |
28 |
11 |
– |
5000 |
127P |
8P |
24P |
12P |
Positive controls, –S9 |
Name |
SA |
SA |
2-NF |
9-AA |
Concentrations (μg/plate) |
5 |
2.5 |
2.5 |
40 |
|
Mean No. of colonies/plate (average of 3) |
575 |
267 |
123 |
125 |
|
+ |
untreated |
134 |
7 |
26 |
13 |
+ |
Ethanol |
155 |
11 |
27 |
10 |
+ |
5000 |
128P |
10P |
31P |
13P |
Positive controls, + S9 |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
839 |
126 |
657 |
76 |
SA = sodium azide
2-NF = 2-nitrofluorene
9-AA = 9-aminoacridine
2-AA = 2-Aminoanthracene
P = Precipitate
Table 1: Ames Test Results after treatment with C10-18 triglycerides
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||
TA97 |
TA98 |
TA100 |
||
- |
0 (vehicle) |
96.0 |
31.6 |
120.0 |
- |
1.6 |
80.7 |
21.7 |
131.3 |
- |
8 |
103.3 |
23.7 |
117.5 |
- |
40 |
88.3 |
17.0 |
141.0 |
- |
200 |
94.0 |
23.3 |
144.3 |
- |
1000 |
92.7ST |
16.0P |
124.0P |
Positive controls - S9 |
Name |
9AA |
2NF |
SA |
Concentrations (μg/plate) |
50 |
50 |
2 |
|
Number of colonies/plate |
446.4 |
920.4 |
886.8 |
|
+ |
0 (vehicle) |
132.2 |
46.8 |
138.8 |
+ |
1.6 |
145.0 |
34.3 |
152.7 |
+ |
8 |
152.0 |
36.7 |
140.3 |
+ |
40 |
165.7 |
37.0 |
141.3 |
+ |
200 |
142.3 |
44.7 |
143.7 |
+ |
1000 |
142.0P |
52.3P |
139.7P |
Positive controls + S9 |
Name |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
5 |
5 |
5 |
|
Number of colonies/plate |
499.8 |
689.2 |
888.4 |
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
SA = sodium azide
9AA = 9-aminoacridine
P= Precipitation observed
ST= signs of toxicity observed (thinning of background layer and/or presence of microcolonies)
1st experiment |
number of revertants: mean value of negative control |
number of revertants: mean value of positive control |
max. number of revertants: mean value of test material [µg/plate] |
without S9-mix |
|
|
|
TA 1535 |
14 ±2 |
399 ±25 |
12±2.5 [39] |
TA 100 |
103±5.0 |
825 ± 34.6 |
113 ± 9.7 [4.9] |
TA 1537 |
11±1.5 |
1989± 52.0 |
13±0.6 [10] |
TA 98 |
19 ±1.7 |
561 ± 19 |
18 ±4.4 [1.2] |
WP2uvrA |
25 ±4.0 |
243 ±24.7 |
30 ±2.6 [5000] |
with S9-mix |
|
|
|
TA 1535 |
11± 0.6 |
399 ±25 |
12 ±2.5 [20] |
TA 100 |
111 ± 6.1 |
825 ± 34.6 |
126 ±7.5 [39] |
TA 1537 |
17 ±4.7 |
1989± 52.0 |
20±3.8 [20] |
TA 98 |
29±1.5 |
561 ± 19 |
28 ±5.0 [39] |
WP2uvrA |
28 ±0.6 |
243 ±24.7 |
33±7.0 [5000] |
2nd experiment |
number of revertants: mean value of negative control |
number of revertants: mean value of positive control |
max. number of revertants: mean value of test material [µg/plate] |
without S9-mix |
|
|
|
TA 1535 |
10±2.3 |
388± 16.4 |
8 ±0.6 [10] |
TA 100 |
121 ± 6.0 |
780 ±7.1 |
114 ±7.8 [20] |
TA 1537 |
11 ±1.0 |
2057±200.9 |
15 ±0 [4.9] |
TA 98 |
17 ±1.2 |
577 ±32.8 |
20±1.5 [1.2] |
WP2uvrA |
20 ±2.0 |
219 ±28:1 |
27 ±4.6 [1250] |
with S9-mix |
|
|
|
TA 1535 |
9 ±2.1 |
388 ±16.4 |
11 ±3.2 [78] |
TA 100 |
114 ±12.7 |
780 ±7.1 |
117±9.3 [10] |
TA 1537 |
23 ±4.7 |
2057 ±200:9 |
21±2.3 [10] |
TA 98 |
25±3.8 |
577 ±32.8 |
29 ±2.6 [39] |
WP2uvrA |
25± 3.2 |
219 ±28:1 |
35 ±2.1 [1250] |
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Analogue justification
No data on the in vitro genetic toxicity of target substance Glycerol trimyristate (CAS 555-45-3) are available. The genetic toxicity assessment was therefore based on studies conducted with analogue (source) substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read across approach is provided in the technical dossier (see IUCLID Section 13).
Genetic toxicity (mutagenicity) in bacteria in vitro
CAS 142-18-7
A bacterial gene mutation assay with 2,3-dihydroxypropyl laurate was conducted following a protocol similar to OECD guideline 471 and GLP (WoE, 1995). Two independent experiments were conducted both in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were treated with concentrations ranging from 8 to 5000 µg/plate. In the second experiment, concentrations ranging from 6.25 to 100 µg/plate were tested. In both experiments, cytotoxicity was observed at concentrations ≥ 40 µg/plate in the bacterial strains. The mean number of revertants was not increased at any concentrations tested. The positive and negative controls included revealed the expected results. Under the experimental conditions reported, the test substance did not induce mutations in the selected strains of S. typhimurium with or without metabolic activation.
CAS 91052-13-0
A bacterial gene mutation assay with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was performed according to OECD guideline 471 and GLP (WoE, 2010). Two independent experiments were performed both in the presence or absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA. In the preliminary toxicity screening, growth inhibitory effects were observed at ≥ 20 µg/plate in S. typhimurium TA 98 and TA 1537 (without metabolic activation), at ≥ 78 µg/plate in S. typhimurium TA 100 and TA 1535 (without metabolic activation), and at ≥ 313 µg/plate in all S. typhimurium strains with metabolic activation. Based on these results, concentrations ranging from 0.61 to 78 µg/plate were used for the tester strains TA 100, TA 1535, TA98 and TA 1537 in the absence of metabolic activation, whereas concentrations ranging from 10 to 313 µg/plate were applied for treatment of the tester strains TA 100, TA 1535, TA 98 and TA 1537 in the presence of metabolic activation. Since no cytotoxicity was seen in E. coli WP2 uvrA, the maximum test concentration of 5000 µg/plate and concentrations of 2500, 1250, 625 and313 µg/plate were selected for treatment in the main assay. Precipitation of the test substance was observed on the plates with E. coli WP2 uvrA at test concentrations ≥ 1250 µg/plate without metabolic activation and at ≥ 2500 µg/plate with metabolic activation in both experiments. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance in the presence or absence of metabolic activation. The positive and negative controls revealed the expected results. Under the conditions of this assay, the test substance did not induce gene mutations in the selected strains of S. typhimurium and in E. coli WP2 uvrA in the absence and presence of metabolic activation, respectively.
CAS 555-43-1
The potential mutagenicity of Glycerol tristearate was assessed in a bacterial mutation assay performed with a protocol similar to OECD guideline 471 and in compliance with GLP (WoE, 1997). S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were exposed to concentrations of 5000 µg/plate with or without S9-mix in two independent experiments. No cytotoxicity and no increase in the mean number of revertants were observed. The solvent and positive control values were within the historical control values. Based on the results of this study, the test substance did not induce mutagenicity in the selected strains of S. typhimurium in the presence and absence of metabolic activation. As only a single concentration was used, this study may not be used for classification purposes.
CAS 67701-26-2
The potential mutagenicity of C12-C18 trialkyl glyceride was assessed in a bacterial mutation assay performed following a protocol similar to OECD guideline 471 and under GLP conditions (WoE, 1987). S. typhimurium TA 97, TA 98 and TA 100 were tested in the presence or absence of a liver metabolic activation system (S9-mix). In a preliminary experiment, the Salmonella strain TA 100 was exposed to concentrations of 8-5000 µg/plate. No cytotoxicity occurred up to 5000 µg/plate, but precipitation of the test substance was observed at this test concentration. Although limited solubility was also observed at 1000 µg/plate, precipitation of the test substance at this concentration did not interfere with colony counting. Concentrations ranging from 1.6 to 1000 µg/plate were selected for treatment in the main assay (plate incorporation method), with and without metabolic activation. No increase in the mean number of revertants per plate was observed when compared to controls. The positive and negative controls included for each test strain were shown to be valid. The test substance was considered to be non-mutagenic under the conditions of the study, with and without metabolic activation.
Overall conclusion for genetic toxicity
There are no available studies on the genetic toxicity of Glycerol trimyristate (CAS 555-45-3). Analogue read-across from 4 source substances was applied from in vitro studies in bacterial cells and the results of the available in vitro studies were consistently negative. Based on the available data and following the analogue approach, Glycerol trimyristate is not expected to be mutagenic in vitro in bacterial cells.
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Glycerol trimyristate (CAS 555-45-3) data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.
Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.
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