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EC number: 949-740-4 | CAS number: -
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Reverse gene mutation assay: Negative (Non-mutagenic); OECD 471; T. Morinaga. (2018)
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:
- 02 March - 30 March 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was conducted under GLP in accordance with the international guideline.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes
- 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:
- S9 mix
- Test concentrations with justification for top dose:
- Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate) Based on the outcome of the range finder study in which bacterial growth inhibition was observed at the lowest dose was used as high dose, the doses for the main test were as follows; In the absence of S9 mix:
0, 9.77, 19.5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250 & 5000 μg per plate for WP2 uvrA. In the present of S9 mix:
39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250 & 5000 μg per plate for TA98, TA1535 & WP2 uvrA. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: Preincubation method
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Yes
- Test substance added in medium; preincubation: 45 mL of each tester strain was directly inoculated into 20 mL 0f 2.5% nutrient broth (Nutrient broth No. 2, Oxoid). The resulting mixture was subject to gyratory culture at 50rpm for 10h at 37°C. After incubation, the optical density of the bacteria suspension was measured to confirm that the viable bacterial count was not less than 1.0x10*9/mL using conversion table.
For tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution. For the test with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of 0.1 M Na-phosphate buffer. This was followed by pre-incubation with shaking at 37°C for the 20 mins, then 2.0mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. And 0.1 mL of positive control solution was carried out equally.
For sterility test, 0.1 mL of test solution of the maximum concentration and 0.5 mL of S9 mix were added to each tube followed by 2.0 mL agar then transferred to surface of minimal glucose agar plate.
These preparations took place over UV absorbent filter.
As top agar, 0.5 mM biotin-0.5 mM L-histidine solution and 0.5 mM l-tryptophan solution were added to the safe agar solution (0.6% agar, 0.5% NaCl) by volume of 1/10 for TA and E.Coli strains respectively.
All plates were incubated at 37°C for 48 hrs, the number of revertant colonies were counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 mins
- Exposure duration/duration of treatment: 48 hours at 37°C
- Harvest time after the end of treatment (sampling/recovery times): not stated - Rationale for test conditions:
- The study was based on the in vitro (pre-incubation) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.
- Evaluation criteria:
- To ensure the validity of the study, the following conditions should be met:
1) Colonies on the plate should be countable without contamination of the test plate or other unexpected events.
2) The number of revertant colonies in the negative control and positive control groups should be within the range of the background data (mean +- 3 standard deviations) of the Shiga laboratory, NISSEI BILIS Co., Ltd. Moreover, the number of revertant colonies in each positive control group should be at least twice higher in each negative control.
3) The number of doses without bacterial growth inhibition should be four or more.
4) No contamination should be observed in the sterility test.
For the number of revertant colonies on each plate, the mean values (rounded to integers) in each dose group are obtained. The case where all the following criteria are met, the results are considered to be positive;
1) The number of revertant colonies in the test article group is equal to or greater than twice the mean negative control value.
2) Dose-dependency is observed.
3) Reproducibility was obtained by the dose-finding study and main study. - Statistics:
- Not stated
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS:
- Data on pH:Not specified
- Data on osmolality:Not specified
- Possibility of evaporation from medium:Not specified
- Water solubility:Not specified
- Precipitation and time of the determination: not observed.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no
RANGE-FINDING/SCREENING STUDIES (if applicable): Yes
Grrowth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.
STUDY RESULTS - Concurrent vehicle negative and positive control data: Both within historical control range
For all test methods and criteria for data analysis and interpretation: - Concentration-response relationship where possible: No increase in revertant were observed.
- Statistical analysis; not stated - Any other criteria: not stated Ames test: - Signs of toxicity: Toxicity was observed beginning at 650 μg per plate in all test conditions
- Individual plate counts: yes - Remarks on result:
- other: No mutagenicity
- Conclusions:
- Based on the condition of the study, the test item did not induce mutagenicity in bacterial reverse mutation assay with or without metabolic activation.
- Executive summary:
OECD 471 ( 2011): The test item was tested to evaluate its mutagenic potential using the pre-incubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.
Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate). Based on the outcome of the range finder study in which bacterial growth inhibition was observed the lowest dose inducing bacterial growth inhibition among the doses with growth inhibition was settled as the highest dose in the main study. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does were set. For the tester strains in which bacterial growth inhibition was not observed, 5000 μg per plate was set as the highest dose in accordance with the guideline. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does up to 156 μg per plate . Therefore, the doses for the main test were as follows;
In the absence of S9 mix:
0, 9.77, 19,5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for WP2 uvrA.
In the presence of S9 mix:
39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for TA98, TA1537 & WP2 uvrA.
All criteria for a valid study were met as described in the protocol. There was no increase in the number of revertant colonies observed in both preliminary and main study. From the preliminary study, growth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.
From the main study, growth inhibition was observed at 156 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 μg per plate in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 625 μg per plate or more in TA100 and TA1535 and at 2500 μg per plate in TA1537. No precipitation was also observed with and without metabolic activation.
No dose dependent increases were observed in all tester strains with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.
Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Reference
Table 1. Result of the Dose-finding study
Experimental Period: March 2 – 5th2009 |
||||||
Metabolic activation |
Dose of test item μg per plate |
Number of revertant colonies/plate |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
S9 mix (-) |
Negative control (DMSO) |
100 |
9 |
22 |
11 |
7 |
0.305 |
92 |
11 |
19 |
14 |
11 |
|
1.22 |
105 |
9 |
22 |
14 |
8 |
|
4.88 |
96 |
8 |
20 |
17 |
7 |
|
19.5 |
117 |
7 |
18 |
14 |
12 |
|
78.1 |
106 |
10 |
16 |
13 |
8 |
|
313 |
78* |
8 |
24 |
19 |
2* |
|
1250 |
65* |
4* |
20 |
18* |
3* |
|
5000 |
58* |
5* |
14* |
14* |
2* |
|
S9 mix (+) |
Negative control (DMSO) |
102 |
7 |
21 |
22 |
16 |
0.305 |
109 |
9 |
26 |
31 |
21 |
|
1.22 |
105 |
11 |
19 |
26 |
11 |
|
4.88 |
98 |
8 |
17 |
18 |
11 |
|
19.5 |
96 |
11 |
25 |
27 |
14 |
|
78.1 |
102 |
6 |
28 |
21 |
13 |
|
313 |
97* |
8 |
26 |
23 |
9 |
|
1250 |
68* |
6* |
24 |
24 |
9 |
|
5000 |
44* |
8* |
20 |
23 |
7* |
|
Positive control (-S9) |
Name |
ENNG |
ENNG |
ENNG |
2-NF |
9-AAc |
μg per plate |
3 |
5 |
2 |
1 |
80 |
|
No. of revertant colonies/plate |
620 |
845 |
801 |
461 |
267 |
|
Positive control (+S9) |
Name |
2-AAn |
2-AAn |
2-AAn |
2-AAn |
2-AAn |
μg per plate |
1 |
2 |
10 |
0.5 |
2 |
|
No. of revertant colonies/plate |
810 |
309 |
285 |
542 |
229 |
2-AAn: 2-aminoanthracnen
9-AAc: 9-aminoacridine
2-NF: 2-nitroflurene
ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine
*: Bacterial growth inhibition observed
Table 2. Result of the main study.
Experimental Period: March 10 – 13th2009 |
||||||
Metabolic activation |
Dose of test item μg per plate |
Number of revertant colonies/plate |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
S9 Mix (-) |
Negative control (DMSO) |
100 |
11 |
24 |
21 |
8 |
9.77 |
115 |
|
|
|
10 |
|
19.1 |
114 |
|
|
|
9 |
|
39.1 |
103 |
9 |
|
14 |
8 |
|
78.1 |
110 |
9 |
|
19 |
7 |
|
156 |
74* |
10 |
26 |
16 |
5* |
|
313 |
67* |
10 |
24 |
16 |
3* |
|
625 |
|
8 |
26 |
17 |
|
|
1250 |
|
7* |
22 |
18* |
|
|
2500 |
|
|
26 |
|
|
|
5000 |
|
|
18* |
|
|
|
S9 Mix (+) |
Negative control (DMSO |
104 |
9 |
27 |
25 |
17 |
39.1 |
106 |
9 |
|
|
|
|
78.1 |
113 |
8 |
|
|
|
|
156 |
112 |
12 |
20 |
28 |
14 |
|
313 |
101 |
11 |
21 |
32 |
18 |
|
625 |
78* |
6* |
34 |
30 |
15 |
|
1250 |
63* |
4* |
20 |
31 |
12 |
|
2500 |
|
|
29 |
33 |
6* |
|
5000 |
|
|
20 |
28 |
8* |
|
Positive control (-S9) |
Name |
ENNG |
ENNG |
ENNG |
2-NF |
9-AAc |
μg per plate |
3 |
5 |
2 |
1 |
80 |
|
No. of revertant colonies/plate |
655 |
827 |
914 |
432 |
391 |
|
Positive control (+S9) |
Name |
2-AAn |
2-AAn |
2-AAn |
2-AAn |
2-AAn |
μg per plate |
1 |
2 |
10 |
0.5 |
2 |
|
No. of revertant colonies/plate |
793 |
279 |
419 |
480 |
189 |
2-AAn: 2-aminoanthracnen
9-AAc: 9-aminoacridine
2-NF: 2-nitroflurene
ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine
*: Bacterial growth inhibition observed
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
The test item is not mutagenic and therefore no mode of action is required.
Additional information
OECD 471 ( 2011): The test item was tested to evaluate its mutagenic potential using the pre-incubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.
Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate). Based on the outcome of the range finder study in which bacterial growth inhibition was observed the lowest dose inducing bacterial growth inhibition among the doses with growth inhibition was settled as the highest dose in the main study. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does were set. For the tester strains in which bacterial growth inhibition was not observed, 5000 μg per plate was set as the highest dose in accordance with the guideline. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does up to 156 μg per plate . Therefore, the doses for the main test were as follows;
In the absence of S9 mix:
0, 9.77, 19,5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for WP2 uvrA.
In the present of S9 mix:
39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for TA98, TA1537 & WP2 uvrA.
All criteria for a valid study were met as described in the protocol. There was no increase in the number of revertant colonies observed in both preliminary and main study. From the preliminary study, growth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.
From the main study, growth inhibition was observed at 156 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 μg per plate in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 625 μg per plate or more in TA100 and TA1535 and at 2500 μg per plate in TA1537. No precipitation was also observed with and without metabolic activation.
No dose dependent increases were observed in all tester strains with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.
Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Justification for classification or non-classification
The test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.
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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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