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Registration Dossier
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EC number: 276-538-7 | CAS number: 72252-58-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In a bacterial reverse mutation assay (Ames) no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
The results of the forward gene mutation test at the hprt locus with the test item indicated that the test item was non-mutagenic.
In an in vitro chromosomal aberration study a total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations. There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. The study indicated that the test item was not clastogenic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016 -04-19 till 2016-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Guideline-conform study under GLP without deviations
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/ß-Naphthoflavone induced rat liver S9 in experiment I and non-induced hamster liver S9 in experiment II
- Test concentrations with justification for top dose:
- 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and experiment II
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: [deionised water
- Justification for choice of solvent/vehicle: best suitable solvent - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- congo red
- other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:in agar (plate incorporation); preincubation;
DURATION
- Preincubation period: 30 min
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates
DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Species / strain:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in strain TA 1535, TA 1537 and TA 98
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: yes
- Precipitation:No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 / / / 5000
TA 1537 / 5000 5000 /
TA 98 / 5000 / /
TA 100 / / / /
WP2 uvrA / / / / - Remarks on result:
- other: other: reverse mutation assay
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Executive summary:
This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.
Furthermore in Experiment II precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain
Experiment I
Experiment II
without S9 mix
with S9 mix
without S9 mix
with S9 mix
TA 1535
/
/
/
5000
TA 1537
/
5000
5000
/
TA 98
/
5000
/
/
TA 100
/
/
/
/
WP2 uvrA
/
/
/
/
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanodal-Schwarz 2LW at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- According to OECD and GLP guidelines
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Cell cultures were exposed to the test item for 3 hours both in the presence and absence of exogenous metabolic activation and for 21 hours in the absence of metabolic activation. Following exposure of cell cultures to the test item, they were treated with Colchicine to arrest the cells in a metaphase-like stage of mitosis (c-metaphase). Cells were then harvested and chromosome preparations made. Preparations were stained and metaphases were analyzed for chromosomal aberrations.
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 homogenate
- Test concentrations with justification for top dose:
- Experiment No. Test Concentrations
Experiments 1 & 2: a) 12 b) 60 and c) 300 µg/mL (factor of 5)
Experiment 3: a) 6 b) 32 and c) 160 µg/mL (factor of 5) - Vehicle / solvent:
- sterile water was used as the vehicle control.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- - Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA
- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen. - Evaluation criteria:
- - Evaluation and Interpretation : When all the validity criteria are fulfilled:
a. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
• The increase is dose-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data
b. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data
c. The results will be considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required to clarify such results. - Statistics:
- Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: strain/cell type:
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item was not clastogenic in CHO-K1 cells at the tested concentration (up to 300 µg/mL) and under the conditions of testing employed - Executive summary:
The clastogenic potential of the test item to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water (SW) at 50 mg/mL.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, exhibited required level of cytotoxicity as RICC between 160 and 500 µg/mL, compared to the SW control, both in the presence and absence of metabolic activation with 3-hour exposure. In the absence of metabolic activation with 21-hour exposure, required level of cytotoxicity was observed between 40 and 320 µg/mL compared to the SW. Based on these observations, a maximum of 300 µg/mL in experiments 1 and 2 and a maximum of 160 µg/mL in experiment 3 of the chromosomal aberration assay was tested.
In the definitive chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for 3 hours at concentrations of 12, 60 and 300mg/mL in experiments 1 and 2 (presence and in the absence of metabolic activation, respectively). Similarly, in experiment 3, CHO-K1 cells were exposed to the test item in duplicate for 21 hours at concentrations of 6, 32, and 160 µg/mL in the absence of metabolic activation. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the respective highest concentrations tested, the reduction in cell growth as RICC was 52, 58 and 52% in Experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.
There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.
The study indicated that the test item was not clastogenic at the concentrations tested and under the conditions of testing.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- according to OECD and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 homogenate
- Test concentrations with justification for top dose:
- A) 62.5 B) 125 C) 250 and D) 500 µg/mL (factor of 2)
- Vehicle / solvent:
- sterile water was used as the vehicle control.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Details on test system and experimental conditions:
- - Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA
- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen. - Evaluation criteria:
- - Evaluation and Interpretation : When all the validity criteria are fulfilled:
a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data
b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:
•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data
c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05). - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: strain/cell type:
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
It is concluded that the test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed. - Executive summary:
The genotoxic potential of the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.
The study consisted of a preliminary cytotoxicity test and a definitive gene mutation assay. The gene mutation assay comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The test item formed a free flowing suspension in sterile water (SW) at 50 mg/mL.
In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 19 and 15 % at the 500 µg/mL, in the presence and absence of metabolic activation, respectively. The test item neither precipitated in the test medium at the highest concentration nor showed any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of
500 µg/mL was tested in the gene mutation assay.In the gene mutation assay, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 62.5, 125, 250 and 500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (SW) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.
There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.
The results of the forward gene mutation test at the hprt locus with the test item indicated that the test item was non-mutagenic under the conditions of this study.
Referenceopen allclose all
Summary Tabellen
Table1 Summary of Experiment I
Study Name: 1763502 |
Study Code: Envigo 1763502 |
Experiment: 1763502 VV Plate |
Date Plated: 19.04.2016 |
Assay Conditions: |
Date Counted: 26.04.2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
Without Activation |
Deion. water |
|
11 ± 4 |
9 ± 2 |
29 ± 4 |
186 ± 16 |
54 ± 6 |
Untreated |
|
12 ± 3 |
9 ± 4 |
32 ± 6 |
157 ± 14 |
52 ± 11 |
|
Sanodal- |
3 µg |
9 ± 1 |
10 ± 3 |
34 ± 4 |
172 ± 4 |
49 ± 11 |
|
Schwarz 2LW |
10 µg |
15 ± 5 |
7 ± 2 |
26 ± 5 |
172 ± 17 |
51 ± 7 |
|
|
33 µg |
11 ± 1 |
9 ± 1 |
27 ± 6 |
157 ± 6 |
41 ± 4 |
|
|
100 µg |
13 ± 6 |
11 ± 5 |
28 ± 6 |
166 ± 28 |
61 ± 6 |
|
|
333 µg |
12 ± 3 |
6 ± 1 |
29 ± 3 |
168 ± 12 |
48 ± 5 |
|
|
1000 µg |
10 ± 2D M |
8 ± 4D M |
26 ± 7D M |
149 ± 4D M |
43 ± 6D M |
|
|
2500 µg |
10 ± 3D M |
8 ± 3D M |
19 ± 5D M |
146 ± 7D M |
36 ± 4D M |
|
|
5000 µg |
8 ± 2D M |
7 ± 1D M |
15 ± 1D M |
135 ± 5D M |
36 ± 3D M |
|
NaN3 |
10 µg |
1382 ± 52 |
|
|
2415 ± 69 |
|
|
4-NOPD |
10 µg |
|
|
403 ± 23 |
|
|
|
4-NOPD |
50 µg |
|
75 ± 7 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
1075 ± 78 |
|
|
|
|
|
|
|
|
|
With Activation |
Deion. water |
|
10 ± 2 |
9 ± 3 |
40 ± 6 |
170 ± 20 |
63 ± 5 |
Untreated |
|
13 ± 4 |
10 ± 5 |
45 ± 7 |
165 ± 20 |
61 ± 14 |
|
Sanodal- |
3 µg |
10 ± 1 |
11 ± 4 |
44 ± 6 |
167 ± 8 |
62 ± 8 |
|
Schwarz 2LW |
10 µg |
12 ± 5 |
9 ± 3 |
48 ± 6 |
172 ± 18 |
53 ± 10 |
|
|
33 µg |
8 ± 2 |
11 ± 2 |
37 ± 5 |
172 ± 16 |
52 ± 4 |
|
|
100 µg |
10 ± 0 |
8 ± 3 |
40 ± 10 |
173 ± 21 |
69 ± 11 |
|
|
333 µg |
9 ± 5 |
11 ± 4 |
34 ± 2 |
174 ± 25 |
54 ± 13 |
|
|
1000 µg |
10 ± 1D M |
8 ± 2D M |
27 ± 4D M |
135 ± 6D M |
42 ± 1D M |
|
|
2500 µg |
7 ± 3D M |
5 ± 2D M |
23 ± 6D M |
115 ± 8D M |
40 ± 2D M |
|
|
5000 µg |
5 ± 2D M |
3 ± 1D M |
17 ± 2D M |
106 ± 7D M |
30 ± 4D M |
|
|
2-AA |
2.5 µg |
415 ± 21 |
219 ± 43 |
6586 ± 361 |
4872 ± 201 |
|
|
2-AA |
10.0 µg |
|
|
|
|
414 ± 15 |
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
D M |
Densely coloured plate Manual count |
Table2 Summary of Experiment II
Study Name: 1763502 |
Study Code: Envigo 1763502 |
Experiment: 1763502 HV2 Pre |
Date Plated: 12.05.2016 |
Assay Conditions: |
Date Counted: 18.05.2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
Without Activation |
Deion. water |
|
10 ± 1 |
13 ± 3 |
29 ± 2 |
146 ± 22 |
51 ± 9 |
Untreated |
|
12 ± 7 |
12 ± 2 |
32 ± 8 |
169 ± 11 |
52 ± 7 |
|
Sanodal- |
3 µg |
11 ± 1 |
11 ± 1 |
26 ± 9 |
161 ± 8 |
41 ± 1 |
|
Schwarz 2LW |
10 µg |
12 ± 6 |
10 ± 2 |
25 ± 6 |
167 ± 13 |
42 ± 2 |
|
|
33 µg |
13 ± 3 |
12 ± 6 |
28 ± 2 |
148 ± 22 |
50 ± 5 |
|
|
100 µg |
13 ± 3 |
11 ± 3 |
27 ± 12 |
154 ± 8 |
37 ± 3 |
|
|
333 µg |
14 ± 4P |
10 ± 1P |
32 ± 9P |
167 ± 7P |
32 ± 8P |
|
|
1000 µg |
7 ± 3P M |
8 ± 2P M |
16 ± 2P M |
159 ± 6P M |
29 ± 4M P |
|
|
2500 µg |
7 ± 2P M |
8 ± 2P M |
14 ± 2P M |
157 ± 14P M |
28 ± 7M P |
|
|
5000 µg |
7 ± 2P D M |
4 ± 1P D M |
15 ± 3P D M |
143 ± 6P D M |
28 ± 9M P D |
|
NaN3 |
10 µg |
1140 ± 35 |
|
|
1849 ± 49 |
|
|
4-NOPD |
10 µg |
|
|
447 ± 25 |
|
|
|
4-NOPD |
50 µg |
|
72 ± 12 |
|
|
|
|
MMS |
2 µL |
|
|
|
|
879 ± 65 |
|
|
|
|
|
|
|
|
|
With Activation |
Deion. water |
|
18 ± 4 |
25 ± 3 |
50 ± 2 |
140 ± 32 |
46 ± 4 |
Untreated |
|
18 ± 4 |
23 ± 9 |
57 ± 10 |
123 ± 19 |
50 ± 11 |
|
Sanodal- |
3 µg |
22 ± 0 |
29 ± 2 |
57 ± 3 |
147 ± 14 |
51 ± 3 |
|
Schwarz 2LW |
10 µg |
20 ± 4 |
30 ± 6 |
55 ± 13 |
150 ± 11 |
48 ± 3 |
|
|
33 µg |
22 ± 6 |
31 ± 10 |
55 ± 6 |
177 ± 13 |
42 ± 4 |
|
|
100 µg |
13 ± 1 |
30 ± 6 |
49 ± 8 |
167 ± 32 |
45 ± 11 |
|
|
333 µg |
16 ± 5P |
29 ± 4P |
51 ± 4P |
143 ± 7P |
49 ± 4P |
|
|
1000 µg |
13 ± 3P M |
30 ± 8P M |
36 ± 3P M |
139 ± 12P M |
31 ± 5P M |
|
|
|
2500 µg |
11 ± 3P M |
21 ± 3P M |
29 ± 4P M |
132 ± 11P M |
33 ± 8P M |
|
|
5000 µg |
8 ± 2P D M |
18 ± 3P D M |
23 ± 3P D M |
123 ± 2P D M |
26 ± 9P D M |
|
2-AA |
2.5 µg |
|
|
|
963 ± 99 |
|
|
2-AA |
2.5 µg |
329 ± 27 |
115 ± 14 |
|
|
|
|
2-AA |
10 µg |
|
|
|
|
1043 ± 60 |
|
Congo red |
500 µg |
|
|
328 ± 10 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD Congo red MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate |
P M D |
Precipitate Manual count Densely coloured plate |
TABLE 1. Preliminary Cytotoxicity Test - pH of Test Medium
Treatment (mg/mL) |
pH at the beginning of exposure to treatment |
pH at the end of exposure to treatment |
||
With S9 |
Without S9 |
With S9 |
Without S9 |
|
SW |
7.02 |
7.02 |
7.05 |
7.16 |
5 |
7.07 |
7.13 |
7.08 |
7.11 |
10 |
7.06 |
7.17 |
7.01 |
7.19 |
20 |
7.08 |
7.18 |
7.07 |
7.19 |
40 |
7.10 |
7.19 |
7.02 |
7.15 |
80 |
7.10 |
7.18 |
7.06 |
7.17 |
160 |
7.08 |
7.19 |
7.02 |
7.20 |
320 |
7.10 |
7.20 |
7.02 |
7.15 |
500 |
7.08 |
7.17 |
7.09 |
7.15 |
Note: No precipitation was observed both in the presence and absence of metabolic activation at any of the tested concentrations at 3 hours post treatment.
TABLE 2. Preliminary Cytotoxicity Test - Osmolality of Test Medium
Treatment (mg/mL) |
Osmolality at the beginning of exposure to treatment (OSMOL/kg) |
Osmolality at the end of exposure to treatment (OSMOL/kg) |
||
With S9 |
Without S9 |
With S9 |
Without S9 |
|
SW |
0.317 |
0.311 |
0.329 |
0.320 |
500 |
0.319 |
0.311 |
0.328 |
0.321 |
TABLE 3. Results of Preliminary Cytotoxicity Test
Treatment (mg/mL) |
Presence of metabolic activation (3-hour exposure) |
Absence of metabolic activation (3-hour exposure) |
Absence of metabolic activation (21-hour exposure) |
||||||
Final – initial cell count (1x106/flask) |
Cell growth index RICC (%) |
Cell growth inhibition (%) |
Final – initial cell count (1x106/flask) |
Cell growth index RICC (%)
|
Cell growth inhibition (%) |
Final – initial cell count (1x106/flask) |
Cell growth index RICC (%) |
Cell growth inhibition (%) |
|
SW |
1.425 |
100 |
0 |
1.175 |
100 |
0 |
1.1375 |
100 |
0 |
5 |
1.175 |
82 |
18 |
1.025 |
87 |
13 |
0.9125 |
80 |
20 |
10 |
1.0125 |
71 |
29 |
0.925 |
79 |
21 |
0.775 |
68 |
32 |
20 |
0.925 |
65 |
35 |
0.8875 |
76 |
24 |
0.6875 |
60 |
40 |
40 |
0.8625 |
61 |
39 |
0.7875 |
67 |
33 |
0.650 |
57 |
43 |
80 |
0.80 |
56 |
44 |
0.675 |
57 |
43 |
0.55 |
48 |
52 |
160 |
0.75 |
53 |
47 |
0.6125 |
52 |
48 |
0.525 |
46 |
54 |
320 |
0.60 |
42 |
58 |
0.4875 |
41 |
59 |
0.075 |
7 |
93 |
500 |
0.425 |
30 |
70 |
0.30 |
26 |
74 |
0.0875 |
8 |
92 |
Note: Initial cell count (No. of cells at the beginning of treatment): 1.45 x 106cells/flask .
TABLE 4. Summary Results of Chromosomal Aberration Assay - Experiment 1
Treatment (µg/mL) |
No. of metaphases scored |
No. (%) of metaphases with aberrations@ |
Total No.(%) of aberrant metaphases* |
Cell Growth Inhibition (%) |
|||||||
Gaps |
Breaks |
Exchanges |
Including Gaps |
Excluding Gaps |
|||||||
Cs |
Ct |
Cs |
Ct |
Cs |
Ct |
RC |
|||||
SW
|
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
12 |
300 |
2 (0.7) |
0 |
0 |
0 |
0 |
0 |
0 |
2 (0.7) |
0 |
17 |
60 |
300 |
0 |
1 (0.3) |
0 |
0 |
0 |
0 |
0 |
1 (0.3) |
0 |
39 |
300 |
300 |
1 (0.3) |
2 (0.7) |
0 |
0 |
0 |
0 |
0 |
3 (1.0) |
0 |
52 |
CPA 55 |
300 |
0 |
0 |
32 (10.7) |
71 (23.7) |
24 (8.0) |
24 (8.0) |
0 |
141 (47.0) |
+ 141 (47.0) |
21 |
*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type RC: Ring chromosome
CPA: Cyclophosphamide monohydrate +: Significantly higher than control (p <0.05) by Fischer exact test
Note: There were no incidences of polyploidy and endoreduplicated cells.
TABLE 5. Summary Results of Chromosomal Aberration Assay - Experiment 2
Treatment (µg/mL) |
No. of metaphases scored |
No. (%) of metaphases with aberrations@ |
Total No.(%) of aberrant metaphases* |
Cell Growth Inhibition (%) |
|||||||
Gaps |
Breaks |
Exchanges |
Including Gaps |
Excluding Gaps |
|||||||
Cs |
Ct |
Cs |
Ct |
Cs |
Ct |
RC |
|||||
SW
|
300 |
0
|
2 (0.7) |
0
|
0
|
0
|
0
|
0
|
2 (0.7) |
0
|
0 |
12 |
300 |
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
19 |
60 |
300 |
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
43
|
300 |
300 |
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
58 |
*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type RC: Ring chromosome
Note: There were no incidences of polyploidy and endoreduplicated cells
TABLE 6. Summary Results of Chromosomal Aberration Assay - Experiment 3
Treatment (µg/mL) |
No. of metaphases scored |
No. (%) of metaphases with aberrations@ |
Total No.(%) of aberrant metaphases* |
Cell Growth Inhibition (%) |
|||||||
Gaps |
Breaks |
Exchanges |
Including Gaps |
Excluding Gaps |
|||||||
Cs |
Ct |
Cs |
Ct |
Cs |
Ct |
RC |
|||||
SW
|
300 |
1 (0.3) |
1 (0.3) |
0
|
2 (0.7) |
0
|
0
|
0
|
4 (1.3) |
2 (0.7) |
0 |
6 |
300 |
0
|
2 (0.7) |
0
|
1 (0.3) |
0
|
0
|
0
|
3 (1.0) |
1 (0.3) |
21 |
32 |
300 |
1 (0.3) |
1 (0.3) |
0
|
2 (0.7) |
0
|
0
|
0
|
4 (1.3) |
2 (0.7) |
43 |
160 |
300 |
1 (0.3) |
0
|
0
|
0
|
0
|
0
|
0
|
1 (0.3) |
0
|
52 |
EMS600 |
300 |
8 (2.7) |
9 (3.0) |
41 (13.7) |
75 (25.0) |
52 (17.3) |
28 (9.3) |
0
|
151 (50.3) |
+ 151 (50.3) |
33 |
*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type RC: Ring chromosome
EMS: Ethyl methanesulfonate +: Significantly higher than control (p <0.05) by Fischer exact test
Note: There were no incidences of polyploidy and endoreduplicated cells
TABLE 1. Determination of pH of Test Medium
Treatment (mg/mL) |
pH at the beginning of exposure to treatment |
pH at the end of exposure to treatment |
||
With S9 |
Without S9 |
With S9 |
Without S9 |
|
SW |
7.12 |
7.15 |
7.08 |
7.16 |
5 |
7.14 |
7.16 |
7.10 |
7.19 |
10 |
7.16 |
7.18 |
7.10 |
7.22 |
20 |
7.16 |
7.18 |
7.11 |
7.24 |
40 |
7.17 |
7.16 |
7.10 |
7.23 |
80 |
7.18 |
7.20 |
7.12 |
7.24 |
160 |
7.18 |
7.23 |
7.10 |
7.24 |
320 |
7.19 |
7.25 |
7.09 |
7.23 |
500 |
7.20 |
7.25 |
7.08 |
7.24 |
Note: There was no precipitation of the test item at any of tested concentrations either in the presence or absence of metabolic activation at 3 hours post treatment.
TABLE 2. Determination of Osmolality of Test Medium
Treatment (mg/mL) |
Osmolality at the beginning of exposure to treatment (OSMOL/kg) |
Osmolality at the end of exposure to treatment (OSMOL/kg) |
||
With S9 |
Without S9 |
With S9 |
Without S9 |
|
SW |
0.317 |
0.311 |
0.329 |
0.320 |
500 |
0.319 |
0.311 |
0.328 |
0.321 |
TABLE 3. Results of Preliminary Cytotoxicity Test
Treatment (mg/mL) |
3-hour exposure with metabolic activation |
3-hour exposure without metabolic activation |
||||||
Cloning Efficiency (CE) |
Cells at end of treatment (1x105/mL) |
Adjusted Cloning Efficiency |
Relative Survival (%RS)* |
Cloning Efficiency (CE) |
Cells at end of treatment (1x105/mL) |
Adjusted Cloning Efficiency |
Relative Survival (%RS)* |
|
SW |
0.97 |
12.90 |
1.44 |
100 |
0.97 |
12.45 |
1.39 |
100 |
5 |
0.88 |
12.00 |
1.21 |
84 |
0.94 |
11.80 |
1.27 |
91 |
10 |
0.82 |
10.95 |
1.03 |
72 |
0.86 |
11.35 |
1.12 |
81 |
20 |
0.80 |
10.35 |
0.95 |
66 |
0.79 |
10.50 |
0.95 |
68 |
40 |
0.74 |
9.15 |
0.78 |
54 |
0.76 |
9.30 |
0.81 |
58 |
80 |
0.65 |
7.05 |
0.53 |
37 |
0.71 |
8.50 |
0.69 |
50 |
160 |
0.62 |
6.90 |
0.49 |
34 |
0.66 |
7.20 |
0.55 |
40 |
320 |
0.57 |
5.15 |
0.34 |
24 |
0.58 |
4.90 |
0.33 |
24 |
500 |
0.47 |
4.95 |
0.27 |
19 |
0.48 |
3.80 |
0.21 |
15 |
TABLE 4. Parallel Cytotoxicity Test Results from Experiment 1
Treatment µg/mL |
No. of Colonies /Flask |
CE* |
ACE |
RS % |
||
1 |
2 |
3 |
||||
SW |
191 |
189 |
195 |
0.968 |
1.41 |
100 |
197 |
193 |
197 |
||||
62.5 |
137 |
141 |
135 |
0.707 |
0.72 |
51 |
145 |
147 |
143 |
||||
125 |
138 |
131 |
136 |
0.670 |
0.55 |
39 |
129 |
132 |
138 |
||||
250 |
126 |
127 |
130 |
0.633 |
0.46 |
33 |
136 |
121 |
119 |
||||
500 |
111 |
118 |
99 |
0.547 |
0.28 |
20 |
106 |
109 |
113 |
||||
3-MCA |
136 |
148 |
131 |
0.716 |
0.72 |
51 |
152 |
143 |
149 |
||||
CE: Cloning Efficiency ; ACE: Adjusted CE; RS: Relative Survival
TABLE 5. Parallel Cytotoxicity Test Results from Experiment 2
Treatment µg/mL |
No. of Colonies /Flask |
CE* |
ACE |
RS % |
||
1 |
2 |
3 |
||||
SW |
197 |
195 |
196 |
0.974 |
1.49 |
100 |
191 |
197 |
193 |
||||
62.5 |
153 |
160 |
149 |
0.763 |
0.77 |
52 |
147 |
156 |
151 |
||||
125 |
141 |
138 |
135 |
0.69 |
0.62 |
42 |
143 |
140 |
131 |
||||
250 |
121 |
127 |
120 |
0.616 |
0.50 |
34 |
119 |
129 |
123 |
||||
500 |
110 |
113 |
97 |
0.533 |
0.24 |
16 |
107 |
101 |
112 |
||||
CE: Cloning EfficiencyACE: Adjusted CE RS: Relative Survival
* calculated from the mean values of the replicates of each group and rounded off to two decimal places
TABLE 6. Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)
Treatment µg/mL |
Mutation Assay Flasks |
Cloning Efficiency of Mutant Colonies |
Cloning Efficiency Flasks |
6-TG Mutants per 106Clonable Cells (MF) |
||||||||
TG Colonies/Flask |
No. of Colonies/Flask |
|||||||||||
1 |
2 |
3 |
4 |
5 |
Total |
1 |
2 |
3 |
CE* |
|||
SW |
1 |
1 |
3 |
1 |
1 |
16 |
0.000008 |
188 |
189 |
193 |
0.95 |
8.42 |
2 |
3 |
1 |
1 |
2 |
191 |
194 |
190 |
|||||
62.5 |
0 |
1 |
2 |
1 |
1 |
12 |
0.000006 |
183 |
172 |
176 |
0.89 |
6.74 |
1 |
2 |
1 |
1 |
2 |
181 |
179 |
174 |
|||||
125 |
3 |
0 |
2 |
1 |
1 |
12 |
0.000006 |
158 |
162 |
171 |
0.83 |
7.23 |
1 |
1 |
1 |
1 |
1 |
172 |
170 |
164 |
|||||
250 |
1 |
1 |
1 |
1 |
2 |
12 |
0.000006 |
157 |
149 |
158 |
0.77 |
7.79 |
0 |
2 |
2 |
1 |
1 |
146 |
151 |
159 |
|||||
500 |
2 |
2 |
1 |
0 |
0 |
11 |
0.0000055 |
141 |
136 |
133 |
0.67 |
8.21 |
2 |
1 |
1 |
1 |
1 |
133 |
130 |
135 |
|||||
3-MCA |
36 |
34 |
38 |
29 |
26 |
315 |
0.0001575 |
148 |
150 |
141 |
0.75 |
210+ |
28 |
29 |
31 |
34 |
30 |
153 |
157 |
146 |
|||||
Positive Control: 8 µg/mL 3-MCA Vehicle Control: DMSO CE: Cloning Efficiency MF: Mutant Frequency
TABLE 7. Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)
Treatment µg/mL |
Mutation Assay Flasks |
Cloning Efficiency of Mutant Colonies |
Cloning Efficiency Flasks |
6-TG Mutants per 106Clonable Cells (MF) |
||||||||
TG Colonies/Flask |
No. of Colonies/Flask |
|||||||||||
1 |
2 |
3 |
4 |
5 |
Total |
1 |
2 |
3 |
CE* |
|||
SW |
2 |
2 |
2 |
3 |
1 |
22 |
0.000011 |
191 |
196 |
187 |
0.95 |
11.58 |
2 |
3 |
2 |
2 |
3 |
189 |
190 |
192 |
|||||
62.5 |
1 |
1 |
2 |
2 |
2 |
17 |
0.0000085 |
178 |
180 |
191 |
0.92 |
9.24 |
2 |
3 |
1 |
1 |
2 |
184 |
185 |
183 |
|||||
125 |
0 |
3 |
1 |
1 |
2 |
15 |
0.0000075 |
163 |
167 |
160 |
0.83 |
9.04 |
4 |
2 |
0 |
1 |
1 |
169 |
165 |
166 |
|||||
250 |
1 |
1 |
1 |
1 |
2 |
14 |
0.000007 |
148 |
153 |
154 |
0.76 |
9.21 |
2 |
1 |
2 |
2 |
1 |
150 |
157 |
152 |
|||||
500 |
1 |
1 |
2 |
2 |
1 |
14 |
0.000007 |
143 |
149 |
137 |
0.71 |
9.86 |
2 |
3 |
1 |
1 |
0 |
135 |
141 |
145 |
|||||
Vehicle Control: DMSO CE: Cloning Efficiency MF: Mutant Frequency
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
No relevant findings observed.
Additional information
Justification for classification or non-classification
No classification
All studies (Ames, HPRT, Chromosomal Aberration) did not afford any indication of mutagenicity.
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