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Registration Dossier
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EC number: 227-030-9 | CAS number: 5610-94-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
OECD 471 (RL1): not mutagenic in the bacterial test system with and without S9 mix
OECD 473 (RL1): not clastogenic in mammalian cells with and without S9 mix
OECD 476 (RL1): not mutagenic in mammalian cells with and without S9 mix
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:
- 1982
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- No historical control range data given
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- yes
- Remarks:
- No historical control range data given
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protected from light - Target gene:
- his operon, trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- 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 male rats, treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Based on a range-finding study (= Experiment 1, performed in all tester strains; doses applied: 4 - 10 000 μg/mL), the following concentrations were used in the main experiment (= Experiment 2): 0.8, 4, 20, 100, 500 and 2500 μg/plate with and without metabolic activation.
The top dose was selected based on precipitation observed at ≥ 2500 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene (2AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))
DURATION
- Exposure duration: 48 - 72 h
NUMBER OF REPLICATIONS: triplicates
DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn
The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance. - Evaluation criteria:
- A chemical is considered to have a positive response, if it shows a significant increase in the number of induced revertants and/or shows dose-dependent effect.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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 applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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 applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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 applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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 applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- 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 applicable
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, observed at ≥ 2500 μg/plate with and without S9 mix in the plates of all tested strains - Conclusions:
- It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.
- Executive summary:
The test item was tested for mutagenicity with Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 and E. coli WP2uvrA with and without the addition of a metabolic activation system according to OECD Guideline 471. The test compound was tested at doses of 0.4 to 10000 µg/plate and proved to be not toxic. Visible precipitation on the plates has been observed at 2500 µg/plate. For mutagenicity testing 2500 µg/plate was chosen as the highest dose.
The test compound did not cause a significant increase in the number of colonies with any of the tester strains either in the absence or presence of S9 mix. No dose dependent effect was obtained.
It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 - 26 Aug 1987
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- No historical control range data; less metaphases scored (200/concentration); only 1 concentration with and without metabolic activation, respectively, for the 6- and 28-h fixation times. Treatment time was 2 h and expression time 4 h, 16 h and 26 h.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- yes
- Remarks:
- No historical control range data; less metaphases scored (200/concentration); only 1 concentration with and without metabolic activation, respectively, for the 6- and 28-h fixation times. Treatment time was 2 h and expression time 4 h, 16 h and 26 h.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at 4 °C, protected from light - Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory of Genetic Toxicology Hoechst Aktiengesellschaft, Frankfurt (Main), Germany
- Suitability of cells: yes
- Methods for maintenance in cell culture if applicable: propagated at 37°C in 25 cm² flasks, cells were subcultured twice a week
MEDIA USED
- Type and identity of media including CO² concentration if applicable: MEM (Minimal essential medium) with 25 mM Hepes-buffer, supplemented with 10% FCS
- Properly maintained: yes - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Spindle inhibitor: colcemid (0.04 µg/mL medium)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 2 h treatment, 6 h fixation time and 2 h treatment, 28 h fixation time: 75 μg/mL (without metabolic activation), 150 μg/mL (with metabolic activation)
2 h treatment, 18 h fixation time: 25, 50 and 75 μg/mL (without metabolic activation), 37.5, 75 and 150 μg/mL (with metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- untreated cells (18 h treatment)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO (all treatments)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
Cell density at seeding:
6 h fixation.: 1 - 2 x 10^6 cells/ flask (80 cm² flask)
18 h fixation: 4 - 6 x 10^5 cells/ flask (25 cm² flask)
28 h fixation.: 2 - 4 x 10^5 cells/ flask (25 cm² flask)
DURATION
- Exposure duration: 2 h
- Expression time (cells in growth medium): 4 h, 16 h and 26 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6, 18 and 28 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.04 µg/mL medium)
STAIN (for cytogenetic assays): 2% orcein solution
DOUBLING TIME: 12 - 16 h
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were detached from the plate by treatment with trypsin solution. A potassium chloride solution (0.075 M) was added, the cells were resuspended and incubated for 10 min in a water bath at 37 °C. 1.5 mL of fixative was added and a flow through with air was performed. After centrifugation the cells were covered with fixative (methanol:glacial acetic acid (3:1)) for 20 min. The supernatant was removed carefully and the cells were resuspended in fixative and incubated for further 30 min. After another centrifugation step the fixative was exchanged and the cells incubated for at least 12 h at 4 °C (overnight). Following centrifugation and resuspension in a small amount of fresh fixative, a drop of the cell suspension was placed on a slide and was air-dried and then stained for 10 min in 2 % orcein solution. After several washing steps in water, acetone and xylene they were embedded in EntellanR and EukittR before being examined microscopically. 2 - 5 slides were prepared from each flask.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases per plate (200 metaphases/ concentration)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, in 1000 cells - Rationale for test conditions:
- The test conditions are based on the results of the range-finding study.
- Evaluation criteria:
- The test chemical is to be considered mutagenic (clastogenic) in this assay if it induces a significantly increased chromosomal aberration rate in at least one tested concentration compared to the negative control or an chromosomal aberration rate clearly exceeding the one induce by the negative control. Additionally, the increase should be in a reproducible or concentration-dependent manner. For a classification as non mutagenic, the results of treatment with and without metabolic activation should be negative.
- Statistics:
- Binominal statistics with Fisher's exact test
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- After treatment with 25 µg/mL of the test substance, a significant increase in abberant cells (with gaps) compared to the negative control was observed, but it can be disregarded as this effect was neither concentration-dependent nor reproducible.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: The highest concentration without visible precipitation was found to be 300 µg/mL. The test substance produced significant cytotoxicity in the absence of metabolic activation starting at a concentration of 70 µg/mL. This effect was not observed in the presence of metabolic activation, however at 240 and 300 µg/mL no metaphases were detectable.
At > 300 µg/mL precipitation was observed, which was not the case for the main experiment.
ADDITIONAL INFORMATION ON GENOTOXICITY:
None of the tested concentrations (after a fixation time of 6, 18 and 28 h, with and without S9 mix) induced an increase in the chromosal aberration rate. (For details see Table 1 in "Any other information on results incl. tables") - Conclusions:
- In conclusion, the test item did not induce a repeatable, significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, it is considered not clastogenic in this test system.
- Executive summary:
The test substance was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).
A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect without metabolic activation from 75 µg/mL up to the limit of solubility (300 µg/mL). No cytotoxic effect was observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent cell cultures with and without metabolic activation were used. At dose levels of 300 and 240 µg/mL with metabolic activation were no metaphases to evaluate. For main experiment dose levels of 25, 50, 75 µg/mL in the absence and 37.5, 75, 150 µg/mL in the presence of S9-mix metabolic acitvation were used.
The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.
In conclusion does not induce chromosome mutations (= aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.
- 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
- Study period:
- 2019-01-17 until 2019-02-07
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: according to OECD 476
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
The test item was supplied by or on behalf of the Sponsor including the following information:
Identification: Diazo PW 980
Batch: DEAA087916
Purity: 93.1% (HPLC)
Molecular Weight: 926 g/mol
Expiry / Retest Date: 13 November 2019
Storage Conditions: At room temperature, protected from light*
Physical state / Appearance: Solid, yellow
Stability in Solvent: Not indicated by the sponsor
* only valid for storage condition, not for test performance
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment (immediately before treatment), the test item was suspended in deionized water. The final concentration of deionized water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
OTHER SPECIFICS: No - Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- without S9 Mix: 0.8, 1.6, 3.1, 6.3, 12.5 (P), 25.0 (P), 50.0 (P), 100.0 (P) µg/mL
with S9 mix: 0.8, 1.6, 3.1, 6.3, 12.5 (P), 25.0 (P), 50.0 (P), 100.0 (P) µg/mL
P = Precipitation visible to the unaided eye at the end of treatment
The cultures at 25.0, 50.0, and 100.0 µg/mL with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations. - Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water (local tap water, deionised at Envigo CRS GmbH)
- Justification for choice of solvent/vehicle: solubility properties - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation,
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 9 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concen¬trations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Results of Linear Regression:
experimental group without S9 mix: p-value 0.879
experimental group with S9 mix: p-value 0.106
The p-value was calculated for the mean mutant frequencies of culture I and II
A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.39 in the solvent control versus pH 7.40 at 200.0 µg/mL)
- Effects of osmolality: No relevant increase (289 mOsm in the solvent control versus 286 mOsm at 200 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 12.5 µg/mL and above in the main experiment
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 1.6 µg/mL and 200 µg/mL were used. The highest concentration was chosen based on the solubility properties of the test item.
No cytotoxic effect, indicated by a relative cloning efficiency of approx. 50% or below was observed up to the highest concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 12.5 µg/mL and above in the absence of metabolic activation, and at 25.0 µg/mL and above in the presence of metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment no cytotoxic effect indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation. - Remarks on result:
- other: strain/cell type: Chinese hamster lung fibroblasts (V79)
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Diazo PW 980 is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The study was performed to investigate the potential of Diazo PW 980 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The treatment period was 4 hours with and without metabolic activation.
The maximum test item concentration of the pre-experiment (200 µg/mL) was based on the solubility properties of the test item. The highest concentration in the main experiment (100.0 µg/mL) was limited by precipitation observed in the pre-experiment.
No increase in the mean mutant colony numbers/106cells was observed in the main experiment up to the maximum concentration.
No cytotoxic effects indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
Referenceopen allclose all
Table 1: Summary of test results (Experiment 1; dose-finding assay (Plate Incorporation Method)
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||||
Frameshift type |
Base-pair substitution type |
||||||
TA 98 |
TA 1537 |
TA 1538 |
TA 100 |
TA 1535 |
WP2 uvr A |
||
– |
Solvent control (DMSO) |
27 |
8 |
27 |
121 |
27 |
36 |
4 |
30 |
11 |
12 |
90 |
33 |
33 |
|
20 |
25 |
9 |
13 |
107 |
27 |
40 |
|
100 |
37 |
9 |
20 |
120 |
31 |
37 |
|
500 |
26 |
5 |
22 |
102 |
26 |
30 |
|
2500 |
24 P |
7 P |
14 P |
115 P |
27 P |
35 P |
|
10000 |
21 P |
5 P |
5 P |
83 P |
23 P |
41 P |
|
Positive controls (µg/plate) |
2NF (5) |
9AA (50) |
2NF (5) |
SA (1) |
SA (1) |
MNNG (10) |
|
Mean (No. of colonies/plate) |
204 |
81 |
319 |
833 |
617 |
1610 |
|
+ |
Solvent control (DMSO) |
39 |
3 |
24 |
99 |
11 |
33 |
4 |
35 |
14 |
8 |
115 |
11 |
38 |
|
20 |
40 |
5 |
24 |
164 |
15 |
62 |
|
100 |
36 |
4 |
18 |
125 |
5 |
41 |
|
500 |
41 |
5 |
21 |
119* |
13 |
44 |
|
2500 |
40 P |
9 P |
17 P |
135 P |
8 P |
47 P |
|
10000 |
33 P |
8 P |
15 P |
125 P |
9 P |
40 P |
|
Positive controls (µg/plate) |
2AA (1) |
2AA |
|||||
Mean (No. of colonies/plate) |
917 |
56 |
786 |
894 |
113 |
241 |
|
Positive controls (µg/plate) |
B(a)P (10) |
||||||
Mean (No. of colonies/plate) |
605 |
121 |
243 |
607 |
19 |
93 |
|
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
P = precipitate
SA = sodium azide
Table 2: Summary of test results (Experiment 2, main assay (Plate Incorporation Method))
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||||
Frameshift type |
Base-pair substitution type |
||||||
TA 98 |
TA 1537 |
TA 1538 |
TA 100 |
TA 1535 |
WP2 uvr A |
||
– |
Solvent control (DMSO) |
25 |
10 |
13 |
120 |
29 |
32 |
0.8 |
28 |
8 |
13 |
133 |
23 |
23 |
|
4 |
20 |
7 |
12 |
128 |
23 |
26 |
|
20 |
28 |
10 |
10 |
137 |
23 |
24 |
|
100 |
30 |
8 |
13 |
127 |
24 |
26 |
|
500 |
28 |
9 |
11 |
131 |
21 |
21 |
|
2500 |
27 P |
8 P |
14 P |
125 P |
23 P |
26 P |
|
Positive controls (µg/plate) |
2NF (5) |
9AA (50) |
2NF (5) |
SA (1) |
SA (1) |
MNNG (10) |
|
Mean (No. of colonies/plate) |
204 |
81 |
319 |
833 |
617 |
1610 |
|
+ |
Solvent control (DMSO) |
31 |
14 |
17 |
125 |
13 |
24 |
0.8 |
41 |
11 |
17 |
111 |
10 |
27 |
|
4 |
34 |
12 |
20 |
111 |
13 |
32 |
|
20 |
39 |
10 |
21 |
126 |
11 |
30 |
|
100 |
34 |
8 |
19 |
123 |
15 |
25 |
|
500 |
37 |
11 |
19 |
123 |
13 |
37 |
|
2500 |
30 P |
10 P |
20 P |
110 P |
12 P |
35 P |
|
Positive controls (µg/plate) |
2AA (1) |
2AA |
|||||
Mean (No. of colonies/plate) |
917 |
56 |
786 |
894 |
113 |
241 |
|
Positive controls (µg/plate) |
B(a)P (10) |
||||||
Mean (No. of colonies/plate) |
605 |
121 |
243 |
607 |
19 |
93 |
|
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
P = precipitate
SA = sodium azide
Table 1: Test results
Test item |
Concentration |
Mitotic Index |
Aberrant cells |
||
|
|
mean in % |
with gaps |
without gaps |
type of abberation |
Exposure period 2 h, fixation time 6 h, without S9 mix |
|||||
DMSO |
- |
8.7 |
3 |
2 |
iG, iD, Di |
Test substance |
75 |
8.1 |
1 |
1 |
iB |
Exposure period 2 h, fixation time 6 h with S9 mix |
|||||
DMSO |
- |
11.3 |
3 |
0 |
G |
Test substance |
150 |
8.5 |
3 |
0 |
G, iG |
Exposure period 2 h, fixation time 18 h, without S9 mix |
|||||
Untreated cells |
- |
16.1 |
4 |
0 |
G, iG |
DMSO |
- |
11.7 |
1 |
0 |
G |
EMS |
10 mM |
11.1 |
17* |
9* |
G, iG, B, F, iD, Ex, Di |
Test substance |
25 µg/mL |
12.2 |
7* |
1 |
G, iB |
50 µg/mL |
10.1 |
4 |
1 |
G, iG, M |
|
75 µg/mL |
10.9 |
4 |
1 |
G, iG, M |
|
Exposure period 2 h, fixation time 18 h, with S9 mix |
|||||
Untreated cells |
- |
12.8 |
1 |
0 |
G |
DMSO |
- |
11.6 |
2 |
0 |
iG |
CP |
2.7 µg/mL |
14.7 |
24* |
13* |
G, iG, B, iB, F, iF, D, iD, Ex, Di, M |
Test substance |
37.5 µg/mL |
11.9 |
1 |
0 |
iG |
75 µg/mL |
13.7 |
6 |
0 |
G |
|
150 µg/mL |
12.4 |
5 |
3 |
iG, F |
|
Exposure period 2 h, fixation time 28 h, without S9 mix |
|||||
DMSO |
- |
9.5 |
1 |
0 |
G |
Test substance |
75 µg/mL |
11.2 |
3 |
1 |
G, iG, iF |
Exposure period 2 h, fixation time 28 h, with S9 mix |
|||||
DMSO |
- |
8.9 |
2 |
0 |
G, iG |
Test substance |
150 µg/mL |
11.0 |
2 |
0 |
G, iG |
EMS = ethylmethanesulfonate
CP = cyclophosphamide
G = gap
iG = isochromatide gap
B = break
iB = isochromatide break
F = fragment
iF = isochromatide fragment
D = deletion
iD = isochromatide deletion
Ex = exchanges including intrachanges
Di = dicentrics
M = minute
* = p < 0.05
Summary Table / Mean values of culture I and II
| conc. µg/mL | S9 mix | relavtive CE I |
relative cell density | rel. adj. CE I | mutant colonies/106 cells | 95% conf. interval | |
|
|
|
|
|
|||
| Solvent control (deion. water) | - | 100.0 | 100.0 | 100.0 | 10.3 | 2.8 - 30.9 | |
| Positive control (EMS) | 300.0 | - | 84.4 | 94.8 | 80.0 | 154.7 | 2.8 - 30.9 |
Test Item |
0.8 | - | 98.8 | 100.7 | 99.3 | 5.7 | 2.8 - 30.9 |
| Test Item | 1.6 | - | 84.4 | 83.7 | 70.6 | 14.5 | 2.8 - 30.9 |
| Test Item | 3.1 | - | 105.4 | 90.8 | 96.0 | 17.9 | 2.8 - 30.9 |
| Test Item | 6.3 | - | 84.3 | 82.1 | 69.1 | 8.4 | 2.8 - 30.9 |
| Test Item | 12.5 | - | 81.9 | 76.6 | 62.7 | 12.0 | 2.8 - 30.9 |
| Test Item | 25.0 | - | # | # | # | # | # |
| Test Item | 50.0 | - | # | # | # | # | # |
| Test Item | 100.0 | - | # | # | # | # | # |
| Solvent control (deion. water) | + | 100.0 | 100.0 | 100.0 | 6.9 | 3.1 - 30.7 | |
| Positive control (DMBA) | 2.3 | + | 93.2 | 70.9 | 65.7 | 54.6 | 3.1 - 30.7 |
| Test Item | 0.8 | + | 94.2 | 99.3 | 93.7 | 8.7 | 3.1 - 30.7 |
| Test Item | 1.6 | + | 83.3 | 93.0 | 77.5 | 8.8 | 3.1 - 30.7 |
| Test Item | 3.1 | + | 85.0 | 121.1 | 103.1 | 7.2 | 3.1 - 30.7 |
| Test Item | 6.3 | + | 79.8 | 91.3 | 72.3 | 8.8 | 3.1 - 30.7 |
| Test Item | 12.5 | + | 89.4 | 108.7 | 98.0 | 10.0 | 3.1 - 30.7 |
| Test item | 25.0 | + | # | # | # | # | # |
| Test item | 50.0 | + | # | # | # | # | # |
| Test item | 100.0 | + | # | # | # | # | # |
P = precipitation visible at the end of treatment
# culture was not continued to avoid too many concentrations with precipitation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on the data provided, the test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.
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.
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