Registration Dossier
Registration Dossier
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EC number: 309-913-1 | CAS number: 101357-16-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
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:
- 9 November 2009 - 3 December 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test method according to OECD Guideline 471. GLP study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- 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:
- Without metabolic activation (all strains): 0.610, 1.22, 2.44, 4.88, 9.77 and 19.5 μg/plate.
With metabolic activation (TA100, TA1535, TA98 and TA1537): 19.5, 39.1, 78.1, 156, 313 and 625 μg/plate.
With metabolic activation (WP2uvrA): 156, 313, 625, 1250, 2500 and 5000 μg/plate. - Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamida (AF2)
- Remarks:
- TA100 (0.1 µg/mL), WP2uvrA (0.2 µg/mL), TA98 (1 µg/mL)
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- sodium azide
- Remarks:
- TA1535 (5 µg/mL)
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- other: 9-aminoacridine hydrochloride hydrate (9AA)
- Remarks:
- TA1537 (800 µg/mL)
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- TA100 (10 µg/mL), TA1535 (20 µg/mL), WP2uvrA (100 µg/mL), TA98 (5 µg/mL), TA1537 (20 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min at 37 ºC
- Exposure duration: 48 hours at 47 ºC
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED:
DETERMINATION OF CYTOTOXICITY: Yes - Evaluation criteria:
- Results are found to be positive when revertant frequency increase 2 times or more compared with vehicle control, with a dose-response relationship and the results are reproducible.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation was observed in the doses with metabolic activation
RANGE-FINDING/SCREENING STUDIES:
First dose-finding test: With and without metabolic activation, for all test strains. Dosages: 5, 15, 50, 150, 1500 and 5000 μg /plate.
Second dose-finding test: Without metabolic activation, for all test strains. Dosages: 0.384, 0.960, 0.240, 6 and 15 μg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
Negative and positive controls according with historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytototixicy was observed at highest dose both with and without metabolic activation. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative (with and without metabolic activation)
Test item did not induce gene mutation in bacteria in a reverse mutation assay with and without metabolic activation. - Executive summary:
A bacterial reverse mutation assay was performed on test item according to OECD Guideline 471 preincubation method. Based on preliminary works, Salmonella typhimurium TA100, TA1535, TA98, TA1537 strains and Escherichia coli WP2uvrA strain were exposed up to 19.5 µg/mL without metabolic activation and up to 625 µg/mL (S. typhimurium strains) and 5000 µg/mL (WP2 uvrA) with metabolic activation. Negative solvent and positive controls were performed satisfactorily. Precipitation of the test item and cytotoxicity was detected in some concentrations. Test item was determined to be nonmutagenic under test conditions since a 2 -fold or greater and dose-dependent increase in revertant colonies was not observed in any test strain with or without metabolic activation. Based on increase in revertant colonies compared to the negative control, test item was determined to be nonmutagenic under test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: genome mutation
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: Test method according to Standard NTP Protocol (with variations). No data on GLP. Only two strains tested. Toxicity and precipitation observed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (only two strains tested, no data on validity criteria)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His+
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Induced male Sprague Dawley rat liver S9 and Syrian hamster liver S9.
- Test concentrations with justification for top dose:
- 1, 3, 10, 33, 100, 333, 666, 1000, 1666, 3333 and 6666 μg /plate (see details in “Any other information from results").
- Vehicle / solvent:
- Dimethyl Sulfoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA100 without metabolic activation
- Positive control substance:
- other: 4-nitro-o-phenylenediamine
- Remarks:
- TA98 without meatabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- TA98 and TA100 with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation (Standart NTP protocol with variations).
Test chemical (0.05 mL), Salmonella culture (0.10 ml), and S-9 mix or buffer (0.50 ml) were incubated at 37 ºC, without shaking, for 20 min. The top agar was added and the contents of the tubes were mixed onto the surface of petri dishes containing Voger-Bonner medium. The arising colories were counted following two days of incubation at 37 ºC. Plates were machine counted unless precipitate was present or the color of the test item interfered the count. Initial testing was in strains TA98 and TA100 without activation and with 30 % rat and hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the test were negative, the other strains were used with 30% and 10% S-9. 5% S-9 was also used. At least five doses of each chemical were tested in triplicate. - Evaluation criteria:
- Significant difference in the growth of revertant colonies compared with the negative control. A chemical was judged mutagenic (+) or weakly mutagenic (-W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, increases in his+ revertants did not meet the criteria for a "+W " response, or if only single doses produced increases in his+ revertants in repeated trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionnable response. A chemical was not designated nonmutagenic unless it had been tested in strains TA98, TA100, TA1535 and TA97 and/or TA 1537, without activation and with 10% and 30% rant and hamster S-9.
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- with 30% induced male Syrian hamster liver S9
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of thest item was observed (see details below).
RANGE-FINDING/SCREENING STUDIES:
Chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed (see details below). - Conclusions:
- Interpretation of results (migrated information):
positive with metabolic activation (with induced Spregue Dawley rat liver S9 at 30%)
The test substance showed gene mutation on Salmonella typhimurium T98 strain with induced Spregue Dawley rat liver S9 at 30%. - Executive summary:
A Salmonella typhimurium mutagenicity test was performed on test item according to NTP's preincubation protocol (test method similar to OECD Guideline 471) with variations. The Salmonella typhimurium strains TA98 and TA100 either with metabolic activation (5, 10 and 30% of S9 mix from Aroclor 1254-induced male Sprague-Dawley rat and Syrian hamster liver) and without metabolic activation were exposed up to 6666 µg/plate tes item in DMSO. Negative solvent and positive controls were performed satisfactorily. No mutagenic responses were observed in TA100 with and without metabolic activation. Test item was found mutagenic in the strain TA98 in the presence of induced Sprague Dawley rat liver S9 at 30%, but no increase in revertant colonies was observed whem treated in the presence of induced male Syrian hamster liver S9 at 30% or without exogenous metabolic activation. It should be mentioned that toxicity and precipitation was observed in the positive results.
- 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
- Study period:
- 10 November 2009 - 22 December 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test method according to OECD Guideline 473. GLP study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- mammalian cell line, other: Chine hamster lung (CHL/IU) cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (rat liver)
- Test concentrations with justification for top dose:
- Short-term treatment (6-18h, -S9mix): 79.0, 119, 178, 267, 400 and 600 µg/mL
Short-term treatment (6-18h, +S9mix): 28.1, 56.3, 113, 225, 450 and 900 µg/mL
Continuous treatment (24h, -S9mix): 35.1, 52.7, 79.0, 119, 178 and 267 μg/mL - Vehicle / solvent:
- - Vehicle: 0.5% CMC (carboxymethyl cellulose) - Sodium aqueous solution.
- Justification for choice of solvent/vehicle: Test item was insoluble in water and dimethyl sulfoxide. In DMSO it was not uniformly suspended at 100 mg/mL. - Negative solvent / vehicle controls:
- yes
- Remarks:
- wtith and without metabolic activation
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- mitomycin C
- Remarks:
- 1.5 µg/mL (6-18h), 0.5 µg/mL (24h)
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 2000 µg/mL (6-18h)
- Details on test system and experimental conditions:
- DURATION
- Exposure duration:
(1) Short-term treatment: with S9 metabolic activation; 24h without S9 metabolic activation
(2) Continuous treatment: 24h
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: 100-200 per replicate
DETERMINATION OF CYTOTOXICITY: relative total growth
EXAMINATIONS:
Chromosomal aberrations: Chromatid and chromosome breaks, chromatid and chromosome exchanges, chromatid and chromosome gaps, fragmentation, multiple aberration, pulverization, C-mitosis.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- Incidence of cells with chromosome aberration (Clastogenicity and polyploidy): <5% negative; 5-10% equivocal; >= 10% positive.
- Key result
- Species / strain:
- mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation was observed.
RANGE-FINDING/SCREENING STUDIES:
Range finding preliminary test was performed up to 5 mg/mL with metabolic activation (6 -18h exposure) and without metabolic activation (6 -18h and 24h exposure).
COMPARISON WITH HISTORICAL CONTROL DATA:
Positive and negative controls were within historical control data. - Conclusions:
- Interpretation of results (migrated information):
negative (with and without metabolic activation)
No increase in chromosomal aberrations was observed with or without metabolic activation under test contidions. - Executive summary:
An in-vitro chromosomal aberration test was performed in chinese hamster lung (CHL/IU) cells according to OECD Guideline 473 (GLP study). Based on preliminary results, cells were exposure for 6 -18h to 9.0, 119, 178, 267, 400 and 600 µg/mL without metabolic activation and 28.1, 56.3, 113, 225, 450 and 900 µg/mL with metabolic activation. Moreover, cells were exposure during 24 hours to 35.1, 52.7, 79.0, 119, 178 and 267 μg/mL test item. Between 100 and 200 cells were evaluated for both estructural aberration (clastogenicity) and numerical aberrations (polyploidy). Study was performed in triplicate. Solvent negative and positive controls were performed in paralelle. Test item did not induce chromosomal aberrations in cultured mammalian cells, regardless of the presence or absence of metabolic activation or treatment length. The substance was determined to be non-clastogenic.
- 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:
- July 10, 2012 - August 23, 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test method according to EU Method B.17 and OECD Guideline 476. GLP study.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/-mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (Rat Liver Homogenate S9 Fraction)
- Test concentrations with justification for top dose:
- Assay 1, 3-hour treatment with metabolic activation: 1000, 500, 250, 125, 93.75, 62.5, 31.25, 15.625 and 7.813 μg/mL
Assay 1, 3-hour treatment without metabolic activation: 500, 250, 125, 93.75, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL
Assay 2, 3-hour treatment with metabolic activation: 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL
Assay 2, 24-hour treatment without metabolic activation: 60, 50, 40, 30, 20, 10, 5, 2.5, 1.25 and 0.625 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the result of a short solubility test, the test tem was insoluble in Distilled water, but the 100 mg/mL formulation was achievable using Dimethyl sulfoxide (DMSO) as solvent (higher concentration formulations of 500 mg/mL or 250 mg/mL were not suitable for the test). Therefore, DMSO was selected for vehicle (solvent) of the study. The selected vehicle (solvent) was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Remarks:
- Untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO)
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.15 μg/mL (3h treatment) and 0.1 μg/mL (24h treatment) in DMSO
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- cyclophosphamide
- Remarks:
- 4 μg/mL in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (RPMI-5 medium)
DURATION
- Exposure duration: Assay 1: 3h (with and without metabolic activation); Assay 2: 3h (with metabolic activation), 24h (without metabolic activation)
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment
NUMBER OF CELLS EVALUATED: Total number of wells per assay: 768
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival
OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.
Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies were calculated. - Evaluation criteria:
- The test item was considered to be mutagenic if all the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency observed in treated cultures compared to the corresponding negative (solvent) control values at one or more concentrations.
3. Increases in mutation frequency reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. Significant concentration-relationship indicated by the linear trend analysis (p < 0.05).
5. Mutation frequency at the test concentration showing the largest increase at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (solvent) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. - Statistics:
- Statistical significance of mutant frequencies was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (see details below)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS: In Assays 1 and 2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was detected in the final treatment medium at the end of the treatment in the 1000-31.25 μg/mL and 500-31.25 μg/mL concentration range in Assay 1 (experiment with and without metabolic activation, respectively); and in the 100-30 μg/mL and 60-20 μg/mL concentration range in Assay 2 (experiment with and without metabolic activation, respectively).
RANGE-FINDING/SCREENING STUDIES: Insolubility and cytotoxicity was detected in the preliminary experiments. Therefore, concentrations up to the cytotoxicity limit were selected for the main experiments according to the recommendations of the relevant OECD guideline. Lower test concentrations were separated by factor of two. More closely spaced concentrations were used in the expected cytotoxic concentration range. At least nine concentrations were selected for the main experiments in each assay.
COMPARISON WITH HISTORICAL CONTROL DATA: The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 1000, 500, 250, 125 and 93.75 μg/mL concentrations, cells of these samples did not survive the expression period. In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 500, 250, 125 and 93.75 μg/mL concentrations, cells of this sample did not survive the expression period. In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity of the test item was observed at 100, 90 and 80 μg/mL concentrations, cells of these samples did not survive the expression period. In Assay 2, following a 24-hour treatment, excessive cytotoxicity of the test item was observed at 60, 50, 40 30 and 20 μg/mL concentrations, cells of these samples did not survive the expression period. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative (with and without metabolic activation)
no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. - Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of acetone oxime to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Dimethyl sulfoxide was used as the solvent of the test item in this study. The test item was examined up to 2000 μg/mL in the Preliminary Toxicity Test (the highest achievable concentration based on the solubility of the test item). Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays: Assay 1, 3-hour treatment with metabolic activation: 1000, 500, 250, 125, 93.75, 62.5, 31.25, 15.625 and 7.813 μg/mL; Assay 1, 3-hour treatment without metabolic activation: 500, 250, 125, 93.75, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL; Assay 2, 3-hour treatment with metabolic activation: 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL; Assay 2, 24-hour treatment without metabolic activation: 60, 50, 40, 30, 20, 10, 5, 2.5, 1.25 and 0.625 μg/mL. In Assays 1 and 2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was detected in the final treatment medium at the end of the treatment in the 1000-31.25 μg/mL and 500-31.25 μg/mL concentration range in Assay 1 (experiment with and without metabolic activation, respectively); and in the 100-30 μg/mL and 60-20 μg/mL concentration range in Assay 2 (experiment with and without metabolic activation, respectively). In both assays cytotoxicity was observed and therefore, an evaluation was made using data of the next concentrations. All the validity criteria were fulfilled and the overall study was considered to be valid. In conclusion, no mutagenic effect was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay (see details above).
Referenceopen allclose all
Results obtained in the main test:
Metabolic activation |
µg/plate |
Revertant colonies / plate (triplicate, average) |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Without |
DMSO |
89 ± 10 |
7 ± 1 |
25 ± 2 |
19 ± 4 |
7 ± 3 |
0.610 |
89 ± 6 |
4 ± 1 |
21 ± 2 |
23 ± 6 |
4 ± 1 |
|
1.22 |
93 ± 11 |
6 ± 2 |
24 ± 4 |
18 ± 4 |
5 ± 1 |
|
2.44 |
95 ± 3 |
8 ±2 |
21 ± 7 |
17 ± 2 |
5 ± 3 |
|
4.88 |
72 ± 3 |
6 ± 1 |
21 ± 5 |
16 ± 9 |
6 ± 2 |
|
9.77 |
86 ± 11* |
4 ± 3 * |
24 ± 4 * |
21 ± 3 * |
5 ± 3 * |
|
19.5 |
82 ± 9 * |
6 ± 1 * |
25 ± 3 * |
21 ± 2 * |
2 ± 1 * |
|
AF-2 |
409 ± 16 |
-- |
427 ± |
551 ± 21 |
-- |
|
NaN3 |
-- |
251 ± 5 |
-- |
-- |
-- |
|
9AA |
-- |
-- |
-- |
-- |
404 ± 65 |
|
With |
DMSO |
98 22 |
7 ± 2 |
28 ± 4 |
30 ± 13 |
9 ± 1 |
19.5 |
118 ± 2 |
9 ± 4 |
-- |
44 ± 6 |
8 ± 3 |
|
39.1 |
117 ± 13 |
9 ± 3 |
-- |
43 ± 5 |
9 ± 1 |
|
78.1+ |
97 ± 5 |
7 ± 4 |
-- |
33 ± 6 |
8 ± 2 |
|
156 + |
103 ± 14 |
5 ± 1 |
29 ± 3 |
33 ± 3 |
6 ± 3 |
|
313 + |
101 ± 2 |
7 ± 2 |
27 ± 3 |
24 ± 2 |
6 ± 1 |
|
625 + |
115 ± 5 * |
8 ± 3 * |
23 ± 6 |
26 ± 9 * |
7 ± 4 * |
|
1250 + |
-- |
-- |
21 ± 2 |
-- |
-- |
|
2500 + |
-- |
-- |
18 ± 1 |
-- |
-- |
|
5000 + |
-- |
-- |
17 ± 5 |
-- |
-- |
|
2AA |
687 ± 41 |
147 ± 10 |
193 ± 18 |
548 ± 27 |
189 ± 10 |
|
(--) Not tested
(+) Precipitation
(*) Cytotoxicity
TA100
Dose |
No Activation |
5% RLI |
30% RLI |
30% RLI |
30% HLI |
10% RLI |
||||||
(Negative) |
(Negative) |
(Equivocal) |
(Negative) |
(Negative) |
(Negative) |
|||||||
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
||||||
ug/Plate |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
0 |
135 |
2.3 |
149 |
8.8 |
130 |
7.8 |
142 |
5.6 |
159 |
.6 |
160 |
.9 |
1 |
|
|
161 |
8.1 |
|
|
154 |
11.7 |
|
|
137 |
13.3 |
3 |
|
|
139 |
2 |
|
|
121 |
13.7 |
|
|
151 |
3.7 |
10 |
163 |
1.9 |
147 |
8.4 |
|
|
165 |
9.7 |
|
|
145 |
2.7 |
33 |
138 |
7.2 |
134 |
6.2 |
200 |
2 |
150 |
4.6 |
|
|
136 |
3.3 |
100 |
125 p |
1.5 |
136 p |
6.4 |
196 p |
17.6 |
153 p |
12 |
133 p |
5.8 |
127 p |
1.2 |
333 |
94 p |
5.2 |
|
|
137 p |
5.8 |
|
|
115 p |
14.1 |
|
|
666 |
45 x |
2.7 |
|
|
|
|
|
|
|
|
|
|
1000 |
|
|
|
|
117 p |
9.2 |
|
|
112 p |
17.7 |
|
|
1666 |
|
|
|
|
126 p |
8.6 |
|
|
|
|
|
|
3333 |
|
|
|
|
|
|
|
|
122 p |
7.4 |
|
|
6666 |
|
|
|
|
|
|
|
|
93 x |
46.5 |
|
|
Positive Control |
620 |
21 |
516 |
14.1 |
392 |
33 |
787 |
42.9 |
529 |
8.4 |
374 |
16.5 |
TA98
Dose |
No Activation |
30% RLI |
30% RLI |
30% RLI |
30% HLI |
|||||
(Negative) |
(Positive) |
(Positive) |
(Positive) |
(Negative) |
||||||
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
|||||
ug/Plate |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
Mean |
±SEM |
0 |
17 |
3.2 |
29 |
2.3 |
26 |
3.1 |
29 |
.6 |
34 |
3.3 |
1 |
|
|
|
|
25 |
6.1 |
21 |
1.2 |
|
|
3 |
|
|
|
|
27 |
2.3 |
34 |
3.8 |
|
|
10 |
16 |
2.7 |
|
|
40 x |
20 |
47 |
3.2 |
|
|
33 |
16 |
1 |
117 |
10.8 |
99 |
3.4 |
84 |
6.1 |
|
|
100 |
16 p |
1.5 |
77 x |
38.9 |
56 x |
28.5 |
119 p |
6.9 |
27 p |
2.7 |
333 |
14 p |
1.5 |
46 p |
3.5 |
|
|
|
|
24 p |
1.3 |
666 |
13 p |
1.5 |
|
|
|
|
|
|
|
|
1000 |
|
|
23 p |
2.2 |
|
|
|
|
15 p |
2.5 |
1666 |
|
|
18 p |
4 |
|
|
|
|
|
|
3333 |
|
|
|
|
|
|
|
|
20 p |
2.5 |
6666 |
|
|
|
|
|
|
|
|
15 p |
1.5 |
Positive Control |
645 |
32 |
115 |
7.3 |
159 |
17.1 |
161 |
14.8 |
251 |
.9 |
Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; t = Toxic; c = Contamination
Results:
Treatment (h) |
S9mix |
Dose µg/mL |
Number of cells |
Estructural ab. (% frequenc) |
Judgment |
Chrom. Ab. (gaps) |
Growth rate (%) |
Polyploid (% frequenc) |
Judgment |
6-18 |
- |
Negative |
100 |
0 |
-- |
0 |
100 |
0 |
-- |
100 |
1 |
0 |
1 |
||||||
200 |
1 (0.5) |
0 |
1 (0.5) |
||||||
6-18 |
- |
79.0 |
NT |
-- |
-- |
-- |
92.0 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
- |
119 |
NT |
-- |
-- |
-- |
86.7 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
- |
178 |
NT |
-- |
-- |
-- |
92.0 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
- |
267 |
100 |
0 |
Negative |
0 |
76.7 |
0 |
Negative |
100 |
0 |
0 |
0 |
||||||
200 |
0 (0) |
0 |
0 (0.0) |
||||||
6-18 |
- |
400 |
100 |
0 |
Negative |
0 |
61.3 |
1 |
Negative |
100 |
1 |
0 |
0 |
||||||
200 |
1 (0.5) |
0 |
1 (0.5) |
||||||
6-18 |
- |
600 |
100 |
0 |
Negative |
0 |
58.7 |
2 |
Negative |
100 |
0 |
0 |
0 |
||||||
200 |
0 (0) |
0 |
2 (1.0) |
||||||
6-18 |
- |
Positive |
100 |
26 |
-- |
0 |
66.7 |
0 |
-- |
100 |
26 |
0 |
0 |
||||||
200 |
52 (26.0) |
0 |
0 (0.0) |
Treatment (h) |
S9mix |
Dose µg/mL |
Number of cells |
Estructural ab. (% frequency) |
Judgment |
Chrom. Ab. (gaps) |
Growth rate (%) |
Polyploid (% frequenc) |
Judgment |
6-18 |
+ |
Negative |
100 |
0 |
-- |
0 |
100 |
0 |
-- |
100 |
0 |
0 |
1 |
||||||
200 |
0 (0) |
0 |
1 (0.5) |
||||||
6-18 |
+ |
28.1 |
NT |
-- |
-- |
-- |
98.6 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
+ |
56.3 |
NT |
-- |
-- |
-- |
108.0 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
+ |
113 |
NT |
-- |
-- |
-- |
105.1 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
6-18 |
+ |
225 |
100 |
0 |
Negative |
0 |
102.9 |
0 |
Negative |
100 |
0 |
0 |
1 |
||||||
200 |
0 (0) |
0 |
1 (0.5) |
||||||
6-18 |
+ |
450 |
100 |
4 |
Negative |
0 |
88.4 |
1 |
Negative |
100 |
1 |
0 |
0 |
||||||
200 |
5 (2.5) |
0 |
1 (0.5) |
||||||
6-18 |
+ |
900 |
100 |
4 |
Negative |
0 |
44.9 |
0 |
Negative |
100 |
2 |
0 |
1 |
||||||
200 |
6 (3) |
0 |
1 (0.5) |
||||||
6-18 |
+ |
Positive |
100 |
27 |
-- |
1 |
73.9 |
0 |
-- |
100 |
22 |
0 |
1 |
||||||
200 |
49 (24.5) |
1 |
1 (0.5) |
Treatment (h) |
S9mix |
Dose µg/mL |
Number of cells |
Estructural ab. (% frequency) |
Judgment |
Chrom. Ab. (gaps) |
Growth rate (%) |
Polyploid (% frequenc) |
Judgment |
24-0 |
- |
Negative |
100 |
0 |
-- |
0 |
100 |
1 |
-- |
100 |
0 |
0 |
1 |
||||||
200 |
0 (0) |
0 |
2 (1.0) |
||||||
24-0 |
- |
35.1 |
NT |
-- |
-- |
-- |
94.5 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
24-0 |
- |
52.7 |
NT |
-- |
-- |
-- |
82.8 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
24-0 |
- |
79.0 |
NT |
-- |
-- |
-- |
71.7 |
- |
-- |
NT |
-- |
-- |
- |
||||||
NT |
-- |
-- |
- |
||||||
24-0 |
- |
119 |
100 |
0 |
Negative |
0 |
69.0 |
1 |
Negative |
100 |
0 |
0 |
0 |
||||||
200 |
0 (0) |
0 |
1 (0.5) |
||||||
24-0 |
- |
178 |
100 |
0 |
Negative |
0 |
62.1 |
1 |
Negative |
100 |
0 |
0 |
1 |
||||||
200 |
0 (0) |
0 |
2 (1.0) |
||||||
24-0 |
- |
267 |
100 |
0 |
Negative |
0 |
53.1 |
0 |
Negative |
100 |
1 |
0 |
0 |
||||||
200 |
1 (0.5) |
0 |
0 (0) |
||||||
24-0 |
- |
Positive |
100 |
14 |
-- |
0 |
76.6 |
0 |
-- |
100 |
18 |
0 |
0 |
||||||
200 |
32 (16.0) |
0 |
0 (0) |
NT: Not tested.
Precipitation was observed.
In Assay 1, following a 3-hour treatment with metabolic activation, an evaluation was made using the concentration of 62.5 μg/mL (relative survival: 23%, relative total growth: 1%) and three lower concentrations (a total of four concentrations). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as negative.
In Assay 1, following a 3-hour treatment without metabolic activation, an evaluation was made using data of following concentration of 62.5 μg/mL (relative survival of 5%, relative total growth: 1%) and the next four concentrations (a total of five concentrations). Statistically significant and biologically relevant increase in the mutation frequency was observed at 31.25 μg/mL concentration. Dose response to the treatment was also indicated by the linear trend analysis. This experiment was considered as being slight positive (requiring confirmation).
In Assay 2, following a 3-hour treatment with metabolic activation, an evaluation was made using data of the following concentration of 70 μg/mL (relative survival: 23%, relative total growth: 1%) and the next seven concentrations (a total of eight concentrations). Statistically significant increase in the mutation frequency was observed at 50 μg/mL concentration. However, the difference between the observed value and the relevant vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically not relevant increase. Dose response to the treatment was indicated by the linear trend analysis, but based on the individual mutation frequency values; this fact had no biological relevance. Overall this experiment was considered as negative, thus confirming the results of the first experiment with metabolic activation.
In Assay 2, following a 24-hour treatment, an evaluation was made using data of following concentration of 10 μg/mL (relative survival: 18%, relative total growth: 6%) and the next four concentrations (a total of five concentrations). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative, thus the observed slight positive results in the first assay using the short treatment without metabolic activation was not confirmed. As the apparent slight effect in Assay 1 was not repeatable in Assay 2, it is concluded that there was no mutagenic effect attributable to the test item.
The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
Additional information
Genetic toxicity in vitro: Bacterial reverse mutation assay:
Key study: Experimental data.
Test method according to OECD 471. GLP study. The test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay.
Genetic toxicity in vitro: Chromosomal aberrations in mammalian cells:
Key study: Experimental data.
Test method according to OECD 473. GLP study. The test substance was not clastogenic in Chinese Hamster Ovary cells.
Genetic toxicity in vitro: Gene mutation in mammalian cells:
Key study: Experimental data.
Test method according to OECD 476. GLP study. The test substance was not mutagenic in the mouse lymphoma assay.
Justification for selection of genetic toxicity endpoint
No study was selected since the results obtained in the in vitro studies were negative.
Short description of key information:
The test item is not genotox since the results obtained in the in-vitro studies, Ames test (OECD 471, GLP), Chromosome aberration test (OECD 473, GLP) and Mammalian cell gene mutation assay (OECD 476, GLP) were negative.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Based on the available data, the substance is not classified for genetic toxicity according to CLP 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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