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EC number: 485-270-1 | CAS number: -
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames study is available performed according to OECD/EC guidelines and GLP principles. The results showed that the substance was positive without metabolic activation in tester strain TA100.
In addition, an in vitro chromosome aberration test is available performed in Chinese Hamster V79 cells according to OECD/EC guidelines and GLP principles. The results do not indicate that the test item is clastogenic.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 December 2006 - 14 May 2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2000
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Storage: at room temperature
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix
- Test concentrations with justification for top dose:
- Duplicate cultures were treated at each concentration. The selection of the concentrations was based on the results from the pre-test (see below).
Experiment I:
without metabolic activation: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 µg/mL
with metabolic activation: 200, 400, 600, 750, 850 and 1000 µg/mL
Experiment II:
without metabolic activation: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 µg/mL
with metabolic activation: 150, 250, 350, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL - Vehicle / solvent:
- Ethanol
The test item could not be dissolved in DMSO, but the test item was found to dissolve in ethanol.
Precipitation of the test item was noted at 400 µg/mL and higher. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Details on test system and experimental conditions:
- Exposure period experiment I: 4 hours (with and without metabolic activation)
Exposure period experiment II: 4 hours (with metabolic activation, 20 hours (with metabolic activation)
Seeding of Cultures:
3 or 4 days old stock cultures with higher than 50% confluency were trypsinised at 37°C for 5 min. by adding a trypsin solution in Ca-Mg-free PBS solution. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment.
The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). Into each chamber 1 x 10^4 - 5x 10^5 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.
Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation)
The treatment with metabolic activation:
2 days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 µL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without exogenic metabolic activation.
4 hours after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
In the experiment without metabolic activation, 2 days after seeding the cells were incubated with the test item in complete medium (MEM with 10% FCS) for 20 hours. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.
All cultures were incubated at 37°C in a humidified atmosphere with 5.0% CO2 (95.0% air).
The cytotoxicity was determined by the mitotic index (% cells in mitosis) by counting the number of mitotic cells per 1000 cells. As an additional parameter the relative cell density was calculated as the mean of twenty cell counts per test group. - Rationale for test conditions:
- A pre-test was conducted under identical conditions as described for the main experiment. The following concentrations were tested:
Without S9-mix: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 µg/mL
With S9-mix: 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL - Evaluation criteria:
- Criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary (3)(10). [...]
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance. - Statistics:
- The non-parametric Fisher's exact test was used.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The following concentrations were selected for microscopic analyses:
Experiment I
without metabolic activation: 10, 12, 14 and 16 µg/mL
with metabolic activation: 400, 600 and 750 µg/mL
Experiment I
without metabolic activation: 26, 28 and 30 µg/mL
with metabolic activation: 750, 800, 850 and 900 µg/mL
Precipitation: Precipitate was noted in all dose groups with metabolic activation. No precipitate of the test item was observed in the dose groups without metabolic activation.
Observations:
No biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
No increase in the frequencies of polyploid cells was found.
The results are included below in tabular form (including historical control data). - Conclusions:
- Based on the results of an in vitro chromosome aberration test in Chinese Hamster V79 cells according to OECD/EC guidelines and GLP principles, the test item is considered to be non-clastogenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 January 2007 - 04 June 2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2000
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Stability: stable, if solvent or vehicle is anhydrous
Storage: at room temperature - Target gene:
- histidine (S. typhimurium) or tryptophan (E.coli)
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix
- Test concentrations with justification for top dose:
- Pre-test (TA 98 and TA 100): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (reported as part of main experiment I);
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate;
Experiment II: 31.6, 10, 316, 1000, 2500 and 5000 µg/plate (with S9), 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535 and TA 1537 without S9);
Experiment III: 250, 350, 750, 1500, 3000 and 5000 µg/plate (TA 100 without S9);
Experiment IV: 110, 130, 150, 175, 250 and 300 µg/plate (TA 100 with S9) - Vehicle / solvent:
- Solvent: DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- With metabolic activation
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- E.coli WP2 uvrA
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other:
- Remarks:
- TA98, TA1537, without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA100, TA1535, without metabolic activation
- Details on test system and experimental conditions:
- For each strain and dose level three plates were used.
Evaluation of cytotoxicity was done based on characterization of the background lawn and colony morphology. - Evaluation criteria:
- A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants over at least two increasing concentrations occurs without concomitant cytotoxicity in at least one of these groups, and/or
- a biologically relevant positive response for at least one of the non-cytotoxic dose groups occurs.
A biologically relevant increase is described as follows:
- if in tester strains TA100 and E.coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA1535, TA1537 and TA98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- A statistical evaluation was not performed.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 250 µg/ plate and above
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 1000 µg/ plate and higher, without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/ plate and higher, without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 316 µg/ plate and higher, without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The results are attached below in tabular format.
The reference mutagens induced a clear increase of revertant colonies verifying the validity of the experimental set-up.
No biologically revertant increases in revertant colony numbers were noted in tester strains TA98, TA1535, TA1537 and E.coli WP2 uvrA in absence or presence of metabolic activation.
Biologically relevant increases of revertant colony numbers were observed without metabolic activation in tester strain TA100 in three independent experiments. The threshold of 3.2 was reached at a dose of 5000 µg/ plate in the second experiment. Moreover, a dose-response relationship was found in each experiment. - Conclusions:
- In an Ames study performed according to OECD/EC guidelines and GLP principles, the substance was found to be positive without metabolic activation in tester strain TA100.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
An in vivo Micronucleus study is available, which shows that the substance is not clastogenic in vivo. As the substance was tested positive in an AMES study, a test proposal for an in vivo COMET assay is included.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- November 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EEC Directive 2000/32 L 136 Annex 4C, B 12
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Winkelmann
- Age at start of treatment: minimum 7 weeks
- Weight at study initiation: male: 30.7 - 38.2g; female: 23.2 - 32.3g
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex per cage (Macrolon Type III; Hereto)
- Diet: Altromin 1324 maintenance diet for rats and mice, TPF
- Water: tap water, ad libitum
- Acclimation period: adequate acclimatisation period
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- rel. Humidity (%): 55±10%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00 - Route of administration:
- intraperitoneal
- Vehicle:
- Cotton Seed Oil
- Details on exposure:
- The dosing formulations were prepared 1 hour before treatment (positive control formulation was prepared on the same day). All animals received a single volume of 10 mL/ kg bw.
- Duration of treatment / exposure:
- Single dose
- Post exposure period:
- Sampling of the peripheral blood was done at 44 (all groups, including solvent control and positive control) and 68 hours (solvent control and high dose group only) after treatment.
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 (main study)
6 males and 3 females (pre-experiment) - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 40 mg/kg bw cyclophosphamide dissolved in physiological saline. Intraperitoneal injection at 10 mL/kg bw
- Tissues and cell types examined:
- Blood from tail vein.
- Details of tissue and slide preparation:
- Blood was immediately fixed in ultracold methanol. At least 16 hours after fixation blood cells were washed in Hank's balanced salt solution, centrifuged at 600 g for 5 minutes. Blood cell populations were discriminated against using CD71 and CD61 and DNA content of micronuclei was determine dby the use of a DNA specific strain (propidium iodid).
- Evaluation criteria:
- Evaluation was performed using FACScan BD flow cytometer. At least 10,000 immature erythrocytes/animals were scored for the incidence of micronucleated immature erythrocytes.
To assess cytotoxicity, the ratio between immature and mature erythrocytes was determined and expressed as relative PCE (proportion of polychromatic (immature) erythrocytes among total erythrocytes.
Criteria for determining a positive result:
- dose related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups
A test item is considered negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level. - Statistics:
- The nonparametric Mann-Whitney Test was used.
The data generated were considered acceptable, if:
- at the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20% of the mean weight of each sex,
- the background frequency of micronucleated cells is in the normal range as reported in the literature or within the laboratory's historical range,
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Reduction rel. PCE
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The results are included in tabular form in the attached document.
The results of the positive control group and the solvent control group were within the range of the corresponding historical control data.
The relative PCE values observed in the male and female group were reduced compared to the corresponding negative control. Although the reduction was not statistically significant, the outcome was considered to be a good indication of systemic exposure of the mice.
The mean values of micronuclei observed after treatment were within the range of the negative control, except for the high dose group samples 68 hours after treatment. The values for males and females were slightly increased compared to the negative control (0.26% (0.25%) in the male (female) controls vs. 0.35% (0.30%) for the high dose males (females). As the increase was minimal and not statistically significant this was not considered to be toxicologically significant.
Historical control data ranges are attached below. - Conclusions:
- Based on the results of an in vivo Micronucleus study performed according to OECD/EC guideline and GLP principles, the test substance is considered to be non-cytogenic.
Reference
Results pre-experiment to find MTD:
Two males died within 72 hours after application of the test item. They showed toxic symptoms as reduction of spontaneous activity, rough fur, constricted opistosoma, palpebral closure and loss of weight.
All other animals survived.
The females and three males did not show signs of toxicity.
One male mouse showed toxic symptoms including reduction of spontaneous activity, rough fur, constricted opisthosoma, palpebral closure and loss of weight.
Endpoint conclusion
- Endpoint conclusion:
- no study available (further information necessary)
Additional information
Justification for classification or non-classification
Based on the available data, the test substance is not classified as a mutagen according to Regulation (EC) No 1272/2008, however it is proposed to conduct an in vivo study to clarify this endpoint.
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