Registration Dossier
Registration Dossier
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EC number: 291-907-2 | CAS number: 90506-45-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: other: clastogenicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-09-14 to 2012-02-23
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL
Experiment I:
without metabolic activation: 15.6, 250, 500 and 1000 µg/mL
with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL
Experiment II:
without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL
with metabolic activation: 900, 1600 and 1800 µg/mL - Vehicle / solvent:
- -Vehicle (s)/solvent(s) used: cell culture medium (MEM)
-Justification for choice of solvent/vehicle: The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity. - Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 400 and 900 µg/mL
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation Migrated to IUCLID6: 0.83 µg/mL
- Details on test system and experimental conditions:
- TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)
FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density - Evaluation criteria:
- There are several 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 (0.0% - 4.0% aberrant cells (with and without metabolic activation)). - Statistics:
- 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.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Afilan V5756 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item Afilan V5756 is considered to be non-clastogenic in this chromosome aberration test. - Executive summary:
To investigate the potential of Afilan V5756 to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitrochromosome aberration assay was carried out.
The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.
Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.
The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended.
The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:
Experiment I:
without metabolic activation: 15.6, 250, 500 and 1000 µg/mL
with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL
Experiment II:
without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL
with metabolic activation: 900, 1600 and 1800 µg/mL
Precipitation of the test item was observed with and without metabolic activation in both experiments.
Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.
In both experiments, 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.
In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
The positive controls induced the appropriate responses.
There was no evidence of Afilan V5756 induced over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2011-09-26 to 2012-02-14
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: read across study
- Justification for type of information:
- Justification for read-across:
The read-across approach is appropriate due to similar composition of source substance and registered substance. From the available data it can be concluded that the potential of genic toxicity of substances with different alkyl moieties (C12 and C16) is comparable.
Data for source substance are conducted on alkyl phosphoric esters, whereas the substance to be registered is neutralized with potassium hydroxide. As both substances dissociates in aqueous media, there are no reasons to assume a different profile for this endpoint. - Reason / purpose for cross-reference:
- read-across source
- 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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- -Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Pre-experiment for experiment I (without metabolic activation):
15.6, 31.3, 62.5, 125, 500, 1000 and 2500 µg/mL
Pre-experiment for experiment I (with metabolic activation):
31.3, 62.5, 125, 250, 500, 1000, 1750 and 2500 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
125, 250, 500, 750, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600 and 2800 µg/mL
Experiment I
without metabolic activation: 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL
and with metabolic activation: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL
Experiment II
without metabolic activation: 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL
and with metabolic activation: 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL - Vehicle / solvent:
- Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was supended in cell culture medium and processed by ultrasound for 5 min.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 300 µg/mL
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week
SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth; cloning efficiency - Evaluation criteria:
- A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- yes (Experiment I without S9: ≥ 1500 μg/mL; experiment I with S9: ≥ 1000 μg/mL; Experiment II without S9: ≥ 75 μg/mL; Experiment II with S9:≥ 1200 μg/mL
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Afilan V5756 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster. - Executive summary:
In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to Afilan V5756 suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of
- 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I)
- 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I)
- 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II)
- 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II).
Afilan V5756 was tested up to cytotoxic concentrations.
Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.
In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.
In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%.
In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%.The positive controls did induce the appropriate response.
There was no evidence(or)evidence of a concentration related positive responseof induced mutant colonies over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.
- 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:
- 2012-06-20 to 2012-06-29
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535: hisG46, uvrB, rfa. - Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix, Aroclor induced
- Test concentrations with justification for top dose:
- first experiment: 5.009, 1.507, 0.500, 0.151, 0.051 mg/plate.
second experiment: 5.001, 2.502, 1.255, 0.625, 0.314 mg/plate.
As no complete dissolution was possible, undissolved particles were visible on the plates. - Vehicle / solvent:
- vehicle: sterile water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) first experiment; preincubation 2nd experiment;
DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): histidin
NUMBER OF REPLICATIONS: 4 plates/ concentration
Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar. - Evaluation criteria:
- A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at
least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. - Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 of S. typhimurium were exposed to Silastol H 200 TS, at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; Aroclor induced rat liver).
The initial Mutation Test was performed as plate incorporation method; the confirmatory and complementary assays were performed according to the pre-incubation method.
No signs of toxicity towards the bacteria could be observed. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn't show any mutagenic effects in both experiments.
The test item Silastol H 200-TS is considered as "not mutagenic under the conditions of the test".
- 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:
- 2012-03-20 to 2012-07-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- -Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Pre-experiment for experiment I (with and without metabolic activation):
50, 100, 250, 500, 750, 1000, 2500 µg/mL
Experiment I
without metabolic activation: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 650, 1000 and 1750 µg/mL
and with metabolic activation: 0.010, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/mL
Experiment II
without metabolic activation: 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 650 µg/mL
and with metabolic activation: 0.75, 2.5, 7.5, 25, 75, 250, 750 and 1500 µg/mL - Vehicle / solvent:
- Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was suspended in cell culture medium and processed by ultrasound for 20 min.
- Untreated negative controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 300 µg/mL
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week
SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth - Evaluation criteria:
- A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment I without S9: ≥ 316 μg/mL; Experiment II without S9: ≥ 100 μg/mL
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster. - Executive summary:
In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to the test item suspended in (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of
- 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 650, 1000 and 1750 µg/mL (without metabolic activation, Experiment I)
- 0.010, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/mL (with metabolic activation, Experiment I)
- 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 650 µg/mL (without metabolic activation, Experiment II)
- 0.75, 2.5, 7.5, 25, 75, 250, 750 and 1500 µg/mL (with metabolic activation, Experiment II).
The test item was tested up to cytotoxic concentrations.
A biologically relevant growth inhibition was observed in experiment I and II without metabolic activation. In experiment I without metabolic activation the relative growth was 17.5% for the highest concentration (1750 µg/mL) evaluated. In experiment II without metabolic activation the relative growth was 10.2% for the highest concentration (650 µg/mL) evaluated.
No biologically relevant growth inhibition was observed in experiment I and II with metabolic activation. In experiment I the highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 80.8%. In experiment II the highest concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 76.2%.
In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.53 was found at a concentration of 3.16 µg/mL with a relative growth of 124.0%.
In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.93 was found at a concentration of 3.16 µg/mL with a relative growth of 105.6%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.63 was found at a concentration of 0.0316 µg/mL with a relative growth of 104.3%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.00 was found at a concentration of 0.75 µg/mL with a relative growth of 98.7%.The positive controlsdidinduce the appropriate response.
There was no evidence of a concentration related positive responseof induced mutant colonies over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.
Referenceopen allclose all
| Results of chromosome analysis | |||||||||||||||
| without metabolic activation | |||||||||||||||
| Cytotoxicity | Chromatid aberrations | Isochromatid aberrations | rel. Mitotic index (%) | rel. Cell density (%) | Poly-ploidy | mean % aberrant cells | |||||||||
| Scored cells | gaps | breaks | inter-changes | other | gaps | breaks | inter-changes | other | incl. Gaps | excl. Gaps | |||||
| Experiment I | |||||||||||||||
| negative control | 200 | - | 5 | 4 | 0 | 0 | 0 | 0 | 1 | 1 | 100 | 100 | 2 | 4.5 | 2.5 |
| 15.6 µg/mL | 200 | no | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 103 | 96 | 3 | 2.5 | 0.0 |
| 250 µg/mL | 200 | no | 6 | 2 | 0 | 1 | 0 | 0 | 0 | 0 | 81 | 78 | 3 | 3.5 | 1.5 |
| 500 µg/mL | 200 | yes | 4 | 3 | 2 | 0 | 0 | 0 | 2 | 0 | 53 | 55 | 1 | 5.0 | 3.0 |
| 1000 µg/mL | 200 | yes | 4 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 51 | 44 | 0 | 3.0 | 0.5 |
| EMS (900 µg/mL) | 200 | - | 7 | 10 | 8 | 2 | 0 | 1 | 2 | 0 | 85 | 94 | 3 | 12.5 | 9.5 |
| Experiment II | |||||||||||||||
| negative control | 200 | - | 1 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 100 | 100 | 2 | 2.5 | 2.0 |
| 15.6 µg/mL | 200 | no | 6 | 3 | 0 | 1 | 0 | 0 | 0 | 0 | 76 | 102 | 3 | 4.5 | 2.0 |
| 31.3 µg/mL | 300 | no | 5 | 7 | 1 | 1 | 0 | 0 | 0 | 0 | 74 | 94 | 1 | 4.7 | 3.0 |
| 62.5 µg/mL | 200 | yes | 2 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 51 | 89 | 1 | 2.5 | 1.5 |
| 125.0 µg/mL | 200 | yes | 1 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 30 | 57 | 1 | 2.0 | 1.5 |
| EMS (400 µg/mL) | 200 | - | 7 | 17 | 3 | 3 | 3 | 0 | 0 | 0 | 72 | 103 | 0 | 13.0 | 10.5 |
| Results of chromosome analysis | |||||||||||||||
| with metabolic activation | |||||||||||||||
| Cytotoxicity | Chromatid aberrations | Isochromatid aberrations | rel. Mitotic index (%) | rel. Cell density (%) | Poly-ploidy | mean % aberrant cells | |||||||||
| Scored cells | gaps | breaks | inter-changes | other | gaps | breaks | inter-changes | other | incl. Gaps | excl. Gaps | |||||
| Experiment I | |||||||||||||||
| negative control | 200 | - | 6 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 100 | 100 | 0 | 4.0 | 1.0 |
| 15.6 µg/mL | 300 | no | 4 | 3 | 3 | 1 | 0 | 0 | 2 | 0 | 96 | 102 | 1 | 4.0 | 3.0 |
| 500 µg/mL | 200 | no | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 90 | 86 | 0 | 2.0 | 1.0 |
| 1000 µg/mL | 200 | yes | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 62 | 85 | 1 | 1.5 | 0.0 |
| 1500 µg/mL | 200 | yes | 2 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 54 | 63 | 1 | 2.0 | 0.5 |
| CPA (0.83 µg/mL) | 200 | - | 3 | 12 | 11 | 1 | 0 | 1 | 1 | 0 | 104 | 85 | 3 | 12.0 | 10.5 |
| Experiment II | |||||||||||||||
| negative control | 200 | - | 4 | 5 | 0 | 1 | 1 | 0 | 1 | 0 | 100 | 100 | 2 | 5.5 | 3.5 |
| 900 µg/mL | 200 | no | 1 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 71 | 88 | 0 | 2.0 | 1.5 |
| 1600 µg/mL | 200 | yes | 3 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 54 | 75 | 0 | 2.5 | 1.5 |
| 1800 µg/mL | 200 | yes | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 1 | 45 | 56 | 0 | 1.5 | 1.5 |
| CPA (0.83 µg/mL) | 200 | - | 3 | 9 | 3 | 3 | 1 | 1 | 2 | 1 | 96 | 100 | 0 | 10.0 | 9.0 |
First Experiment
Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was suspended in deionised water. All concentrations were weighed directly. As no complete dissolution was possible, undissolved particles were visible on the plates. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions.
To verify this result, a second experiment was performed using the pre-incubation method.
Second Experiment
Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was suspended in deionised Water. All concentrations were weighed directly. As no complete dissolution was possible, undissolved particles were visible on the plates. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames Test
In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 of S. typhimurium were exposed to the read-across substance1-Octadecanol, phosphate, potassium salt(Silastol H 200 TS), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. No signs of toxicity towards the bacteria could be observed. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. 1-Octadecanol, phosphate, potassium salt didn't show any mutagenic effects in both experiments
Mammalian Cell Gene Mutation Test
Anin vitromammalian cell assay according to OECD Guideline 476
was performed in V79, (HPRT locus), to test the potential of the read-across substance Phosphoric acid, dodecyl ester, potassium salt (Afilan V5756) to cause gene mutation and/or chromosome damage. Cells were exposed to the test substance in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) up to cytotoxic concentrations, with and without metabolic activation.There was no evidence of a concentration related positive response of induced mutant colonies over background.
The same study was conducted with the read-across substance hexadecyl dihydrogen phosphate. As well, in this studythere was no evidence of a concentration related positive response of induced mutant colonies over background.
Chromosomal Aberration Test
The potential of the read-across substance Phosphoric acid, dodecyl ester, potassium salt (Afilan V5756) to induce structural chromosome aberrations in Chinese hamster V79 cells, was investigated in an in vitro chromosome aberration assay, according to OECD guideline 479.
The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.
In both experiments, 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.
In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Afilan V5756 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, Phosphoric acid, dodecyl ester, potassium salt (Afilan V5756) is considered to be non-clastogenic in this chromosome aberration test.
Conclusion
All in vitro and in vivo tests in genetic toxicity showed negative results. There is no reason to believe that the negative results would not be relevant to humans. No further testing is required.
Justification for read-across:
The read-across approach is appropriate due to similar composition of source substance and registered substance. From the available data it can be concluded that the potential of genetic toxicity of substances with different alkyl moieties (C12 and C16) is comparable.
Data for source substance are conducted on alkyl phosphoric esters, whereas the substance to be registered is neutralized with potassium hydroxide. As both substances dissociates in aqueous media, there are no reasons to assume a different profile for this endpoint.
Justification for selection of genetic toxicity endpoint
Reliable data are available from the full in-virto test set required under REACH regulation.
Short description of key information:
Negative data are available from a gene mutation assay in bacteria, from a chromosomal aberration test and two mammalian cell gene mutation assays with the read-across substances.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
No classification is required based on negative in-vitro data set.
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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