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Registration Dossier
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EC number: 500-465-4 | CAS number: 160901-28-0 1 - 2.5 moles ethoxylated
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to genetic toxicity will be updated once all ongoing studies have been finalised.
Bacterial reverse mutation assay (Ames / OECD 471): negative
In vitro mammalian cell gene mutation assay (MLA / OECD 476): negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.
- Test concentrations with justification for top dose:
- plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9 mix: Sodium azide: 10 µg/plate (TA 100, TA 1535), 4-NOPD: 10 µg/plate (TA 98)/40 µg/plate (TA1537), MMS: 1 µL/plate (TA 102); +S9 mix: 2-AA: 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) / 10 µg/plate (TA 102)
- Details on test system and experimental conditions:
- A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I.
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation
The initial plate incorporation test was conducted as follows:
Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate.
The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.
DETERMINATION OF CYTOTOXICITY
The reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity. - Evaluation criteria:
- The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by the laboratories’ own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience
3. The bacteria strains TA 98, TA 100 and TA 102 demonstrate their typical response to ampicillin.
4. Corresponding background growth on negative control, solvent control and test plates is oberved
A clear and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. - Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- beginning at 1000 µg/plate in the plate incorporation experiment and 316 µg/plate in the preincubation experiment.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation was observed in any tester strain at any concentration.
- Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- (1997)
- 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
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidin kinase-locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone.
- Test concentrations with justification for top dose:
- Experiment I:
with S9 mix: 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM (incubation period: 4 h)
without S9 mix: 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM (incubation period: 4 h)
Experiment II:
with S9 mix: 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM (incubation period: 4 h)
without S9 mix: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM(incubation period: 24 h) - Vehicle / solvent:
- Based on a solubility test RPMI (+5% horse serum) was used as solvent.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Ethylmethanesulfonate (EMS): 200 + 300 µg/mL; Methylmethanesulfonate (MMS): 10 µg/mL; Benzo(a)pyrene (BaP): 2.5 µg/mL
- Details on test system and experimental conditions:
- Experiment I with and without S9 mix and Experiment II with S9 mix (incubation period: 4 h)
1 x 10E+7 cells were suspended in medium and exposed to the test item either in the presence or absence of metabolic activation in the mutation experiment for 4 h. Thereafter the test item was removed, the cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.
Experiment II without S9 mix (incubation period: 24 h)
5 x 10E+6 cells were suspended in medium and exposed to the test item in the absence of metabolic activation. After 24 h the test item was removed, cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.
After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in selective medium with TFT. The plates were scored after an incubation period of approx. 14 days at 37 °C in humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequency is expressed as “mutants per 10E+6 viable cells”.
Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation (GEF = 126 for the microwell method). - Evaluation criteria:
- A mutation assay is considered acceptable if:
- At least 3/4 plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E+6 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period or 32-180 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E+6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E+6 cells.
- The RTG is greater than 10%.
The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E+6 cells.
- A dose-dependent increase in mutant frequency is detected. - Statistics:
- The Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation was observed at any concentration. The pH-value detected in medium containing the test item was within the physiological range.
- Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Target gene:
- his-operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.
- Test concentrations with justification for top dose:
- plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9 mix: Sodium azide: 10 µg/plate (TA 100, TA 1535), 4-NOPD: 10 µg/plate (TA 98)/40 µg/plate (TA1537), MMS: 1 µL/plate (TA 102); +S9 mix: 2-AA: 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) / 10 µg/plate (TA 102)
- Details on test system and experimental conditions:
- A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I.
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation
The initial plate incorporation test was conducted as follows:
Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate.
The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.
DETERMINATION OF CYTOTOXICITY
The reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity. - Evaluation criteria:
- The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by the laboratories’ own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience
3. The bacteria strains TA 98, TA 100 and TA 102 demonstrate their typical response to ampicillin.
4. Corresponding background growth on negative control, solvent control and test plates is oberved
A clear and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. - Statistics:
- Mean values and standard deviation were calculated.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- beginning at 1000 µg/plate in the plate incorporation experiment and 316 µg/plate in the preincubation experiment.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation was observed in any tester strain at any concentration.
- Conclusions:
- negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Target gene:
- Thymidin kinase-locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor supplemented S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone.
- Test concentrations with justification for top dose:
- Experiment I:
with S9 mix: 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM (incubation period: 4 h)
without S9 mix: 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM (incubation period: 4 h)
Experiment II:
with S9 mix: 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM (incubation period: 4 h)
without S9 mix: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM(incubation period: 24 h) - Vehicle / solvent:
- Based on a solubility test RPMI (+5% horse serum) was used as solvent.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Ethylmethanesulfonate (EMS): 200 + 300 µg/mL; Methylmethanesulfonate (MMS): 10 µg/mL; Benzo(a)pyrene (BaP): 2.5 µg/mL
- Details on test system and experimental conditions:
- Experiment I with and without S9 mix and Experiment II with S9 mix (incubation period: 4 h)
1 x 10E+7 cells were suspended in medium and exposed to the test item either in the presence or absence of metabolic activation in the mutation experiment for 4 h. Thereafter the test item was removed, the cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.
Experiment II without S9 mix (incubation period: 24 h)
5 x 10E+6 cells were suspended in medium and exposed to the test item in the absence of metabolic activation. After 24 h the test item was removed, cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.
After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in selective medium with TFT. The plates were scored after an incubation period of approx. 14 days at 37 °C in humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequency is expressed as “mutants per 10E+6 viable cells”.
Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation (GEF = 126 for the microwell method). - Evaluation criteria:
- A mutation assay is considered acceptable if:
- At least 3/4 plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E+6 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period or 32-180 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E+6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E+6 cells.
- The RTG is greater than 10%.
The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E+6 cells.
- A dose-dependent increase in mutant frequency is detected. - Statistics:
- The Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation was observed at any concentration. The pH-value detected in medium containing the test item was within the physiological range.
- Conclusions:
- negative
Referenceopen allclose all
Table 1: Test results of experiment 1 (plate incorporation).
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|
without S9-Mix |
|||||
0 |
107 ± 7.5 |
6 ± 3.1 |
175 ± 10.6 |
25 ± 10.4 |
9 ± 2.5 |
3.16 |
128 ± 14.6 |
10 ± 6.7 |
196 ± 13.1 |
27 ± 6.4 |
14 ± 4.5 |
10.0 |
134 ± 4.2 |
7 ± 0.6 |
174 ± 13.4 |
21 ± 3.1 |
12 ± 3.1 |
31.6 |
134 ± 17.3 |
8 ± 1.7 |
185 ± 25.1 |
25 ± 2.5 |
9 ± 0.6 |
100 |
120 ± 15.0 |
8 ± 0.0 |
164 ± 15.1 |
28 ± 7.5 |
12 ± 2.5 |
316 |
95 ± 8.5 |
10 ± 3.2 |
182 ± 15.1 |
27 ± 9.7 |
8 ± 2.5 |
1000 |
74 ± 13.2 B |
10 ± 4.2 |
215 ± 10.8 |
21± 3.1 |
9± 4.2 |
2500 |
38 ± 3.0 B |
0± 0.0 B |
179± 10.8 |
19± 6.5 B |
6± 4.6 B |
5000 |
0± 0.6 B |
0± 0.0 B |
148± 11.0 |
14± 2.5 B |
0± 0.0 B |
Positive controls: |
Na-azide |
MMS |
4-NOPD |
||
Concentration (μg/plate) |
10 |
1 µL/plate |
10 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
934 ± 22.0 |
758 ± 220.3 |
1782 ± 345.7 |
461 ± 23.8 |
119 ± 7.8 |
With S9-Mix |
|||||
0 |
124 ± 13.8 |
7 ± 4.0 |
253 ± 5.3 |
26 ± 1.0 |
7 ± 3.5 |
3.16 |
120 ± 20.6 |
7 ± 7.1 |
272 ± 21.0 |
35 ± 5.2 |
11 ± 2.5 |
10.0 |
120 ± 8.1 |
10 ± 3.6 |
262 ± 2.5 |
35 ± 2.9 |
13 ± 4.5 |
31.6 |
113 ± 12.5 |
10 ± 3.2 |
262 ± 7.2 |
40 ± 7.2 |
10 ± 3.5 |
100 |
133 ± 16.7 |
7 ± 1.7 |
248 ± 19.7 |
35 ± 7.6 |
9 ± 1.0 |
316 |
121 ± 4.6 |
9 ± 1.0 |
277 ± 7.8 |
32 ± 3.5 |
12 ± 1.2 |
1000 |
117 ± 29.5 |
11 ± 1.7 |
274 ± 7.0 |
36 ± 5.3 |
13 ± 3.8 |
2500 |
100 ± 9.9 |
7 ± 1.5 |
233 ± 8.4 |
31 ± 3.1 |
6 ± 2.5 |
5000 |
61 ± 4.4 B |
4 ± 1.0 B |
238 ± 28.6 |
20 ± 5.5 |
9 ± 1.5 |
Positive controls: |
2-AA |
||||
Concentrations (μg/plate) |
2.5 |
10 |
2.5 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1581 ± 229.7 |
102 ± |
516 ± |
1959 ± 504.5 |
261 ± |
MMS = Methylmethanesulfonate
4-NOPD = 4-Nitro-o-phenylene diamine
2-AA = 2-Aminoanthracene
B = Reduction of background lawn
Table 2: Test results of experiment 2 (pre-incubationtion).
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|
without S9-Mix |
|||||
0 |
115± 23.1 |
10± 4.0 |
270± 12.2 |
22± 3.2 |
7± 1.5 |
3.16 |
129± 14.4 |
4± 2.1 |
241± 29.2 |
24± 1.2 |
10± 2.0 |
10.0 |
133± 9.0 |
9± 2.3 |
264± 29.4 |
31± 2.5 |
10± 6.0 |
31.6 |
119± 4.4 |
5± 1.0 |
278± 29.7 |
23± 2.6 |
12± 6.4 |
100 |
114± 15.9 |
8± 2.0 |
283± 23.1 |
26± 1.0 |
9± 3.2 |
316 |
100± 6.1 |
7± 1.2 |
262± 9.8 |
25± 2.1 |
8± 1.2 B |
1000 |
58± 9.1 B |
0± 0.0 B |
280± 22.8 |
25 ± 0.6 B |
0 ± 0.0 B |
2500 |
0 ± 0.0 B |
0 ± 0.0 B/N |
155 ± 37.0 |
22 ± 6.1 B |
0 ± 0.0 N |
5000 |
0 ± 0.0 N |
0 ± 0.0 N |
125 ± 45.0 B |
0 ± 0.0 B |
0 ± 0.0 N |
Positive controls: |
Na-azide |
MMS |
4-NOPD |
||
Concentration (μg/plate) |
10 |
1 µL/plate |
10 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
965± 120.2 |
1038± 227.1 |
2158± 67.5 |
389± 30.1 |
107± 7.0 |
With S9-Mix |
|||||
0 |
108± 12.7 |
10± 2.5 |
292± 7.8 |
38± 4.5 |
9± 3.2 |
3.16 |
137± 30.7 |
5± 2.1 |
289± 15.9 |
39± 5.6 |
7± 2.1 |
10.0 |
139± 4.2 |
5± 2.5 |
323± 36.5 |
37± 2.0 |
4± 2.9 |
31.6 |
121± 14.0 |
7± 3.1 |
310± 21.2 |
32± 4.5 |
5± 4.9 |
100 |
134± 14.7 |
7± 1.5 |
297± 6.1 |
31± 6.0 |
7± 2.6 |
316 |
116± 7.6 |
8± 1.5 |
286± 24.0 |
31± 6.7 |
4 ± 1.2 |
1000 |
101± 15.5 |
7± 0.6 |
310± 7.4 |
30± 12.7 |
12± 3.5 |
2500 |
85± 11.8 B |
4± 2.5 B |
284± 15.0 |
22± 4.2 B |
7± 3.5 B |
5000 |
70± 4.6 B |
0± 0.0 B |
273± 10.7 |
21± 10.4 B |
6± 3.6 B |
Positive controls: |
2-AA |
||||
Concentrations (μg/plate) |
2.5 |
10 |
2.5 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
789± |
60 ± |
628 ± |
2943 ± |
128 ± |
MMS = Methylmethanesulfonate
4-NOPD = 4-Nitro-o-phenylene diamine
2-AA = 2-Aminoanthracene
B = Reduction of background lawn
N = No background lawn
None of the experimental results met the criteria for considering the test substance mutagenic, except for the highest dose level in experiment I with metabolic activation. There a significantly increased number of mutants at 0.48 mM and a dose dependency were seen. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (RTG below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.
Table 1: Experiment I - 4 h exposure – With and without Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
|
+S9 mix |
||||
Control 1 |
0 |
82.9 |
100 |
73.7 |
- |
Control 2 |
0 |
70.6 |
92.8 |
||
Test Item |
0.01 |
73.7 |
90.7 |
89.2 |
5.9 |
0.05 |
79.3 |
107.8 |
66.8 |
-16.5 |
|
0.24 |
71.7 |
97.2 |
93.8 |
10.6 |
|
0.28 |
80.5 |
93.1 |
98.1 |
14.9 |
|
0.32 |
72.7 |
78.0 |
88.4 |
5.1 |
|
0.36 |
80.5 |
60.1 |
70.2 |
-13.0 |
|
0.40 |
72.7 |
40.4 |
104.3 |
21.0 |
|
0.44 |
93.5 |
27.3 |
141.3 * |
58.0 |
|
0.48 |
82.9 |
9.4 |
217.2 * |
134.0 |
|
Benzo[a]pyrene |
2.5 |
58.8 |
48.3 |
829.8 * |
746.5 |
|
-S9 mix |
||||
Control 1 |
0 |
92.1 |
100.0 |
76.3 |
/ |
Control 2 |
0 |
84.1 |
83.3 |
/ |
|
Test Item |
0.01 |
98.0 |
107.4 |
85.9 |
6.1 |
|
0.02 |
85.4 |
101.2 |
73.3 |
-6.5 |
|
0.05 |
82.9 |
109.0 |
82.8 |
2.9 |
|
0.10 |
96.5 |
110.7 |
87.0 |
7.2 |
|
0.20 |
110.1 |
79.6 |
70.8 |
-9.0 |
|
0.24 |
102.9 |
42.3 |
93.5 |
13.7 |
|
0.28 |
132.6 |
31.0 |
137.4* |
57.6 |
|
0.32 |
114.0 |
23.6 |
126.4* |
46.5 |
EMS |
300 |
77.0 |
63.5 |
713.3* |
633.5 |
MMS |
10 |
79.3 |
64.3 |
447.5* |
367.7 |
*: p <0.05
EMS Ethyl methane sulphonate, MMS Methyl methane sulphonate
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Table 2: Experiment II - 4 h Exposure - With Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
Control 1 |
0 |
68.7 |
100.0 |
115.5 |
/ |
Control 2 |
0 |
86.6 |
75.6 |
/ |
|
Test Item |
0.15 |
93.5 |
121.1 |
89.6 |
-5.9 |
0.35 |
67.7 |
77.0 |
130.3 |
34.7 |
|
0.39 |
92.1 |
82.2 |
94.5 |
-1.0 |
|
0.43 |
93.5 |
60.7 |
118.5 |
22.9 |
|
0.45 |
116.0 |
60.8 |
102.5 |
7.0 |
|
0.47 |
108.2 |
36.1 |
148.6* |
53.0 |
|
0.49 |
89.3 |
18.9 |
169.4* |
73.9 |
|
0.53 |
89.3 |
11.1 |
220.6* |
125.1 |
|
Benzo[a]pyrene |
2.5 |
68.7 |
68.3 |
691.7* |
596.1 |
*: p <0.05
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Table 3: Experiment II - 24 h exposure - Without Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
Control 1 |
0 |
93.5 |
100.0 |
75.3 |
/ |
Control 2 |
0 |
95.0 |
108.4 |
/ |
|
Test Item |
0.0005 |
118.1 |
124.7 |
77.4 |
-14.4 |
0.001 |
118.1 |
130.7 |
82.6 |
-9.2 |
|
0.005 |
89.3 |
98.1 |
74.9 |
-16.9 |
|
0.01 |
106.4 |
98.3 |
72.7 |
-19.1 |
|
0.05 |
127.4 |
94.1 |
69.3 |
-22.5 |
|
0.10 |
125.0 |
84.9 |
69.5 |
-22.4 |
|
0.15 |
89.3 |
43.7 |
92.0 |
0.2 |
|
0.20 |
92.1 |
26.0 |
113.6 |
21.8 |
|
0.25 |
80.5 |
7.9 |
188.6* |
96.8 |
|
EMS |
200 |
38.9 |
12.2 |
3002.5* |
2910.7 |
MMS |
10 |
34.2 |
10.1 |
2321.3* |
2229.5 |
*: p <0.05
EMS Ethyl methane sulphonate; MMS Methyl methane sulphonate
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Table 1: Test results of experiment 1 (plate incorporation).
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|
without S9-Mix |
|||||
0 |
107 ± 7.5 |
6 ± 3.1 |
175 ± 10.6 |
25 ± 10.4 |
9 ± 2.5 |
3.16 |
128 ± 14.6 |
10 ± 6.7 |
196 ± 13.1 |
27 ± 6.4 |
14 ± 4.5 |
10.0 |
134 ± 4.2 |
7 ± 0.6 |
174 ± 13.4 |
21 ± 3.1 |
12 ± 3.1 |
31.6 |
134 ± 17.3 |
8 ± 1.7 |
185 ± 25.1 |
25 ± 2.5 |
9 ± 0.6 |
100 |
120 ± 15.0 |
8 ± 0.0 |
164 ± 15.1 |
28 ± 7.5 |
12 ± 2.5 |
316 |
95 ± 8.5 |
10 ± 3.2 |
182 ± 15.1 |
27 ± 9.7 |
8 ± 2.5 |
1000 |
74 ± 13.2 B |
10 ± 4.2 |
215 ± 10.8 |
21± 3.1 |
9± 4.2 |
2500 |
38 ± 3.0 B |
0± 0.0 B |
179± 10.8 |
19± 6.5 B |
6± 4.6 B |
5000 |
0± 0.6 B |
0± 0.0 B |
148± 11.0 |
14± 2.5 B |
0± 0.0 B |
Positive controls: |
Na-azide |
MMS |
4-NOPD |
||
Concentration (μg/plate) |
10 |
1 µL/plate |
10 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
934 ± 22.0 |
758 ± 220.3 |
1782 ± 345.7 |
461 ± 23.8 |
119 ± 7.8 |
With S9-Mix |
|||||
0 |
124 ± 13.8 |
7 ± 4.0 |
253 ± 5.3 |
26 ± 1.0 |
7 ± 3.5 |
3.16 |
120 ± 20.6 |
7 ± 7.1 |
272 ± 21.0 |
35 ± 5.2 |
11 ± 2.5 |
10.0 |
120 ± 8.1 |
10 ± 3.6 |
262 ± 2.5 |
35 ± 2.9 |
13 ± 4.5 |
31.6 |
113 ± 12.5 |
10 ± 3.2 |
262 ± 7.2 |
40 ± 7.2 |
10 ± 3.5 |
100 |
133 ± 16.7 |
7 ± 1.7 |
248 ± 19.7 |
35 ± 7.6 |
9 ± 1.0 |
316 |
121 ± 4.6 |
9 ± 1.0 |
277 ± 7.8 |
32 ± 3.5 |
12 ± 1.2 |
1000 |
117 ± 29.5 |
11 ± 1.7 |
274 ± 7.0 |
36 ± 5.3 |
13 ± 3.8 |
2500 |
100 ± 9.9 |
7 ± 1.5 |
233 ± 8.4 |
31 ± 3.1 |
6 ± 2.5 |
5000 |
61 ± 4.4 B |
4 ± 1.0 B |
238 ± 28.6 |
20 ± 5.5 |
9 ± 1.5 |
Positive controls: |
2-AA |
||||
Concentrations (μg/plate) |
2.5 |
10 |
2.5 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
1581 ± 229.7 |
102 ± |
516 ± |
1959 ± 504.5 |
261 ± |
MMS = Methylmethanesulfonate
4-NOPD = 4-Nitro-o-phenylene diamine
2-AA = 2-Aminoanthracene
B = Reduction of background lawn
Table 2: Test results of experiment 2 (pre-incubationtion).
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
|
without S9-Mix |
|||||
0 |
115± 23.1 |
10± 4.0 |
270± 12.2 |
22± 3.2 |
7± 1.5 |
3.16 |
129± 14.4 |
4± 2.1 |
241± 29.2 |
24± 1.2 |
10± 2.0 |
10.0 |
133± 9.0 |
9± 2.3 |
264± 29.4 |
31± 2.5 |
10± 6.0 |
31.6 |
119± 4.4 |
5± 1.0 |
278± 29.7 |
23± 2.6 |
12± 6.4 |
100 |
114± 15.9 |
8± 2.0 |
283± 23.1 |
26± 1.0 |
9± 3.2 |
316 |
100± 6.1 |
7± 1.2 |
262± 9.8 |
25± 2.1 |
8± 1.2 B |
1000 |
58± 9.1 B |
0± 0.0 B |
280± 22.8 |
25 ± 0.6 B |
0 ± 0.0 B |
2500 |
0 ± 0.0 B |
0 ± 0.0 B/N |
155 ± 37.0 |
22 ± 6.1 B |
0 ± 0.0 N |
5000 |
0 ± 0.0 N |
0 ± 0.0 N |
125 ± 45.0 B |
0 ± 0.0 B |
0 ± 0.0 N |
Positive controls: |
Na-azide |
MMS |
4-NOPD |
||
Concentration (μg/plate) |
10 |
1 µL/plate |
10 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
965± 120.2 |
1038± 227.1 |
2158± 67.5 |
389± 30.1 |
107± 7.0 |
With S9-Mix |
|||||
0 |
108± 12.7 |
10± 2.5 |
292± 7.8 |
38± 4.5 |
9± 3.2 |
3.16 |
137± 30.7 |
5± 2.1 |
289± 15.9 |
39± 5.6 |
7± 2.1 |
10.0 |
139± 4.2 |
5± 2.5 |
323± 36.5 |
37± 2.0 |
4± 2.9 |
31.6 |
121± 14.0 |
7± 3.1 |
310± 21.2 |
32± 4.5 |
5± 4.9 |
100 |
134± 14.7 |
7± 1.5 |
297± 6.1 |
31± 6.0 |
7± 2.6 |
316 |
116± 7.6 |
8± 1.5 |
286± 24.0 |
31± 6.7 |
4 ± 1.2 |
1000 |
101± 15.5 |
7± 0.6 |
310± 7.4 |
30± 12.7 |
12± 3.5 |
2500 |
85± 11.8 B |
4± 2.5 B |
284± 15.0 |
22± 4.2 B |
7± 3.5 B |
5000 |
70± 4.6 B |
0± 0.0 B |
273± 10.7 |
21± 10.4 B |
6± 3.6 B |
Positive controls: |
2-AA |
||||
Concentrations (μg/plate) |
2.5 |
10 |
2.5 |
||
Mean No. of colonies/plate (average of 3 ± SD) |
789± |
60 ± |
628 ± |
2943 ± |
128 ± |
MMS = Methylmethanesulfonate
4-NOPD = 4-Nitro-o-phenylene diamine
2-AA = 2-Aminoanthracene
B = Reduction of background lawn
N = No background lawn
None of the experimental results met the criteria for considering the test substance mutagenic, except for the highest dose level in experiment I with metabolic activation. There a significantly increased number of mutants at 0.48 mM and a dose dependency were seen. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (RTG below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.
Table 1: Experiment I - 4 h exposure – With and without Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
|
+S9 mix |
||||
Control 1 |
0 |
82.9 |
100 |
73.7 |
- |
Control 2 |
0 |
70.6 |
92.8 |
||
Test Item |
0.01 |
73.7 |
90.7 |
89.2 |
5.9 |
0.05 |
79.3 |
107.8 |
66.8 |
-16.5 |
|
0.24 |
71.7 |
97.2 |
93.8 |
10.6 |
|
0.28 |
80.5 |
93.1 |
98.1 |
14.9 |
|
0.32 |
72.7 |
78.0 |
88.4 |
5.1 |
|
0.36 |
80.5 |
60.1 |
70.2 |
-13.0 |
|
0.40 |
72.7 |
40.4 |
104.3 |
21.0 |
|
0.44 |
93.5 |
27.3 |
141.3 * |
58.0 |
|
0.48 |
82.9 |
9.4 |
217.2 * |
134.0 |
|
Benzo[a]pyrene |
2.5 |
58.8 |
48.3 |
829.8 * |
746.5 |
|
-S9 mix |
||||
Control 1 |
0 |
92.1 |
100.0 |
76.3 |
/ |
Control 2 |
0 |
84.1 |
83.3 |
/ |
|
Test Item |
0.01 |
98.0 |
107.4 |
85.9 |
6.1 |
|
0.02 |
85.4 |
101.2 |
73.3 |
-6.5 |
|
0.05 |
82.9 |
109.0 |
82.8 |
2.9 |
|
0.10 |
96.5 |
110.7 |
87.0 |
7.2 |
|
0.20 |
110.1 |
79.6 |
70.8 |
-9.0 |
|
0.24 |
102.9 |
42.3 |
93.5 |
13.7 |
|
0.28 |
132.6 |
31.0 |
137.4* |
57.6 |
|
0.32 |
114.0 |
23.6 |
126.4* |
46.5 |
EMS |
300 |
77.0 |
63.5 |
713.3* |
633.5 |
MMS |
10 |
79.3 |
64.3 |
447.5* |
367.7 |
*: p <0.05
EMS Ethyl methane sulphonate, MMS Methyl methane sulphonate
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Table 2: Experiment II - 4 h Exposure - With Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
Control 1 |
0 |
68.7 |
100.0 |
115.5 |
/ |
Control 2 |
0 |
86.6 |
75.6 |
/ |
|
Test Item |
0.15 |
93.5 |
121.1 |
89.6 |
-5.9 |
0.35 |
67.7 |
77.0 |
130.3 |
34.7 |
|
0.39 |
92.1 |
82.2 |
94.5 |
-1.0 |
|
0.43 |
93.5 |
60.7 |
118.5 |
22.9 |
|
0.45 |
116.0 |
60.8 |
102.5 |
7.0 |
|
0.47 |
108.2 |
36.1 |
148.6* |
53.0 |
|
0.49 |
89.3 |
18.9 |
169.4* |
73.9 |
|
0.53 |
89.3 |
11.1 |
220.6* |
125.1 |
|
Benzo[a]pyrene |
2.5 |
68.7 |
68.3 |
691.7* |
596.1 |
*: p <0.05
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Table 3: Experiment II - 24 h exposure - Without Metabolic Activation
Test group |
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants/1E+06 surviving cells |
Induced mutant frequency |
Control 1 |
0 |
93.5 |
100.0 |
75.3 |
/ |
Control 2 |
0 |
95.0 |
108.4 |
/ |
|
Test Item |
0.0005 |
118.1 |
124.7 |
77.4 |
-14.4 |
0.001 |
118.1 |
130.7 |
82.6 |
-9.2 |
|
0.005 |
89.3 |
98.1 |
74.9 |
-16.9 |
|
0.01 |
106.4 |
98.3 |
72.7 |
-19.1 |
|
0.05 |
127.4 |
94.1 |
69.3 |
-22.5 |
|
0.10 |
125.0 |
84.9 |
69.5 |
-22.4 |
|
0.15 |
89.3 |
43.7 |
92.0 |
0.2 |
|
0.20 |
92.1 |
26.0 |
113.6 |
21.8 |
|
0.25 |
80.5 |
7.9 |
188.6* |
96.8 |
|
EMS |
200 |
38.9 |
12.2 |
3002.5* |
2910.7 |
MMS |
10 |
34.2 |
10.1 |
2321.3* |
2229.5 |
*: p <0.05
EMS Ethyl methane sulphonate; MMS Methyl methane sulphonate
Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Mammalian Bone Marrow Chromosome Aberration Test (CA / OECD 475): negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- chromosome aberration assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- According to Guideline.
- Route of administration:
- oral: gavage
- Vehicle:
- Distilled water
- Duration of treatment / exposure:
- n.a.
- Frequency of treatment:
- Once
- Post exposure period:
- test group: 10, 24 and 48 h
positive control: 26 h - Remarks:
- Doses / Concentrations:
0, 1000, 2000 mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide (50 mg/kg bw)
- Tissues and cell types examined:
- Femoral bone marrow cells
- Details of tissue and slide preparation:
- SAMPLING TIMES
test groups: 10, 24 and 48 h after treatment
positive control group: 26 h after treatment - Evaluation criteria:
- According to Guideline.
- Statistics:
- Yes
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- negative
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- According to Guideline.
- Route of administration:
- oral: gavage
- Vehicle:
- Distilled water
- Duration of treatment / exposure:
- n.a.
- Frequency of treatment:
- Once
- Post exposure period:
- test group: 10, 24 and 48 h
positive control: 26 h - Remarks:
- Doses / Concentrations:
0, 1000, 2000 mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide (50 mg/kg bw)
- Tissues and cell types examined:
- Femoral bone marrow cells
- Details of tissue and slide preparation:
- SAMPLING TIMES
test groups: 10, 24 and 48 h after treatment
positive control group: 26 h after treatment - Evaluation criteria:
- According to Guideline.
- Statistics:
- Yes
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- negative
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to genetic toxicity will be updated once all ongoing studies have been finalised.
No data on genetic toxicity are available for AES (C9-11, 1-2.5 EO) Na (CAS 160901-28-0). Therefore this endpoint is covered by read across from structurally related AES, i.e. AES (C8-10, 1-2.5 EO) Na and AES (C12-14; 1-2.5 EO) Na (CAS 68891-38-3). The AES reported within the AES category show similar structural, physico-chemical, environmental and toxicological properties. The approach of grouping different AES for the evaluation of their effects on human health and the environment was also made by the Danish EPA (2001) and HERA (2003), supporting the read across approach between structurally related AES. For further details on the suitability of the read-across please refer to the AES Category Approach Justification.
In general a lack of mutagenic activity for the AES category is predictable based on structural and mechanistic considerations. Mutagens are chemicals that either 1) contain highly reactive electrophilic centers capable of interacting with nucleophilic sites on DNA (direct acting agents) or 2) can be metabolized to highly reactive electrophiles. The chemical structures represented by this chemical class do not contain electrophilic functional groups or functional groups capable of being metabolized to electrophiles. AES are readily absorbed in the gastrointestinal tract in human and rat and excreted principally via the urine or faeces depending on the length of the ethoxylate chain but independently of the route of administration. Once absorbed, AES are extensively metabolized by beta- or omega oxidation. The EO-chain seems to be resistant to metabolism. Thus, AES with fully saturated carbon chains are not metabolized to reactive electrophiles.
There are two studies for the read-across substance AES (C8-10, 1-2.5 EO) Na and one study for the read-across substance AES (C12-14; 1-2.5 EO) Na (CAS 68891-38-3) addressing genetic toxicity available.
Mutagenicity of AES (C8-10, 2 EO) Na in bacteria was assessed in a study performed according to OECD Guideline 471 with Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 102 and TA 100 (Z&S, 2012e). The tester strains were treated using the plate incorporation and the pre incubation method both with and without the addition of a rat liver S9-mix. The concentrations for both testing methods was 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Results achieved with vehicle (distilled water) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation while no genotoxicity was observed under both circumstances.
The mutagenicity of AES (C8-10, 2 EO) Na in a mammalian cell line was investigated according to OECD guideline 476 using the mouse lymphoma L5178Y cells with and without metabolic activation (Z&S, 2012f). The test concentrations were 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM without metabolic activation as well as 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM with metabolic activation in the first experiment (4 h incubation). In the second experiment the cells were incubated with concentrations of 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM in the presence of metabolic activation for 4 h and at concentrations of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM in the absence of metabolic activation for 24 h. Results achieved with the vehicle (RPMI medium) and positive controls were valid. Cytotoxicity was seen in presence and absence of metabolic activation. No genotoxicity was observed, except for the highest dose level in experiment I with metabolic activation where a significantly increased number of mutants and a dose dependency were seen at 0.48 mM. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was observed in the highly cytotoxic range only (relative total growth below 10%) and was not verified in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.
The in vivo clastogenic potential of AES (C12-14) Na (CAS 68891-38-3, analytical purity 27-29%, no data on grade of ethoxylation) was assessed in a mammalian bone marrow chromosomal aberration test with CD-1 mouse according to OECD Guideline 475 (BASF, 1995). The test substance was administered via gavage at doses of 1000 and 2000 mg/kg bw to five animals per sex per dose. Distilled water was used as vehicle. The post exposure period were 10, 24 and 48 h for the test group including the vehicle control and 26 h for the positive control group. Results achieved with the negative (distilled water) and positive controls were valid. No signs of toxicity and no increased number of chromosome aberration were seen at 1000 and 2000 mg/kg bw. Thus the test substance did not show clastogenicity at 1000 and 2000 mg/kg bw based on the test material and 270 to 290 and 540 to 580 mg/kg bw based on the active ingredient.
In conclusion, AES and their metabolites lack the structural moieties which confer mutagenic properties. This is reflected by the lack of reliable positive genotoxicity studies within the AES category. This is supported by the conclusions of the HERA report for AES were it is stated that: “In all available in vitro and in vivo genotoxicity assays, there is no indication of genetic toxicity of AES.”
References:
Danish EPA - Environmental and Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products (2001). Environmental Project No. 615, pp. 24-28
HERA (2003). Human & Environmental Risk Assessment on ingredients of European household cleaning products Alcohol Ethoxysulphates, Human Health Risk Assessment Draft, 2003. http: //www. heraproject. com.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.
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