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EC number: 947-574-7 | CAS number: -
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Key studies with an anlaogue substance containing C12 -C15 alkyl lactates.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP applied.
- Justification for type of information:
- The substance tested contained C12-15 alkyl lactates, and thus was a true analogue for the C12 and C14 alkyl lactates present in the substance to be registered here.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late
exponential or early stationary phase of growth (approx. 109 cells/mL). A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
- Properly maintained: yes - Additional strain / cell type characteristics:
- other: All the strains contains mutations in the histidine operon, the deep rough mutation
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- Experiment I (plate incorporation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate ( for TA 98 and TA 102 with and without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation and for TA 1535 and TA 1537 with and without metabolic activation)
Experiment II (preincubation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 with metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for TA 100, TA 1535 and TA 1537 with metabolic activation)
0.0000316, 0.0001000, 0.000316, 0.001000, 0.00316, 0.0100, 0.0316 and 0.100 µL/plate (for TA 100, TA 1535 and TA 1537 without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (for TA 102 with and without metabolic activation). - Vehicle / solvent:
- The test material was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
- Untreated negative controls:
- yes
- Remarks:
- Purified water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
- Details on test system and experimental conditions:
- STRAIN CHARACTERISTICS:
- TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
- TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
- TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
- TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
- TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA.
Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland.
They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at 37 °C for at least 48 h in the dark.
NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.
DATA RECORDING:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.
DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "B" in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control (indicated as "N" in the results tables). - Evaluation criteria:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control [11]. - 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. 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.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- other: S.typhimurium TA 100, TA 1535, TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: other: all strains
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test material did not cause gene mutation by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Ceraphyl 41 is considered to be non-mutagenic in this bacterial reverse mutation assay. - Executive summary:
The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.
The results show that in experiment I and II, the toxic effects of the test item were observed in tester strain (except strain TA 102) with and without metabolic activities depending on the particular tester strains and concentration of the test material (see the section of any other information on results inc tables).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment..
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP applied
- Justification for type of information:
- The substance tested contained C12-15 alkyl lactates, and thus was a true analogue for the C12 and C14 alkyl lactates present in the substance to be registered here.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- Experiment I
without metabolic activation:
0.03125, 0.0625, 0.125, 0.25, 0.50, 0.75 and 1.00 mM
with metabolic activation:
0.25, 0.50, 0.75, 1.00, 1.25, 1.50 and 2.00 mM
Experiment II
without metabolic activation:
0.0156, 0.0313, 0.0625, 0.1250, 0.25, 0.50 and 1.00 mM
with metabolic activation:
0.2, 0.4, 0.6, 0.8 and 1.0 mM - Vehicle / solvent:
- RPMI cell culture medium (RPMI + 0% FBS) were used. This vehicle was compatible with the survival of the cells and the S9 activity.
- Untreated negative controls:
- yes
- Remarks:
- RPMI cell culture medium without FBS for 4h exposure and 15% FBS for 24h exposure.
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- used for experiment without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- RPMI cell culture medium without FBS for 4h exposure and 15% FBS for 24h exposure.
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- used for experiment with metabolic activation
- Details on test system and experimental conditions:
- BLOOD COLLECTION:
Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).
CULTURE MEDIUM:
RPMI 1640 medium supplemented with:
15 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
0.24 g/mL PHA-L
Also used for the long-term treatment (which is 24h) and the post incubation.
Treatment Medium (short-term exposure which is 4h, no FSB )
Complete culture medium without FBS.
All incubations were done at 37 °C in humidified atmosphere with 5% CO2.
MAMMALIAN MICROSOMAL FRACTION S9 HOMOGENATE
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and -naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.
TREATMENT:
Experiment I
Short-term exposure 4 h (without and with S9 mix)
After 48 h the culture medium was replaced with serum-free medium containing the test item (without metabolic activation) and serum-free medium containing the test item with 50 µL/mL S9 mix (with metabolic activation). After 4 h the cells were spun down by gentle centrifugation for 10 min. The supernatant with the dissolved test item was discarded and the cells were resuspended in PBS. The washing procedure was repeated once as described. After washing, the cells were resuspended in complete cell culture medium. The cells were prepared
24 h after the beginning of the treatment.
Experiment II
Long-term exposure 24 h (without S9 mix):
After 48 h the culture medium was replaced with complete medium (with 15% FBS) containing the test item without S9 mix. The treated cells were prepared at the end of the treatment.
Short-term exposure 4 h (with S9 mix):
The cells were treated as described for experiment I (with metabolic activation).
DETERMINATION OF CYTOTOXICITY:
To describe a cytotoxic effect the mitotic index (% cells in mitosis; by counting the number of mitotic cells in 1000 cells) was determined. Additionally the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.
DATA RECORDING:
The data generated were recorded in the raw data file. The results are presented in tables, including experimental groups with the test item, negative, solvent and positive controls. The experimental unit was the cell and therefore, the percentage of cells with structural aberration was evaluated. Different types of chromosome aberrations are listed with their numbers of frequencies for experimental and control groups. Gaps were recorded separately and reported but generally not included in the aberration frequency. Concurrent measurements of cytotoxicity were also recorded.
- 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 (without and with metabolic activation).
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.
- Statistics:
- According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results.
A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- seen at conc > 0.4nM after 4h treatment
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative 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
- Remarks:
- seen at conc of 1.0 nM after 4h treatment
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item Ceraphyl 41 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item Ceraphyl 41 is considered to be non-clastogenic in this chromosome aberration test. - Executive summary:
The test item was investigated for its potential to induce structural chromosome aberrations in human lymphocytes. The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without and 4 h with metabolic activation (experiment II). Duplicate cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.
The results show that in experiment I at concentrations of 0.75 mM and higher without metabolic activation a precipitation was observed. With metabolic activation precipitation was detected in all concentrations. In experiment II with metabolic activation precipitation was noted at concentration of 0.4 mM and higher. Without metabolic activation precipitation was only detected in concentrations higher than evaluated for microscopic analysis.
In experiment I without metabolic activation, toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 1.00 mM. With metabolic activation toxic effects were noted at concentrations of 0.75 mM and higher. In experiment II with metabolic activation, toxic effects were noted at concentration 0.4 mM and higher. In experiment II without metabolic activation no biologically relevant decrease of the relative mitotic index was noted in all dose groups evaluated.
No biologically relevant decreases of proliferation index occurred in either experiment I or II.
In experiment I and II, no biologically relevant increase of the aberration rates was observed after treatment with the test item without or with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
Irrespective of the absence or presence of metabolic activation, biologically relevant increase in the frequencies of polyploid cells was not found after treatment with the test item as compared to the controls in experiment I and II.
Referenceopen allclose all
The results shows that precipitation of the test item was observed in tester strains TA 98 and TA 102 in experiment I at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment II precipitation of the test item was found in tester strain TA 102 at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 0.00100 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were seen at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.0316 μL/plate and higher (with metabolic activation).
The results also shows that precipitation was observed in experiment I at concentrations of 0.75 mM and higher without metabolic activation.With metabolic activation precipitation was detected in all concentrations. In experiment II with metabolic activation precipitation was noted at concentration of 0.4 mM and higher.Without metabolic activation precipitation was only detected in concentrations higher than evaluated for microscopic analysis.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
Bacterial mutagenicity test:
The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The results show that in experiment I and II, the toxic effects of the test item were observed in tester strain (except strain TA 102) with and without metabolic activities depending on the particular tester strains and concentration of the test material (see the section of any other information on results inc tables).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment.
Mamalian genotoxicity test:
The test item was investigated for its potential to induce structural chromosome aberrations in human lymphocytes. The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without and 4 h with metabolic activation (experiment II). Duplicate cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.
In experiment I without metabolic activation, toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 1.00 mM. With metabolic activation, toxic effects were noted at concentrations of 0.75 mM and higher. In experiment II with metabolic activation, toxic effects were noted at concentration 0.4 mM and higher. In experiment II without metabolic activation, no biologically relevant decrease of the relative mitotic index was noted in all dose groups evaluated. No biologically relevant decreases of proliferation index occurred in either experiment I or II.
In experiment I and II, no biologically relevant increase of the aberration rates was observed after treatment with the test item either without or with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
Irrespective of the absence or presence of metabolic activation, biologically relevant increase in the frequencies of polyploid cells was not found after treatment with the test item as compared to the controls in experiment I and II.
Justification for selection of genetic toxicity endpoint
Key studies with an anlaogue substance containing C12 -C15 alkyl lactates.
Justification for classification or non-classification
Longer chain alkyl lactates like C12 -C16 alkyl lactates do not cause gene mutation in the bacterial reverse mutation assay or chromosomal abberration in human lymphocytes.
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