Registration Dossier
Registration Dossier
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EC number: 936-560-6 | CAS number: 212070-45-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1. Key study, Ceramide VI: guideline study, GLP, Reliability 1: non-mutagenic in the bacterial reverse mutation assay (5 strains tested)
2. Supporting study, source substance Ceramide IIIB: guideline study, Reliability 1: non-mutagenicin the bacterial reverse mutation assay (5 strains tested)
3. Supporting study, target substance Ceramide III: guideline study, Reliability 2: non-mutagenic in the bacterial reverse mutation assay (2 strains tested)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-02-01 to 2017-02-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Characteristics: (Off-) white powder
- Storage conditions: at room temperature (+10 °C to +25 °C) - Target gene:
- histidine operon
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: rfa-, pKM 101/pAQ1, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, non-resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, non-resistance to Ampicillin
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany
- Test concentrations with justification for top dose:
- 1, 3.16, 10, 31.6, 100 and 316 µg/plate. Two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) were carried out in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation. Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol (Batch no. F2540; Honeywell Specialty Chemicals)
- Justification for choice of solvent/vehicle: The test item was not soluble in water or dimethylsulfoxide (DMSO). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-amino-anthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 -72 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. - Evaluation criteria:
- Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing facility. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment. Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the vehicle control and/or a scarce background lawn.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2015 to 2017 (most recent background data, not audited by the QAU-department) are given in Table 1 in section “any other details on results incl. tables”. - Statistics:
- Interpretation of results:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: the test item is insoluble in water
- Precipitation: see section “any other information on results incl. tables”.
RANGE-FINDING/SCREENING STUDIES: see section “any other information on results incl. tables”.
HISTORICAL CONTROL DATA see tables 1 and 2 in section “any other information on results incl. tables”. - Conclusions:
- Under the described conditions and tested up to a concentration of 316 µg/plate, that led to test item precipitation, the test substance caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. Therefore, the test substance is considered non-mutagenic in the bacterial reverse mutation assay.
- Executive summary:
The potential of Ceramide IIIB to induce gene mutations was examined in 5 Salmonella typhimurium strains in two independent experiments without and with metabolic activation (plate incorporation test and preincubation test).
The test item was completely dissolved in ethanol for concentrations lower than 1000 µg/plate. The vehicle ethanol was employed as the negative control.
In two preliminary cytotoxicity tests, test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation. Thus, six concentrations ranging from 1.0 to 316 µg/plate were employed in the main experiment.
Cytotoxicity as determined by the reduction of the number of revertants by more than 50% was noted at the top concentration of 316 µg/plate in some of the test strains with and without metabolic activation.
No increase in revertant colony numbers as compared with control up to a concentration of 316 µg/plate was observed in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, the test substance was tested up to a concentration of 316 µg/plate, which led to precipitation but caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- May-1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May, 1983
- Deviations:
- yes
- Remarks:
- only 2 strains tested, no historical data were reported
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted December, 1992
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix (Aroclor 1254-induced rat liver S-9 plus cofactors)
- Test concentrations with justification for top dose:
- Main study (1st and 2nd mutation assay): S. typhimurium strains (TA98 and TA100): 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate with and without mammalian metabolic activation.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol absolute pro analyse (Merck)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- not specified
- Remarks:
- without metabolic activiation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- not specified
- Remarks:
- with metabolic activiation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: mutation assay: plate incorporation
DURATION (plate incorporation)
- Preincubation period: not given
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction of the background lawn, viability or reduction of the spontaneous reversion rate
RANGE FINDING TEST
- no information given - Evaluation criteria:
- Criteria for a Negative Response.
A response is considered to have caused a negative response if all of the strains treated with the test article have mean reversion frequencies that are no greater than twice that of the mean reversion frequencies of the corresponding solvent control plates in any tester strain
Criteria for a Positive Response.
A response is considered to have caused a positive response if any strain has a dose that produces a mean reversion frequency that is two times or more greater than the mean reversion frequency of the corresponding solvent control plates. In addition, the response must be dose dependent or increasing concentrations of the extract must show increasing mean reversion frequencies. In evaluating the results, consideration will be given to the degree of toxicity exhibited by the dose causing the two-fold/three-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.
Criteria for an Equivocal Response.
A response is considered to have caused an equivocal response if it does not fulfill the criteria of either a negative or a positive response and/or the Study Director does not consider the response to be either positive or negative. - Statistics:
- No formal hypothesis testing was done.
- Key result
- Species / strain:
- other: TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Based on the results of this study it is concluded that the test substance Ceramide VI is not mutagenic in the Ames Salmonella/microsome assay.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted May 26, 1983) and EEC Directive 67/548/EEC B.14 (adopted December 1992), two strains of S. typhimurium (TA 98 and TA 100) were exposed to Ceramide VI at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.
Precipitation of Ceramide VI on the plates was observed at the start and at the end of the incubation period at test substance concentrations of 333µg/plate and upwards in the tester strains TA98 ans TA100.
In strain TA98, in the presence of S9 -mix, a reduction in the number of revertants was observed at concentrations of 3300 and 5000µg/plate. In strain TA98, in the absence of S9 -mix, and in strain TA100 no decrease in the number of revertants was observed. No reduction of the bacterial background lawn was observed at all concentrations tested.
Both bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants. The negative and strain-specific positive control values were within our laboratory background historical control datd ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Conclusion: Based on the results of this study it is concluded that Ceramide VI is not mutagenic in the Salmonella typhimurium reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-04-02 to 2019-06-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Characteristics: (Off-) white to light yellow powder
- Storage conditions: at room temperature (+10 °C to +25 °C) - Target gene:
- histidine operon
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: rfa-, pKM 101/pAQ1, resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, non-resistance to Ampicillin
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- other: uvrB-, rfa-, non-resistance to Ampicillin
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany)
- Test concentrations with justification for top dose:
- PC-2019-829 was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced test item precipitation was noted starting at a concentration of 1000 µg PC-2019-829/plate in both experiments. These concentrations were not evaluable.
Hence, 1000 µg PC-2019-829 per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (Batch no. STBG9092; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany)
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents recommended (water, dimethyl sulfoxide (DMSO), ethanol, methanol, acetone, acetonitrile, heptane or N,N-Dimethylformamid (DMF)). A short-lasting stable suspension could be achieved with PC-2019-829 in dimethyl sulfoxide (DMSO). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-amino-anthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 -72 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. - Evaluation criteria:
- Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing facility. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment. Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the vehicle control and/or a scarce background lawn.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2016 to 2018 (most recent background data, not audited by the QAU-department) are given in Table 1 in section “any other details on results incl. tables”. - Statistics:
- Interpretation of results:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: the test item is insoluble in water
- Precipitation: see section “any other information on results incl. tables”.
RANGE-FINDING/SCREENING STUDIES: see section “any other information on results incl. tables”.
HISTORICAL CONTROL DATA see tables 1 and 2 in section “any other information on results incl. tables”. - Conclusions:
- Under the described conditions and tested up to a concentration of 1000 µg/plate, that led to test item precipitation, the test substance caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. Therefore, the test substance is considered non-mutagenic in the bacterial reverse mutation assay.
- Executive summary:
The potential of Ceramide VI to induce gene mutations was examined in 5 Salmonella typhimurium strains in two independent experiments without and with metabolic activation (plate incorporation test and preincubation test).
The test item was not soluble in any of the solvents recommended (water, dimethyl sulfoxide (DMSO), ethanol, methanol, acetone, acetonitrile, heptane or N,N-Dimethyl-formamid (DMF)). A short-lasting stable suspension could be achieved with Ceramide VI in dimethyl sulfoxide (DMSO). A correction factor of 1.07 was used to correct for the purity of the test item of 93.5% only. The vehicle DMSO was employed as the negative control.
Ceramide VI was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced test item precipitation was noted starting at a concentration of 1000 µg Ceramide VI/plate in both experiments. These concentrations were not evaluable.
Hence, 1000 µg Ceramide VI per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Six concentrations ranging from 3.16 to 1000 µg Ceramide VI/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Pronounced test item precipitation was noted at the top concentration of 1000 µg Ceramide VI/plate in both experiments, each carried out without and with metabolic activation (plate incorporation test and preincubation test) in all test strains. The top concentration was not evaluable. No cytotoxicity could be determined due to the intense test item precipitation.
No increase in revertant colony numbers as compared with control counts was observed for Ceramide VI, tested up to a concentration of 1000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the historical control range generated by LPT. Hence, all acceptance criteria are met.
In conclusion, under the present test conditions, Ceramide VI tested up to a concentration of 1000 µg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Referenceopen allclose all
Preliminary test
The test substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation (see tables 2 and 3). Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Main study
Six concentrations ranging from 1.0 to 316 µg of the test item per plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Cytotoxicity
Test item precipitation was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at the top concentration of 316 µg/plate in all test strains. Reduction of the number of revertants by more than 50% was noted in both experiments at the top concentration of 316 µg/plate in the following test strains:
Plate incorporation test:
- S9: TA1535 and TA1537;
+S9: TA1537;
Preincubation test:
- S9: TA98, TA102, TA1535 and TA1537;
+S9: TA1535 and TA1537.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 316 µg/plate, that led to test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data generated by the test facility (see table 1). A summary of the results is given in tables 4 to 7.
Table 1: history profile of negative and positive control values of the years 2015 to 2017 (n= 80 studies). Data obtained from plate incorporation and preincubation tests.
Negative reference item |
||||||||||
Strain |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
|||||
S9-mix |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
Mean |
30.3 |
32.1 |
145.1 |
145.2 |
277.3 |
279.0 |
19.7 |
19.9 |
6.7 |
6.7 |
SD |
5.6 |
5.9 |
18.4 |
19.2 |
16.5 |
17.2 |
4.4 |
4.6 |
1.7 |
1.8 |
Min |
20 |
20 |
107 |
101 |
245 |
203 |
10 |
10 |
2 |
3 |
Max |
49 |
49 |
195 |
198 |
323 |
324 |
34 |
36 |
10 |
10 |
Positive reference item |
||||||||||
Strain |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
|||||
S9-mix |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
|
2-nitro-fluorene |
Benzo[a]-pyrene |
Sodium azide |
2-amino-anthracene |
Mitomycin C |
Benzo[a]-pyrene |
Sodium azide |
2-amino-anthracene |
9-amino-acridine |
Benzo[a]-pyrene |
Mean |
151.2 |
150.4 |
952.9 |
948.8 |
1029.7 |
1024.3 |
135.6 |
135.4 |
76.7 |
77.6 |
SD |
27.9 |
28.8 |
99.7 |
103.7 |
102.3 |
97.1 |
28.8 |
28.4 |
26.5 |
26.4 |
Min |
91 |
96 |
677 |
703 |
756 |
781 |
51 |
49 |
28 |
31 |
Max |
293 |
291 |
1213 |
1195 |
1637 |
1366 |
266 |
270 |
185 |
184 |
Table 2: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.
Test item (µg/plate) |
Background lawn |
Revertants plate 1 |
Revertants plate 2 |
0.316 |
normal |
121 |
127 |
1 |
normal |
144 |
142 |
3.16 |
normal |
116 |
149 |
10 |
normal |
144 |
134 |
31.6 |
normal |
142 |
149 |
100 |
normal |
146 |
146 |
316 |
test item precipitation |
110 |
100 |
1000 |
test item precipitation |
0 |
0 |
3160 |
test item precipitation |
0 |
0 |
5000 |
test item precipitation |
0 |
0 |
Solvent control 100 µL/plate |
normal |
147 |
149 |
Table 3: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.
Test item (µg/plate) |
Background lawn |
Revertants plate 1 |
Revertants plate 2 |
0.316 |
normal |
130 |
120 |
1 |
normal |
163 |
127 |
3.16 |
normal |
120 |
153 |
10 |
normal |
131 |
131 |
31.6 |
normal |
158 |
161 |
100 |
normal |
144 |
143 |
316 |
test item precipitation |
114 |
97 |
1000 |
test item precipitation |
0 |
0 |
3160 |
test item precipitation |
0 |
0 |
5000 |
test item precipitation |
0 |
0 |
Solvent control 100 µL/plate |
normal |
153 |
157 |
Table 4: Plate incorporation test without metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
1.0 |
22.7 |
112.3 |
281.7 |
24.3 |
6.3 |
3.16 |
24.0 |
119.3 |
292.0 |
15.0 |
6.0 |
10.0 |
28.3 |
118.0 |
287.7 |
18.7 |
6.0 |
31.6 |
26.0 |
120.3 |
284.0 |
17.3 |
5.3 |
100 |
25.7 |
128.0 |
251.0 |
16.3 |
6.7 |
316 |
22.7 test item precipitation |
109.0 test item precipitation |
260.7 test item precipitation |
11.0 test item precipitation |
2.0 test item precipitation |
Vehicle control 100 µL/plate |
32.7 |
128.0 |
271.3 |
26.7 |
6.7 |
Positive reference item |
2-nitro-fluorene |
Sodium azide |
Mitomycin C |
Sodium azide |
9-amino-acridine |
Concentration µg/plate |
10 |
10 |
10 |
10 |
100 |
|
193.7 |
993.0 |
1093.3 |
136.0 |
73.3 |
Table 5: Plate incorporation test with metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
1.0 |
29.7 |
119.3 |
255.3 |
23.0 |
4.3 |
3.16 |
29.3 |
132.0 |
293.0 |
19.0 |
6.0 |
10.0 |
24.7 |
119.0 |
264.3 |
22.7 |
4.0 |
31.6 |
24.3 |
132.0 |
277.7 |
16.7 |
5.7 |
100 |
30.7 |
118.7 |
258.3 |
19.0 |
5.0 |
316 |
22.7 test item precipitation |
108.3 test item precipitation |
245.0 test item precipitation |
10.0 test item precipitation |
2.0 test item precipitation |
Vehicle control 100 µL/plate |
30.7 |
120.0 |
274.0 |
17.0 |
5.3 |
Positive reference item |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
Concentration µg/plate |
10 |
2 |
10 |
2 |
10 |
|
184.0 |
992.7 |
1088.3 |
137.7 |
77.3 |
Table 6: Preincubation test without metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
1.0 |
31.7 |
117.3 |
281.7 |
16.7 |
8.0 |
3.16 |
29.3 |
113.0 |
276.0 |
17.0 |
8.3 |
10.0 |
28.7 |
118.7 |
258.7 |
18.0 |
6.3 |
31.6 |
28.3 |
129.0 |
267.0 |
16.0 |
6.7 |
100 |
29.7 |
120.3 |
288.7 |
20.7 |
5.7 |
316 |
12.0 test item precipitation |
79.7 test item precipitation |
126.7 test item precipitation |
9.0 test item precipitation |
2.3 test item precipitation |
Vehicle control 100 µL/plate |
30.7 |
125.0 |
286.7 |
18.3 |
6.0 |
Positive reference item |
2-nitro-fluorene |
Sodium azide |
Mitomycin C |
Sodium azide |
9-amino-acridine |
Concentration µg/plate |
10 |
10 |
10 |
10 |
100 |
|
165.7 |
998.3 |
1036.7 |
139.7 |
32.3 |
Table 7: Preincubation test with metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
1.0 |
32.0 |
116.3 |
267.3 |
22.7 |
9.7 |
3.16 |
32.0 |
118.3 |
273.7 |
23.3 |
9.3 |
10.0 |
33.3 |
109.3 |
276.3 |
26.3 |
6.7 |
31.6 |
32.3 |
118.7 |
271.0 |
20.7 |
4.3 |
100 |
29.0 |
111.3 |
252.3 |
18.3 |
7.0 |
316 |
15.3 test item precipitation |
75.3 test item precipitation |
202.7 test item precipitation |
9.0 test item precipitation |
2.0 test item precipitation |
Vehicle control 100 µL/plate |
22.7 |
131.0 |
302.0 |
25.0 |
7.3 |
Positive reference item |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
Concentration µg/plate |
10 |
2 |
10 |
2 |
10 |
|
163.3 |
1029.7 |
997.3 |
137.7 |
65.0 |
FORMULATION
The test substance was suspended in ethanol absolute pro analyse (Merck). The stock solution was treated with ultra-sonication to obtain a homogeneous suspension. The test substance concentrations were prepared directly prior to use.
PRECIPITATION
The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of Ceramide VI on the plates was observed at the start and at the end of the incubation period at test substance concentrations of 333 µg/plate and upwards in the tester strains TA98 ans TA100.
TOXICITY OF THE TEST SUBSTANCE
In strain TA98, in the presence of S9 -mix, a reduction in the number of revertants was observed at concentrations of 3300 and 5000 µg/plate. In strain TA98, in the absence of S9 -mix, and in strain TA100 no decrease in the number of revertants was observed. No reduction of the bacterial background lawn was observed at all concentrations tested.
NUMBER OF REVERTANTS
Both bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants. The negative and strain-specific positive control values were within our laboratory background historical control datd ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
CONCLUSION
Based on the results of this study it is concluded that Ceramide VI is not mutagenic in the Salmonella typhimurium reverse mutation assay.
Table 1: MUTAGENIC RESPONSE OF CERAMIDE VI IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY
|
Viable counts/plate (duplicate plates) |
|||
Concentration (µg/plate) |
Without S9-mix TA98 TA100 |
With S9-mix TA98 TA100 |
||
Solvent Control (0.1 mL ethanol) |
24 |
97 |
29 |
89 |
3 |
16 |
103 |
23 |
116 |
10 |
21 |
106 |
24 |
114 |
33 |
20 |
93 |
26 |
113 |
100 |
15 |
104 |
24 |
124 |
333 |
19 |
111 |
22 |
110 |
1000 |
18 |
101 |
25 |
108 |
3330 |
16 |
89 |
8 |
100 |
5000 Positive control |
18 648 |
84 862 |
4 105 |
86 320 |
Preliminary test:
PC-2019-829 was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced test item precipitation was noted starting at a concentration of 1000 µg PC-2019-829/plate in both experiments. These concentrations were not evaluable (see tables 1 and 2).
Hence, 1000 µg PC-2019-829 per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Main study:
Six concentrations ranging from 3.16 to 1000 µg PC-2019-829/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
Cytotoxicity:
Pronounced test item precipitation was noted at the top concentration of 1000 µg PC-2019-829/plate in both experiments, each carried out without and with metabolic activation (plate incorporation test and preincubation test) in all test strains. The top concentration was not evaluable. No cytotoxicity could be determined due to the intense test item precipitation.
Mutagenicity:
No increase in revertant colony numbers as compared with control counts was observed for PC-2019-829, tested up to a concentration of 1000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the historical control range generated by LPT. Hence, all acceptance criteria are met.
History profile of negative and positive control values of the years 2016 to 2018 (n= 90 studies). Data obtained from plate incorporation and preincubation tests.
Negative reference item |
||||||||||
Strain |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
|||||
S9-mix |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
Mean |
30.5 |
31.7 |
144.2 |
143.4 |
278.9 |
280.4 |
19.3 |
19.3 |
6.9 |
6.6 |
SD |
5.9 |
6.2 |
21.0 |
20.8 |
17.2 |
18.7 |
4.2 |
4.4 |
1.8 |
1.9 |
Min |
18 |
20 |
95 |
100 |
245 |
203 |
10 |
10 |
2 |
2 |
Max |
48 |
53 |
200 |
209 |
333 |
324 |
32 |
30 |
10 |
10 |
Positive reference item |
||||||||||
Strain |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
|||||
S9-mix |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
-S9 |
+ S9 |
|
2-nitro-fluorene |
Benzo[a]-pyrene |
Sodium azide |
2-amino-anthracene |
Mitomycin C |
Benzo[a]-pyrene |
Sodium azide |
2-amino-anthracene |
9-amino-acridine |
Benzo[a]-pyrene |
Mean |
164.8 |
163.9 |
956.4 |
957.6 |
1017.5 |
1012.9 |
151.0 |
152.7 |
67.7 |
68.0 |
SD |
26.1 |
27.5 |
107.4 |
107.7 |
96.1 |
98.7 |
44.7 |
47.6 |
28.8 |
25.6 |
Min |
102 |
104 |
701 |
703 |
709 |
759 |
62 |
67 |
25 |
24 |
Max |
301 |
304 |
1576 |
1412 |
1437 |
1433 |
406 |
404 |
187 |
184 |
Table 1: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.
Test item (µg/plate) |
Background lawn |
Revertants plate 1 |
Revertants plate 2 |
0.316 |
normal |
108 |
128 |
1 |
normal |
115 |
134 |
3.16 |
normal |
100 |
146 |
10 |
normal |
136 |
100 |
31.6 |
normal |
127 |
107 |
100 |
normal |
115 |
117 |
316 |
normal |
125 |
117 |
1000 |
test item precipitation |
# |
# |
3160 |
test item precipitation |
# |
# |
5000 |
test item precipitation |
# |
# |
Solvent control 100 µL/plate |
normal |
124 |
104 |
# not evaluable due to the intense test item precipitation
Table 2: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.
Test item (µg/plate) |
Background lawn |
Revertants plate 1 |
Revertants plate 2 |
0.316 |
normal |
122 |
134 |
1 |
normal |
121 |
133 |
3.16 |
normal |
124 |
127 |
10 |
normal |
95 |
112 |
31.6 |
normal |
80 |
110 |
100 |
normal |
170 |
153 |
316 |
test item precipitation |
180 |
175 |
1000 |
test item precipitation |
# |
# |
3160 |
test item precipitation |
# |
# |
5000 |
test item precipitation |
# |
# |
Solvent control 100 µL/plate |
normal |
112 |
136 |
# not evaluable due to the intense test item precipitation
Table 3: Plate incorporation test without metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
3.16 |
27.7 |
150.0 |
279.0 |
11.7 |
4.3 |
10.0 |
24.7 |
117.7 |
283.7 |
12.3 |
6.7 |
31.6 |
20.7 |
134.0 |
267.7 |
10.3 |
6.0 |
100 |
23.7 |
144.3 |
291.7 |
16.0 |
7.3 |
316 |
23.0 |
147.7 |
279.7 |
16.7 |
8.3 |
1000 |
# |
# |
# |
# |
|
Vehicle control 100 µL/plate |
28.7 |
133.3 |
284.3 |
14.7 |
6.7 |
Positive reference item |
2-nitro-fluorene |
Sodium azide |
Mitomycin C |
Sodium azide |
9-amino-acridine |
Concentration µg/plate |
10 |
10 |
10 |
10 |
100 |
|
159.7 |
868.0 |
1015.7 |
185.0 |
61.3 |
# not evaluable due to the intense test item precipitation
Table 4: Plate incorporation test with metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
3.16 |
29.3 |
139.7 |
247.7 |
13.0 |
4.0 |
10.0 |
29.3 |
129.3 |
263.3 |
14.7 |
8.0 |
31.6 |
28.7 |
112.0 |
247.7 |
12.7 |
3.7 |
100 |
25.3 |
140.0 |
289.7 |
18.3 |
8.7 |
316 |
27.3 |
157.3 |
266.3 |
17.7 |
6.3 |
1000 |
# |
# |
# |
# |
# |
Vehicle control 100 µL/plate |
28.0 |
154.7 |
269.7 |
15.3 |
6.0 |
Positive reference item |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
Concentration µg/plate |
10 |
2 |
10 |
2 |
10 |
|
155.0 |
875.3 |
949.7 |
164.3 |
67.3 |
# not evaluable due to the intense test item precipitation
Table 5: Preincubation test without metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
3.16 |
26.3 |
144.3 |
290.3 |
15.3 |
5.0 |
10.0 |
41.3 |
128.0 |
312.7 |
13.3 |
6.3 |
31.6 |
39.3 |
123.0 |
280.3 |
14.0 |
5.3 |
100 |
35.7 |
170.7 |
262.3 |
20.0 |
6.3 |
316 |
37.7 |
170.3 |
285.7 |
19.0 |
9.7 |
1000 |
# |
# |
# |
# |
# |
Vehicle control 100 µL/plate |
33.0 |
148.3 |
275.0 |
18.0 |
8.3 |
Positive reference item |
2-nitro-fluorene |
Sodium azide |
Mitomycin C |
Sodium azide |
9-amino-acridine |
Concentration µg/plate |
10 |
10 |
10 |
10 |
100 |
|
166.3 |
992.0 |
1066.0 |
165.7 |
56.7 |
# not evaluable due to the intense test item precipitation
Table 6: Preincubation test with metabolic activation
Test item (µg/plate) |
Number of reverted colonies (mean values, n=3) |
||||
|
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
3.16 |
23.7 |
116.7 |
272.7 |
17.3 |
7.7 |
10.0 |
28.3 |
122.0 |
302.3 |
17.7 |
5.0 |
31.6 |
28.3 |
116.0 |
253.3 |
11.3 |
3.0 |
100 |
25.3 |
112.0 |
260.3 |
13.7 |
4.0 |
316 |
23.0 |
137.7 |
255.3 |
15.7 |
7.3 |
1000 |
# |
# |
# |
# |
# |
Vehicle control 100 µL/plate |
34.3 |
115.7 |
281.0 |
12.7 |
4.7 |
Positive reference item |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
2-amino-anthracene |
Benzo[a]pyrene |
Concentration µg/plate |
10 |
2 |
10 |
2 |
10 |
|
175.3 |
826.0 |
991.0 |
165.7 |
51.7 |
# not evaluable due to the intense test item precipitation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The source substance Ceramide IIIB did not reveal mutagenic potential in a guideline study under GLP.
The target substance Ceramide III was not mutagenic in a study according to the principle described by Ames.
Therefore, based on the information of both source and target substance, Ceramide III ist considered to be non-mutagenic in the bacterial reverse mutation assay.
Justification for classification or non-classification
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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
