Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 and E. coli WP2 uvrA: negative with and without metabolic activation
Read-across from structural analogue source substance glycerides, C8-18 and C18-unsatd. mono- and di-,acetates (CAS 91052-13-0).
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): Chinese hamster lung (CHL/IU) cells: negative with and without metabolic activation
Read-across from structural analogue source substance glycerides, C8-18 and C18-unsatd. mono- and di-,acetates (CAS 91052-13-0).
Gene mutation in mammalian cells (OECD 476, HGPRT test): Chinese hamster lung fibroblasts (V79): negative with and without metabolic activation
Read-across from structural analogue source substance glycerides, C16-C18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6).
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:
- 09 Apr - 24 Apr 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Due to growth inhibition in Salmonella, but not in E.coli strains, only low test concentrations used.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium) and trp operon (for E. coli)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)
- Test concentrations with justification for top dose:
- 0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metabolic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: +S9: 2-Aminoanthracene, 2.0 & 10.0 µg/plate for TA1535 and WP2 uvrA; -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 0.01 & 0.1 µg/plate for TA100, WP2 uvrA & TA98; 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine, 1.0 µg/plate for TA1537
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation);
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition - Evaluation criteria:
- If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.
- Statistics:
- The results at each concentration were demonstrated with the mean and the standard deviation.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True 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
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True 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
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True 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
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL
Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- other: N-ethyl-N-nitrosourea; -S9, 50 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)
- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: duplicates
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)
OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations. - Evaluation criteria:
- The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period
When the above mentioned criteria were not met, the test item was considered as non-mutagenic. - Statistics:
- Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 16 Oct - 18 Apr 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- primary culture, other: human lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL
Only slides from cultures of the following dose groups were selected for metaphase analyses:
First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL
Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: daunomycin, -S9, 0.015 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)
Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)
SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases - Evaluation criteria:
- EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations
For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.
The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.
The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.
Results which failed to meet the above mentioned criteria were considered as equivocal. - Statistics:
- When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.
- Key result
- Species / strain:
- primary culture, other: human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.
Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).
Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.
In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.
Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 May 2010 - 12 Jun 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- 6 h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy - Evaluation criteria:
- The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more - Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher
RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 Jun - 19 Sep 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HGPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines. - Vehicle / solvent:
- - Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation. .
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. - Conclusions:
- Interpretation of results: negative
- Executive summary:
The test item (Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 106cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 106cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- As detailed in the analogue justification, it is considered that the target and the source substance are unlikely to lead to differences in in vitro mammalian chromosome aberration potential. The results of the available in vitro mammalian chromosome aberration test with the two source substances CAS No. 91052 -13 -0 and CAS No. 736150 -63-3 revealed no genotoxic effects. Therefore, the target substance is not expected to show clastogenic properties.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source CAS 91052-13-0
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in in vitro bacterial mutagenicity potential. The result of the available Ames tests with both source substances (CAS No. 91052-13-0 and CAS 91744-23-9) was negative. Therefore, the target substance is also not expected to show mutagenic properties in bacteria.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- Summary of available data used for the endpoint assessment of the target substance
- Adequacy of study:
- key study
- Justification for type of information:
- refer to the analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Remarks:
- HGPRT assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Source Substance CAS 91744-38-6
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- As detailed in the analogue justification, it is considered that the target and the source substance are unlikely to lead to differences in gene mutation in mammalian cells. The results of the available on gene mutation in mammalian cells with the two source substances CAS No. 91744-38-6 and CAS No. 736150 -63-3 revealed no genotoxic effects. Therefore, the target substance is not expected to show mutagenic properties.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 19 - 23 Mar 1996
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- Incomplete strain selection and only 2-AA used as positive control
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- yes
- Remarks:
- incomplete strain selection; only 2-AA used as positive control.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- First mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98.
Second mutation assay (plate incorporation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence of S9-mix in tester strain TA1537.
Third mutation assay (plate incorporation method) = 800, 1600, 2400, 3200 and 4000 µg/plate in the absence and presence of S9-mix in tester strain TA1537.
Fourth mutation assay (pre-incubation method) = 50, 160, 500, 1600 and 5000 µg/plate in the absence and presence of S9-mix in tester strains TA 1535, TA 1537, TA100 and TA 98. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tetrahydrofurane (THF)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water, DMSO and Tetrahydrofurane (THF)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2 Amino-anthracene, +S9, 2.5 µg/plate for all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 30 min
- Exposure duration: 96 h
NUMBER OF REPLICATIONS: 3 replications in 4 independent experiments.
DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined
OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures - Evaluation criteria:
- For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.
- Statistics:
- Mean values and standard deviation were calculated
- Species / strain:
- S. typhimurium, other: TA 98, TA 100, TA 1535 and TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes. at 5000 µg/plate with and without metabolic activation in all strains.
RANGE-FINDING/SCREENING STUDIES: not performed.
ADDITIONAL INFORMATION ON CYTOTOXICTY: None
ADDITIONAL INFORMATION ON MUTAGENICITY: In the first plate incorporation test with TA 13537 (-S9 mix), all plates treated with either the test compound or controls revealed a total absence of the bacterial background lawn. This test was therefore not accepted by the study director and was repeated. In this repeat (second) experiment, a small increase in the revertant frequency was observed at the two top dose levels of 1600 and 5000 µg/plate. At the top dose, this effect however was accompanied by a reduced bacterial background lawn and precipitation of the test compound. For clarification, another repeat (third) experiment with adjusted dose levels was therefore performed. Finally, in the fourth pre-incubation experiment, a precipitation of the test compound was observed at the top dose levels with all bacterial tester strains. This was however not accompanied by any overt signs of toxicity. - Conclusions:
- Interpretation of results: inconclusive
Referenceopen allclose all
Table 1: Results of the Ames Test
1st experiment |
number of revertants: mean value of negative control |
number of revertants: mean value of positive control |
max. number of revertants: mean value of test material [µg/plate] |
without S9-mix |
|
|
|
TA 1535 |
14 ±2 |
399 ±25 |
12±2.5 [39] |
TA 100 |
103±5.0 |
825 ± 34.6 |
113 ± 9.7 [4.9] |
TA 1537 |
11±1.5 |
1989± 52.0 |
13±0.6 [10] |
TA 98 |
19 ±1.7 |
561 ± 19 |
18 ±4.4 [1.2] |
WP2uvrA |
25 ±4.0 |
243 ±24.7 |
30 ±2.6 [5000] |
with S9-mix |
|
|
|
TA 1535 |
11± 0.6 |
399 ±25 |
12 ±2.5 [20] |
TA 100 |
111 ± 6.1 |
825 ± 34.6 |
126 ±7.5 [39] |
TA 1537 |
17 ±4.7 |
1989± 52.0 |
20±3.8 [20] |
TA 98 |
29±1.5 |
561 ± 19 |
28 ±5.0 [39] |
WP2uvrA |
28 ±0.6 |
243 ±24.7 |
33±7.0 [5000] |
2nd experiment |
number of revertants: mean value of negative control |
number of revertants: mean value of positive control |
max. number of revertants: mean value of test material [µg/plate] |
without S9-mix |
|
|
|
TA 1535 |
10±2.3 |
388± 16.4 |
8 ±0.6 [10] |
TA 100 |
121 ± 6.0 |
780 ±7.1 |
114 ±7.8 [20] |
TA 1537 |
11 ±1.0 |
2057±200.9 |
15 ±0 [4.9] |
TA 98 |
17 ±1.2 |
577 ±32.8 |
20±1.5 [1.2] |
WP2uvrA |
20 ±2.0 |
219 ±28:1 |
27 ±4.6 [1250] |
with S9-mix |
|
|
|
TA 1535 |
9 ±2.1 |
388 ±16.4 |
11 ±3.2 [78] |
TA 100 |
114 ±12.7 |
780 ±7.1 |
117±9.3 [10] |
TA 1537 |
23 ±4.7 |
2057 ±200:9 |
21±2.3 [10] |
TA 98 |
25±3.8 |
577 ±32.8 |
29 ±2.6 [39] |
WP2uvrA |
25± 3.2 |
219 ±28:1 |
35 ±2.1 [1250] |
|
|||
Table 1: First Experiment - 4 h exposure - Without Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.36 |
1 |
|
625 |
91 |
116 |
57 |
2.04 |
0.86 |
|
1250 |
108 |
94 |
65 |
2.44 |
1.03 |
|
2500 |
106 |
83 |
36 |
2.66 |
1.12 |
|
3600 |
89 |
76 |
11 |
0.30* |
0.12 |
|
5000 |
74 |
83 |
8 |
0.04* |
0.017 |
|
ENU (50) |
53 |
62 |
45 |
# |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 2: First Experiment - 3 h exposure - With Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.39 |
1 |
|
625 |
75 |
81 |
65 |
2.59 |
1.08 |
|
1250 |
92 |
44 |
46 |
3.04 |
1.27 |
|
2500 |
93 |
30 |
9 |
0.9 |
0.38 |
|
3600 |
90 |
5 |
1 |
0.2 |
0.08 |
|
5000 |
84 |
4 |
1 |
0.47 |
0.2 |
|
DMBA (3.3) |
24 |
50 |
29 |
# |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation |
|
|||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
not reported |
not reported |
|
313 |
82 |
9 |
2 |
not reported |
not reported |
|
625 |
73 |
4 |
1 |
not reported |
not reported |
|
1250 |
68 |
4 |
1 |
not reported |
not reported |
|
2500 |
38 |
4 |
1 |
not reported |
not reported |
|
3600 |
31 |
1 |
0 |
not reported |
not reported |
|
ENU (50) |
53 |
35 |
9 |
not reported |
not reported |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation |
|
|
||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2 |
1 |
|
156 |
83 |
95 |
62 |
1.95 |
0.98 |
|
313 |
84 |
101 |
70 |
1.84 |
0.92 |
|
625 |
82 |
105 |
65 |
1.79 |
0.9 |
|
1250 |
46 |
98.5 |
65 |
1.84 |
0.92 |
|
2500 |
38 |
88 |
37 |
1.93 |
0.97 |
|
3600 |
36 |
4 |
0 |
0 |
0 |
|
DMBA (3.3) |
28 |
52 |
27 |
37.3 |
18.65 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation |
|
|||||
Concentration (µg/mL) |
Cloning efficiency (%) |
Relative Total Growth (%) |
Mutants per 1E+4 surviving cells |
Mutation factor |
|
|
Day 0 |
Day 3 |
|
||||
0 (DMSO) |
100 |
100 |
100 |
2.17 |
1 |
|
`2.5 |
102 |
100 |
62 |
2.16 |
1 |
|
5 |
100 |
114 |
151 |
2.02 |
0.93 |
|
10 |
92 |
95 |
145 |
1.97 |
0.91 |
|
20 |
105 |
100 |
60 |
2.11 |
0.97 |
|
40 |
82 |
95 |
103 |
2.17 |
1 |
|
80 |
69 |
111 |
97 |
1.82 |
0.84 |
|
160 |
25 |
44 |
16 |
2.32 |
1.07 |
|
320 |
11 |
31 |
6 |
1.94 |
0.89 |
|
ENU (50) |
16 |
31 |
9 |
51.8 |
23.87 |
|
ENU: N-ethyl-N-nitrosourea
DMBA: 7,12-dimethyl-1,2-benzanthracene
*: statistically significant with p < 0.01
#: could not be calculated as all wells contained mutant colonies
Table 1. Test results of the preliminary toxicity test
Treatment (µg/mL) |
Mitotic index (MI) |
|||||
Without S9 |
With S9 |
|||||
Individual values |
Relative mean MI (%) |
Individual values |
Relative mean MI (%) |
|||
0 |
4.8 |
5.3 |
100 |
3.8 |
2.9 |
100 |
313 |
4.8 |
4.3 |
90 |
3.1 |
3.5 |
99 |
625 |
4.3 |
4.1 |
83 |
2.9 |
3.5 |
96 |
1250 |
3.4 |
3.7 |
70 |
3.4 |
2.8 |
93 |
2500 |
3.7 |
3.3 |
69 |
2.1 |
1.8 |
58 |
5000 |
3.6 |
2.9 |
64 |
0.4 |
0.3 |
10 |
Mitotic index: percentage of cells at metaphase
Individual values: values for each of the duplicate
Relative mean MI: relative mean mitotic index for the duplicates
Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)
Treatment (µg/mL) |
S9 mix |
Relative mean MI (%) |
No. aberrant metaphases |
Number and types of aberrations |
Number of polyploid metaphases |
||
Gaps |
Breaks |
Exchanges |
|
||||
0 |
- |
100 |
1 / 0 |
2 / 1 |
1 / 0 |
|
1 / 0 |
1250 |
- |
110 |
1 / 1 |
0 / 2 |
2 / 1 |
|
|
2500 |
- |
75 |
1 / 4 |
1 / 3 |
1 / 4 |
|
|
5000 |
- |
70 |
2 / 3 |
2 / 1 |
2 / 4 |
|
|
Daunomycin (0.015 µg/mL) |
- |
91 |
17 / 14 ** |
9 / 5 |
20 / 14 |
2 / 1 |
|
0 |
2% |
100 |
0 / 0 |
1 / 2 |
0 / 0 |
|
|
625 |
2% |
67 |
0 / 1 |
0 / 0 |
0 / 1 |
|
|
1250 |
2% |
51 |
0 / 0 |
2 / 1 |
0 / 0 |
|
|
2500 |
2% |
42 |
0 / 1 |
1 / 1 |
0 / 1 |
|
|
Cyclophosphamide (6 µg/mL) |
2% |
20 |
33a /19b ** |
16 / 3 |
36 / 20 |
15 / 9 |
|
Relative mean: relative mean mitotic index for the duplicates
No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates
** Statistically significant with p < 0.01
a: only 59 scoreable metaphases
b: only 30 scoreable metaphases
The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)
Table 3. Test results of the second main test (repeat test)
Treatment (µg/mL) |
S9 mix |
Relative mean MI (%) |
No. aberrant metaphases |
Number and types of aberrations |
Number of polyploid metaphases |
||
Gaps |
Breaks |
Exchanges |
|
||||
0 |
- |
100 |
0 / 3 |
1 / 5 |
0 / 3 |
|
|
40 |
- |
84 |
1 / 4 |
3 / 6 |
1 / 4 |
|
1 / 0 |
80 |
- |
59 |
1 / 2 |
0 / 2 |
1 / 2 |
|
|
160 |
- |
49 |
0 / 2 |
3 / 1 |
0 / 2 |
|
0 / 1 |
Daunomycin (0.015 µg/mL) |
- |
102 |
13 / 14 ** |
6 / 9 |
13 / 13 |
1 / 1 |
|
0 |
4% |
100 |
2 / 1 |
3 / 2 |
1 / 1 |
1 / 0 |
|
625 |
4% |
66 |
2 / 4 |
3 / 1 |
5 / 1 |
|
0 / 1 |
1250 |
4% |
61 |
1 / 1 |
3 / 4 |
1 / 1 |
|
|
2500 |
4% |
33 |
5 / 1 |
3 / 0 |
3 / 4 |
|
|
Cyclophosphamide (6 µg/mL) |
4% |
77 |
36 / 33 ** |
8 / 8 |
37 / 33 |
11 / 11 |
|
Relative mean: relative mean mitotic index for the duplicates
No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates
** Statistically significant with p < 0.01
a: only 59 scoreable metaphases
b: only 30 scoreable metaphases
The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)
Table 4. Historical Data (n = 9 previous study)
Treatment (µg/mL) |
S9 mix |
Frequency of metaphases with aberrant chromosomes excluding gaps (%) |
Number of cultures |
|||
Mean |
SD |
Mimimum |
Maximum |
|
||
Negative control |
- |
0.8 |
0.8 |
0 |
3 |
32 |
Daunomycin (0.015 µg/mL) |
- |
15.1 |
7.4 |
7 |
34 |
32 |
Negative control |
+ |
0.7 |
0.9 |
0 |
3 |
32 |
Cyclophosphamide (6 µg/mL) |
+ |
43.0 |
13.6 |
23 |
70 |
32 |
Table 1. Effects of the test substance on viability and chromosome aberrations.
Test item |
Concentration |
Cell viability |
Aberrant cells in % |
|
|
in µg/mL |
in % |
Numerical |
Structural including gaps |
Exposure period 6h without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.0 |
1.5 |
MMC |
0.010 |
105.5 |
0.0 |
39.5 |
Test substance |
0.020 |
101.0 |
0.5 |
0.0 |
0.039 |
98.0 |
1.0 |
1.5 |
|
0.078 |
88.0 |
0.0 |
0.0 |
|
0.156 |
82.5 |
0.5 |
1.0 |
|
Exposure period 6h with S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
2.0 |
Solvent |
-- |
100 |
0.0 |
0.5 |
B(a)P |
7.000 |
96.5 |
0.0 |
28.5 |
Test substance |
0.020 |
96.0 |
0.0 |
2.0 |
0.039 |
92.0 |
0.0 |
0.0 |
|
0.078 |
86.0 |
1.0 |
0.5 |
|
0.156 |
84.5 |
0.5 |
1.0 |
|
Exposure period 24h without S9 mix |
||||
Untreated |
-- |
-- |
1.0 |
0.5 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
102.0 |
0.0 |
41.5 |
Test substance |
0.078 |
90.5 |
0.0 |
0.5 |
0.156 |
82.5 |
0.0 |
1.0 |
|
0.313 |
76.0 |
1.0 |
1.0 |
|
0.625 |
66.0 |
0.0 |
0.5 |
|
1.250 |
66.0 |
0.5 |
2.0 |
|
2.500 |
84.5 |
0.5 |
0.0 |
|
5.000 |
92.0 |
0.0 |
1.0 |
|
Exposure period 48h without S9 mix |
||||
Untreated |
-- |
-- |
0.0 |
0.0 |
Solvent |
-- |
100 |
0.5 |
0.0 |
MMC |
0.050 |
94.0 |
0.0 |
58.5 |
Test substance |
0.078 |
99.0 |
0.0 |
1.0 |
0.156 |
86.5 |
0.0 |
0.5 |
|
0.313 |
74.5 |
1.0 |
0.0 |
|
0.625 |
70.0 |
0.0 |
0.0 |
|
1.250 |
79.5 |
0.5 |
0.0 |
|
2.500 |
87.5 |
0.5 |
0.0 |
|
5.000 |
92.5 |
0.5 |
1.0 |
|
MMC: Mitomycin
B(a)P: Benz(a)pyrene
Summary Table
| relative | relative | relative | mutant | relative | relative | relative | mutant | ||||||
| conc. | P | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
| µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | ||
| % | % | % | % | % | % | ||||||||
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| Experiment I / 4 h treatment | culture I | culture II | |||||||||||
| Solvent control with THF | - | 100.0 | 100.0 | 100.0 | 12.3 | 1.0 | 100.0 | 100.0 | 100.0 | 13.8 | 1.0 | ||
| Positive control (EMS) | 150.0 | - | 84.1 | 132.6 | 113.8 | 70.9 | 5.8 | 80.4 | 76.7 | 63.8 | 114.8 | 8.3 | |
| Test item | 4.7 | - | 97.9 | culture was not continued# | 87.8 | culture was not continued# | |||||||
| Test item | 9.4 | - | 84.6 | 104.9 | 118.8 | 13.4 | 1.1 | 79.6 | 106.1 | 55.7 | 24.3 | 1.8 | |
| Test item | 18.8 | - | 88.6 | 142.2 | 100.7 | 9.8 | 0.8 | 98.8 | 105.7 | 31.0 | 47.0 | 3.4 | |
| Test item | 37.5 | - | 80.4 | 155.1 | 139.6 | 12.3 | 1.0 | 83.9 | 76.0 | 39.0 | 25.3 | 1.8 | |
| Test item | 75.0 | P | - | 68.5 | 70.8 | 113.2 | 8.3 | 0.7 | 82.0 | 96.4 | 42.7 | 25.1 | 1.8 |
| Test item | 150.0 | P | - | 80.7 | 90.3 | 126.2 | 7.4 | 0.6 | 69.8 | 80.6 | 35.6 | 84.9 | 6.1 |
| Experiment I / 4 h treatment | culture I | culture II | |||||||||||
| Solvent control with THF | + | 100.0 | 100.0 | 100.0 | 11.9 | 1.0 | 100.0 | 100.0 | 100.0 | 10.7 | 1.0 | ||
| Positive control (DMBA) | 1.1 | + | 58.3 | 70.4 | 70.2 | 802.4 | 67.3 | 85.0 | 48.2 | 119.1 | 274.5 | 25.5 | |
| Test item | 4.7 | + | 81.4 | culture was not continued# | 113.4 | culture was not continued# | |||||||
| Test item | 9.4 | + | 77.8 | 120.4 | 92.1 | 28.2 | 2.4 | 96.0 | 60.9 | 109.9 | 13.3 | 1.2 | |
| Test item | 18.8 | + | 80.5 | 105.1 | 100.7 | 10.3 | 0.9 | 119.3 | 87.5 | 92.3 | 17.5 | 1.6 | |
| Test item | 37.5 | + | 78.5 | 130.0 | 100.3 | 11.5 | 1.0 | 119.0 | 97.9 | 105.2 | 8.5 | 0.8 | |
| Test item | 75.0 | P | + | 72.4 | 100.9 | 119.0 | 9.5 | 0.8 | 128.4 | 77.6 | 109.0 | 7.4 | 0.7 |
| Test item | 150.0 | P | + | 72.6 | 98.2 | 107.7 | 6.7 | 0.6 | 131.9 | 76.2 | 121.7 | 15.1 | 1.4 |
| Experiment II / 24 h treatment | culture I | culture II | |||||||||||
| Solvent control | - | 100.0 | 100.0 | 100.0 | 20.4 | 1.0 | 100.0 | 100.0 | 100.0 | 9.7 | 1.0 | ||
| Positive control (EMS) | 150.0 | - | 108.1 | 79.6 | 86.7 | 381.1 | 18.7 | 105.1 | 126.4 | 103.5 | 422.4 | 43.7 | |
| Test item | 4.7 | - | 109.1 | culture was not continued# | 106.8 | culture was not continued# | |||||||
| Test item | 9.4 | - | 98.6 | 108.0 | 84.6 | 26.2 | 1.3 | 105.6 | 95.6 | 117.2 | 23.1 | 2.4 | |
| Test item | 18.8 | - | 98.8 | 92.3 | 92.4 | 34.1 | 1.7 | 109.3 | 75.7 | 98.4 | 20.2 | 2.1 | |
| Test item | 37.5 | P | - | 102.2 | 93.8 | 85.5 | 14.7 | 0.7 | 106.1 | 93.8 | 99.1 | 9.9 | 1.0 |
| Test item | 75.0 | P | - | 97.2 | 104.0 | 84.7 | 24.0 | 1.2 | 105.8 | 144.5 | 113.3 | 13.2 | 1.4 |
| Test item | 150.0 | P | - | 99.0 | 115.4 | 79.0 | 30.3 | 1.5 | 103.9 | 94.1 | 95.9 | 2.6 | 0.3 |
| Experiment II / 4 h treatment | |||||||||||||
| Solvent control with THF | + | 100.0 | 100.0 | 100.0 | 35.2 | 1.0 | 100.0 | 100.0 | 100.0 | 21.0 | 1.0 | ||
| Positive control (DMBA) | 1.1 | + | 103.0 | 120.7 | 67.8 | 732.9 | 20.8 | 100.0 | 105.0 | 93.9 | 348.7 | 16.6 | |
| Test item | 4.7 | + | 104.4 | culture was not continued# | 101.6 | culture was not continued# | |||||||
| Test item | 9.4 | + | 123.9 | 109.0 | 98.6 | 14.8 | 0.4 | 108.7 | 107.6 | 95.2 | 4.5 | 0.2 | |
| Test item | 18.8 | + | 133.2 | 89.6 | 93.2 | 29.6 | 0.8 | 112.2 | 109.0 | 98.7 | 10.8 | 0.5 | |
| Test item | 37.5 | + | 94.0 | 127.6 | 79.8 | 26.9 | 0.8 | 100.9 | 93.4 | 101.3 | 16.4 | 0.8 | |
| Test item | 75.0 | P | + | 97.8 | 95.3 | 74.5 | 23.4 | 0.7 | 105.7 | 123.3 | 107.4 | 15.7 | 0.7 |
| Test item | 150.0 | P | + | 106.3 | 104.3 | 74.7 | 15.7 | 0.4 | 105.3 | 99.5 | 94.7 | 14.2 | 0.7 |
# culture was not continued since a minimum of only four analysable concentrations is required
P precipitation observed at the end of treatment
Besides the key study with source substance CAS 91052 -13 -0 there is supporting data from a cytogenicity test with source substance CAS 736150 -63 -3 available. The latter test was performed in primary human lymphocytes in the presence and absence of metabolic activation. In the first experiment the cells were exposed for 3 h in the absence and absence of S9 mix, in the second experiment the cells were exposed for 3 h with S9 mix and for 20 h without S9 mix. Metaphases were sampled 20 h after start of exposure and scored for chromosomal aberrations. Cytotoxicity was noted at at 5000 µg/mL in the presence and absence of metabolic activation. There was no increase in the frequency of aberrant metaphase cells at any concentration, neither in the presence, nor in the absence of S9 mix. Results of the vehicle and the positive controls were as expected, thus confirming the validity of the test and demonstrating the functionality of the S9 mix.
The study with source substance CAS 91052 -13 -0 was selected as key study because of a higher structural similarity with the target substance EC 701 -358 -7 and because the study period was more recent than for the study with source substance CAS 736150 -63 -3.
Besides the key study with source substance CAS 91052 -13 -0 there is supporting data from a study with source substance CAS 91744 -23 -9 available. The Ames test with source substance CAS 91744 -23 -9 was performed with S. typhimuirum strains TA 98, TA 100, TA 1535 and TA 1537 in the presence and absence of metabolic activation. Test item concentrations ranged from 50 - 5000 µg/plate for all strains. There was no cytotoxicity and no precipitation observed up to the highest tested concentration. In addition, there was no increase in the number of revertant colonies noted for any strain at any concentration. Results of the positive and vehicle controls were valid. The Ames test conducted with source substance CAS 91744 -23 -9 was considered to provide supporting information as there was no strain for the detection of cross-linking mutagens such as S. typhimurium TA 102 or E. coli WP2 uvrA included.
Besides the key study with CAS 91744 -38 -6 there is supporting data from a Mouse Lymphoma Assay with source substance CAS 736150 -63 -3 available. The latter test was performed in mouse lymphoma L5178 cells in the presence and absence of metabolic activation. The cells were exposed for 3 h in the presence and for 4 h in the absence of S9 mix (1st experiment) and for 24 h in the absence of S9 mix (2nd experiment). In the absence of S9 mix, cytotoxicity was noted at ≥ 3600 µg/mL after 4 h exposure and at ≥ 160 µg/mL after 24 h exposure. In the presence of S9 mix cytotoxicity was noted at ≥ 2500 µg/plate. There was no statistically significant increase in the number of mutant colonies at any concentration. The vehicle and the positive control showed the expected results, thus confirming the validity of the test and demonstrating the functionality of the S9 mix.
The study with source substance CAS 91744 -38 -6 was selected as key study because of a higher structural similarity with the target substance EC 701 -358 -7and because the study period was more recent than for the study with CAS 736150 -63 -3.
Table 1. Test results of experiment 1 (plate incorporation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
0 |
146±16 |
8±3 |
16±3 |
0±0A |
– |
50 |
166±1 |
6±1 |
14±2 |
0±0A |
– |
160 |
136±16 |
7±1 |
18±1 |
0±0A |
– |
500 |
182±13 |
10±3 |
15±7 |
0±0A |
– |
1600 |
175±11 |
7±1 |
18±7 |
0±0A |
– |
5000 |
166±18P |
7±2P |
21±4P |
0±0A,P |
Positive controls, –S9 |
Name |
SA |
SA |
2NF |
9-AC |
Concentrations (μg/plate) |
5.0 |
2.5 |
2.5 |
25 |
|
Mean No. of colonies/plate (average of 3) |
499±38 |
242±34 |
134±25 |
0A |
|
+ |
0 |
168±10 |
12±3 |
48±10 |
13±3 |
+ |
50 |
163±8 |
9±2 |
39±8 |
16±5 |
+ |
160 |
168±3 |
15±4 |
50±5 |
15±8 |
+ |
500 |
172±9 |
12±2 |
49±8 |
15±4 |
+ |
1600 |
163±15 |
12±5 |
59±9 |
21±2 |
+ |
5000 |
179±8P |
18±4P |
55±4P |
20±4 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
2028±39 |
215±14 |
1561±88 |
154±13 |
|
SA = Sodium azide
9NF = 2-Nitro-fluorene
2AA = 2-Aminoanthracene
9-AC = 9-Amino-acridine
P = Precipitate
A = Absence of background lawn
Table 2. Test results of experiment 2 (plate incorporation)
Without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
Frameshift type |
||
TA1537 |
||
– |
0 |
7±1 |
– |
50 |
10±3 |
– |
160 |
11±2 |
– |
500 |
11±1 |
– |
1600 |
15±3 |
– |
5000 |
13±1B,P |
Positive controls, –S9 |
Name |
9-AC |
Concentrations (μg/plate) |
25 |
|
Mean No. of colonies/plate (average of 3) |
49±14 |
9-AC = 9-Amino-acridine
P = Precipitate
B = Background lawn reduced
Table 3. Test results of experiment 3 (plate incorporation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
Frameshift type |
||
TA1537 |
||
– |
0 |
15±3 |
– |
800 |
12±4 |
– |
1600 |
16±6 |
– |
2400 |
12±4 |
– |
3200 |
11±3 |
– |
4000 |
16±2P |
Positive controls, –S9 |
Name |
9-AC |
Concentrations (μg/plate) |
25 |
|
Mean No. of colonies/plate (average of 3) |
40±10 |
|
+ |
0 |
14±5 |
+ |
800 |
11±5 |
+ |
1600 |
13±3 |
+ |
2400 |
12±4 |
+ |
3200 |
15±4 |
+ |
4000 |
23±4P |
Positive controls, +S9 |
Name |
2AA |
Concentrations (μg/plate) |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
233±35 |
9-AC = 9-Amino-acridine
2-AA = 2-Amino-anthracene
P = Precipitate
Table 4. Test results of experiment 4 (pre-incorporation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
|||
Base-pair substitution type |
Frameshift type |
||||
TA 100 |
TA1535 |
TA98 |
TA1537 |
||
– |
0 |
81±8 |
9±2 |
21±5 |
8±2 |
– |
50 |
101±16 |
7±3 |
24±4 |
7±2 |
– |
160 |
88±10 |
6±3 |
19±4 |
6±2 |
– |
500 |
66±2 |
4±2 |
24±4 |
10±3 |
– |
1600 |
82±4 |
7±3 |
24±3 |
7±1 |
– |
5000 |
80±10P |
10±6P |
23±9P |
6±4P |
Positive controls, –S9 |
Name |
SA |
SA |
2NF |
9-AC |
Concentrations (μg/plate) |
5.0 |
2.5 |
2.5 |
25 |
|
Mean No. of colonies/plate (average of 3) |
539±17 |
327±46 |
103±17 |
55±22 |
|
+ |
0 |
84±9 |
6±3 |
22±6 |
11±4 |
+ |
50 |
77±11 |
9±2 |
23±6 |
9±3 |
+ |
160 |
81±11 |
8±2 |
17±6 |
11±3 |
+ |
500 |
82±8 |
9±2 |
20±3 |
13±1 |
+ |
1600 |
93±15 |
9±3 |
17±4 |
14±2 |
+ |
5000 |
104±20P |
11±1P |
19±3P |
11±3 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3) |
1033±163 |
220±49 |
1059±330 |
143±35 |
|
SA = Sodium azide
9NF = 2-Nitro-fluorene
2AA = 2-Aminoanthracene
9-AC = 9-Amino-acridine
P = Precipitate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for read-across
There are no data on genetic toxicity available for glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7). The assessment of genetic toxicity in vitro was therefore based on studies conducted with analogue substances as part of a read across approach, in order to fulfil the standard requirements set out in Annex VII-VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.
For gene mutation in bacteria, read-across was conducted with the structurally related analogue substances glycerides, C8-18 and C18-unsatd. mono- and di-,acetates (CAS. 91052-13-0) and glycerides, C16-18 and C18-hydroxy mono- and di- (CAS 91845-19-1). For cytogenicity, read-across was conducted with the structurally related analogue substances glycerides, C8-18 and C18-unsatd. mono- and di-,acetates (CAS 91052-13-0) and castor-oil mono-, hydrogenated, acetates (CAS 736150-63-3. For gene mutation in mammalian cells, read-across was conducted with the structurally related analogue substances glycerides, C16-C18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) and glycerides, castor-oil mono-, hydrogenated, acetates (CAS 736150-63-3). A statement on the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).
Gene mutation in bacteria
CAS 91052-13-0
A bacterial gene mutation assay with glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was performed according to OECD guideline 471 and GLP (Riken, 2010d). Two independent experiments were performed both in the presence or absence of metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E. coli WP2 uvrA. In the preliminary toxicity screening, growth inhibitory effects were observed at ≥ 20 µg/plate in S. typhimurium TA 98 and TA 1537 (without metabolic activation), at ≥ 78 µg/plate in S. typhimurium TA 100 and TA 1535 (without metabolic activation), and at ≥ 313 µg/plate in all S. typhimurium strains with metabolic activation. Based on these results, concentrations ranging from 0.61 to 78 µg/plate were used for the tester strains TA 100, TA 1535, TA98 and TA 1537 in the absence of metabolic activation, whereas concentrations ranging from 10 to 313 µg/plate were applied for treatment of the tester strains TA 100, TA 1535, TA 98 and TA 1537 in the presence of metabolic activation. Since no cytotoxicity was seen in E. coli WP2 uvrA, the maximum test concentration of 5000 µg/plate and concentrations of 2500, 1250, 625 and313 µg/plate were selected for treatment in the main assay. Precipitation of the test substance was observed on the plates with E. coli WP2 uvrA at test concentrations ≥ 1250 µg/plate without metabolic activation and at ≥ 2500 µg/plate with metabolic activation in both experiments. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance in the presence or absence of metabolic activation. The positive and negative controls revealed the expected results. Under the conditions of this assay, the test substance did not induce gene mutations in the selected strains of S. typhimurium and in E. coli WP2 uvrA in the absence and presence of metabolic activation, respectively.
CAS 91744-23-9
A bacterial gene mutation assay with glycerides, C16-18 and C18-unsatd. mono-, di- and tri-, citrates, potassium salts was performed according to OECD guideline 471 and GLP (Sasol, 1996). Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were treated with the test substance by the Ames test plate incorporation as well as the pre-incubation method test. Five dose levels covering the range between 50 and 5000 µg/plate in solvent (tetrahydrofurane), in triplicate both with and without the addition of a metabolising system (Acolor 1254 induced rat liver S9 mix) was used. All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in the acceptable range. Mutagenicity activity of the test substance to one or more of the tester strains was not observed in either experiment with and without metabolic activation. Due to the limited information given on materials and methods, the results are considered inconclusive.
Cytogenicity in mammalian cells in vitro
CAS 91052-13-0
The clastogenic potential of glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was assessed in a GLP-study in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 (Riken, 2010e). In a preliminary cell growth inhibition test with concentrations ranging from 9.8 to 5000 µg/mL, no significant inhibition of cell growth was observed after 4 h exposure in the presence and absence of metabolic activation (S9 mix). However, a moderate reduction of cell growth of ca. 30-40% compared to controls was observed at concentrations ranging from 156-1250 µg/mL after 24 and 48 h continuous exposure. Based on the results of this study, concentrations of 20, 39, 78 and 156 µg/mL (± S9 mix) were used for the analysis of chromosomal aberrations after short-term exposure (6 h), whereas concentrations of 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL (± S9 mix) were chosen for analysis chromosomal aberrations after continuous exposure (24 and 48 h). No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. No cytotoxic effects were observed in any of the experiments performed. An oily precipitation of the test substance was observed at concentrations ≥ 78 µg/mL, but did not interfere with chromosomal analysis of the cells. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung (CHL/IU) cells in the presence and absence of metabolic activation.
CAS 736150-63-3
An in vitro mammalian chromosome aberration test was performed with glycerides, castor-oil mono, hydrogenated, acetates in human lymphocytes according to OECD guideline 473 and in compliance with GLP (DuPont, 2004). An initial cytotoxicity test was performed with concentrations of 313, 625, 1250, 2500 and 5000 µg/mL in the presence or absence of metabolic activation (S9-mix) for of 3 h. A dose-related toxicity was observed. At the highest concentration (5000 µg/mL), the mitotic index was reduced to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation, respectively. Based on these results, concentrations of 625, 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250, 2500, 3600 and 5000 µg/mL (with 2% S9-mix) were chosen for treatment of cells in the main assay for an exposure period of 3 h. Metaphase analysis was performed at concentrations of 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250 and 2500 µg/mL (with 2% S9-mix). In the second main assay, cells were exposed to concentrations of 313-5000 µg/mL (without S9 mix) or 625-5000 µg/mL (with 4% S9 mix) for either 3 or 20 h, respectively. As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups, this part of the experiment was repeated with lower concentrations ranging from 2.5 to 320 µg/mL. Based on the cytotoxicity data obtained, concentrations of 40, 80 and 160 µg/mL (without S9) and 625, 1250 and 2500 µg/mL (with 4% S9-mix) were used for metaphase analysis. The chromosome analysis of all experiments showed no treatment-related increase in the number of cells with chromosomal aberrations compared to controls. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values and the positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations. Based on the results of this chromosome aberration test, the test substance was not clastogenic in human lymphocytes in the presence or absence of metabolic activation under the experimental conditions chosen.
Gene mutation in mammalian cells in vitro
CAS 91744-38-6
The test item (glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (Cremer, 2012). The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration was limited by the solubility of the test item in THF and aqueous medium. Precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL in the first experiment with and without metabolic activation. In the second experiment, precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation. No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data in the second culture of the first experiment without metabolic activation at 18.8 and 150.0 µg/mL. However, the increase at 18.8 µg/mL was marginal (induction factor of 3.4) and was not reproduced in the parallel culture under identical conditions. The increase at 150 µg/mL was substantial (induction factor of 6.1) but again, not reproduced in the parallel culture under identical experimental conditions. Furthermore, the concentration of 150 µg/mL was the second precipitating concentration so, the irreproducible increase of the mutation frequency was judged as irrelevant precipitation artefact. The minor increase at 18.8 µg/mL was judged as biologically irrelevant fluctuation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was detected in both cultures of the first experiment without metabolic activation. The trend observed in culture I however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. The trend observed in culture II was judged irrelevant as it was based on a precipitation artefact at 150 µg/mL as described above. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 35.2 mutants per 10^6 cells; the range of the groups treated with the test item was from 2.6 up to 84.9 mutants per 10^6 cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies
CAS 736150-63-3
An in vitro mammalian cell gene mutation assay was performed with glycerides, castor-oil, mono, hydrogenated, acetates according to OECD guideline 476 and under GLP conditions (DuPont, 2002). In the first experiment, mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated at concentrations of 625, 1250, 2500, 3600 and 5000 µg/mL. In this experiment, cells were exposed to the test material for a period of 3 h in the presence and for 4 h in the absence of metabolic activation (S9-mix). At 3600 µg/mL, the relative total growth was 10-20% compared to the negative controls, thus providing an appropriate maximum concentration for treatment in the second experiment. In this experiment, cells were exposed without S9-mix to concentrations ranging from 313 to 3600 µg/ for a period of 24 h, whereas in the presence of S9-mix, cells were treated with concentrations of 156-3600 µg/mL for a period of 4 h. Since the relative growth with S9-mix was very low (0-2%) at all test concentrations, the 24-h treatment of cells in the absence of S9-mix was repeated with concentrations ranging from 2.5-320 µg/mL, which resulted in appropriate levels of cytotoxicity (10-20% relative growth) at 160 µg/mL. In the presence of metabolic activation, the relative total growth was 9% at 2500 µg/mL in the first experiment and 37 and 0% at 2500 and 3600 µg/mL in the second experiment, respectively. After a 3-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose concentration. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.
Overall conclusion for genetic toxicity
There are no available studies on the genetic toxicity of the target substance glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7). Therefore, read-across from source substances was applied from in vitro studies on cytogenicity and in vitro gene mutation in bacteria and mammalian cells, using 4 source substances. The reliable data available for the read-across analogue substances glycerides, C8-18 and C18-unsatd. mono- and di-,acetates (CAS 91052-13-0), glycerides, C16-18 and C18-hydroxy mono- and di- (CAS 91845-19-1), glycerides, C16-C18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) and glycerides, castor-oil mono-, hydrogenated, acetates (CAS 736150-63-3) did not indicate a genotoxic effect in vitro, neither in the presence, nor in the absence of metabolic activation. Therefore, glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7) is not expected to be mutagenic and not clastogenic in vitro.
Justification for classification or non-classification
Based on read-across from structurally similar substances, 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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