Registration Dossier
Registration Dossier
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EC number: 292-960-4 | CAS number: 91031-57-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, 1537, 98 and 100, and E. coli WP2 uvr A
Chromosome aberration (OECD 473, read-across): negative in primary human peripheral lymphocytes with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, read-across): negative in mouse lymphoma L5178Y cells with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 24 - 26 Jul 1985 and 13 - 18 Aug 1985 (First and second test series)
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no E.coli or TA102 strain tested, lack of information on test substance
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1983
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- yes
- Remarks:
- no E.coli or TA102 tested, no test substance purity reported, no historical control data, only one positive control (2-AA) with S9
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Remarks:
- and TA1538
- Details on mammalian cell type (if applicable):
- 0.5 mL of frozen bacteria was thawed in 20 mL standard nutrient broth, mixed and incubated for 10h by shaking at 37 degree Celsius. Cell titre was approx. 10e9.
MEDIA
Nutrient broth: standard (Merck)
Top agar: 9 g Bacto Agar (Difco) and 5 g NaCl (Merck) was dissolved in 1000 mL water and autoclaved.
Petri dishes: 30 mL of 1.5% Bacto Agar in Vogel-Bonner-Medium with 2% glucose (Merck) - Metabolic activation:
- with and without
- Metabolic activation system:
- Postmitochondrial supernatant fluids from liver of male rats treated with Aroclor 1254 (S9 mix)
- Test concentrations with justification for top dose:
- 8, 40, 200, 1000 and 5000 µg/plate
- Vehicle / solvent:
- Tween 80 in water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 2-aminoanthracene for all 5 strains with S9
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenyldiamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
48 h incubation at 37 °C
NUMBER OF REPLICATIONS: 2 test series, each in triplicates
DETERMINATION OF CYTOTOXICITY
background lawn
- OTHER:
Counting of colonies with BioTran-III-Counter (Biotronik) - Evaluation criteria:
- - no cytotoxicity as determined by assessment of bacterial lawn
- spontaneous mutation rates within the characteristic ranges as given in report
- positive control mutation rates at least 2-fold as compared to TA100 and at least 3-fold as compared to other strains at at least two different tested concentrations - Statistics:
- None
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- 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
- Positive controls validity:
- valid
- Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 09 Apr - 10 May 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9-mix
- Test concentrations with justification for top dose:
- 3, 10 and 33 µg/mL
At a concentration of 33 µg/mL and above precipitation was seen in the culture medium. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with rat liver S9-mix; 10 µg/mL
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without rat liver S9-mix; 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3, 24 and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 3h treatment: 24 and 48h; 24h treatment: 24h; 48h treatment: 48h
SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water
NUMBER OF REPLICATIONS: 2 (duplicates at the 3h-exposure time)
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- Chi-square test, one-sided, p < 0.05
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- 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
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium
- Other confounding effects: 2-ethylhexyl oleate showed no significant effects on the number of polyploid cells
RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the duplicate cultures of the 3h exposure time. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 09 Apr - 27 Jul 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine Kinase locus (TK gene)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes were prepared from adult male Wistar rats which were treated with phenobarbital (89 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).
- Test concentrations with justification for top dose:
- 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL
Precipitation in the exposure medium was seet at concentrations of 100 µg/mL and above. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation; 7.5 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Without metabolic activation; 15 and 5 µg/mL for a 3 and 24 hours treatment period
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
For 3-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum.
For 24-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT). Non selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) .
DURATION
- Exposure duration: In experiment 1 - 3 hours. Experiment 2 - 24 hours (- S9 mix) and 3 hours (+S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2-yl]-2,5- diphenyltetrazolium bromide (MTT).
NUMBER OF REPLICATIONS: 1
NUMBER OF CELLS EVALUATED: Whole wells counted
DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth - Evaluation criteria:
- In addition to the criteria stated below, any increase of the mutation frequency will be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evalulation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered postive (mutagenic) in the mutation assay if it induces a mutation factor (MF) of more than MF(control) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. The test substance will be considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
Thes test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The resullts are confirmed in an indepedently repeated test.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 2-Ethylhexyl oleate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL.
- Precipitation: 2-Ethylhexyl oleate precipitated in the exposure medium at concentrations of 100 µg/mL and above.
RANGE-FINDING/SCREENING STUDIES: In the dose finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24-hour treatment period and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours treatment and 24 and 48 hours subculture, no toxicity in the suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix.
After 24 hours of treatment and 24-hour subculture, in the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentration of 333 µg/mL compared to the solvent control.
COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the
minimum and maximum value of the historical control data range. See Table 1 to 4. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 11 Feb - 20 Apr 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - 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:
- lymphocytes: cultured human peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: F10 complete culture medium, containing Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin.
- 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
- Test concentrations with justification for top dose:
- Experiment 1:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
48 h treatment, 48 h harvest time, without metabolic activation: 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 48 h harvest time, with metabolic activation: 1000 µg/mL
Experiment 2:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.9% DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycine C (0.2 µg/mL, 24 h treatment time, -S9; 0.1 µg/mL, 48 h treatment time, -S9) and cyclophosphamide (15 µg/mL in nutrient medium, 3 h treatment time, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h with metabolic activation, 24 and 48 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h fixation time with and without metabolic activation
SPINDLE INHIBITOR (cytogenetic assays): 0.5 µg/mL colcemid, added 3 hours before harvest time (21 h with metabolic activation, 21 and 45 h without metabolic activation)
STAIN (for cytogenetic assays): 5% (v/v) Gimsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 (100 per culture, duplicate cultures)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
OTHER:
blood samples were taken from healthy, adult male volunteers by venapuncture, using a sterile vessel containing sodium heparine. The blood samples were stored at 4-25 ºC. Cultures were started within 4 h after blood collection. The average generation time of the cells is 13.6-14.1 h. Cultures were incubated at 37 ºC for 48 h prior to exposure to the test substance. - Evaluation criteria:
- A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range (min = 0, max = 5 (mean = 0.9, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0, max = 5 (mean = 0.7, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded)
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
c) A homogeneous response between the replicate cultures is observed.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- 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
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at 1000 µg/mL.
RANGE-FINDING/SCREENING STUDIES:
A range-finding test was performed to select the dose levels for the main experiment, using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation. The test substance precipitated in the culture medium at 1000 µg/mL (see Table 1).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
no cytotoxicity was observed at any concentration, with or without metabolic activation (see Table 2 and 3). - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- 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:
- lymphocytes: cultured human peripheral lymphocytes
- 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
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- 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
- Positive controls validity:
- valid
- Remarks on result:
- other: Source:
- Remarks:
- CAS 26399-02-0 and 93803-87-3
- Conclusions:
- An analogue substance approach was used for Read Across to the target substance.
In an in vitro mammalian chromosome aberration test (OECD GL 473) in cultured human peripheral lymphocytes with the analogue substance 2-ethylhexyl oleate (CAS 26399-02-0) no genotoxicity was found at concentrations up to 33 µg/mL (precipitation at higher doses).
In an in vitro mammalian chromosome aberration study (OECD GL 473) in primary peripheral human lymphocytes with the analogue substance 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) no genotoxicity was found at concentrations up to 1000 µg/mL.
For the target substance no genotoxic effects on mammalian cells are anticipated.
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Positive controls validity:
- valid
- Remarks on result:
- other: Source:
- Remarks:
- CAS 26399-02-0
- Conclusions:
- An analogue substance approach was used for Read Across to the target substance.
In an in vitro mammalian cell gene mutation test (OECD GL 476) in mouse lymphoma cells with the analogue substance 2-ethylhexyl oleate (CAS 26399-02-0) no genotoxicity was found at concentrations up to 100 µg/mL (precipitation at higher doses).
For the target substance no genotoxic effects on mammalian cells are anticipated.
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008. - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 - 25 Jul 2022
- 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:
- adopted in 1997; corrected in 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains)
trp operon (for the E.coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- other: rfa (deep rough (defective lipopolysaccharide cellcoat)), gal (mutation in galactose metabolism); chl (mutation in nitrate reductase); bio (defective biotin synthesis); uvrB (loss of excision repair system (deletion of ultraviolet-repair B gene))
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- other: The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH (Giessen, Germany); prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA, USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components, 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively. - Test concentrations with justification for top dose:
- Experiment 1 (Direct plate assay):
- all strains: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation,
Experiment 2 (Pre-incubation assay):
- all strains: 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation
5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances according to OECD 471. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol (Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle: Ethanol was selected for use in the assay as it has been routinely used and validated for use in the bacterial reverse mutation assay. The test material formed a clear colourless solution in ethanol. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- other: ICR-191; in DMSO; -S9 / TA1537 / 2.5 µg/plate (Exp 1); Methylmethanesulfonate - MMS; in DMSO; -S9 / TA 100 / 650 µg/plate (Exp 1 and 2);
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three replicate plates / dose
- Number of independent experiments: 2 (direct plate and pre-incubation assay)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in agar (plate incorporation; Experiment 1); pre-incubation (Experiment 2)
TREATMENT AND HARVEST SCHEDULE:
Direct plate assay
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
Pre-incubation assay
- Preincubation period: 30 ± 2 min at 37.0 ± 1.0 °C
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies per plate.
METHODS FOR MEASUREMENTS OF GENOTOXICITY
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
OTHER
- The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- The Escherichia coli WP2uvrA strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year. - Rationale for test conditions:
- Test conditions according to OECD 471
- Evaluation criteria:
- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment. - Statistics:
- Mean values and standard deviations were calculated.
- 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
- Remarks:
- Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation at concentrations of 512 μg/plate and upwards in Experiment 1 and 1600 μg/plate and upwards in Experiment 2 with and without S9-mix.
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation at concentrations of 512 μg/plate and upwards in Experiment 1 (with S9-mix) and 1600 μg/plate and upwards in Experiment 1 (without S9-mix) and in Experiment 2.
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.
- 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:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2 with and without S9-mix.
- 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
- The test material formed a clear colourless solution in ethanol.
- Precipitation: In Experiment 1, precipitation of the test material on the plates was observed at concentrations of 512 μg/plate and upwards in tester strains TA100 (absence and presence of S9-mix) and TA98 (presence of S9-mix). Precipitation of the test material on the plates was observed at concentrations of 1600 μg/plate and upwards in the other tester strains. In Experiment 2, precipitation of the test material on the plates was observed at concentrations of 1600 μg/plate and upwards in all tester strains in the absence and presence of S9-mix.
STUDY RESULTS:
In the Direct Plate Assay (Experiment 1) and Pre-Incubation Assay (Experiment 2), there was no increase in the number of revertants observed upon treatment with the test material under all conditions tested (refer to 'Any other information on results incl. tables', table 1 and 2).
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants observed upon treatment with the test material under all conditions tested.
HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 3 and 4. - Conclusions:
- Under the present test conditions, Fatty acids, C16-18, isononyl esters showed negative mutagenic responses in all bacterial strains over the entire dose-range.
Referenceopen allclose all
First test series 24 Jul – 26 Jul 1985
TA1535 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
15 |
14 |
8 |
21 |
10 |
13 |
|
16 |
20 |
11 |
11 |
16 |
12 |
Sodium acide, 2 µg/plate |
420 |
404 |
382 |
189 |
181 |
206 |
2-Aminoanthracene, 2.5 µg/plate |
9 |
15 |
12 |
117 |
111 |
127 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
9 |
10 |
17 |
12 |
9 |
14 |
1000 |
13 |
14 |
20 |
11 |
16 |
12 |
200 |
9 |
13 |
8 |
16 |
13 |
15 |
40 |
7 |
9 |
8 |
12 |
13 |
12 |
8 |
9 |
8 |
10 |
16 |
14 |
9 |
TA100 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
88 |
79 |
77 |
85 |
81 |
67 |
|
82 |
86 |
88 |
80 |
90 |
80 |
Sodium acide, 2 µg/plate |
400 |
260 |
374 |
263 |
255 |
308 |
2-Aminoanthracene, 5 µg/plate |
71 |
63 |
75 |
1789 |
1735 |
1653 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
70 |
113 |
74 |
123 |
97 |
105 |
1000 |
99 |
103 |
112 |
107 |
94 |
94 |
200 |
92 |
108 |
67 |
106 |
98 |
99 |
40 |
88 |
73 |
61 |
100 |
103 |
104 |
8 |
68 |
69 |
60 |
88 |
84 |
85 |
TA1537 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
5 |
5 |
4 |
6 |
6 |
5 |
|
6 |
9 |
5 |
8 |
7 |
7 |
9-aminoacridine, 80 µg/plate |
289 |
304 |
354 |
345 |
362 |
337 |
2-Aminoanthracene, 2.5 µg/plate |
6 |
6 |
7 |
145 |
153 |
167 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
7 |
10 |
4 |
8 |
6 |
7 |
1000 |
5 |
6 |
6 |
5 |
6 |
8 |
200 |
5 |
4 |
6 |
6 |
5 |
10 |
40 |
4 |
6 |
7 |
5 |
7 |
9 |
8 |
8 |
4 |
7 |
6 |
9 |
7 |
TA1538 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
17 |
10 |
11 |
24 |
25 |
17 |
|
10 |
14 |
6 |
13 |
18 |
17 |
4-Nitro-o-phenyldiamin, 40 µg/plate |
460 |
552 |
555 |
1081 |
917 |
1034 |
2-Aminoanthracene, 5 µg/plate |
18 |
16 |
15 |
1568 |
2028 |
1722 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
12 |
7 |
15 |
17 |
16 |
23 |
1000 |
17 |
14 |
13 |
25 |
13 |
21 |
200 |
13 |
18 |
9 |
13 |
26 |
15 |
40 |
9 |
8 |
12 |
13 |
15 |
19 |
8 |
18 |
15 |
12 |
13 |
20 |
12 |
TA98 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
19 |
18 |
18 |
22 |
31 |
22 |
|
16 |
29 |
20 |
16 |
30 |
24 |
4-Nitro-o-phenyldiamin, 40 µg/plate |
360 |
333 |
342 |
651 |
689 |
685 |
2-Aminoanthracene, 5 µg/plate |
17 |
18 |
26 |
1474 |
1424 |
1647 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
28 |
22 |
28 |
17 |
14 |
24 |
1000 |
24 |
30 |
18 |
21 |
13 |
13 |
200 |
20 |
24 |
21 |
16 |
18 |
27 |
40 |
24 |
25 |
25 |
21 |
21 |
23 |
8 |
17 |
21 |
20 |
18 |
33 |
26 |
Second test series 13 Aug – 15 Aug 1985
TA1535 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
8 |
11 |
7 |
4 |
9 |
14 |
|
9 |
7 |
12 |
13 |
7 |
5 |
Sodium acide, 2 µg/plate |
326 |
392 |
396 |
245 |
257 |
278 |
2-Aminoanthracene, 2.5 µg/plate |
11 |
6 |
4 |
257 |
262 |
256 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
7 |
8 |
8 |
13 |
8 |
11 |
1000 |
8 |
11 |
7 |
11 |
9 |
11 |
200 |
6 |
9 |
12 |
5 |
13 |
4 |
40 |
11 |
6 |
16 |
5 |
8 |
7 |
8 |
10 |
7 |
7 |
10 |
12 |
16 |
TA100 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
51 |
37 |
66 |
87 |
79 |
90 |
|
53 |
65 |
70 |
68 |
69 |
70 |
Sodium acide, 2 µg/plate |
217 |
211 |
198 |
95 |
159 |
232 |
2-Aminoanthracene, 5 µg/plate |
42 |
39 |
53 |
1828 |
1001 |
1672 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
57 |
62 |
47 |
82 |
67 |
91 |
1000 |
46 |
58 |
41 |
96 |
73 |
83 |
200 |
46 |
53 |
56 |
93 |
49 |
62 |
40 |
48 |
61 |
52 |
71 |
92 |
61 |
8 |
48 |
39 |
44 |
82 |
74 |
67 |
TA1537 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
2 |
3 |
7 |
10 |
13 |
7 |
|
3 |
3 |
7 |
5 |
13 |
11 |
9-aminoacridine, 80 µg/plate |
183 |
229 |
224 |
183 |
137 |
145 |
2-Aminoanthracene, 2.5 µg/plate |
6 |
4 |
4 |
234 |
215 |
239 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
6 |
4 |
1 |
6 |
4 |
6 |
1000 |
5 |
4 |
3 |
5 |
3 |
6 |
200 |
3 |
5 |
2 |
11 |
5 |
7 |
40 |
1 |
3 |
3 |
6 |
3 |
2 |
8 |
5 |
6 |
5 |
7 |
5 |
5 |
TA1538 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
12 |
13 |
14 |
26 |
21 |
12 |
|
10 |
13 |
13 |
27 |
20 |
26 |
4-Nitro-o-phenyldiamin, 40 µg/plate |
538 |
550 |
305 |
998 |
1043 |
998 |
2-Aminoanthracene, 5 µg/plate |
20 |
26 |
30 |
1742 |
1839 |
1822 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
7 |
10 |
20 |
17 |
26 |
30 |
1000 |
9 |
16 |
12 |
27 |
28 |
23 |
200 |
12 |
5 |
11 |
23 |
25 |
25 |
40 |
13 |
9 |
16 |
22 |
19 |
22 |
8 |
12 |
15 |
15 |
20 |
29 |
22 |
TA98 |
Without S9 Mix |
|
|
With S9 Mix |
|
|
Negative control |
22 |
20 |
17 |
27 |
29 |
29 |
|
21 |
17 |
18 |
28 |
39 |
25 |
4-Nitro-o-phenyldiamin, 40 µg/plate |
361 |
323 |
354 |
741 |
669 |
782 |
2-Aminoanthracene, 5 µg/plate |
32 |
21 |
22 |
2236 |
2424 |
2102 |
Test material [µg/plate] |
|
|
|
|
|
|
5000 |
17 |
13 |
24 |
22 |
26 |
25 |
1000 |
12 |
21 |
15 |
21 |
20 |
32 |
200 |
16 |
29 |
20 |
24 |
34 |
31 |
40 |
13 |
21 |
19 |
33 |
33 |
33 |
8 |
15 |
25 |
19 |
30 |
34 |
31 |
Table 1: Cytotoxic and Genotoxic observations
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
|
in µg/mL |
in % |
with gaps |
without gaps |
Exposure period 3 h, fixation time 24 h, without S9 mix |
||||
Ethanol |
1.0% (v/v) |
100 |
2 |
2 |
MMC |
0.5 |
67 |
31 |
30 |
Test substance |
3 |
99 |
1 |
1 |
10 |
98 |
2 |
2 |
|
33 |
92 |
1 |
1 |
|
Exposure period 3 h, fixation time 24 h, with S9 mix |
||||
Ethanol |
1.0% (v/v) |
100 |
1 |
1 |
CP |
0.5 |
51 |
38 |
38 |
Test substance |
3 |
101 |
1 |
1 |
10 |
108 |
0 |
0 |
|
33 |
103 |
3 |
3 |
|
Exposure period 24 h, fixation time 24 h, without S9 mix |
||||
Ethanol |
1.0% (v/v) |
100 |
1 |
1 |
MMC |
0.2 |
54 |
34 |
33 |
Test substance |
3 |
99 |
1 |
1 |
10 |
103 |
3 |
3 |
|
33 |
70 |
4 |
3 |
|
Exposure period 48 h, fixation time 48 h, without S9 mix |
||||
Ethanol |
1.0% (v/v) |
100 |
2 |
0 |
MMC |
0.1 |
120 |
51 |
49 |
Test substance |
3 |
108 |
1 |
0 |
10 |
100 |
0 |
0 |
|
33 |
99 |
2 |
2 |
|
Exposure period 3 h, fixation time 48 h, with S9 mix |
||||
Ethanol |
1.0% (v/v) |
100 |
0 |
0 |
CP |
10.0 |
-- |
44 |
44 |
Test substance |
3 |
100 |
2 |
2 |
10 |
94 |
2 |
2 |
|
33 |
97 |
1 |
0 |
|
MMC: Mitomycin CP: Cyclophosphamide
Table 1. Experiment 1: Cytotoxic and mutagenic response of 2-Ethylhexyl oleate in the mouse lymphoma L5178Y test system.
Dose (µg/mL) |
RSG (%) |
CEday2(%) |
RSday2 (%) |
RTG (%) |
Mutation Frequency x 10-6 |
||
Total |
Small |
Large |
|||||
|
Without metabolic activation (3-hour treatment) |
||||||
SC1 |
100 |
89 |
100 |
100 |
100 |
69 |
28 |
SC2 |
100 |
108 |
100 |
100 |
99 |
67 |
28 |
0.03 |
106 |
105 |
107 |
113 |
87 |
63 |
20 |
0.1 |
102 |
101 |
102 |
104 |
76 |
50 |
23 |
0.3 |
88 |
86 |
88 |
77 |
109 |
71 |
34 |
1 |
107 |
99 |
101 |
108 |
99 |
73 |
22 |
3 |
106 |
97 |
98 |
104 |
93 |
64 |
25 |
10 |
103 |
105 |
107 |
110 |
88 |
62 |
22 |
33 |
82 |
111 |
113 |
92 |
133 |
86 |
38 |
100(1) |
82 |
120 |
121 |
100 |
93 |
67 |
22 |
MMS |
66 |
56 |
57 |
37 |
1463 |
939 |
292 |
|
With 8% (v/v) metabolic activation (3-hour treatment) |
||||||
SC1 |
100 |
65 |
100 |
100 |
68 |
41 |
26 |
SC2 |
100 |
67 |
100 |
100 |
58 |
32 |
25 |
0.03 |
96 |
66 |
100 |
96 |
73 |
45 |
26 |
0.1 |
102 |
63 |
95 |
97 |
71 |
39 |
30 |
0.3 |
93 |
67 |
102 |
94 |
71 |
46 |
24 |
1 |
107 |
66 |
100 |
107 |
71 |
40 |
29 |
3 |
108 |
58 |
88 |
95 |
74 |
53 |
20 |
10 |
107 |
53 |
80 |
85 |
74 |
50 |
22 |
33 |
95 |
62 |
94 |
89 |
74 |
43 |
29 |
100(1) |
100 |
54 |
81 |
81 |
68 |
44 |
23 |
CP |
57 |
44 |
66 |
38 |
752 |
574 |
137 |
Note: all calculations were made without rounding off.
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = Ethanol; MMS Methylmethanesulfonate; CP = Cyclophosphamide.
(1) = 2-Ethylhexyl oleate precipitated in the exposure medium.
Table 2. Experiment 2: Cytotoxic and mutagenic response of 2-Ethyl oleate in the mouse lymphoma L5178Y test system.
Dose (µg/mL) |
RSG (%) |
CEday2(%) |
RSday2 (%) |
RTG (%) |
Mutation Frequency x 10-6 |
||
Total |
Small |
Large |
|||||
|
Without metabolic activation (24-hour treatment) |
||||||
SC1 |
100 |
85 |
100 |
100 |
59 |
35 |
22 |
SC2 |
100 |
93 |
100 |
100 |
63 |
35 |
26 |
0.03 |
119 |
93 |
104 |
124 |
67 |
36 |
29 |
0.1 |
134 |
91 |
103 |
138 |
56 |
34 |
21 |
0.3 |
121 |
115 |
129 |
156 |
40 |
20 |
20 |
1 |
124 |
97 |
109 |
135 |
47 |
35 |
12 |
3 |
105 |
89 |
100 |
105 |
57 |
38 |
18 |
10 |
124 |
94 |
106 |
131 |
52 |
27 |
24 |
33 |
119 |
99 |
112 |
133 |
58 |
31 |
25 |
100(1) |
129 |
97 |
109 |
140 |
44 |
28 |
15 |
MMS |
106 |
81 |
92 |
97 |
503 |
305 |
152 |
|
With 12% (v/v) metabolic activation (3-hour treatment) |
||||||
SC1 |
100 |
76 |
100 |
100 |
102 |
51 |
47 |
SC2 |
100 |
101 |
100 |
100 |
76 |
38 |
35 |
0.03 |
96 |
81 |
92 |
88 |
82 |
44 |
36 |
0.1 |
100 |
80 |
91 |
91 |
82 |
44 |
35 |
0.3 |
103 |
95 |
108 |
111 |
97 |
52 |
41 |
1 |
106 |
101 |
114 |
121 |
70 |
41 |
27 |
3 |
102 |
83 |
94 |
95 |
85 |
40 |
42 |
10 |
96 |
88 |
99 |
95 |
76 |
46 |
28 |
33 |
99 |
81 |
92 |
91 |
89 |
55 |
31 |
100(1) |
98 |
86 |
98 |
96 |
73 |
37 |
34 |
MMS |
62 |
66 |
75 |
46 |
1337 |
776 |
310 |
Table 3. Historical control data of the spontaneous mutation frequencies of the solvent controls of the mouse lymphoma assay.
|
-S9-mix |
+S9-mix |
|
3-hour treatment |
24-hour treatment |
|
|
Range |
[51-120] x 10-6 |
[50-127] x10-6 |
[50-170] x 10-6 |
Mean |
77 x 10-6 |
80 x 10-6 |
92 x 10-6 |
SD |
18 x 10-6 |
19 x10-6 |
33 x10-6 |
n |
88 |
82 |
141 |
SD = Standard deviation
n = Number of observation
The above mentioned historical control data range of the solvent controls were obtained by collecting all data of the solvent control values (media, DMSO, and ethanol) over the period of June 2006 to June 2009.
Table 4. Historical control data of the spontaneous mutation frequencies of the positive controls for the mouse lymphoma assay.
|
-S9-mix |
+S9-mix |
|
3-hour treatment |
24-hour treatment |
|
|
Range |
[518-2052] x 10-6 |
[578-1533] x10-6 |
[724-3715] x 10-6 |
Mean |
1004 x 10-6 |
1063 x 10-6 |
1597 x 10-6 |
SD |
356 x 10-6 |
232 x10-6 |
712 x10-6 |
n |
45 |
34 |
81 |
The above mentioned historical control data range of the positive controls were obtained by collecting all data over the period of June 2006 to June 2009.
Table 1: results of range-finding assay
Metabolic activation |
Test substance concentration (μg/mL) |
Mitotic index (%) |
|
|
|
24 h harvesting time |
48 h harvesting time |
- |
Vehicle control (DMSO) |
100 |
100 |
- |
10 |
91 |
98 |
- |
33 |
86 |
98 |
- |
100 |
91 |
105 |
- |
333 |
93 |
105 |
- |
1000 |
100 |
91 |
+ |
Vehicle control (DMSO) |
100 |
|
+ |
10 |
92 |
- |
+ |
33 |
90 |
- |
+ |
100 |
94 |
- |
+ |
333 |
98 |
- |
+ |
1000 |
94 |
- |
Table 1: Chromosome aberrations, summarised data, experiment 1
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
100 |
200 |
7 |
6 |
6 |
5 |
98 |
- |
333 |
200 |
1 |
1 |
1 |
1 |
106 |
- |
1000 |
200 |
3 |
2 |
3 |
2 |
106 |
- |
mitomycin C |
200 |
41 |
37 |
38*** |
34*** |
61 |
48 h treatment, 48 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
1 |
1 |
1 |
1 |
100 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
- |
mitomycin C |
150 |
72 |
65 |
59*** |
55*** |
49 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
4 |
2 |
4 |
2 |
100 |
+ |
100 |
200 |
4 |
4 |
4 |
4 |
68 |
+ |
333 |
200 |
0 |
0 |
0 |
0 |
81 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
73 |
+ |
cyclophosphamide |
200 |
89 |
75 |
71*** |
63*** |
33 |
3 h treatment, 48 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
9 |
9 |
5 |
5 |
100 |
+ |
1000 |
200 |
1 |
1 |
1 |
1 |
81 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 2: Chromosome aberrations, summarised data, experiment 2
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Total number of aberrations1 |
Number of cells with aberrations1 |
Mitotic index (%)2
|
||
|
|
|
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
24 h treatment, 24 h harvesting time |
|||||||
- |
Vehicle control (DMSO 1%) |
200 |
0 |
0 |
0 |
0 |
100 |
- |
100 |
200 |
2 |
2 |
2 |
2 |
99 |
- |
333 |
200 |
4 |
4 |
4 |
4 |
113 |
- |
1000 |
200 |
1 |
1 |
1 |
1 |
97 |
- |
mitomycin C |
150 |
63 |
63 |
53*** |
53*** |
39 |
3 h treatment, 24 h harvesting time |
|||||||
+ |
Vehicle control (DMSO 1%) |
200 |
3 |
3 |
3 |
3 |
100 |
+ |
100 |
200 |
2 |
2 |
2 |
2 |
108 |
+ |
333 |
200 |
2 |
2 |
2 |
2 |
101 |
+ |
1000 |
200 |
2 |
2 |
2 |
2 |
104 |
+ |
Cyclophosphamide |
200 |
111 |
109 |
80*** |
79*** |
32 |
1gaps include chromatid and isochromatid (chromosome) gaps
2percentage of metaphases per 1000 cells, compared to control
* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)
Table 1: Experiment 1 – Direct plate assay
Dose (µg/plate) |
Mean number of revertant colonies / 3 replicate plates (± SD) |
|||||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2 uvrA |
||||||
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
|
Without metabolic activation |
||||||||||
Solvent control |
9 |
4 |
5 |
4 |
20 |
2 |
94 |
20 |
12 |
2 |
1.7 |
8 |
4 |
3 |
2 |
10 |
2 |
94 |
12 |
15 |
4 |
5.4 |
8 |
3 |
3 |
2 |
10 |
2 |
86 |
20 |
17 |
4 |
17 |
7 |
2 |
2 |
1 |
15 |
5 |
90 |
5 |
15 |
3 |
52 |
5 |
1 |
4 |
2 |
19 |
7 |
89 |
2 |
14 |
4 |
164 |
6 |
2 |
3 |
1 |
13 |
2 |
77 |
6 NP |
15 |
1 |
512 |
10 |
2 NP |
2 |
2 NP |
11 |
6 NP |
84 |
14 SP |
18 |
4 NP |
1600 |
6 |
2 SP |
3 |
1 SP |
9 |
3 SP |
93 |
13 SP |
15 |
8 SP |
5000 |
10 |
3 n SP |
4 |
2 n SP |
11 |
3 n SP |
86 |
9 n SP |
22 |
3 n SP |
Positive control |
903 |
83 |
1147 |
129 |
1044 |
242 |
662 |
106 |
984 |
250 |
With metabolic activation |
||||||||||
Solvent control |
9 |
3 |
7 |
4 |
22 |
7 |
59 |
13 |
20 |
2 |
1.7 |
9 |
5 |
2 |
2 |
15 |
7 |
72 |
8 |
17 |
2 |
5.4 |
5 |
3 |
4 |
1 |
17 |
3 |
58 |
7 |
13 |
3 |
17 |
8 |
5 |
7 |
6 |
21 |
6 |
74 |
13 |
21 |
3 |
52 |
5 |
3 |
4 |
1 |
18 |
5 |
77 |
11 |
24 |
6 |
164 |
8 |
3 |
2 |
2 |
19 |
1 NP |
72 |
4 NP |
15 |
1 |
512 |
5 |
3 NP |
2 |
1 NP |
23 |
4 SP |
62 |
6 SP |
17 |
5 NP |
1600 |
9 |
2 SP |
6 |
5 SP |
20 |
3 SP |
78 |
12 SP |
19 |
4 SP |
5000 |
5 |
3 n SP |
4 |
1 n SP |
22 |
11 n SP |
80 |
13 n SP |
20 |
5 n SP |
Positive control |
|
26 |
196 |
24 |
1176 |
196 |
812 |
173 |
339 |
23 |
NP = No precipitate SP = Slight precipitate n = Normal bacterial background lawn
Positive controls (without metabolic activation): TA1535 – sodium azide (SA) TA1537 – ICR-191 TA98 – 2-nitrofluorene (NF) TA100 – methylmethanesulfonate (MMS) WP2 uvrA - 4-nitroquinoline N-oxide (4-NQO)
Positive controls (with metabolic activation) All strains – 2-aminoanthracene (2AA) |
||||||||||
Table 2: Experiment 2 – Pre-incubation assay
Dose (µg/plate) |
Mean number of revertant colonies / 3 replicate plates (± SD) |
|||||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2 uvrA |
||||||
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
Mean |
± SD |
|
Without metabolic activation |
||||||||||
Solvent control |
6 |
5 |
4 |
1 |
13 |
1 |
89 |
10 |
19 |
10 |
17 |
7 |
3 |
4 |
1 |
6 |
3 |
95 |
7 |
19 |
1 |
52 |
9 |
6 |
3 |
2 |
9 |
1 |
95 |
6 |
19 |
3 |
164 |
10 |
1 |
2 |
1 |
12 |
2 |
89 |
10 |
23 |
2 |
512 |
6 |
2 NP |
3 |
2 NP |
8 |
3 NP |
101 |
15 NP |
22 |
11 NP |
1600 |
9 |
6 SP |
6 |
2 SP |
8 |
4 e MC m SP1 |
89 |
8 SP |
15 |
8 SP |
5000 |
10 |
3 n SP |
4 |
1 n SP |
15 |
8 n SP |
116 |
5 n SP |
20 |
9 n SP |
Positive control |
723 |
65 |
1038 |
231 |
1362 |
1552 |
537 |
30 |
187 |
73 |
With metabolic activation |
||||||||||
Solvent control |
9 |
2 |
3 |
2 |
21 |
8 |
94 |
4 |
24 |
6 |
17 |
10 |
3 |
8 |
3 |
16 |
4 |
83 |
13 |
19 |
1 |
52 |
10 |
3 |
4 |
1 |
23 |
3 |
84 |
8 |
27 |
1 |
164 |
8 |
3 |
4 |
4 |
17 |
2 |
72 |
10 |
23 |
4 |
512 |
5 |
2 NP |
5 |
4 NP |
22 |
2 NP |
84 |
10 NP |
26 |
8 NP |
1600 |
11 |
7 SP |
6 |
2 SP |
12 |
3 SP |
91 |
4 SP |
31 |
7 SP |
5000 |
10 |
5 n SP |
4 |
1 n SP |
24 |
10 n SP |
93 |
10 n SP |
27 |
6 n SP |
Positive control |
125 |
50 |
259 |
110 |
614 |
26 |
470 |
400 |
446 |
25 |
1 = Considered a outlier due to effects only seen at one dose in the middle of the tested dose range 2 = Outlier excluded: Mean of two plates MC = Microcolonies NP = No precipitate SP = Slight precipitate e = Bacterial background lawn extremely reduced m = Bacterial background lawn moderately reduced n = Normal bacterial background lawn
Positive controls (without metabolic activation): TA1535 – sodium azide (SA) TA1537 and TA98 – 2-nitrofluorene (NF) TA100 – methylmethanesulfonate (MMS) WP2 uvrA - 4-nitroquinoline N-oxide (4-NQO)
Positive controls (with metabolic activation) All strains – 2-aminoanthracene (2AA) |
||||||||||
Table 3: Historical control data of the solvent control (Jun 2019 – Jun 2022)
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
|||||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Range |
2 – 22 |
3 – 28 |
1 – 24 |
1 – 20 |
3 – 32 |
5 – 62 |
56 – 188 |
34 – 169 |
9 – 54 |
9 – 73 |
Mean |
9 |
10 |
4 |
5 |
12 |
17 |
99 |
86 |
19 |
22 |
SD |
2.6 |
2.8 |
2.1 |
2.3 |
3.6 |
5.1 |
16 |
20 |
5.0 |
6.4 |
Total number of plates |
2242 |
2282 |
2274 |
2323 |
2337 |
2413 |
2347 |
2390 |
2137 |
2158 |
95% upper limit |
14 |
15 |
8.1 |
9.5 |
19 |
27 |
130 |
126 |
29 |
35 |
95% lower limit |
3.9 |
4.5 |
-0.12 |
0.49 |
4.8 |
7.0 |
68 |
46 |
9.2 |
9.5 |
SD = Standard deviation |
||||||||||
Table 4: Historical control data of the positive control (Jun 2019 – Jun 2022)
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2uvrA |
|||||
S9-mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Range |
95 – 1373 |
78 – 1145 |
71 – 2175 |
46 – 1989 |
379 – 2141 |
251 – 3369 |
167 – 1870 |
166 – 2185 |
89 – 2027 |
115 – 1642 |
Mean |
863 |
261 |
863 |
253 |
1393 |
978 |
783 |
1259 |
1281 |
437 |
SD |
176 |
109 |
377 |
164 |
319 |
416 |
189 |
426 |
361 |
209 |
Total number of plates |
2054 |
2107 |
1609 |
2133 |
2180 |
2186 |
2133 |
2212 |
2002 |
1983 |
95% upper limit |
1207 |
474 |
1603 |
574 |
2017 |
1794 |
1152 |
2093 |
1988 |
846 |
95% lower limit |
519 |
48 |
123 |
-68 |
769 |
162 |
414 |
425 |
574 |
28 |
SD = Standard deviation |
||||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
OECD 474 (read-across): negative in mammalian erythrocyte micronucleus assay
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 Feb-28 Mar 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: Swiss, CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 31.2-32.4 (males, range of mean group values), 24.2-25.8 (females, range of mean group values)
- Assigned to test groups randomly: yes, assigned to treatment groups as they came to hand from delivery boxes
- Fasting period before study: no
- Housing: Five animals per sex per group were housed in labelled polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, the Netherlands)
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBV, Oud-Turnhout, Belgium)
- Water: tap water
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
dosing solutions were prepared directly prior to administration by dissolving the test substance in corn oil. The dosing volume was 10 mL/kg bw. - Duration of treatment / exposure:
- Not applicable
- Frequency of treatment:
- Single treatment
- Post exposure period:
- 24 or 48 h
- Dose / conc.:
- 500 mg/kg bw (total dose)
- Remarks:
- nominal
- Dose / conc.:
- 1 000 mg/kg bw (total dose)
- Remarks:
- nominal
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Remarks:
- nominal
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- The positive control animals were dosed with cyclophosphamide.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg bw - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A suitable dose range for the micronucleus test was selected based on the results of a dose range-finding study. Two dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The animals were observed for 3 days, during which mortality and physical condition were recorded daily.
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The animals were dosed once and sacrificed by cervical dislocation at 24 or 48 h (2000 mg/kg bw test substance and vehicle control) or 24 h only (500 and 1000 mg/kg bw test substance, and positive control group)
DETAILS OF SLIDE PREPARATION:
The femur bone was removed, and the bone marrow canal exposed and flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were then air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.The slides were automatically stained using the 'Wright-stain-procedure' in an 'Ames' HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
METHOD OF ANALYSIS:
Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. - Evaluation criteria:
- A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision. - Statistics:
- The Wilcoxon Rank Sum Test; two-sided test (P < 0.05) was for all statistical calculations.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: no effects were observed
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes from the bone marrow of test substance treated animals (see Table 1 and 2). The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes, showing the positive control was valid.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups that were treated with the test substance did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. As the highest dose of the test substance was the recommended limit dose according to the guideline, the study result is acceptable, despite the lack of toxicity at the highest dose level. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. - Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- 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
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- Source: CAS 93803-87-3
- Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
The available data on a suitable source substance did not show any cytogenic effect in mammalian erythrocytes in vivo. Therefore, the target substance Fatty acids, C16-18, isononyl esters is not predicted to be genotoxic in mammalian cells in vivo.
Referenceopen allclose all
Table 1: Results of the in vivo micronucleus assay in male animals
Exposure group |
Sampling time (h) |
Number of animals |
Number of micronucleated PCEs/ 2000 PCEs (mean±SD) |
Ratio PCEs/ NCEs (mean±SD) |
Vehicle (corn oil) |
24 |
5 |
0.4±0.5 |
0.99±0.02 |
Vehicle (corn oil) |
48 |
5 |
0.2±0.4 |
1.01±0.04 |
500 mg/kg bw |
24 |
5 |
0.8±0.8 |
1.00±0.03 |
1000 mg/kg bw |
24 |
5 |
0.2±0.4 |
0.99±0.01 |
2000 mg/kg bw |
24 |
5 |
0.6±0.9 |
1.01±0.04 |
2000 mg/kg bw |
48 |
5 |
0.4±0.5 |
1.00±0.01 |
Cyclophosphamide |
48 |
5 |
25.0±8.0* |
0.51±0.20 |
PCE = polychromatic erythrocyte
NCE = normochromatic erythrocytes
*Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P < 0.05)
Table 2: Results of the in vivo micronucleus assay in female animals
Exposure group |
Sampling time (h) |
Number of animals |
Number of micronucleated PCEs/ 2000 PCEs (mean±SD) |
Ratio PCEs/ NCEs (mean±SD) |
Vehicle (corn oil) |
24 |
5 |
0.6±0.9 |
1.01±0.02 |
Vehicle (corn oil) |
48 |
5 |
0.0±0.0 |
0.99±0.02 |
500 mg/kg bw |
24 |
5 |
1.2±0.8 |
1.02±0.02 |
1000 mg/kg bw |
24 |
5 |
0.6±0.5 |
0.98±0.02 |
2000 mg/kg bw |
24 |
5 |
0.4±0.5 |
0.99±0.07 |
2000 mg/kg bw |
48 |
5 |
0.4±0.5 |
1.00±0.02 |
Cyclophosphamide |
48 |
5 |
13.2±2.8* |
0.56±0.05 |
PCE = polychromatic erythrocyte
NCE = normochromatic erythrocytes
*Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P < 0.05)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Read Across Justification
Limited data on the genotoxic potential of the target substance Fatty acids, C16-18, isononyl esters (CAS 91031-57-1) were available, comprising two bacterial reverse mutation tests.
The assessment was therefore supported using studies conducted with analogue substances as read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. 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. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).
Genetic toxicity (mutagenicity) in bacteria in vitro
CAS 91031-57-1
The ability of the registration substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA, either in the presence or absence of a metabolic activation system (S9-mix) was examined according to OECD 471 (Bacterial Reverse Mutation Assay) and GLP principles (Key, 2022). The test was performed in two independent experiments: a direct plate assay (Experiment 1) and a pre-incubation assay (Experiment 2). The vehicle/solvent in the study was ethanol. In Experiment 1, the test item was tested at concentrations of 1.7 - 5000 µg/plate in all strains. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards. No toxicity was observed at any of the dose levels tested and no biologically relevant decrease in the number of revertants was observed. In Experiment 2, the test item was tested at concentrations of 17 - 5000 µg/plate in all strains. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. No toxicity was observed at any of the dose levels tested and no biologically relevant decrease in the number of revertants was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a biologically relevant, dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (S. typhimurium TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain E. coli WP2uvrA both in the absence and presence of S9-metabolic activation. Based on the results of this study it was concluded that the registration substance was not mutagenic in the bacterial reverse mutation assay.
The in vitro genetic toxicity of Fatty acids, C16-18, isononyl esters (CAS 91031-57-1) was also assessed in a bacterial reverse mutation study (Ames test), performed under GLP conditions according to a protocol similar to OECD guideline 471 (WoE, 1985). S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test substance at concentrations up to 5000 µg/plate. No TA 102 or E. coli strain was included. The vehicle and positive controls were valid in the presence and absence of metabolic activation. The test substance did not induce reversions in the S. typhimurium strains, with or without metabolic activation.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
CAS 26399-02-0
An in vitro mammalian cell gene mutation assay was performed with 2-ethylhexyl oleate (CAS 26399-02-0) according to OECD guideline 476 (WoE, 2010, MLA). Two independent experiments (with 3 or 24 hours of exposure) were performed in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) with test substance concentrations up to 100 μg/mL dissolved in ethanol. Precipitation was seen at concentrations of 100 µg/mL and higher. The positive and vehicle controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
CAS 26399-02-0
The cytogenetic potential of 2-ethylhexyl oleate (CAS 26399-02-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 (WoE, 2010, CA). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation. In the first experiment, cells were incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with a 24 hours fixation time in the absence and presence of a metabolic activation system. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and for 48 hours following 48 hours expression time, all without metabolic activation. In the presence of metabolic activation 2-ethylhexyl oleate was also tested with 3, 10 and 33 µg/mL for 3 hours followed by 48 hours expression time. 33 µg/mL was the maximum concentration due to the limited solubility of the test substance. The highest concentration of the test substance caused modest cytotoxicity. The vehicle (solvent) and positive controls were shown to be valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation.
CAS 93803-87-3
The cytogenetic potential of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD Guideline 473 and under GLP conditions (WoE, 1998, CA). Duplicate cultures of cultured human peripheral lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation. In the first experiment, cells were incubated with test substance concentrations of 100, 333 and 1000 µg/mL for 24 hours with a 24-hour fixation time and at 1000 µg/mL for 48 hours with a 48-hour fixation time, in the absence of a metabolic activation system. The first experiment was also performed with cells exposed to 100, 333 and 1000 µg/mL for 3 hours with a 24-hour fixation time and at 1000 µg/mL for 3 hours with a 48-hour fixation time in the presence of metabolic activation. In the second experiment cells were incubated with 100, 333 and 1000 µg/mL for 24 hours followed by a 24-hour expression time, without metabolic activation. In the presence of metabolic activation cell were exposed to 100, 333 and 1000 µg/mL for 3 hours followed by a 24-hour expression time. No cytotoxicity was observed. At 1000 µg/mL, precipitation was observed in the culture medium. The vehicle and positive controls were valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation.
Genetic toxicity (cytogenicity) in vivo
An in vivo mammalian somatic cell study (erythrocyte micronucleus test) was conducted with 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) according to OECD guideline 474 (MN, 1998, RL1). Male and female CD-1 mice (5 per sex and dose group) were treated with doses of 0, 500, 1000, and 2000 mg/kg by single intraperitoneal injection. Corn oil was used as vehicle. At 24 and 48 hours after injection the number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes per animal. No significant increase (Wilcoxon Rank Sum Test; two-sided test (P < 0.05) in the frequency of micronucleated polychromatic erythrocytes was observed as compared to the vehicle control. The positive control (cyclophosphamide) was valid. No in vivo genotoxicity was detected.
Overall conclusion for genetic toxicity
The analogue read-across from source substances was applied from in vitro studies on cytogenicity, and in vitro studies on gene mutation in mammalian cells, and in vivo studies on mammalian cytogenicity. The results of the available studies were consistently negative. Based on the available data from analogue substances and the bacterial reverse mutation assays performed with the target substance, no mutagenic or clastogenic potential is expected for Fatty acids, C16-18, isononyl esters (CAS 91031-57-1).
Justification for classification or non-classification
According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids, C16-18, isononyl esters (CAS 91031-57-1), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.
Therefore, based on the analogue read-across approach and data on the target substance Fatty acids, C16-18, isononyl esters (CAS 91031-57-1), the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.
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