Registration Dossier
Registration Dossier
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EC number: 241-644-4 | CAS number: 17671-26-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation
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:
- key study
- Study period:
- 15 Mar - 24 May 2017
- 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:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Departement of Health of the Government of the United Kingdom
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon, trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats
- Test concentrations with justification for top dose:
- Following concentrations were used in the main experiments:
First experiment (plate ioncorporation, all strains): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (pre-incubation, all strains): 15, 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation (based on results of first experiment, tested up to the limit concentration) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ditilled water and DMSO, acetone was selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation) (first experiment); preincubation (second experiment)
DURATION
- Preincubation period: 20 min
- Exposure duration: approx. 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn - Evaluation criteria:
- Acceptance criteria
The study was considered valid if:
- the bacterial strains demonstrate the required strain characteristics
- the number of revertant colonies of the negative (solvent) and positive controls are in the historical control range in all strains of the main tests
- all tester strain cultures should be in the range of 0.9 - 9 x 10^9 bacteria/mL.
- diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix
- a minimum of four non-toxic test item dose levels
- no evidence of excessive contamination
Evaluation criteria
A positive result is determined by any, one or all of the following:
- dose-related increase in mutant frequency over the dose range tested
- a reproducible increase at one or more concentrations
- biological relevance against in-house historical control ranges
- statistical analysis of data as determined by UKEMS
- at least 2-fold increase compared to the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
In some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: started at a concentration of 1500 μg/plate in all strains in both experiments
RANGE-FINDING/SCREENING STUDIES: first main test was used to detremine concentrations of secon main test - Conclusions:
- Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.
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:
- key study
- Study period:
- 09 Apr - 09 Jul 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- 200 instead of 300 metaphases counted
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- yes
- Remarks:
- 200 instead of 300 metaphases counted
- 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:
- key study
- 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)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted in 2016
- 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 seen 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. - Remarks on result:
- other: strain/cell type: mouse lymphoma L5178Y cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Referenceopen allclose all
Table 1: Summary of test results (main experiment 1 (Plate Incorporation Method)
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA 98 |
TA 1537 |
TA 100 |
TA 1535 |
WP2 uvrA |
||
– |
Solvent control (Acetone) |
22 ± 7.1 |
10 ± 2.3 |
101 ± 11.0 |
16 ± 5.0 |
39 ± 12.0 |
1.5 |
19 ± 2.5 |
12 ± 3.0 |
103 ± 10.6 |
20 ± 5.0 |
44 ± 3.5 |
|
5 |
22 ± 1.2 |
11 ± 0.6 |
*122 ± 12.3 |
18 ± 1.5 |
38 ± 3.8 |
|
15 |
19 ± 7.0 |
11 ± 4.7 |
114 ± 13.9 |
18 ± 2.5 |
36 ± 4.2 |
|
50 |
18 ± 2.6 |
10 ± 0.0 |
108 ± 5.2 |
19 ± 2.1 |
41 ± 4.0 |
|
150 |
18 ± 4.7 |
12 ± 3.2 |
116 ± 14.2 |
19 ± 2.9 |
37 ± 4.2 |
|
500 |
15 ± 4.2 |
13 ± 1.7 |
*125 ± 3.8 |
18 ± 0.6 |
38 ± 8.4 |
|
1500 |
20 ± 3.2 P |
15 ± 4.0 P |
*127 ± 6.8 P |
17 ± 1.0 P |
43 ± 10.5 P |
|
500 |
19 ± 2.1 P |
12 ± 4.6 P |
**129 ± 6.1 P |
21 ± 3.8 P |
38 ± 2.5 P |
|
Positive controls (µg/plate) |
4NQO (0.2) |
9AA (80) |
ENNG (3) |
ENNG (5) |
ENNG (2) |
|
Mean (No. of colonies/plate) |
202 ± 3.8 |
232 ± 30.5 |
496 ± 25.1 |
437 ± 56.9 |
724 ± 33.0 |
|
+ |
Solvent control (Acetone) |
35 ± 3.2 |
10 ± 1.2 |
121 ± 5.6 |
11 ± 2.0 |
47 ± 6.0 |
1.5 |
30 ± 7.0 |
11 ± 4.5 |
107 ± 8.7 |
10 ± 3.2 |
43 ± 6.5 |
|
5 |
30 ± 6.8 |
11 ± 2.3 |
122 ± 6.8 |
11 ± 0.6 |
50 ± 4.5 |
|
15 |
23 ± 3.5 |
15 ± 1.5 |
121 ± 8.5 |
13 ± 0.0 |
42 ± 6.0 |
|
50 |
28 ± 6.7 |
13 ± 1.2 |
107 ± 13.3 |
10 ± 1.2 |
49 ± 1.2 |
|
150 |
26 ± 3.6 |
14 ± 2.5 |
122 ± 16.5 |
11 ± 1.2 |
46 ± 4.0 |
|
500 |
24 ± 8.2 |
11 ± 0.6 |
123 ± 8.5 |
14 ± 4.6 |
45 ± 8.2 |
|
1500 |
25 ± 5.5 P |
15 ± 4.2 P |
97 ± 15.4 P |
11 ± 2.0 P |
48 ± 7.0 P |
|
500 |
19 ± 3.1 P |
12 ± 3.5 P |
100 ± 4.9 P |
9 ± 2.1 P |
41 ± 1.5 P |
|
Positive controls (µg/plate) |
B(a)P (5) |
2AA (2) |
2AA (1) |
2AA (2) |
2AA |
|
Mean (No. of colonies/plate) |
316 ± 22.5 |
394 ± 64.0 |
1215 ± 120.6 |
261 ± 3.1 |
374 ± 23.1 |
|
2AA = 2-aminoanthracene
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
ENNG = N-ethyl-N-nitro-N-nitrosoguanidine
P = precipitate
* = p ≤ 0.05
** = p ≤ 0.01
Table 2: Summary of test results (main experiment 2 (Pre-Incubation Method))
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA 98 |
TA 1537 |
TA 100 |
TA 1535 |
WP2 uvrA |
||
– |
Solvent control (Acetone) |
19 ± 2.3 |
9 ± 1.2 |
107 ± 21.9 |
13 ± 3.0 |
31 ± 3.0 |
15 |
21 ± 0.6 |
8 ± 2.3 |
115 ± 9.7 |
8 ± 0.0 |
34 ± 3.2 |
|
50 |
20 ± 6.8 |
10 ± 3.5 |
110 ± 5.5 |
9 ± 1.7 |
36 ± 1.2 |
|
150 |
25 ± 4.7 |
8 ± 2.1 |
111 ± 16.0 |
13 ± 5.0 |
30 ± 7.6 |
|
500 |
22 ± 1.2 |
10 ± 2.5 |
120 ± 8.2 |
12 ± 1.0 |
30 ± 3.8 |
|
1500 |
24 ± 5.8 P |
9 ± 2.1 P |
102 ± 5.0 P |
13 ± 1.7 P |
32 ± 2.1 P |
|
500 |
18 ± 2.6 P |
10 ± 4.4 P |
104 ± 3.1 P |
10 ± 0.0 P |
35 ± 4.0 P |
|
Positive controls (µg/plate) |
4NQO (0.2) |
9AA (80) |
ENNG (3) |
ENNG (5) |
ENNG (2) |
|
Mean (No. of colonies/plate) |
235 ± 33.6 |
261 ± 57.0 |
840 ± 44.8 |
557 ± 25.6 |
811 ± 64.8 |
|
+ |
Solvent control (Acetone) |
28 ± 1.7 |
10 ± 2.5 |
109 ± 17.5 |
12 ± 3.2 |
42 ± 5.5 |
15 |
29 ± 8.4 |
11 ± 1.2 |
102 ± 5.2 |
11 ± 3.1 |
43 ± 5.9 |
|
50 |
24 ± 4.9 |
13 ± 1.2 |
121 ± 5.8 |
12 ± 3.8 |
35 ± 11.7 |
|
150 |
30 ± 1.5 |
13 ± 1.5 |
116 ± 13.1 |
10 ± 2.0 |
41 ± 0.6 |
|
500 |
22 ± 4.4 |
12 ± 1.5 |
127 ± 2.6 |
13 ± 4.9 |
44 ± 6.6 |
|
1500 |
27 ± 7.8 P |
12 ± 1.5 P |
119 ± 13.2 P |
10 ± 2.6 P |
40 ± 4.7 P |
|
500 |
25 ± 0.6 P |
9 ± 0.6 P |
125 ± 13.6 P |
14 ± 0.6 P |
42 ± 2.0 P |
|
Positive controls (µg/plate) |
B(a)P (5) |
2AA (2) |
2AA (1) |
2AA (2) |
2AA |
|
Mean (No. of colonies/plate) |
135 ± 14.4 |
301 ± 26.2 |
1296 ± 155.1 |
206 ± 15.8 |
200 ± 24.3 |
|
2AA = 2-aminoanthracene
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
ENNG = N-ethyl-N-nitro-N-nitrosoguanidine
P = precipitate
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.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test (similar to OECD 474): negative in male and female mice after single intraperitoneal dosing at up to 4300 mg/ kg bw
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 17 Jul - 28 Aug 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Lack of details on test substance; no analytical purity given, 1000 erythrocytes counted for incidence of micronuclei
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1983
- Deviations:
- yes
- Remarks:
- analytical purity of test substance not specified
- Principles of method if other than guideline:
- The incidence of micronuclei was counted in 1000 erythrocytes.
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 7 - 12 weeks
- Assigned to test groups randomly: yes
- Housing: 5 animals of each sex in polycarbonate cages
- Acclimation period: 7 - 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Details on exposure:
- Dosing volume: 10 mL/kg
- Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- Up to 72 h
- Dose / conc.:
- 1 075 mg/kg bw/day
- Remarks:
- 1250 µL/kg bw given, calculated with density 860 mg/mL
- Dose / conc.:
- 2 150 mg/kg bw/day
- Remarks:
- 2500 µL/kg bw given, calculated with density 860 mg/mL
- Dose / conc.:
- 4 300 mg/kg bw/day
- Remarks:
- 5000 µL/kg bw given, calculated with density 860 mg/mL
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 30 mg/kg bw - Tissues and cell types examined:
- Erythrocytes of the bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Results of a pretest with 50, 160, 500, 1600 and 5000 µL/kg and the sponsor´s specification
DETAILS OF SLIDE PREPARATION: Immediately after sacrife, both femurs of the animal were removed and flushed with a few drops of fetal calf serum onto a clean microscope slide. A second slide was inverted and placed to the first slide. Using a circular motion, the two slides were rubbed together until the bone marrow was evenly dispersed. The two slides were gently pulled appart, air dried, fixed in methanol for approx. 10 min and again air dried.The slides were stained with Giemsa for 5 min and differentiated in distilled water afterwards.
METHOD OF ANALYSIS: at least 1000 polychromatic erythrocytes were counted for each category. - Evaluation criteria:
- There should be a statistically significant increase in the number of micronucleated polychromatic erythrocytes in the treatment group over the negative control group.
- Statistics:
- The two-tailed Student´s t-test was applied to compare the frequency of micronucleated cells to the respective vehicle control.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- 1000 erythrocytes counted
- Conclusions:
- CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Reference
Table 1: Mean numbers of micronucleated cells
Treatment |
Micronucleated cells |
|
mean |
SD |
|
Vehicle control |
2.26 |
1.41 |
5000 µL/kg |
1.66 |
1.15 |
2500 µL/kg |
1.93 |
1.17 |
1250 µL/kg |
1.80 |
0.81 |
Positive control |
17.20 |
2.62 |
There was no significant increase of micronucleated cells in the treatment groups compared to the negative control. The positive control was valid. No indications for mutagenicity in vivo were given.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity (mutagenicity) in bacteria in vitro
Reliable data on bacterial mutagenicity is available for Lauryl nonanoate (CAS 17671-26-0). The in vitro genetic toxicity of Lauryl nonanoate (CAS 17671-26-0) was assessed in a bacterial reverse mutation assay (Ames test) according to OECD 471 under GLP conditions (key study, 2017). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains including TA 98, 100, 1535 and 1537 and in the E. coli strain WP2 uvr A at concentrations up to 5000 µg/plate in 2 independent experiments, including the plate incorporation and pre-incubation method. Precipitates were visible in all tested strains and tests starting at a concentration of 1500 µg/plate. The test substance did not exhibit mutagenic properties, i.e. a reproducible at least 2-fold increase in revertant colonies, in the absence or presence of metabolic activation. Cytotoxicity was not observed up to precipitation and limit concentration. The vehicle and positive controls were valid and lay within the range of the historical control data, proving validity of the assay. Based on the results of the conducted study, Lauryl nonanoate is not considered to exhibit mutagenic properties in bacterial cells.
Read across justification
With exception for an in vitro gene mutation study in bacteria (Ames test) no data on the potential for genetic toxicity of Lauryl nonanoate (CAS 17671-26-0) are available.
The assessment was therefore based on studies conducted with analogue substances as part of a 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 (cytogenicity) in mammalian cells in vitro
CAS 26399-02-0
The cytogenicity 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 (key study, 2010). 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 substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. Based on the results of the conducted study, the test substance is not considered to exhibit clastogenic properties in mammalian cells.
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 (key study, 2010). 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 negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed. Based on the results of the conducted study, the test substance is not considered to exhibit mutagenic properties in mammalian cells.
Genetic toxicity (cytogenicity) in vivo
CAS 135800-37-2
An in vivo mammalian somatic cell study (erythrocyte micronucleus test) was conducted with Fatty acids, C8-16(even numbered), 2-ethylhexyl esters (CAS 135800-37-2) equivalent to OECD guideline 474 (supporting study, 1991). Male and female CD-1 mice (5 per sex, dose group and necropsy time) were treated with doses of 0, 1075, 2150, and 4300 mg/kg bw by single intraperitoneal injection. Corn oil was used as vehicle. At 24, 48 and 72 hours after injection 1000 polychromatic erythrocytes were counted per animal. No significant increase (2-tailed Student’s t-test) in the frequency of micro-nucleated polychromatic erythrocytes was detected as compared to the vehicle control. The positive control (cyclophosphamide) was valid. No in vivo genotoxicity was detected. Based on the results of the conducted study, the test substance is not considered to exhibit genotoxic properties in vivo.
Overall conclusion for genetic toxicity
Based on the negative outcome of the conducted Ames test with the target substance Lauryl nonanoate (CAS 17671-26-0), it is not considered to exhibit mutagenic properties in bacterial cells.
Analogue read-across from source substances was applied for in vitro cytogenicity, in vitro mutagenicity in mammalian cells and for in vivo cytogenicity. The results of the available in vitro and in vivo studies with the source substances were consistently negative. Based on the available data and following the analogue approach, absence of mutagenic or clastogenic potential can be anticipated for Lauryl nonanoate (CAS 17671-26-0).
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 Lauryl nonanoate (CAS 17671-26-0), 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, 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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