Registration Dossier
Registration Dossier
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EC number: 284-864-6 | CAS number: 84988-75-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and TA1538 with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, Mouse lymphoma assay): negative in mouse lymphoma L5178Y cells with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in Chines hamster lung fibroblasts (V79 cells) with and without metabolic activation
The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.
For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.
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:
- 05 Jun - 28 Jun 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- No S. typhimurium TA102 or E. coli strain was included.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May 26, 1983 (2)
- Deviations:
- yes
- Remarks:
- no S. typhimurium TA102 or E. coli strain was included
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted March 2, 1983
- Deviations:
- yes
- Remarks:
- no S. typhimurium TA102 or E. coli strain was included
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- 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:
- First and second experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Suspension medium Tween 80/bidest. water
- Justification for choice of solvent/vehicle: According to the author, the suspension medium was chosen based on the solubility properties preliminary tested before start of the study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Tween 80/bidest. water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (2 µg/plate, ±S9, TA1535 and TA100); 9-Aminoacridine (80 µg/plate,±S9, TA1537); 4-Nitro-o-phenylendiamine (40 µg/plate, ±S9,TA98 and TA1538); 2 Amino-anthracene (2.5 or 5 µg/plate, ±S9, TA1535 and TA1357; TA100, TA1538 and TA98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments - Evaluation criteria:
- The test material may be considered positive in this test system if a combination of the following criteria are met:
- the plate background of non-reverted bacteria does not show any growth reduction versus the respective negative controls.
- the spontaneous mutation rates of each tester strain per plate are within the characteristic spontaneous mutation range.
- the positive controls show mutation rates exceeding the control values of TA100 at least two fold and those of the other strains at least by the factor 3.
- at more than one dose tested, at least a two-fold (or more) increase in comparison with the negative controls in the tester strain TA100. For the other strains, an increase in the mutation rate of more than 3 above the corresponding negative controls. - Statistics:
- Mean values and standard deviation were calculated.
- 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:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Contamination of 1 plate of strain TA98 in the second experiment (1000 µg/plate), therefore only 2 individual values were used for calculation of the mean number of revertant colonies at this concentration.
COMPARISON WITH HISTORICAL CONTROL DATA:
- see Table 1 and Table 2 under "Any other information on results incl. tables". - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- No discussion of results and no historical control data.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- no discussion of results and no historical control data.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- no discussion of results and no historical control data.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
MEM medium supplemented with 2 mM L-Glutamine and
- 4% (v/v) fetal calf serum (MEM4) or
- 0% (v/v) fetal calf serum (MEM0)
- 100 IU/mL penicillin/streptomycin
During exposure to the test substancewith S9 mix, MEM0 medium was used and replaced by MEM4 after 3 h after test substance administration. - Additional strain / cell type characteristics:
- other: modal chromosome number of 22 and a cell cycle length of approx. 16.5 h
- 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:
- 18h treatment: 10, 40 and 80 µg/mL (without metabolic activation)
18h treatment: 10, 60, 80 and 100 µg/mL (with metabolic activation)
28h treatment: 80 µg/mL (without metabolic activation)
28h treatment: 100 µg/mL (with metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- MEM4 medium (resp. MEM0 medium in the test with S9 mix) containing 1% (v/v) ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: cyclophosphamide, 3 and 4 µg/mL in MEM0 medium, +S9; mitomycin C, 0.03 and 0.04 µg/mL in MEM4 medium, -S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 18 and 28 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h treatment: 18 h; 28 h treatment: 28 h
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 replications each in one (28 h treatment) or two (18 h treatment) independent experiment
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells per slide
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplicated cells: yes - Evaluation criteria:
- The test chemical is to be considered clastogenic in this assay if:
- it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
- the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system
- positive results can be verified in an independent experiment. - Statistics:
- The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test ( Richardson, C., Williams, D. A., Allen, J. A., Amphlett, G., Chanter, D. O. and Phillips, B. Analysis of Data from In Vitro Cytogenetic Assays, in: Statistical Evaluation of Mutagenicity Test Data, Kirkland, D. J., (ed) Cambridge University Press, Cambridge, pp. 41-64., 1990 ). The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- systematic influence of the test compound which led to a reduction in the mitotic index from 10 µg/mL.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was soluble in ethanol, in MEM4 medium containing 1% ethanol the solubility limit of the test substance was determined to be 100 µg/mL (homogeneous emulsion).
- Other confounding effects: In the first test without metabolic activation the negative controls exhibited only a mitotic index of 2.0%. Therefore, test 1 without metabolic activation was completely repeated with the same doses and named test #1a. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 17 Jun 2010 - 17 Aug 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP-Guideline study, tested with the source substance Fatty acids, C16-18, esters with ethylene glycol(CAS 91031-31-1). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin amd 10% (v/v) heat-inactivated horse serum (24 h exposure); for 3 h exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium and 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphtoflavone-induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1:
With and without S9-mix: 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL (3 h)
Experiment 2:
In the absence of S9-mix: 3.0, 10.0, 33.0, 100.0, 125.0, 140.0 and 175.0 µg/mL for 24h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL for 3h - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: cyclophosphamide, 7.5 µg/mL, with S9; methylmethanesulfonate, 15 µg/mL and 5 µg/mL without S9, for 3 h and 24 h treatment, respectively
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 48 h
SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
- Selection time (if incubation with a selection agent): 11-12 days
NUMBER OF REPLICATIONS: 2 replications each in 5 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth, relative survival, suspension growth, relative suspension growth, growth rate - Evaluation criteria:
- A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50E-06 and ≤ 170E-06.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500E-06, and fro CP not below 700E-06.
The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: The test substance precipitated in the exposure medium at concentrations of 100 µg/mL and above. The test substance 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 ending test was 333 µg/mL.
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 66% at the test substance concentration of 333 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, no toxicity in the relative 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.
COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
COMPARISON WITH HISTORICAL DATA
Table 1. Characteristic spontaneous mutation range of the test batches without S9-mix.
Tester strains |
TA1535 |
TA100 |
TA1537 |
TA1538 |
TA98 |
Historical laboratory values (Mean values) Extreme values |
6 1-25 |
87 35-201 |
7 1-24 |
15 6-27 |
20 5-39 |
Ames et al.* (Mean values) Extreme values |
20 5-50 |
160 60-220 |
7 3-25 |
25 5-40 |
40 15-75 |
*Ames, BN, McCann, J, Yamasaki, E 1977, ´Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test.`in Kilbey et al. Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 1 -17.
Table 2. Characteristic spontaneous mutation range of the test batches containing S9-mix.
Tester strains |
TA1535 |
TA100 |
TA1537 |
TA1538 |
TA98 |
Historical laboratory values (Mean values) Extreme values |
8 1-25 |
105 54-252 |
6 1-23 |
18 3-48 |
27 7-76 |
STUDY RESULTS
Table 3. Test results of experiment 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA1535 |
TA100 |
TA1537 |
TA1538 |
TA98 |
||
– |
Negative control |
9.7 ± 3.1 |
97.7 ± 2.1 |
6.3 ± 3.2 |
6.7 ± 1.2 |
46.7 ± 3.2 |
– |
Solvent control |
7.7 ± 3.5 |
94.3 ± 17.4 |
8.0 ± 1.7 |
7.3 ± 2.3 |
46.3 ± 6.0 |
– |
8 |
8.3 ± 2.1 |
70.0 ± 7.8 |
9.0 ± 1.0 |
6.7 ± 1.2 |
33.7 ± 0.6 |
– |
40 |
11.0 ± 2.7 |
74.3 ± 3.2 |
7.3 ± 2.1 |
5.3 ± 0.6 |
40.3 ± 4.9 |
– |
200 |
12.0 ± 4.6 |
67.3 ± 11.2 |
6.3 ± 1.2 |
5.7 ± 1.2 |
37.3 ± 3.2 |
– |
1000 |
10.0 ± 4.0 |
62.7 ± 10.8 |
10.3 ± 1.2 |
4.3 ± 1.5 |
42.7 ± 3.1 |
– |
5000 |
9.0 ± 1.7 |
83.0 ± 6.9 |
5.7 ± 1.5 |
4.3 ± 0.6 |
48.3 ± 3.5 |
Positive controls, –S9 |
Name |
SA |
SA |
9AA |
4NPD |
4NPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
744.7 ± 49.3 |
894.0 ± 62.0 |
939.7 ± 181.4 |
1541.0 ± 96.0 |
1410.7 ± 154.4 |
|
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Concentrations (μg/plate) |
2.5 |
5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
9.0 ± 1.7 |
88.7 ± 17.7 |
7.0 ± 2.7 |
10.3 ± 1.5 |
46.0 ± 8.7 |
|
+ |
Negative control |
5.7 ± 0.6 |
80.3 ± 15.3 |
8.7 ± 2.9 |
11.3 ± 2.3 |
53.3 ± 3.2 |
+ |
Solvent control |
9.7 ± 4.6 |
80.3 ± 14.4 |
9.3 ± 1.2 |
13.7 ± 2.3 |
44.3 ± 6.7 |
+ |
8 |
12.3 ± 3.1 |
82.3 ± 12.1 |
7.7 ± 3.5 |
14.7 ± 2.9 |
49.7 ± 4.2 |
+ |
40 |
11.3 ± 4.6 |
92.0 ± 11.8 |
6.3 ± 0.6 |
13.0 ± 4.2 |
47.7 ± 4.0 |
+ |
200 |
8.0 ± 2.0 |
84.3 ± 4.9 |
8.0 ± 1.0 |
16.3 ± 1.5 |
53.3 ± 4.0 |
+ |
1000 |
10.0 ± 2.0 |
70.7 ± 9.7 |
9.3 ± 3.8 |
13.0 ± 1.0 |
50.5 ± 3.5C |
+ |
5000 |
10.0 ± 3.6 |
74.7 ± 11.0 |
7.0 ± 1.7 |
13.0 ± 3.0 |
54.0 ± 7.0 |
Positive controls, +S9 |
Name |
SA |
SA |
9AA |
4NPD |
4NPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
213.3 ± 19.8 |
230.0 ± 4.6 |
283.3 ± 27.8 |
1434.0 ± 68.9 |
1307.6 ± 68.3 |
|
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Concentrations (μg/plate) |
2.5 |
5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
186.3 ± 19.4 |
1328.3 ± 166.5 |
52.3 ± 1.5 |
1711.3 ± 25.0 |
1587.7 ± 237.7 |
|
SA = sodium azide
4NPD = 4-nitro-o-phenylendiamine
9AA = 9-aminoacridine
2AA = 2-Amino-anthracene
C = Contamination, only 2 individual values
Table 4. Test results of experiment 2 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA1535 |
TA100 |
TA1537 |
TA1538 |
TA98 |
||
– |
Negative control |
13.3 ± 1.5 |
117.7 ± 10.0 |
7.7 ± 2.9 |
9.0 ± 4.4 |
33.0 ± 1.0 |
– |
Solvent control |
10.3 ± 5.8 |
111.7 ± 5.0 |
7.3 ± 0.6 |
9.3 ± 1.5 |
30.0 ± 6.3 |
– |
8 |
11.3 ± 2.3 |
118.3 ± 12.5 |
7.3 ± 1.5 |
13.0 ± 2.0 |
32.0 ± 12.5 |
– |
40 |
14.0 ± 2.7 |
105.0 ± 17.4 |
9.3 ± 0.6 |
9.3 ± 0.6 |
25.0 ± 6.2 |
– |
200 |
13.0 ± 1.0 |
97.7 ± 2.9 |
8.0 ± 1.7 |
7.7 ± 2.1 |
32.0 ± 6.6 |
– |
1000 |
13.3 ± 2.1 |
111.7 ± 9.0 |
9.7 ± 4.0M |
8.7 ± 4.0 |
41.3 ± 4.2 |
– |
5000 |
12.0 ± 1.0 |
125.3 ± 4.9 |
7.3 ± 2.1 |
11.0 ± 4.6 |
30.7 ± 2.5 |
Positive controls, –S9 |
Name |
SA |
SA |
9AA |
4NPD |
4NPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
807.3 ±10.6 |
945.3 ± 37.5 |
1156.0 ± 216.5 |
2159.3 ± 106.6 |
226.7 ± 24.7 |
|
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Concentrations (μg/plate) |
2.5 |
5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
12.3 ± 5.5 |
110.3 ± 13.1 |
9.3 ± 3.8 |
22.0 ± 6.1 |
29.3 ± 3.8 |
|
+ |
Negative control |
12.3 ± 5.8 |
121.7 ± 10.3 |
9.3 ± 2.3 |
14.0 ± 7.2 |
39.7 ± 3.1 |
+ |
Solvent control |
12.3 ± 3.1 |
120.3 ± 17.0 |
8.7 ± 1.2 |
11.3 ± 1.5 |
45.0 ± 1.0 |
+ |
8 |
15.0 ± 3.5 |
122.3 ± 12.7 |
6.7 ± 2.5 |
9.7 ± 1.2 |
40.1 ± 2.3 |
+ |
40 |
13.3 ± 2.3 |
123.0 ± 7.6 |
8.7 ± 5.0 |
13.7 ± 6.7 |
41.3 ± 8.3 |
+ |
200 |
16.0 ± 3.0 |
122.3 ± 13.3 |
9.0 ± 3.6 |
9.7 ± 6.4 |
45.0 ± 10.2 |
+ |
1000 |
16.0 ± 4.6 |
104.0 ± 19.3 |
8.3 ± 1.2 |
12.3 ± 3.2 |
41.7 ± 6.4 |
+ |
5000 |
16.0 ± 1.7 |
95.0 ± 8.5 |
11.7 ± 3.5 |
12.0 ± 5.6 |
39.0 ± 13.1 |
Positive controls, +S9 |
Name |
SA |
SA |
9AA |
4NPD |
4NPD |
Concentrations (μg/plate) |
2 |
2 |
80 |
40 |
40 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
232.0 ± 15.1 |
286.0 ± 29.2 |
748.0 ± 139.2 |
455.3 ± 14.2 |
531.3 ± 22.7 |
|
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Concentrations (μg/plate) |
2.5 |
5 |
2.5 |
5 |
5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
131.0 ± 5.2 |
1735.0 ± 84.5 |
81.3 ± 8.4 |
585.3 ± 32.1 |
594.3 ± 56.6 |
|
SA = sodium azide
4NPD = 4-nitro-o-phenylendiamine
9AA = 9-aminoacridine
2AA = 2-Amino-anthracene
M = Manual evaluation
Table 1. Summary of data obtained in experiment #1a.
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
|
in µg/mL |
in % mean |
with gaps |
without gaps |
Exposure period 18h, without S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
10.7 |
2.0 |
0.0 |
MMC |
0.03 |
2.1 |
24.6 |
18.5* |
MMC |
0.04 |
2.9 |
40.0 |
31.9* |
Test substance |
10 |
8.0 |
3.5 |
1.5 |
40 |
6.7 |
3.0 |
0.5 |
|
80 |
5.3 |
5.0 |
0.0 |
|
Exposure period 18h, with S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
7.0 |
6.5 |
4.0 |
CP |
3.0 |
2.7 |
45.0 |
34.0* |
Test substance |
10 |
6.7 |
3.5 |
1.5 |
60 |
6.1 |
4.5 |
1.0 |
|
100 |
5.7 |
4.0 |
2.0 |
|
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
*: significant, no statistical evaluation
Table 2. Summary of data obtained in experiment #2.
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
|
in µg/mL |
in % mean |
with gaps |
without gaps |
Exposure period 18h, without S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
7.9 |
6.0 |
3.5 |
MMC |
0.03 |
3.4 |
23.5 |
14.0* |
Test substance |
10 |
7.5 |
4.0 |
0.5 |
40 |
8.0 |
9.5 |
3.0 |
|
80 |
5.8 |
5.5 |
0.5 |
|
Exposure period 18h, with S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
6.3 |
8.0 |
2.0 |
CP |
3.0 |
1.6 |
42.0 |
33.3* |
Test substance |
10 |
6.5 |
5.5 |
0.0 |
60 |
5.7 |
4.5 |
1.5 |
|
100 |
6.7 |
6.5 |
1.5 |
|
Exposure period 28h, without S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
7.9 |
3.5 |
0.0 |
MMC |
0.03 |
4.4 |
43.5 |
32.5* |
Test substance |
80 |
5.8 |
4.5 |
0.5 |
Exposure period 28h, with S9 mix |
||||
Control, Ethanol |
1.0% (v/v) |
9.2 |
6.5 |
1.5 |
CP |
3.0 |
6.0 |
36.5 |
27.5* |
Test substance |
100 |
8.4 |
3.5 |
1.0 |
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
*: significant, no statistical evaluation
Table 1: Cytotoxic and mutagenic responses
Treatment |
Concentration [µg/mL] |
Cloning efficiency [%] |
Relative total growth [%] |
Mutation frequency x 10-6
|
||
total |
small colonies |
large colonies |
||||
3 h treatment without S9-mix |
||||||
Solvent control (mean) |
-- |
86 |
100 |
61 |
42 |
19 |
Test substance |
0.1 |
101 |
114 |
56 |
29 |
25 |
0.3 |
101 |
108 |
52 |
28 |
23 |
|
1.0 |
118 |
140 |
66 |
48 |
16 |
|
3.0 |
91 |
109 |
63 |
46 |
15 |
|
10.0 |
95 |
108 |
66 |
32 |
33 |
|
33.0 |
97 |
104 |
76 |
46 |
28 |
|
100.0* |
91 |
97 |
81 |
32 |
46 |
|
333.0* |
102 |
77 |
64 |
48 |
14 |
|
MMS |
15 |
60 |
45 |
685 |
521 |
118 |
3 h treatment with 8% (v/v) S9-mix |
||||||
Solvent control (mean) |
-- |
91 |
100 |
67 |
36 |
29 |
Test substance |
0.1 |
108 |
111 |
74 |
47 |
25 |
0.3 |
89 |
94 |
71 |
45 |
24 |
|
1.0 |
108 |
113 |
64 |
42 |
20 |
|
3.0 |
98 |
107 |
78 |
53 |
23 |
|
10.0 |
95 |
100 |
87 |
54 |
29 |
|
33.0 |
108 |
96 |
55 |
32 |
22 |
|
100.0* |
98 |
89 |
83 |
60 |
21 |
|
333.0* |
94 |
92 |
63 |
23 |
39 |
|
CP |
7.5 |
60 |
32 |
1074 |
829 |
144 |
24 h treatment without S9-mix |
||||||
Solvent control (mean) |
-- |
115 |
100 |
51 |
26 |
23 |
Test substance |
3 |
135 |
92 |
58 |
42 |
14 |
10 |
137 |
109 |
51 |
39 |
11 |
|
33 |
133 |
85 |
71 |
32 |
35 |
|
100* |
110 |
30 |
100 |
35 |
60 |
|
125* |
118 |
31 |
70 |
31 |
36 |
|
140* |
118 |
20 |
103 |
51 |
47 |
|
175* |
102 |
9 |
134 |
53 |
72 |
|
MMS |
5 |
101 |
73 |
865 |
463 |
233 |
3 h treatment with 12% (v/v) S9-mix |
||||||
Solvent control (mean) |
-- |
109 |
100 |
72 |
47 |
23 |
Test substance |
0.1 |
110 |
100 |
73 |
49 |
21 |
0.3 |
123 |
117 |
72 |
47 |
23 |
|
1.0 |
104 |
105 |
82 |
52 |
27 |
|
3.0 |
97 |
104 |
94 |
62 |
29 |
|
10.0 |
115 |
126 |
87 |
57 |
26 |
|
33.0 |
107 |
108 |
85 |
59 |
23 |
|
100.0* |
131 |
123 |
80 |
51 |
26 |
|
333.0* |
97 |
83 |
92 |
64 |
24 |
|
CP |
7.5 |
70 |
59 |
979 |
621 |
221 |
*precipitation of test substance in the exposure medium
MMS = methylmethanesulfonate
CP = cyclophosphamide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Cytogenicity in vivo (OECD 474, Micronucleus test): negative in the bone marrow of male and female mice
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 17 Oct 1989 - 30 Jan 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Analytical purity of the test substance not specified.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- analytical purity of test substance not specified
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- analytical purity of test substance not specified
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: outbred albino mice, strain CFW 1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 20.7 - 32.8 g (males) and 19.5-24.8 g (females)
- Fasting period before study: animals were fasted overnight prior to dosing and until approximately 3-4 h after administration of test substance and control material.
- Housing: male mice were housed individually in Macrolon cages type I, female mice were housed in groups up to three in Macrolon cages type II.
- Diet: standard animal diet Altromin No. 1314 with 10 mm pellet diameter (Altrogge Spezialfutter), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days (dose range finding study) and at least 4 - 6 days (main study)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: arachis oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Concentration of test material in vehicle: 300, 400 and 500 mg/mL (dose range finding study) and 500 mg/mL (main study) - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in arachis oil (80°C and applied after cooling at a temperature of approx. 35°C) at an application volume of 10 mL/kg. The test substance concentrations were prepared immediately before use. Homogeneity was maintained during application using a magnetic stirrer. - Duration of treatment / exposure:
- 24, 48 and 72 h
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24, 48 and 72 h
- Dose / conc.:
- 5 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 2 (dose range finding study)
6 (main study) - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg bw in water, application volume 10 mL/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:
As the acute oral toxicity (LD50 mouse) was determined to be higher than 5000 mg/kg bw according to acute toxicity studies, the following doses were chosen initially to determine the maximum tolerated dose: 3000, 4000 and 5000 mg/kg bw. According to the results of the dose range finding study, a dose level of 5000 mg/kg was chosen for the main study, because it is generally recommended to use the maximum tolerated dose for the micronucleus test.
TREATMENT AND SAMPLING TIMES:
The animals were sacrificed 24, 48 or 72 h after treatment.
DETAILS OF SLIDE PREPARATION:
The slides were fixed with methanol, air dried at least overnight and stained with Giemsa according to modification of Gollapudi and Kamra.
METHOD OF ANALYSIS:
Three slides per animal were prepared, one slide was randomly chosen and analysed. The slides of 5 males and 5 females per treatment group were analysed and the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time.
- Evaluation criteria:
- The test is considered acceptable if the positive control substance induced statistically significant increase in polychromatic erythrocytes and the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory. The test is considered positive if the test substance induced biologically as well as statistically significant (p < 0.05) increase in the frequency of micronuclei at any dose either in the male or in the female groups. The test is considered negative if the test substance did not induce any biologically as well as statistically significant (p < 0.05) increase in micronuclei at any dose either in the male or in the female groups.
- Statistics:
- Statistical significance of test substance values versus negative controls were calculated with the aid of tables of Kastenbaum and Bowman.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000 and 5000 mg/kg bw
- Solubility: soluble
- Harvest times: 24 h
- Clinical signs of toxicity in test animals: no findings were observed in any animals up to 72 h after administration.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant induction
- Clinical signs of toxicity in test animals: signs at clinical examination were not noticed
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant changes were observed
- Statistical evaluation: the investigated sample did not induce a statistically significant (time dependent) increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of male or female mice. - Conclusions:
- Interpretation of results: negative
Reference
Table 1: Mean values per group of PCE and PCE/NCE
Treatment group (sampling time |
Species and sex |
Dose |
Mean of |
|
Micronucleated cells/ 1000 PCE |
Ratio of PCE/NCE |
|||
Negative control (vehicle) (24 h) |
male mice |
10 mL/kg |
1.6 |
1.0 |
female mice |
1.8 |
0.9 |
||
Positive control (24 h) |
male mice |
10 mg/kg |
9.8 |
1.1 |
female mice |
7.4 |
1.0 |
||
Test substance |
|
|||
24 h |
male mice |
5000 mg/kg |
2.4 |
1.1 |
female mice |
1.6 |
1.0 |
||
48 h |
male mice |
5000 mg/kg |
1.2 |
1.0 |
female mice |
1.4 |
1.0 |
||
72 h |
male mice |
5000 mg/kg |
1.2 |
1.3 |
female mice |
1.4 |
1.2 |
||
PCE: Polychromatic erythrocytes
NCE: Nonchromatic erythrocytes
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.
For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.
Genetic toxicity (mutagenicity) in bacteria in-vitro
CAS 627-83-8
A bacterial gene mutation assay (Ames test) was performed with ethylene distearate following OECD guideline 471 and in compliance with GLP ( Labor L+S AG, 1994). The plate incorporation procedure was performed with Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 in the absence and presence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). Two independent experiments were conducted each in triplicates at concentrations from 8 to 5000 µg/plate (vehicle: DMSO). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results, however instead of the solvent control DMSO, water was included as negative control substance. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
CAS 68583-51-7
Two bacterial gene mutation assays were performed with Decanoic acid, mixed diesters with octanoic acid and propylene glycol following OECD guideline 471 and in compliance with GLP (Henkel, 1991; Hüls AG, 1995). The strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested in two independent experiments according to the plate incorporation procedure at concentrations from 8 to 5000 µg/plate (vehicle: suspension medium Tween 80/bidest. water) with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any strain of bacteria tested, with and without a metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results and were therefore considered as valid. Under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested (Banduhn, 1991). A further experiment in the tester strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 confirmed the above described results (Ebert, 1995). In concentrations from 50 to 5000 µg/plate with and without a metabolic activation system, no cytotoxicity was apparent and no increase in the number of revertant colonies was observed in any of the strains tested.
Thus, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
CAS 84988-75-0
A study investigating the genetic toxicity in vitro of Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol is available. The study was conducted according to OECD guideline 471 under GLP conditions (Henkel, 1991). In two independent experiments, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested according to the plate incorporation procedure. Concentrations from 8 to 5000 µg/plate were investigated with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results and were therefore considered as valid. Thus, under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.
CAS 624-03-3
No studies are available investigating the gene mutation properties of ethane-1,2-diyl palmitate in bacteria. However, there are available data on the category members ethylene distearate (CAS 627-83-8) and Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1). The studies of the category members were considered for assessment and read-across was conducted based on a category and weight of evidence approach.
The gene mutation properties of the category member Fatty acids, C16-18, esters with ethylene glycol were determined according to OECD guideline 471 under GLP conditions (Henkel, 1991). The tester strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. The main study was performed in triplicates each in two independent experiments according to the plate incorporation procedure at concentrations from 8 to 5000 µg/plate (vehicle: Tween 80; 1:1 (w/w) dilution with water) with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. Precipitation was noted at the highest test concentration of 5000 µg/plate. The positive and negative controls showed the expected results and were therefore considered as valid. Under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.
One bacterial gene mutation assay was performed with ethylene distearate following OECD guideline 471 and in compliance with GLP. The study of the category member showed no induction of gene mutations in the bacterial mutation test and is discussed under the respective CAS number.
All available data on the category members were consistently negative. Thus, the available data on the category members, do not provide evidence for gene mutation properties of ethane-1,2-diyl palmitate in bacteria.
Genetic toxicity (cytogenicity) in mammalian cells in-vitro
CAS 627-83-8, CAS 68583-51-7, CAS 84988-75-0, CAS 624-03-3
Within the Glycol Ester category, one study investigating the cytogenicity in mammalian cells is available. Therefore, the study of the category substance Butylene glycol dicaprylate / dicaprate (CAS 853947-59-8) was considered for assessment and read-across was conducted based on a category approach.
An in vitro mammalian chromosome aberration test was conducted with Butylene glycol dicaprylate / dicaprate in accordance with OECD guideline 473 under GLP conditions (Hüls AG, 1997). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung fibroblasts (V79) cells, incubated for 18 and 28 h with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-100 µg/mL (18 h incubation) and 80 and 100 µg/mL (28 h incubation) of the test substance in the vehicle ethanol were applied. The solubility limit of the test substance in the vehicle ethanol in the culture medium was determined to be 100 µg/mL. In the first experiment without metabolic activation, the negative controls exhibited a mitotic index of 2.0% only and the experiment was therefore repeated. Thereafter, the negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploidy cells with and without metabolic activation was within the expected range (< 10%). In the experiments both with and without metabolic activation, a systematic influence of the test substance was observed, which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.
Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in Chinese hamster lung fibroblasts in vitro.
Genetic toxicity (mutagenicity) in mammalian cells in-vitro
CAS 627-83-8, CAS 68583-51-7, CAS 84988-75-0, CAS 624-03-3
Within the Glycol Ester category, one study investigating the gene mutation properties in mammalian cells is available. Therefore, the study of the category member Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) was considered for assessment and read-across was conducted based on a category approach.
The in vitro mammalian cell gene mutation study of Fatty acids, C16-18, esters with ethylene glycol was carried out according to OECD guideline 476 under GLP conditions (NOTOX B.V., 2010). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/β-naphtoflavone-induced rat liver S9). In the first experiment, cells were exposed for 3 h to test substance at concentrations of 0.1-333 µg/mL (in DMSO) with and without metabolic activation. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3-175 µg/mL and with metabolic activation (3 h; 12% S9-mix) from 0.1-333 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. No cytotoxicity was observed up to the precipitating concentration of 100 µg/mL and up to 333 µg/mL, respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Fatty acids, C16-18, esters with ethylene glycol did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.
Genetic toxicity in vivo
CAS 627-83-8, CAS 68583-51-7, CAS 84988-75-0, CAS 624-03-3
Within the Glycol Ester category, one study investigating the cytogenetic properties of Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol (CAS 151661-88-0) in vivo is available. Therefore, the study was considered for assessment and read-across was conducted based on a category approach.
The in vivo micronucleus assay of Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol was carried out according to OECD guideline 474 under GLP conditions (Henkel, 1990). Based on the results of a preliminary dose range finding study, the test substance diluted in arachis oil was administered at 3000, 4000 and 5000 mg/kg bw as single oral doses to groups of 6 male and female CFW1 mice, observed for 24, 48 and 72 h post-dose. A concurrent negative control with the vehicle alone and a positive control group given cyclophosphamide was included in the study. No mortality and no signs at clinical examinations were reported. The test substance did not induce a statistically significant increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of the animals. The negative and positive controls showed the expected results. Therefore, under the conditions of the study, Fatty acids, C18 and C18 unsatd., epoxidized, ester with ethylene glycol did not induce chromosomal mutations in the bone marrow of mice.
Additional data
Three bacterial gene mutation studies are available for the category members C8-C10-1,3-Butandiolester (CAS 853947-59-8), Stearic acid, monoester with propane-1,2-diol (CAS 1323-39-3) and Fatty acids, C18 and C18 unsatd. epoxidized, ester with ethylene glycol (CAS 151661-88-0) all showing negative results.
Conclusion for genetic toxicity
In summary, five studies investigating the genetic mutation in bacteria in-vitro are available within the Glycol Ester category for Ethylene distearate (CAS 627-83-8), Decanoic acid, mixed diesters with octanoic acid and propylene glycol (CAS 68583-51-7), Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol Fatty acids (CAS 84988-75-0) and C16-18, esters with ethylene glycol (CAS 91031-31-1), all providing negative results. Furthermore, no cytogenicity in mammalian cells in-vitro (CAS 853947-59-8), no mutagenicity in mammalian cells in-vitro (CAS 151661-88-0) and no Genetic toxicity in-vivo (CAS 91031-31-1) was observed with members of the Glycol Ester category.
Therefore, no properties for genetic toxicity were observed within the Glycol Ester group for any member.
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 group concept is applied to the members of the Glycol Ester Category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labeled on this basis.
Therefore, based on the group concept, all available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.
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