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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material did not induce mutagenic effects in an Ames test performed according to OECD 471.

The test substance is assumed to be not genotoxic, as deduced from an in vitro mammalian cell gene mutation test in CHO cells performed according to OECD 476 with structural analogue CAS 10042-59-8.

The test substance is assumed not to induce structural chromosomal aberrations, as deduced from an in vitro mammalian chromosome aberration test in human lymphocytes performed according to OECD 473 with structural analogue CAS 2425-77-6.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only four strains tested, 2-aminoanthracene was the sole indicator of the efficacy of the S9-mix)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): C13 - C15 - Alkohol
- Physical state: colorless liquid
- Analytical purity: 99.3% alcohols (0.5% low boiling substances, 0.2% high boiling substances)
- Composition of test material, percentage of components: 29.5% i-C13-alcohol, 33% n-C13-alcohol, 19% i-C15-alcohol, 17.8% n-C15-alcohol
- Lot/batch No.: from continuous production
- Sustance number: 88/577
- Storage: room temperature
Target gene:
his-gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of Aroclor 1254 induced rat livers (Sprague-Dawley)
Test concentrations with justification for top dose:
20 - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see Details on test system and conditions for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

Standard plate test:
The experimental procedure is based on the method of Ames et al ., 1975

Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:

0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test:
The experimental procedure is based on the method described by Yahagi et al. (1977) and Matsushima et al. (1980).

0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.

Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate

After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:

with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535

without S-9 mix:
5 µg N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535,
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98,
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537

NUMBER OF REPLICATIONS: triplicate




Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control);
- dose-response relationship;
- reproducibility of the results;
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Standard plate test (20 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 23 1.0 (20/500/2500/5000) no negative 29 (NPD)
  yes 34 1.1 (20/100) no negative 34.7 (2-AA)
TA 100 no 114 0.9 (100) no negative 14.8 (MNNG)
  yes 103 1.3 (100) no negative 17.2 (2-AA)
TA 1537 no 9 1.3 (20) no negative 47 (AAC)
  yes 12 0.9 (20/100/500) no negative 11.7 (2-AA)
TA 1535 no 15 1.0 (20) no negative 128.4 (MNNG)
  yes 15 1.2 (2500) no negative 10.5 (2-AA)
Preincubation test (20 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 24 1.1 (500) no negative 39.9 (NPD)
  yes 35 1.2 (500) no negative 26.5 (2-AA)
TA 100 no 110 1.1 (500) no negative 10.6 (MNNG)
  yes 125 1.1 (20) no negative 9.5 (2-AA)
TA 1537 no 8 1.2 (100) no negative 43.9 (AAC)
  yes 12 0.8 (20/100/2500) no negative 9.3 (2-AA)
TA 1535 no 19 1.2 (5000) no negative 59 (MNNG)
  yes 18 1.0 (20/5000) no negative 7.1 (2-AA)
2-AA = 2-aminoanthracene
NPD = N-nitro-o-phenylendiamine
MNNG = N-methyl-N-nitro-N-nitrosoguanidine
AAC = 9-aminoacridine chloride monohydrate
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See 'Attached justification'.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: Result from read-across CAS 10042-59-8
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The genetic toxicity for alcohols C13 - 15 branched and linear (CAS 85566-16-1) was determined in an Ames test. Additionally, the genetic toxicity was determined in an HPRT-assay and an in vitro chromosome aberration assay for the structural analogues CAS 10042-59-8 (BASFSE, 2011) and CAS 2425-77-6 (Sasol/Cognis, 2009), respectively. Furthermore, toxicity data on "alcohols C9 -11-iso-, C10-rich" with CAS number 68526-85-2 was obtained from its disseminated dossier.

In an Ames-test performed according to OECD guideline study 471, Salmonella strains S. typhiriumTA 1535, TA 1537, TA 98 and TA 100 were exposed to the test material in doses of 20 - 5000 µg/plate via a standard plate test and a preincubation test in triplicate with and without the presence of S-9 mix. DMSO was used as vehicle. No cytotoxicity was observed and the test material did not induce mutagenic effects.

 

Furthermore, a GLP-compliant in vitro mammalian cell gene mutation test was performed according to OECD guideline 476 with structural analogue CAS 10042-59-8. In this study, chinese hamster ovary (CHO) cells were exposed in medium to concentrations of 3.1, 6.3, 12.5, 25, 50, 100 µg/mL for 4 hours in the presence and absence of a metabolic activation system (S9-mix). In a second experiment, cells were exposed to the previously mentioned concentrations for 24 hours where in addition cells were exposed to concentrations of 10, 20, 40, 60, 80, 100 µg/mL for 4 hours in the presence of S9-mix. In a third experiment in the presence of S9 mix, cells were exposed to concentrations of 2.5, 5, 10, 20, 40, 60, 80 µg/mL for 4 hours. For all experiments duplicate cultures were used. The expression time was 7 - 9 days and the selection time was 6 - 7 days. Cytotoxicity was determined based on the cloning efficiency. No genotoxicity was observed in any experiment performed and all controls were considered valid. Cytotoxic effects were observed in all experiments in the presence and absence of S9-mix at least in the highest applied concentration.

 

Additionally, a GLP- compliant in vitro mammalian chromosome aberration test performed according to OECD 473 was conducted with structural analogue CAS 2425-77-6. In the first test of this study, human lymphocytes were exposed to test substance concentrations of 16, 22 and 24 µg/mL (no metabolic activation system) and 25, 65 and 80 µg/mL (including S9-mix as metabolic activation system) for 3 hours. In the second test, human lymphocytes were exposed to concentrations of 9, 15 and 17 µg/mL (no metabolic activation system; exposure duration 21 hours) and 120, 130 and 150 µg/mL (including S9-mix as metabolic activation system; exposure duration 3 hours). DMSO was used as vehicle. The mitotic index was determined as basis for cytotoxicity in addition to determination of polyploidy. No genotoxic effects were observed at any test concentration in the presence or absence of a metabolic activation system. The test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Supporting information is dissiminated on the ECHA Homepage for a structurally similar substance, CAS 68526 -85 -2. In a GLP-compliant study performed according to OECD guideline 473, chinese hamster ovary cells were exposed to test concentrations of 5, 10, 20, 30, 40, 80, and 160 µg/mL (test 1) and 5, 10, 20, 30, 40, 50, 60, 70, 80 and 100 µg/mL (test 2) in the presence and absence of S9-mix (functioning as metabolic activation system). DMSO was used as vehicle. The mitotic index was determined as the basis for cytotoxicity. At the end of the study period, cytotoxicity was observed and all controls were considered valid. No genotoxicity was observed in any of the previously described test conditions. In addition an in vitro gene mutation was conducted for this substance, performed according to OECD guideline 476 and GLP-compliant. In this test, mouse lymphoma L5178Y cells were exposed to test concentrations of 0, 0.45, 0.9, 1.8, 3.6, 7.2, 14, 21, 29, 42 µg/mL (test 1) and 0, 2.5, 4.9, 9.8, 14, 20, 29, 32, 35, 39, 44 µg/mL (test 2) in the presence and absence of S9 -mix, that functioned as metabolic activation system. Cells were exposed for 4 or 24 hours and the expression time was 2 days, followed by selection for 10-14 days. The relative total growth was determined as the basis for cytotoxictiy. Overall, all controls were considered valid and cytotoxicity was observed in doses above 21 µg/mL. No genotoxicity was observed. It was concluded that the test material is not a mutagenic agent.

Justification for classification or non-classification

Based on the available information the substance does not need to be classified for genetic toxicity, as in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.