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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxicity:

Bacterial reverse mutation assay (Ames test / OECD 471): negative

In vitro mammalian cell gene mutation assay (MLA / OECD 476): negative

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No E.coli strains tested.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 11, 56, 280, 1400, 7000 µg/plate
Vehicle / solvent:
water
Negative solvent / vehicle controls:
yes
Remarks:
destilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine, sodium azide, 2-nitrofluorene, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Evaluation criteria:
According to Guideline.
Statistics:
yes
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct. 1994 - 25 Nov. 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of five young male Sprague-Dawley rats treated with phenobarbitone and betanaphthoflavone.
- quality controls of S9: The efficacy was checked in an Ames test and produced acceptable responses with 2-aminoanthracene and benzo(a)pyrene in S. typhimurium TA100; protein content (28.4 ± 0.37 and 24.2 ± 4.19 mg/mL) and aminopyrine demethylase activity (2.59 ± 0.08 and 3.48 ± 0.13 µM/g liver/5 min formaldehyde production) checked.




Test concentrations with justification for top dose:
-S9: 2.44, 4.88, 9.76, 19.5, 39.1, 58.6 µg/mL
+S9: 2.44, 4.88, 9.76, 19.5, 39.1, 78.1, 117 µg/mL

The selection of the concentrations used in the main experiments was based on data from a preliminary cytotoxicity assay. Severe toxicity was observed at the seven highest dose-levels (with and without S9). Based on these results maximum concentrations of 58.6 and 117 µg/mL were selected for treatments in the absence and presence of S9 mix, respectively.
Vehicle / solvent:
Since no solvent vehicle was employed in this study the negative controls consisted of untreated cultures.
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
no
Remarks:
no vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : single
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10E6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h at 37 °C

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 72 h
- Selection time (if incubation with a selective agent): 12 - 16 days
- Method used: agar
- Selection agent: Trifluorothymidine (final concentration 4.0 µg/mL)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 500,000 cells; after incubation, the plates are scored either manually or using a calibrated Artek Model 890 Automatic colony counter; the mutatioh frequency at each test point is calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Percentage survival relative to the solvent controls is calculated for each treatment in a preliminary cytotoxicity experiment. Dose-levels giving a predicted 20% survival are estimated with and without S9. The estimated concentrations are chosen as the highest dose-levels for the mutation assays.
Evaluation criteria:
For a test substance to be considered mutagenic in this assay, it is required that:
(i) There is a two-fold (or more) increase in mutation frequency compared with the solvent control values, over two consecutive test substance treatment levels. If only the highest practicable dose-level (or the highest dose-level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
(ii) The increases must be reproduced in an independent experiment.
(iii) There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).
Statistics:
The results of the experiments were subjected to an Analysis of Variance, in which the contribution of experiment number and dose-level to the observed variation was examined.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 58.6 µg/mL -S9 and 177 µg/mL + S9
Vehicle controls validity:
not examined
Remarks:
no vehicle used
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The addition of the test substance solution did not have any obvious effect on the pH of the treatment medium.
- Data on osmolality: The addition of the test substance solution did not have any obvious effect on the osmolality of the treatment medium.
- Possibility of evaporation from medium: The test substance is not volatile.
- Water solubility: The test substance was soluble in complete medium at a concentration of 100 mg/mL. On the basis of these results, a maximum concentration of 10,000 µg/mL was selected for the cytotoxicity test.

RANGE-FINDING/SCREENING STUDIES (if applicable):
The test substance was assayed at a maximum concentration of 10,000 µg/mL and 8 lower dose levels spaced at two-fold intervals. Following treatment both in the absence and presence of S9 mix, severe toxicity was observed at the 7 highest dose levels, reducing total suspension growth to below the limit of detection. In the absence of S9 mix, at the next lower dose level (78.1 µg/mL) the total suspension growth value was reduced to 2% of the negative control. In the presence of S9 mix, no toxicity was observed at the next lower dose level (78.1 µg/mL). Maximum concentrations of 58.6 and 117 µg/mL were selected for the main study in the absence and presence of S9 mix, respectively.
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo genotoxicity:

Mammalian Bone Marrow Chromosome Aberration Test (OECD 475): negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Oct. 1994 - 6 Feb. 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Como, Italy
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 25 - 32 g (males); 20 - 27 g (females)
- Assigned to test groups randomly: yes, immediately after arrival at test facility
- Fasting period before study: yes, overnight
- Housing: 5 animals/cage, separated by sexes, in clear polycarbonate cages (type 2b: Techniplast) with a stainless steel mesh lid and floor; each cage equiped with absorbent bedding which was inspected daily and changed as necessary.
- Diet: Altromin MT, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATE: 26 Oct. 1994
Route of administration:
oral: gavage
Vehicle:
Vehicle(s)/solvent(s) used: injectable grade sterile distilled water
- Lot/batch no.: 22143-lB (Don Baxter S.p.A., Trieste, italy)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Fresh solutions of the test substance were prepared for each day's work; solutions were prepared on a weight/volume basis. Solutions of the test substance, following corrections of active constituent were prepared in injectable grade sterile distilled water.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
Test groups: 10, 24 and 48 h
Positive control group: approx. 26 h
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
corrected for a.i. content of test material
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
corrected for a.i. content of test material; represents the recommended limit dose for administration periods < 14 days according to OECD TG 475
No. of animals per sex per dose:
Test groups: 5 for each sampling time (10, 24 and 48 h)
Positive control group: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg bw)
Tissues and cell types examined:
Tissue: femoral bone marrow
Cell type: femoral bone marrow cells
Details of tissue and slide preparation:
SAMPLING TIMES
Test groups: 10, 24 and 48 h after treatment
Positive control group: 26 h after treatment

CRITERIA FOR DOSE SELECTION:
Range finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Following treatment and prior to sample collection, animals are injected intraperitoneally with 4 mg/mL colchicine and samples are collected 3 h thereafter. Cells are harvested from the bone marrow, swollen, fixed and stained, and analysed for chromosomal aberrations.

DETAILS OF SLIDE PREPARATION:
Slides were fixed with a methanol:acetic acid solution, air-dried and stained with Giemsa.

METHOD OF ANALYSIS:
50 metaphase spreads per animal were examined microscopically at high magnification for the presence of chromosomal aberrations. Only metaphases containing 40 chromosomes are scored for aberrations. The number of chromosomes, the specific types and numbers of aberrations are recorded. The Vernier readings of aberrant or equivocal metaphases are recorded.

OTHER:
The mitotic index is calculated. This is based on the number of metaphases observed per 1000 cells and is expressed as a percentage.
Evaluation criteria:
A test substance is considered to have clastogenic properties if a statistically significant increase in the incidence of cells bearing aberrations is observed at any test point, excluding gaps. The lesions observed must be consistent with those expected at the sampling time at which the increases are observed. The internal consistency of the findings within groups are also taken into account.
Statistics:
The experimental unit investigated in the present study is the proportion of cells from an animal which exhibit chromosomal aberrations. It is therefore assumed that the data has a binomial error structure, each cell out of a total of 'n' for each animal either bearing or not-bearing aberrations.
A logistic-linear model is fitted to the data adding the factors 'Sex', 'Treatment' and 'Sex-Treatment Interaction' in turn. The change in deviance with each factor is compared with the Chi-squared distribution to determine the level of significance. The deviance associated with the residual term is compared with the Chi-squared distribution to determine the goodness-of-fit of the logistic-linear model. This allows the construction of a table similar to a classical analysis of variance.
Data from in vivo cytogenetic assays often contain extra-binomial variation, which may result in part from animal-to-animal variation, or from the responses of different populations of cells within animals. This situation will be highlighted by a significant lack of fit of the logistic-linear model. When this is the case, an allowance for the extra-binomial variation can be made using a generalised 'heterogeneity factor' to weight the logistic-linear model variables.
The statistical analysis is performed using the values for the total number of aberration-bearing cells observed for each animal, excluding gaps from consideration.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No clinical signs were observed at any dose level tested.
- Evidence of cytotoxicity in tissue analysed: No reduction in the mitotic index was observed in any treatment group.
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The genetic toxicity of alcohol ethoxysulfates (AES) was evaluated in several in vitro and in vivo genotoxicity tests. In summary, all Ames tests conducted with the substance AES (C12-14, 1-2.5 EO) Na (CAS 68891-38-3) using several strains of S. typhimurium showed a negative result. Testing for mammalian genotoxicity in vitro revealed a negative outcome as well. The result of in vivo testing revealed that the substance AES (C12-14, 1-2.5 EO) Na (CAS 68891-38-3) has no clastogenic potential. According to Regulation (EC) No. 1907/2006 (REACH) VIII 8.4.2, column 2, an in vitro cytogenicity study in mammalian cells does not need to be conducted if adequate data from an in vivo cytogenicity test are available. Based on the outcome of these tests, AES (C12-14, 1-2.5 EO) Na (CAS 68891-38-3) can be considered as non-genotoxic.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.