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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
EC Number:
239-032-7
EC Name:
Sodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
Cas Number:
14960-06-6
Molecular formula:
C18H35NO4.Na
IUPAC Name:
sodium 3-[(2-carboxyethyl)(dodecyl)amino]propanoate
Specific details on test material used for the study:
- Name of test material (as cited in study report): DERIPHAT 160 C
- Physical state:Solid (lyophilized), white
- Analytical purity: 97% The test substance was characterized analytically (for details see the analytical report; project no.: AU 122958-1)
- Lot/batch No.:4986V1
- Expiration date of the lot/batch: until 03 Jun 2014
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage condition of test material:Room temperature (avoid temperatures > 40°C)

Method

Target gene:
X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital- and ß-naphthoflavone induced rats
Test concentrations with justification for top dose:
1st Experiment, without S9 mix: 0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 µg/mL
1st Experiment, with S9 mix: 0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL

2nd Experiment, without S9 mix: 0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL
2nd Experiment, with S9 mix: 0; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0 µg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Negative controls, with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups.
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Attachment period:20 - 24 hours
- Exposure duration:4 hour, removal of test substance by intense washing (4 hour exposure)
- Expression time (cells in growth medium):3 days (4-hour treatment)
- Selection time (if incubation with a selection agent):6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):10-11 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium with 6-thioguanine (10 μg/mL), stable glutamine (200 mM), fetal calf serum (FCS)
STAIN (for cytogenetic assays):colonies were fixed with methanol, stained with Giemsa

NUMBER OF REPLICATIONS:Duplicate cultures were used for all experimental
groups.

NUMBER OF CELLS EVALUATED: Cloning efficiency 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL. For selection of the mutants, six 75 cm2 flasks with 3x105 cells each from every treatment group, if possible, were seeded in 10 mL selection medium.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The cloning efficiency (CE, %) was calculated for each test group as follows:
CEabsolute = total number of colonies in the test group/total number of seeded cells in the test group x 100

The uncorrected mutant frequency (MFuncorr.) per 106 cells was calculated for each test group
as follows:
MFuncorr. = total number of mutant colonies / number of seeded cells x 106
MFcorr. = MFuncorr. / CE2 absolut x 100

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 6).
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 in highest tested concentration
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In both experiments, in the presence and absence of S9 mix, after 4 hours treatment the morphology and attachment of the cells were adversely influenced in at least the highest applied concentration

Applicant's summary and conclusion