Fatty acid chlorides, C12-18 (even numbered) and C18 unsatd., reaction products with sodium N-methyltaurinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliance to GLP and test guideline; coherence between data, results and conclusion

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-[methyloleoylamino]ethane-1-sulphonate

EC Number:

205-285-7

EC Name:

Sodium 2-[methyloleoylamino]ethane-1-sulphonate

Cas Number:

137-20-2

Molecular formula:

C21H41NO4S.Na

IUPAC Name:

sodium 2-[methyl(oleoyl)amino]ethanesulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appereance supplied by the Sponsor: yellowish pale powder
- Description at first use: yellowish powder
- Storage condition of test material: room temperature

Method

Target gene:

hypoxanthine-guaninphosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:

EMEM Minimal Medium
EMEM Complete Medium (10% Fetal Bovine Serum)

DMEM Minimal Medium
DMEM Complete Medium (10% Fetal Bovine Serum)

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction from rats induced with phenobarbitone and beta-naphthoflavone (Mixed Induction).

Test concentrations with justification for top dose:

Experiments without S9 mix:
32.3, 23.1, 16.5, 11.8, 8.40 and 6.00 ug/mL for the first experiment
28.2, 21.7, 16.7, 12.8 and 9.87 ug/mL for the second experiment

Experiments with S9 mix:
600, 462, 355, 273, 210 and 162 ug/mL for the first experiment
500, 458, 382, 318 and 265 ug/mL for the second experiment

Vehicle / solvent:

Minimal Culture Medium.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 3 hours in the absence and presence of S9 metabolism
- Expression time (cells in growth medium): day 6 and day 9
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): 6 -thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS: Two replicate cultures for each experimental point; two indipendent mutation experiments

NUMBER OF CELLS EVALUATED: 1x10^5 cells/ 100 mm tissue culture petri dishes (five plates, a total of 5 x10^5 cells)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative cloning efficiency;

DETERMINATION OF MUTATION
- Method: Induced mutant frequency

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:

(i) There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item. 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) There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

The “five-fold increase” in mutant frequency above the concurrent negative control is used as arbitrary criteria for positive response and it was established in our laboratory based on analysis of variation of negative control data.
If spontaneous mutation frequency is in the upper part of the historical range, significance of mutation increase is evaluated case by case.
Historical control data are used to demonstrate biological relevance of the results obtained.

Statistics:

ANOVA analysis for effect of replicate culture, expression time and dose level

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation of the test item was noted upon addition of the test item to the culture in all treatment series and by the end of treatment incubation period.
The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

RANGE-FINDING/SCREENING STUDIES:
Two preliminary cytotoxicity assays were performed.
In the first experiment the test item was assayed at a maximum dose level of 5000 g/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 µg/mL. In the absence of S9 metabolism, no cell survived after treatment at the seventh highest dose levels, while marked toxicity was noted at 39.1 µg/mL reducing survival to 16% of the negative control value. In the presence of S9 metabolism, no cell survived at the three highest dose levels. Marked toxicity was observed at the next lower dose level (625 µg/mL) reducing survival to 10% of the concurrent negative control value, while no toxicity was observed over the remaining dose levels.
Since the plating efficiency obtained in this experiment did not meet the acceptability criteria, a second preliminary cytotoxicity assay was performed. Taking into account the results obtained in the first experiment, the following dose levels were selected:
without S9 mix: 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.977, 0.488 and 0.244 µg/mL
with S9 mix: 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 µg/mL
In the absence of S9 metabolism, obvious and dose related toxicity was observed at the three highest dose levels. At 62.5 and 31.3 µg/mL survival was reduced to near the limit of detection, while at 15.6 µg/mL it was reduced to 38% of the concurrent negative control value. In the presence of S9 metabolic activation, survival was reduced to below the limit of detection at the highest dose level, while at the next lower concentration survival was 23% of the concurrent negative control value.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control treatments were included in the mutation experiments in the absence and presence of S9 metabolism. The mutant frequwensy in the solvent control cultures fell within the normal range based on the laboratory historical control data. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON CYTOTOXICITY AND MUTAGENICITY:
On the basis of the cytotoxicity results, two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described below:
Experiments without S9 mix:
32.3, 23.1, 16.5, 11.8, 8.40 and 6.00 ug/mL for the first mutation assay
28.2, 21.7, 16.7, 12.8 and 9.87 ug/mL for the second mutation assay

Experiments with S9 mix:
600, 462, 355, 273, 210 and 162 ug/mL for the first mutation assay
500, 458, 382, 318 and 265 ug/mL for the second mutation assay

In the first mutation assay, following treatment in the absence of S9 metabolism, a severe toxic effect was observed at the highest dose level (32.3 ug/mL) reducing survival to 6% of the concurrent negative control value. At the next lower concentration, survival was reduced to 30%, while no relevant toxicity was observed at the remaining dose levels. In the presence of S9 metabolic activation, survival was reduced to below the limit of detection at the highest dose level of 600 ug/mL, while the two next lower concentrations (462 and 355 ug/mL) yielded 34% and 58% relative survival, respectively.
In the second mutation assay, in the absence of S9 metabolism, a severe reduction in cell survival was observed at 28.2 ug/mL (9%). At the next lower dose level (21.7 ug/mL) survival was reduced to 38% of the concurrent negative control value, while no relevant toxicity was observed at the remaining dose levels. In the presence of S9 metabolism, marked toxicity was observed at the highest dose level (5% percentage survival) while the next lower concentration (458 ug/mL) yielded 41% relative survival. No relevant toxicity was observed over the remaining dose levels.

No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism.

Any other information on results incl. tables

No reproducible five-fold or greater increase in mutant frequency was observed either in the absence or presence of metabolic activation at any test point. No statistically significant effect of dose level in the ANOVA analysis was observed. It is concluded that HOSTAPON TPHC does not induce gene mutation in Chinese hamster V79 cellsin vitro.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that HOSTAPON TPHC does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

This report describes experiments performed to assess the mutagenic activity of the test item by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells afterin vitrotreatment (in the absence and presence of S9 metabolic activation).

The Study was performed in compliance with:

-  OECD Guidelines for the testing of chemicals No. 476 (Adopted July 1997).

No reproducible five-fold or greater increase in mutant frequency was observed either in the absence or presence of metabolic activation at any test point. No statistically significant effect of dose level in the ANOVA analysis was observed.

It is concluded that HOSTAPON TPHC does not induce gene mutation in Chinese hamster V79 cellsin vitro.