[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.07.2004 - 07.09.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Study according to OECD Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Glyceryl Behenate/eicosandioate

IUPAC Name:

Glyceryl Behenate/eicosandioate

Constituent 2

Reference substance name:

-

EC Number:

478-910-6

EC Name:

-

Cas Number:

215322-01-3

Molecular formula:

C114H218O12 for the stoichiometric product

IUPAC Name:

Reaction product of Eicosanedioic acid, Docosanoic acid and Glycerin

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Nomcort HK-G
- Substance type: organic
- Physical state: solid
- Analytical purity: 100 %
- Lot/batch No.: 4-1-B
- Expiration date of the lot/batch: April 29, 2007
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: room temperatur

Method

Target gene:

his- (S. typhimurium) and trp+ (E. Coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix: Rat liver fraction induced by phenobarbital/beta- Naphthoflavone

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33 ... 5000 µg/plate

Vehicle / solvent:

Solvent: THF
Justification: The solvent was chosen becuase of its solubility properties and its relative non-toxicity to the baceria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth/colony number

Evaluation criteria:

Acceptability
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of results
a test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (Strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentraiont is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertnant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the histroical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

exceptions see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 2500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that during the dexcribed mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pir changes or frameshifts in the genome of the strains used. Therefore, Nomcort HK-G is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Nomcort HK-G to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was perforemed in two independent experiments both with and without liver microsomal activation. Each concentration, uncluding the controls, was tested in triplicate. The test item was tested at the following concentrations:

33, 100, 333, 1000, 2500 and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants, occurred in experiment I in strains TA 1535 and TA 98 with metabolic activation at 2500 and 5000 µg/plate. A reduction in the number of revertants was also observed in strain TA 98 without metabolic activation at 5000 µg/Plate in experiment I.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Nomcort HK-G at any dose level, neither in the presence nor absence of metablic activation (S9 -mix). There was also no tnedency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.