[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

010-01-12 - 2010-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Recombinant spider silk protein eADF4(C16) derived from E. Coli by fermentation

Molecular formula:

not applicable, UVCB

IUPAC Name:

Recombinant spider silk protein eADF4(C16) derived from E. Coli by fermentation

Test material form:

liquid

Method

Target gene:

uvrB gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µl./plate

Vehicle / solvent:

0.9% NaCl

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

Pre-Experiment for Toxicity
The toxicity of the lest item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre¬experiment were the same as described below for the main experiment 1 (plate incorporation test).
Toxicity may be delected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.
The test item was tested in the pre-experirnent with the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0. 2.5 and 5.0 µL/plate

Exposure Concentrations
The lest item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5.0 µL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Experimental Performance
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µl: Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control).
500 µL: S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation).
100 µL: Bacteria suspension (cf. Preparation of Bacteria, pre¬culture of the strain).
2000 µL: Overlay agar.
For the pre-incubation method 100µ L of the test item preparation was pre¬incubated with the tester strains (100 pL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes al 37 °C prior Io adding the overlay agar (2000µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtoCOL counter. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation criteria:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in al least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 100 and TA 102 the number of reversions is al least twice as high
- if in tester strains TA 98. TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, eADF4(C16) did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, eADF4(C16) is considered to be non-mutagenic in this bacterial reverse mutation assay.