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Toxicological information

Genetic toxicity: in vitro

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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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP applied.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: Ceraphyl 41
Product Code: 826791
Batch No.: 0001749388
Physical State: liquid
Colour: clear, water white to straw coloured
Storage Conditions: room temperature
Retest Date: June 2015
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late
exponential or early stationary phase of growth (approx. 109 cells/mL). A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
- Properly maintained: yes
Additional strain / cell type characteristics:
other: All the strains contains mutations in the histidine operon, the deep rough mutation
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment I (plate incorporation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate ( for TA 98 and TA 102 with and without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation and for TA 1535 and TA 1537 with and without metabolic activation)

Experiment II (preincubation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 with metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for TA 100, TA 1535 and TA 1537 with metabolic activation)
0.0000316, 0.0001000, 0.000316, 0.001000, 0.00316, 0.0100, 0.0316 and 0.100 µL/plate (for TA 100, TA 1535 and TA 1537 without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (for TA 102 with and without metabolic activation).

Vehicle / solvent:
The test material was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Remarks:
Purified water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
STRAIN CHARACTERISTICS:
- TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
- TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
- TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
- TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
- TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA.
Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland.
They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DATA RECORDING:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). 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.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "B" in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control (indicated as "N" in the results tables).
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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 at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control [11].
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S.typhimurium TA 100, TA 1535, TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Remarks on result:
other: other: all strains
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The results shows that precipitation of the test item was observed in tester strains TA 98 and TA 102 in experiment I at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment II precipitation of the test item was found in tester strain TA 102 at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 0.00100 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were seen at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.0316 μL/plate and higher (with metabolic activation).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material did not cause gene mutation by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Ceraphyl 41 is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.

The results show that in experiment I and II, the toxic effects of the test item were observed in tester strain (except strain TA 102) with and without metabolic activities depending on the particular tester strains and concentration of the test material (see the section of any other information on results inc tables).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment..