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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
9th addendum
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:040802#
- Expiration date of the lot/batch:06.08.2016
- Purity test date:08/01/2015
- Purity: 99.17%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature
- Stability under test conditions:stable
- Solubility and stability of the test substance in the solvent/vehicle:stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:the test item is dissolved in Aqua destillata and diluted prior to treatment. Aqua distilla is compatible with bacteria and the S9 activity.
- Final dilution of a dissolved solid, stock liquid or gel:different concentration tested (see below)

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

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):
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.
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for
histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the
polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell
permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a
protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting
many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes
(bacteria require biotin for growth).
The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains
are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor
parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an errorprone
DNA repair system which is normally present in these organisms.
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and
tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly
according to Ames et al.In this way it is ensured that the experimental conditions set up by
Ames are fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
EXPERIMENT I (Plate incorporation method) 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate and 1.00 µg/plate only for TA 1535 and TA1537
EXPERIMENT II (pre incubation method) 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
Justification of top dose: for soluble non toxic test compounds the recommended maximum test concentration is 5 µl/plate and a pre-experiment test was performed for toxicity evaluation showing no background lawn from 2500 µg/plate in one strain
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water (Aqua Destillata)
- Justification for choice of solvent/vehicle:The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
The solvent and the negative controls were the same.
True negative controls:
no
Positive controls:
yes
Remarks:
4 different positive controls depending on the metabolic activation or not and the tester strains.
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):approx. 10^9 cells/ml

DURATION
- Preincubation period:60 min in experiment II
- Exposure duration:48h for both experiments

NUMBER OF REPLICATIONS:3

DETERMINATION OF CYTOTOXICITY
- Method: detected by a clearing / diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of 0.5 or less in relation to the solvent control


OTHER EXAMINATIONS:
colonies counted using a ProtoCOL counter (Meintrup DWS Laborgäte GmbH) or manually (for TA 1535 and 1537 or if precipitation occurred)

- OTHER:- preparation of bacteria: each strain was grown by culturing for 12h at 37°C in Nutrient Broth (8g/l Nutrient Broth + 5 g/l NaCl). Ampicillin (10 mg/ml) was added to TA 98, TA 100 and TA 102.
- Agar plates: Vogel-Bonner Medium E agar plates with 2% glucose, sterilized 20 min at 121°C in autoclave.
- Overlay agar: contains agar-agar, NaCl, LhistidinexHClxH2O, biotin, sterilized 20 min at 121°C in autoclave
- Metabolic activation system: S9 liver microsomal fraction (from Male Wistar rats and Sprague Dawley rats (phenobarbital/Bêta-naphthoflavone)) in a S9 mix preparation with MgCl2, KCl, glucose-6-phosphate and NADP, stored on ice.
- For the test without metabolic activation, S9 mix is replaced by a sterilized phosphate buffer solution stored at 4°C
Evaluation criteria:
Mutation factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.

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
Statistics:
Not necessary because the biological relevance of the results is the criterion for the interpretation of the results

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentration with toxic effects depends on experimental conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentration with toxic effects depends on experimental conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentration with toxic effects depends on experimental conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentration with toxic effects depends on experimental conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentration with toxic effects depends on experimental conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-Precipitation: no precipitation observed


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data:
mean values of the spontaneous reversion frequency are within the historical control data range(2012 -2014):
- S9 + S9
min max min max
TA 98 13 48 13 61
TA 100 61 182 68 194
TA 1535 4 35 4 34
TA 1537 2 27 3 31
TA 102 136 415 91 495

Any other information on results incl. tables

Concentrations were toxic effects where observed:

      EXPERIMENT I     EXPERIMENT II
   with (+S9)  without (-S9)  with (+S9)  without (-S9)
 TA98  ≥1000  ≥1000  2500  ≥ 316
TA100   ≥ 1000  ≥ 1000  ≥ 1000 2500 
 TA102  ≥ 1000  ≥1000  ≥ 316 ≥ 316
 TA1535  ≥ 1000 2500   ≥ 1000 ≥  316
 TA1537 2500  ≥ 100 ≥  316≥ ≥ 100

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with X330 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 experiments.

      

 

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions and during the described mutagenicity test, X330 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore X330 is considered to be non mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of X330 for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains 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.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with X330 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 experiments.

In conclusion, under these experimental conditions and during the described mutagenicity test, X330 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore X330 is considered to be non mutagenic in this bacterial reverse mutation assay.

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