Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Conformity All procedures will be according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: not applicable
- Activation: not required
water as a additive
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Method

Target gene:
Name Category Effect
hisD6610 frame shift histidine deficiency
hisD3052 frame shift histidine deficiency
hisG46 base pair substitution histidine deficiency
hisG428 base pair substitution histidine deficiency
uvrB deletion UV sensitivity, biotin deficiency
rfa deletion lipopolysaccharide side chain deficiency
pKM101 plasmid ampicillin resistance
pAQ1 plasmid tetracycline resistance
Species / strainopen allclose all
Species / strain / cell type:
other: TA97A
Details on mammalian cell type (if applicable):
hisD6610 frame shift histidine deficiency
uvrB deletion UV sensitivity, biotin deficiency
rfa deletion lipopolysaccharide side chain deficiency
pKM101 plasmid ampicillin resistance
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
hisD3052 frame shift histidine deficiency
uvrB deletion UV sensitivity, biotin deficiency
rfa deletion lipopolysaccharide side chain deficiency
pKM101 plasmid ampicillin resistance
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
hisG46 base pair substitution histidine deficiency
uvrB deletion UV sensitivity, biotin deficiency
rfa deletion lipopolysaccharide side chain deficiency
pKM101 plasmid ampicillin resistance
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
hisG428 base pair substitution histidine deficiency
rfa deletion lipopolysaccharide side chain deficiency
pKM101 plasmid ampicillin resistance
pAQ1 plasmid tetracycline resistance
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
hisG46 base pair substitution histidine deficiency
uvrB deletion UV sensitivity, biotin deficiency
rfa deletion lipopolysaccharide side chain deficiency
Metabolic activation:
with and without
Metabolic activation system:
Specification S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
Test concentrations with justification for top dose:
5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate
Vehicle / solvent:
In a non-GLP pre-test, the solubility of the test item was determined in demineralised wa-ter, acetone and dimethyl sulfoxide (DMSO). The test item is soluble in a concentration of 50 mL/L in DMSO.
Based on these results, DMSO will be used as solvent in the experiments.
Therefore, a test item solution containing 50 ± 5 mL/L in DMSO will be prepared in the initial experiment.
Controls
Untreated negative controls:
other: determination of spntaneous revertants
Remarks:
spontaneous revertants of the negative control are compared to the historical data of the laboratory
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO) for test item and the positive controls nitrophenylendiamine, benzo-a-pyrene and 2-amino-anthracene Demineralised water for the positive control sodium azide.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: Nitrophenylendiamine
Details on test system and experimental conditions:
VALIDITY
Genotype
Each strain must fulfil the requirements concerning the presence (or absence) of specific mutations. The following genotype specifications of the test system were tested for each batch (procedure is laid down in corresponding SOP):
• histidine deficiency
• Ampicillin-/Tetracycline-resistance (pKM101 and pAQ1; for TA97a, TA98, TA100, TA102; control : TA1535)
• Sensitivity against crystal violet (deep rough/rfa, for TA 97a, TA98, TA100, TA102, TA1535)
• UV-sensitivity (uvrB, only TA102 may show growth)

Spontaneous Revertants
If the spontaneous revertants of the negative control were in the range of the historical data of the laboratory.

Positive Controls
Mutagenicity of all positive controls must be detected. The revertant numbers was compared with the history of revertants based on the tests performed in the test facility.

Titre Determination
The titre determination showed a density of at least 109 bacteria/mL. If the detected number is lower but the spontaneous revertants lie within the normal range, the test may be stated as valid.

Sterility Control
No growth of bacteria should be detected.
Rationale for test conditions:
The study is implemented with the following guidelines:
• OECD Guidelines for the Testing of Chemicals, Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“
• Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”
Additional information was taken from:
• K. Mortelmans, Errol Zeiger: “The Ames Salmonella/microsome mutagenicity assay”, Mutation Research 455 (2000) 29-60, Elsevier Science B.V.
• D.M. Maron, B.N. Ames: “Revised methods for the Salmonella mutagenicity test”, Mutation Research 113 (1983) 173-215, Elsevier Biomedical Press
Corresponding SOP of LAUS GmbH
• SOP 118 008 03 edition 13, valid from 24. Jul. 2017
“Bestimmung des erbgutverändernden Potentials mit dem Bacterial-Reverse-Mutation-Test (Ames-Test)“
Evaluation criteria:
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Tables and, if applicable, graphs of the values will be included in the final report. It will be stated, at which concentration (µL or nL/plate) mutagenicity could be observed. The lowest concentration which showed mutagenicity is decisive of the assessment of the test item.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Additional information on results:
Detailed results not available at the moment. Test item is cytotoxic
VALIDITY
Genotype
Each strain must fulfil the requirements concerning the presence (or absence) of specific mutations. The following genotype specifications of the test system will be tested for each batch (procedure is laid down in corresponding SOP):
• histidine deficiency
• Ampicillin-/Tetracycline-resistance (pKM101 and pAQ1; for TA97a, TA98, TA100, TA102; control : TA1535)
• Sensitivity against crystal violet (deep rough/rfa, for TA 97a, TA98, TA100, TA102, TA1535)
• UV-sensitivity (uvrB, only TA102 may show growth)

Spontaneous Revertants
If the spontaneous revertants of the negative control are not in the range of the historical data of the laboratory and the mutagenicity of the test item is not clear, the experiment will be repeated.

Positive Controls
Mutagenicity of all positive controls must be detected. The revertant numbers will be com-pared with the history of revertants based on the tests performed in the test facility. If no mutagenicity is detected, the experiment will be repeated with the respective strain.

Titre Determination
The titre determination should show a density of at least 109 bacteria/mL. If the detected number is lower but the spontaneous revertants lie within the normal range, the test may be stated as valid. This decision is responsibility of the study director.

Sterility Control
No growth of bacteria should be detected.

Applicant's summary and conclusion

Conclusions:
not mutagenic but cytotoxic at tested concentrations
Executive summary:

In in vitro study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU B.13/14

the substance was tested with 5 strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102, TA1535), without and with S-9 mix Aroclor 1254 induced rat liver metabolic activation system, to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.

In the Bacterial Reverse Mutation Test, the test item didn´t show any mutagenic potential.