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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Juli 21st, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 30th, 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8887
- Physical state: clear yellowish liquid
- Analytical purity: 78.1g/100g (for details see analytical report, study code 11L00317)
- Purity test date: 2011-11-03
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-08-03
- Storage condition of test material: room temperature in the dark

Method

Target gene:
his (salmonella)
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:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st experiment
0; 33; 100; 333; 1 000; 3 250 and 6 500 μg/plate
doses were adjusted to purity (highest dose equals 5mg pure substance/plate)
2nd experiment (pre-incubation)
0; 10; 33; 100; 333; 1 000 and 3 000 μg/plate
doses were adjusted to purity (highest dose equals 2300mg pure substance/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance is almost insoluble in water, DMSO has been demonstrated to be suitable for this test and historical control data with this vehicle are available.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
TA 1535, TA 100 (without S9)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
TA 98 (without S9)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S9)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E.coli WP2 uvrA (without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (no preincubation), in suspension (preincubation)

DURATION
- Preincubation period: 20min
- Exposure duration: 48 - 72h

SELECTION AGENT (mutation assays): minimal agar (0.5M his + 0.5M biotin (salmonella) or 0.5M trp (e.coli))

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: titer determination (no. of bacterial colonies after incubation with test substance without selection agent)

OTHER:
The Salmonella strains are checked for the following characteristics at regular intervals:
deep rough character (rfa); UV sensitivity (Ä uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA is checked for UV sensitivity.
Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.
Evaluation criteria:
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 1 billion cells per mL were used. For this approval the titer of viable bacteria has to be ≥ 100 million colonies per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
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:
cytotoxicity
Remarks:
from app. 1000µg/plate
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 found
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

Thus, under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.