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Toxicological information

Genetic toxicity: in vitro

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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:
2015-07-16 to 2015-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Aqueous solution of beta-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-cocoacyl derivs., monosodium salts

Method

Target gene:
Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed
Additional strain / cell type characteristics:
other: Salmonella strains: rfa-, uvrB-; E. coli strain: uvrA-
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 Mix
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties and relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (without metabolic activation); 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION:plate incorporation (experiment I) and preincubation (experiment II)

DURATION
- Preincubation period: 60 min (only experiment II)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): all Salmonella typhimurium strains: histidine; E. coli strain: tryptophane

NUMBER OF REPLICATES: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants; reduction of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
not mandatory

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA: In experiment I the number of colonies did not quite reach the lower limit of the laboratory's historical control data in the negative control of strain TA 1535 with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Any other information on results incl. tables

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

1000-5000

1000-5000

1000-2500

2500

TA 1537

1000-5000

1000-5000

333-2500

1000-2500

TA 98

1000-5000

1000-5000

333-2500

1000-2500

TA 100

333-5000

333-5000

333-2500

1000-2500

WP2 uvrA

1000-5000

1000-5000

2500

2500

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

1000-5000

2500-5000

1000-2500

2500

TA 1537

1000-5000

2500-5000

1000-2500

2500

TA 98

1000-5000

1000-5000

333-2500

1000-2500

TA 100

33; 333-5000

333-5000

33-2500

333-2500

WP2 uvrA

2500-5000

2500-5000

2500

2500

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, no substantial increase in revertant colony numbers of any of the five tester strains was observed at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, Amphopropionate C12-18 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (July, 1997) and EU Method B. 13/14 (2008) strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed in two independent experiments to Amphopropionate C12 -18 (ca. 40% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation method and at concentrations of 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate using the reincubation method both in the absence and presence of mammalian metabolic activation.

 

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of Amphopropionate C12 -18 did not occur up to the highest investigated dose. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C12 -18 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C12 -18 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  This study is classified as acceptable. It satisfies the requirements for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.