Disodium Capryloyl Glutamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 March 2019 to 06 May 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Also Commission Regulation (EC) n° 440/2008 B.13/14 and EPA Health Effects Test Guidelines, OPPTS 870.5100 were kept in consideration.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt

Cas Number:

167888-81-5

Molecular formula:

Not applicable, see single constituents

IUPAC Name:

Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt

Test material form:

liquid

Remarks:

Water solution

Details on test material:

- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles:
- Crystal structure:
- Coating:
- Surface properties:
- Density:
- Moisture content:
- Residual solvent:
- Activation:
- Stabilisation:
- Other:

Specific details on test material used for the study:

Batch 3747 supplied by sponsor
clear liquid
pH as it is 10 (20 °C)
Dry matter 38.5% (in water) as active ingredient, purity 100%
Soluble in water
NaCl 5.1%
Constituents are in the same range used for registration
Storage condition of test material: room temperature (15 °C - 25 °C)
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
More information can be found on the attached report

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: rfa-, uvrB-, R-factor

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomial fraction is obtained from male Wistar rats induced with phenobarbital (80 mg/kg bw) and Beta-naphthoflavone (100 mg/kg bw) for 3 consecutive days by oral route.

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strainsTA98 and TA100 in a pre-experiment. The following concentrations were tested:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 microl/plate.
According to the obtained results the below concentrations were chosed for the test.
5.0 microl/plate was selected as the maximum concentration and the concentration range covered two logarithmic decades.
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 microl/plate.

Vehicle / solvent:

Purified water for the substance,
purified water or DMSO for positive controls

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or purified water

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

Following dose range finding studies, the test was done as descibed below. For the plate incorporation method the following materials are mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL overlay agar.
For the pre-incubation method 100 µL of the test item-preparation is 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.
For each strain and dose level, including the controls, a minimum of three plates is used.
After solidification the plates are inverted and incubated at 37 °C for at least 48 h in the dark.
Two different experiments were performed, plate incorporation test (experiment I) and pre-incubation test (experiment 2). The colonies are counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precludes automatic counting the revertant colonies are counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 are counted manually.

Rationale for test conditions:

Based on OECD 471 (1997) and also on Commission Regulation (EC) n° 440/2008 B.13/14 and EPA Health Effects Test Guidelines, OPPTS 870.5100.
A dose range finding study was performed.

Evaluation criteria:

Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as N or B respectively 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.

Statistics:

According to the 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.
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 following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.

Any other information on results incl. tables

All tables are 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Bacterial reverse mutation assays use amino-acid requiring strains of Salmonella typhimurium
(S. typhimurium) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of these bacterial reversion assays is that they detect mutations which functionally reverse mutations present in the tester strains and restore the capability to synthesize an essential amino acid.
The purpose of this study is to establish the potential of the test item to induce gene mutations in bacteria by means of a S. typhimurium reverse mutation assay. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentrations spacing and / or the method of treatment (pre-incubation method). In case of severe
toxicity of the test item or the use of e.g. ethanol, acetone or tetrahydrofuran as the most appropriate solvent, the confirmatory experiment is carried out according to the plate incorporation method with a different spacing between dose levels.
The S. typhimurium histidine (his) reversion system measures his- → his+ reversions. The
S. typhimurium strains are constructed to differentiate between base pair (TA100, TA1535, TA102) and frameshift (TA98, TA1537) mutations.
These assays directly measure heritable DNA mutations of a type which is associated with adverse effects. Point mutations are the cause of many human genetic diseases and
there is substantial evidence that somatic cell point mutations in oncogenes and tumour suppressor genes are involved in cancer in humans and experimental systems.
The tester strains have several features that make them more sensitive for the detection of
mutations. The specificity of the strains can provide useful information on the types of mutations that are induced by mutagenic agents.
According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
At least five different concentrations of the test item are tested with approximately half log (i.e. √10)
intervals between test points for an initial test. Narrower spacing between dose levels may be
appropriate when a dose response is investigated. For soluble, non-toxic test compounds the
recommended maximum test concentration is 5 mg/plate or 5 µL/plate.
To validate the test, reference mutagens are tested in parallel to the test item. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.