Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
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:
Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II:
1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Vehicle / solvent:
Distilled water
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle control
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene - with metabolic activation 4-NOPD; 4-nitro-o-phenylene-diamine - without metabolic activation
Details on test system and experimental conditions:
Pre-Experiment for Toxicity

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Exposure Concentrations

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see chapter 12.1.1 Pre-Experiment). 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II:
1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

Experimental Performance

For the plate incorporation method the following materials were 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 was 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, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation of Cytotoxicity

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.
10.8. Criteria of Validity
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the ranges (mean values of the spontaneous reversion frequency within the historical control data range (2016 -2018))
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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 TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to 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.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:
at concentrations of 1000 μg/plate and higher (with and without metabolic activation)
Vehicle 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
Remarks:
In experiment I, toxicity at concentrations of 1000 μg/plate and higher (with and without metabolic act.). In experiment II, toxicity at 1000 μg/plate and higher (without metabolic act.) and at 2500 μg/plate and higher (with metabolic act.).
Vehicle 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
Remarks:
In tester strain TA102 toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation).
Vehicle controls validity:
valid
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:
In experiment I, toxicity at concentrations of 2500 μg/plate and higher (without metabolic act.). In experiment II, toxicity was noted at 100 μg/plate and higher (without metabolic act.) and at 1000 μg/plate and higher (with metabolic act.).
Vehicle 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:
cytotoxicity
Remarks:
In experiment I, toxicity at concentrations of 1000 μg/plate and higher (with and without metabolic act.). In experiment II, toxicity was noted at 100 μg/plate and higher (without metabolic act.) and at 1000 μg/plate and higher (with metabolic act.).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Cocoyl Proline CAS 960241-19-4 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:

Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Experiment II:
1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

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

Toxic effects of the test item were noted in most tester strains evaluated in experiment I and II.
In experiment I toxic effects of the test item were observed in tester strains TA98, TA100 and TA1535 at concentrations of 1000 μg/plate and higher (with and without metabolic activation). In tester strain TA1537 toxic effects of the test item were noted at concentrations of 2500 μg/plate and higher (without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations of 1000 μg/plate and higher (without and with metabolic activation). In tester strain TA100 toxic effects were noted at concentrations of 1000 μg/plate and higher (without metabolic activation) and at concentrations of 2500 μg/plate and higher (with metabolic activation). In tester strains TA1535 and TA1537 toxic effects of the test item were noted at concentrations of 100 μg/plate and higher (without metabolic activation) and at concentrations of 1000 μg/plate and higher (with metabolic activation). In tester strain TA102 toxic effects of the test item were observed at concentrations of 1000 μg/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1535 at a concentration of 31.6 μg/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Cocoyl Proline CAS 960241-19-4 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. Only in experiment II, in tester strain TA1535 (with metabolic activation) a low mutation factor was found (2.2). Nevertheless, compared to the mutation factors found with the test item concentrations the increase can be considered as distinct. Thus, this effect was regarded as not biologically relevant.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Cocoyl Proline CAS 960241-19-4 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Cocoyl Proline CAS 960241-19-4 is considered to be non-mutagenic in this bacterial reverse mutation assay.