Propanoic acid, 2-hydroxy-, C12-15-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP applied.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late
exponential or early stationary phase of growth (approx. 109 cells/mL). A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.
- Properly maintained: yes

Additional strain / cell type characteristics:

other: All the strains contains mutations in the histidine operon, the deep rough mutation

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Experiment I (plate incorporation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate ( for TA 98 and TA 102 with and without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate ( for TA 100 without metabolic activation and for TA 1535 and TA 1537 with and without metabolic activation)

Experiment II (preincubation test): 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 with metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for T98 without metabolic activation)
0.000316, 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate (for TA 100, TA 1535 and TA 1537 with metabolic activation)
0.0000316, 0.0001000, 0.000316, 0.001000, 0.00316, 0.0100, 0.0316 and 0.100 µL/plate (for TA 100, TA 1535 and TA 1537 without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (for TA 102 with and without metabolic activation).

Vehicle / solvent:

The test material was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

Purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

STRAIN CHARACTERISTICS:
- TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
- TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
- TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
- TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
- TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions
Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA.
Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland.
They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 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.
- Exposure duration: at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DATA RECORDING:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "B" 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 (indicated as "N" in the results tables).

Evaluation criteria:

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 TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control [11].

Statistics:

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.

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S.typhimurium TA 100, TA 1535, TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

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

Remarks on result:

other: other: all strains

Remarks:

Migrated from field 'Test system'.

The results shows that precipitation of the test item was observed in tester strains TA 98 and TA 102 in experiment I at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment II precipitation of the test item was found in tester strain TA 102 at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 0.00100 μL/plate and higher (without metabolic activation) and at concentrations of 0.100 μL/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were seen at concentrations of 0.00316 μL/plate and higher (without metabolic activation) and at concentrations of 0.0316 μL/plate and higher (with metabolic activation).

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material did not cause gene mutation by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Ceraphyl 41 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

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 typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

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 results show that in experiment I and II, the toxic effects of the test item were observed in tester strain (except strain TA 102) with and without metabolic activities depending on the particular tester strains and concentration of the test material (see the section of any other information on results inc tables).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment..

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

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) usingSalmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

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 results show thatin experiment I and II, the toxic effects of the test item were observed in tester strain (except strain TA 102) with and without metabolic activities depending on the particular tester strains and concentration of the test material (see the section of any other information on results inc tables).No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment..

Justification for selection of genetic toxicity endpoint
Key study with Ceraphyl 41.

Justification for classification or non-classification

The test material did not cause gene mutation by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Ceraphyl 41 is considered to be non-mutagenic in this bacterial reverse mutation assay.