Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, Current Step 4 version dated November 9, 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

EC Number:

270-279-3

EC Name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Cas Number:

68424-19-1

Molecular formula:

Not applicable as the substance is a UVCB

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18-unsatd., compds. with triethanolamine.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine
- Physical state: Viscous, translucent yellowish waxy paste
- Analytical purity: >95%
- Impurities (identity and concentrations):See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: 01/2013
- Lot/batch No.: DDR02/91
- Expiration date of the lot/batch: October 2014
- Storage condition of test material: At +10° to +25°C
- Other: Manufacturer/Supplier: OLEON NV, Assenedestraat 2, 9940 Ertvelde, Belgium

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
Main test: 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was completely dissolved in dimethyl sulfoxide (DMSO) .

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide in aqua ad iniectabilia

Remarks:

10 µg/plate TA 1535, TA 100 without S9

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene in DMSO

Remarks:

10 µg/plate TA 98 without S9

Controls 3

Positive controls:

yes

Positive control substance:

other: Mitomycin in DMSO

Remarks:

10 µg/plate TA 102 without S9

Controls 4

Positive controls:

yes

Positive control substance:

other: 9-amino-acridine in ethanol, abs.

Remarks:

100µg/plate TA 1537 without S9

Controls 5

Positive controls:

yes

Positive control substance:

other: benzo(a)pyrene in DMSO

Remarks:

10 µg/plate TA 98, TA 102, TA 1537 with S9

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene in DMSO

Remarks:

2 µg/plate TA 100, TA 1535 with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st experiment: in agar (plate incorporation)
2nd experiment: preincubation

DURATION
1st experiment:
- Exposure duration / Selection time: 48 to 72 hours

2nd experiment:
- Preincubation period: 20 minutes
- Exposure duration / Selection time: 48 to 72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Evaluation criteria:

In the laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA98, TA100 and TA102 and 3-fold of the vehicle control for TA1535 and TA1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.
- Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Statistics:

The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance.
The Spearman's rank correlation coefficient may be applied.

Results and discussion

Test results

Species / strain:

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

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
History Profile of Negative and Positive Control Values (n = 37 studies) of the year 2012 is provided in the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains. The reduction of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test without metabolic activation at concentrations of 31.6 and 1000 µg test item/plate and in the preincubation test with metabolic activation at 1000 and 3160 µg/plate is considered to be caused by the high variation in individual counts and not due to cytotoxicity.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the present test conditions the Fatty acids, C16-18 and C18-unsatd., compds. with triethanolaminetested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved in sulfoxide (DMSO). DMSO was used as vehicle control.

Preliminary test

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

 

In conclusion, under the present test conditions the Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.