Lecithins, acetylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 2000/32/EC Annex 4D–B.13/14 Number L 136

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lecithins, acetylated

EC Number:

293-316-5

EC Name:

Lecithins, acetylated

Cas Number:

91053-50-8

Molecular formula:

Not applicable- complex UVCB substance

IUPAC Name:

91053-50-8

Details on test material:

Purity: Unknown variable composition biological substance (UVCBS)
Composition of test material, percentage of components: Unknown variable composition biological substance (UVCBS)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

-For the toxicity-mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate
-For the mutagenicity test: 333, 667, 1000, 3333, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hexane
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Hexane

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene (TA98 +S9); 4-nitroquinoline N-oxide (WP2uvrA -S9); acridine mutagen ICR-191 (TA1537 -S9); sodium azide (TA100 and TA1535 -S9); 2-aminoanthracene (TA100, TA1535, TA1537, and WP2uvrA +S9); 2-nitrofluorene (TA98 -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: The plate incorporation method was applied. In the non-activated assays, 0.5 mL of sham mix and 100 µL of vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain. The test substance dilutions and controls were allowed to flash off in the pre-heated tubes for 10 minutes prior to the addition of the bacterial tester strains. In the S9-activated assays, 100 µL of the vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain and 0.5 mL of S9 mix. The test substance dilutions and controls were allowed to flash off in the pre-heated tubes for 10 minutes prior to the addition of the bacterial tester strains. All mixtures were vortexed and overlaid onto the surface of minimum glucose agar plates. After the overlay solidified, the plates were inverted and incubated for approximately 49-50.5 hours at 37 ± 2°C. Plates that were not evaluated immediately following incubation were stored at approximately 4°C. All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 trials with 3 treatments per concentration

SCORING
The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered as signs of toxicity. Revertant colonies were counted with an automated colony counter. Plates that could not be accurately counted automatically were counted manually.

Evaluation criteria:

-Tester Strain Integrity: To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrA and uvrB mutations, all tester strains cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the pKM101 plasmid, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin.
-Tester Strain Culture Density: To ensure that appropriate numbers of bacteria are plated, all tester strain culture densities must be approximately 1E9 cells per millilitre.
-Negative Control Values : The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions. The acceptable ranges for the mean values of negative controls are as follows:TA98(8-60), TA100(60-240), TA1535(4-45), TA1537(2-25), WP2uvrA( 5-60).
-Positive Control Values: Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative (vehicle) control value for each tester strain.
-Toxicity: A minimum of 3 non-toxic scorable dose levels were required to validate the study. A dose level was considered toxic if it caused: A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibited a dose-dependent drop in the revertant count, or a reduction in the background lawn. In the event that less than 3 non-toxic dose levels were achieved, the affected portion of the test was repeated with an appropriate change in dose levels.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity- Mutation Test
In the toxicity-mutation test, the maximum dose evaluated was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. A >50% reduction in mean number of revertants was observed at 1000 and 66.7 µg/plate for TA1537 with S9 activation; however, this reduction occurred at intermediate dose levels with no dose related correlation. Test substance precipitation was observed starting at 3333 µg/plate in both the non-activated and activated testing system.
Mutagenicity Test
Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 µg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 1000 µg/plate in the non-activated testing system in 2/3 plates with tester strain TA100 and 1/3 plates with tester strain TA1537. For all remaining in all tester strains both with and without S9 activation the test substance precipitation was observed starting at 3333 µg/plate.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Executive summary:

The test substance, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method.  Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance. Hexane was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance formed a hazy amber solution in hexane at 50 mg/mL, the highest stock concentration that was prepared for use on this study. In the toxicity-mutation test, the maximum dose evaluated was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3333 µg/plate in both the non-activated and activated testing system. Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 µg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 1000 and 3333 µg/plate. All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.