3,4-dihydroxy-1-[(Z)-octadec-9-enyl]pyrrolidine-2,5-dione

Genetic toxicity: in vitro

Administrative data

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:

21 November 2008 and 10 December 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a guideline and used GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 19/08/2008. Date of signature 04/03/2009.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Substance type: organic
- Physical state: waxy tan solid
- Analytical purity: >99%
- Impurities (identity and concentrations): Heptane 0.007%, 2,3-dihydroxybutanedioc acid, bis oleyl amine < 1%.
- Lot/batch No.: 55-L-1434-F
- Stability under test conditions: stable
- Storage condition of test material: room temperature in the dark

Method

Target gene:

Not required

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Histidine dependant

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Remarks:

Histidine dependant

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was a complex mixture, insoluble in water, and DMSO was selected as the solvent because the test material was readily soluble in it at the required concentration.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) for WP2uvrA, TA100 and TA1535; 9-Aminoacridine (9AA) for TA1537; 4-Nitroquinoline-1-oxide (4NQO) for TA98; With S9-mix: 2-Aminoanthracene (2AA) for TA100, TA1535 and TA1537, Benzo(a)pyrene for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method: Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptohan supplemented top agar at 45 °C, 0.1 mL of the test material formulation or vehicle control and either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each tube were mixed and equally distributed onto the surface of the agar plates (one tube per plate).
The plates were then incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.
A range-finding study was also performed.

DURATION
- Preincubation period: Overnight sub-cultures of the appropraite coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours.
- Exposure duration: Approxiamtely 48 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable


SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable


NUMBER OF REPLICATIONS: Three replicates of each bacterial strain, each concentration of the test material with and without S9 mix and each control plate were prepared.


NUMBER OF CELLS EVALUATED: Cell viability at the end of pre-culture was in the range of 1 ~ 9.9x 10^9 bacterial per ml.


DETERMINATION OF CYTOTOXICITY
- Method:: Not applicable.


OTHER EXAMINATIONS: Not applicable.
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER: Prior to being used, characterisation checks were carried out on the bacterial strains to determine the amino-acid requirement, presence of rfa, R factors, urvB mutation and the spontaneous reversion rate.
A preliminary study was performed in order to select the appropriate dose levels for use in the main study using the bacterial strains TA100 and WP2uvrA. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical methods recommended by the UKEMS.

Results and discussion

Test results

Test results 1

Species / strain:

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

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: None - the results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
- Effects of pH: No effect on pH.
- Effects of osmolality: No effect on osmolality.
- Evaporation from medium: No data.
- Water solubility: Insoluble
- Precipitation: A particulate precipitate was observed at and above 1500 ug/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA"), 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 ug/plate because of excessive test material precipitation.
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA} The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results for negative, vehicle and positive control were within the expected range as comparing to historical profile.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Non-toxic to any of the bacterial strains, at any dose level either with or without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9­mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	120	112	108	113	98	86	109	102	95	119P	101P
+	TA100	82	115	111	98	97	89	87	102	102	95P	89P
-	WP2uvrA"	19	25	19	14	23	23	14	15	15	15P	17P
+	WP2uvrA"	21	16	23	22	25	26	23	19	23	25P	29P

 

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4.

A history profile of vehicle and positive control values is presented in Appendix 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000mg/plate. A particulate precipitate was observed at and above 1500mg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

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

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLWand MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Council Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A particulate precipitate was observed at and above 1500 ug/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

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