Acetic acid, 2-(ethylamino)-2-oxo-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro study: Bacterial reverse mutation assay /Ames test (equivalent or similar to EU method B.13/14): negative

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:

2008-02-15 to 2008-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Minor deviation from guideline: - justification for choice of vehicle was not clearly provided. It was only stated that the vehicle was selected based on information given by the sponsor.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008-05-31

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-10-15

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

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 prepared from the livers of male Sprague-Dawley rats, which had received phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
- all strains without S9: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- all strains with S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Additional dose levels (5 and 15 µg/plate were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Experiment 2:
- all strains without S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
- all strains with S9: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
An additional dose level (15 µg/plate) was again included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethylsulphoxide was therefore selected as the vehicle.

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment.
Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 X 10°-4 microns.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation; Strains TA100 and TA1535

Migrated to IUCLID6: 3 µg/plate for TA100 and 5 µg/plate for TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic acitivation; strain TA1537

Migrated to IUCLID6: 80 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; strain TA102

Migrated to IUCLID6: 0.5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation; strain TA98

Migrated to IUCLID6: 0.2 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- Aminoanthracene; 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537

Remarks:

with metabolic activation, strain TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation; strain TA98

Migrated to IUCLID6: 5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-dihydroxyanthraquinone; 10 µg/plate

Remarks:

with metabolic activation; strain TA102

Details on test system and experimental conditions:

Experiment 1 and 2:
METHOD OF APPLICATION: in agar (plate incorporation)
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

DURATION
- Exposure duration: all of the plates were incubated at 37°C for approximately 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the frequency of revertant colonies were assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
A preliminary test 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 µg/plate.The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). ten doses 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 supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately48 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.

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are present in the "Attached background material" below with historical control ranges.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 X 10^9 bacteria per mL.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges are presented in "Attached background material" below.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

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.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

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

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material was toxic at and above 1500 and at 5000 µg/plate, in the absence and presence of S9, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test material was toxic to the strain of Salmonella used (TA100) at and above 1500 and at 5000 µg/plate, in the absence and presence of S9, respectively. The test material formulation and the S9 -mix used in this experiment were both shown to be sterile. The number of revertant colonies for the toxicity can be seen in table 1 in the field "Any other information on results incl. tables" below.

COMPARISON WITH HISTORICAL CONTROL DATA: a history profile of vehicle and positive control value was given in the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawns and/or substantial decrease in the frequency of revertant colonies to all of the tester strains in the presence and absence of S9 at 5000 µg/plate. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

NEGATIVE CONTROL.
Results for the negative controls (spontaneous mutation rates) are presented in in table 2 in the field "Any other information on results incl. tables" below. They were acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1

With (+) or without (-) metabolic activation	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	78	75	80	70	93	88	90	92	84	73S	30SP
+	TA100	96	88	82	87	90	76	89	81	87	98	36SP

SP: Sparse bacterial background lawn

P: Precipitate

Table 2 Spontaneous Mutation Rates (concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	TA102	TA98	TA1537
86 88            (87) 86	16 19                 (18) 20	241 266             (260) 273	25 15                 (19) 17	15 15                 (15) 16

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	TA102	TA98	TA1537
102 146            (124) 123	29 26                 (32) 40	375 309             (352) 371	16 25                (19) 15	15 13                 (14) 15

RESULTS POSITIVE CONTROLS:

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.

Conclusions:

Interpretation of results (migrated information):
negative

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

One reliable in vitro study described in Thompson (2008) (equivalent or similar to EU method B.13/14, GLP complaint) is considered to be reliable with restirctions. The results of the test was negative.

Justification for classification or non-classification

Reference Thompson (2008) is considered as the key study and will be used for classification. The test results was negative. Thus, no classification is required for the time being according to Regulation 1272/2008 and subsequent regulations.