2-Pentanone-4methyl-,O,O’,O”-(ethenylsilylidyne)trioxime

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:

3 May to 26 May, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD 471, OECD 472; EU B.14, EU B.13. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-860-7

EC Name:

-

Cas Number:

156145-64-1

Molecular formula:

C20H39N3O3Si

IUPAC Name:

(4E,9E)-7-ethenyl-2,4,10,12-tetramethyl-7-{[(E)-(4-methylpentan-2-ylidene)amino]oxy}-6,8-dioxa-5,9-diaza-7-silatrideca-4,9-diene; (4Z,9E)-7-ethenyl-2,4,10,12-tetramethyl-7-{[(E)-(4-methylpentan-2-ylidene)amino]oxy}-6,8-dioxa-5,9-diaza-7-silatrideca-4,9-diene; (4Z,9Z)-7-ethenyl-2,4,10,12-tetramethyl-7-{[(E)-(4-methylpentan-2-ylidene)amino]oxy}-6,8-dioxa-5,9-diaza-7-silatrideca-4,9-diene; (4Z,9Z)-7-ethenyl-2,4,10,12-tetramethyl-7-{[(Z)-(4-methylpentan-2-ylidene)amino]oxy}-6,8-dioxa-5,9-diaza-7-silatrideca-4,9-diene

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): OS-2200 (489-95A).
- Physical state: Clear to light yellow liquid.
- Analytical purity: >92%.
- Lot/batch No.:38659-14.
- Expiration date of the lot/batch: 1 January 1996.
- Storage condition of test material: room temperature in the dark under nitrogen.

Method

Target gene:

Histidine or tryptophan gene.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: E. coli WP2 uvrA trp

Metabolic activation:

with and without

Metabolic activation system:

liver preparations (S9 mix) from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0 (control), 321.5, 625, 1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: Test item was fully miscible in acetone.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Controls 2

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA 1535, TA 100, WP2 uvrA

Controls 3

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Controls 4

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

2-nitrofluorene

Remarks:

TA 98

Controls 5

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation) (Main test and confirmation test)
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S9 mix or 0.5 mL 0.1 M phosphate buffer (at pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 mL of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.

DURATION
-Exposure duration: 3 days at 37ºC.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Reductions in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
a)If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b)If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
-If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a and b, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
-Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph a), the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
-If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989)

Statistics:

No statistical analysis.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

RANGE-FINDING/SCREENING STUDIES: No toxicity was observed in the preliminary test study up to 5000 µg/plate test item. Therefore 5000µg/plate was chosen as the top dose level in the mutation tests.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean reverntan colonies*:

Test 1:

Material	Concentrat. (µg/plate)	Without metabolic activation					With metabolic activation
		Base pair exchange type			Frameshift type		Base pair exchange type			Frameshift type
		TA100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA100	TA 1535	WP2 uvrA	TA 98	TA 1537
Solvent control		121	17	70	24	13	121	16	69	23	14
OS-2200	5000.0	100	15	39	21	14	111	19	50	24	8
	2500.0	117	14	58	22	13	131	13	64	26	11
	1250.0	118	14	62	19	9	101	14	62	25	11
	625.0	132	11	75	23	11	112	15	66	27	11
	312.5	120	14	61	20	10	119	13	63	26	12
Negative control	0.0	120	13	69	23	10	139	14	72	21	10
Positive control	EENG	355	210	745	-	-	-	-	-	-	-
	NF	-	-	-	191	-	-	-	-	-	-
	9 AC	-	-	-	-	X	-	-	-	-	-
	AA	-	-	-	-	-	354	147	246	106	70

Test 2:

Material	Concentrat. (µg/plate)	Without metabolic activation					With metabolic activation
		Base pair exchange type			Frameshift type		Base pair exchange type			Frameshift type
		TA 100#	TA1535#	WP2uvrA#	TA 98#	TA1537#	TA100#	TA1535#	WP2uvrA#	TA98#	TA1537#
Solvent control		108	12	66	26	10	120	14	75	24	8
OS-2200	5000.0	109	12	53	25	7	120	13	47	14	10
	2500.0	115	11	67	20	8	107	14	81	23	6
	1250.0	85	12	58	20	6	133	18	77	17	7
	625.0	96	13	63	29	6	128	12	76	15	6
	312.5	115	9	66	24	10	120	11	81	20	9
Negative control	0.0	112	9	68	27	8	129	21	90	15	5
Positive control	EENG	312 401$	200	567	-	-	-	-	-	-	-
	NF	-	-	-	181	-	-	-	-	-	-
	9 AC	-	-	-	-	X	-	-	-	-	-
	AA	-	-	-	-	-	205	109	311	77	50

X Too many colonies to count accurately
ENNG N-Ethyl-N’-nitro-N-nitrosoguanidine
9 AC 9-Aminoacridine
NF 2-Nitrofluorene
AA 2-Aminoanthracene
* Values are the mean of 3 plates
# S-9 data obtained from a separate experiment due to a contamination
$ Additional control data obtained from the separate S-9 mix experiment

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with OS-2200 (489-95A) at any dose level, in the presence or absence of S-9 mix, in either mutation test.

Applicant's summary and conclusion

Conclusions:

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

OS-2200 when tested in acetone shows no evidence of mutagenic activity in the described bacterial system.

Executive summary:

An in-vitro bacterial reverse assay (Ames test) was performed on OS-2200 in accordance with OECD Guideline 471, 472 and EU methods B.13/14 with histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA). In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. Therefore, the dose levels tested in the main test were 5000, 2500, 1250, 625, 312.5 µg/plate. Two independent mutation tests were performed, in the presence md absence of liver preparations from Aroclor 1254-induced rats. Negative controls, solvent controls and positive controls were included in each test. No signs of toxicity were observed towards the tester strains in either mutation tests following exposure to OS-2200 . No evidence of mutagenic activity was seen at any concentration of OS-2200 in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It was concluded that OS-2200 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.