2-Propenoic acid, 2-methyl-, tris(1-methylethyl)silyl ester

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:

20 July to 30 August 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent study conducted by a GLP certified laboratory in accordance with a suitable study guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

TIS-M

IUPAC Name:

TIS-M

Details on test material:

- Name of test material: TIS-M
- Lot/batch No.: 3301 MS-0505-4
- Storage condition of test material: Under nitrogen at room temperature, in the dark.

Method

Target gene:

Three mutant strains of Salmonella are incapable of synthesising histidine and are dependant on an external source of this particular amino acid for normal growth. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independant forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of the organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substition in the genetic material (miscoding) or a frame-shift mutation in which the genetic material is inther added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB- Salmonella strains) which renders the organism incapble of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of Escherichia coli (WP2uvrA-) , which requires tryptophan and which can be reverse mutated by base substition to trytophan independence was used to complement Salmonella strains. This strain also has a deletion in an excision repair gene (uvrA-). Since many compounds do not exert a mutagenic effect until this have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria also incubated in the prescence of a (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Incapable of synthesising histidine.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Incapable of synthesising histidine.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal preparation S9-mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main Test: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Information from sponsor advised against the use of sterile distilled water.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

2-Aminoanthracene; Benzo(a)pyrene

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-nitrosoguanidine (ENNG); 9-Aminoacridine; 4-Nitroquinoline-1-oxide.

Details on test system and experimental conditions:

Tester strains:
The Salmonella strains were obtained from the University of California at Berkley on culture discs on August 4 1995 whilst Escherichia coli strain WP2uvra- was obtained from the British Industrial Biological Research Association on 17 August 1987. All strains were sotred at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, prescence of rfa, uvrB or uvrA mutation and the spontaneous reversion rate. In this assay, overnight subcultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Ltd; lot no: 350536 06/09) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Preparation of Test and Control Materials:
The test material was immiscible in dimethyl sulphoxide at 50mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Acetone was used as the vehicle. The test material was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Analysis for concentration, homogenicity and stability of test material formualtions was not a requirement for the guideline so it was not carried out. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e. 2mm pellets with a nominal pore diameter of 4x10^-4 microns.Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive contol and positive control materials used in a series of plates without S9 -mix were as follows:

-N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3µg/plate for TA100, 5µg/plate for TA1535 and 2µg/plate for WP2uvrA-.
-9 -Aminoacridine (9AA): 80µg/plate for TA98

In addition, 2 -Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, which were used in the series of plates with S9 -mix at the following concentrations:
-2AA at 1µg/plate for TA100
-2AA at 2µ/plate for TA1535 and TA1537
-BP at 5µg/plate for TA98.
-2AA at 10µg/plate for WPuvrA-

Microsomal enzyme fraction:
S9 was prepared in-house on 15 May 2005 (preliminary toxicity and range finding tests) and 10 July 2005 (main test) from the livers of male Sprague-dawley rats weighing ~ 250g. These had each orally recieved three consecutive daily doses of phenorbitone/β-napthoflavone (80/100 mg per kg per day) prior to S9 preparation on day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames test.

S9 -Mix and Agar:
The S9 -mix was prepared immediately before using sterilised co-factors and maintained on ice for the duration of thes test.
S9 - 5.0ml
1.65 M KCl/0.4M MgCl2 - 1.0ml
0.1M Glucose-6 -phosphate - 2.5ml
0.1M NADPH - 2.0ml
0.1M NADH - 2.0ml
0.2 M Sodium phosphate buffer (pH 7.4) - 25.0ml
Sterile distilled water - 12.5ml

A 0.5ml aliquot of S9 -mix and 2ml of molten, trace histidine or trytophan supplemente, top agar were overlaid onto a steri;e Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9 -mix. This procedure was repeated, in triplicate, on the day of each experiment. Top agar was prepared using 0.6% Difco Bacto agar (lot no: 4348296 09/09) and 0.5% sodium chloride with 5ml of 1.0mM fistidine and 1.0mM biotin or 1.0mM trytophan solution added to each 100ml of top agar. Vogel-Bonner Minimal agar plates purchased from ILS Ltd. (Lot nos: 899855 -02 01/10 and 903393-02 02/10).

Method: Preliminary toxicity test:
The aim of the test was to select appropriate dose levels for the main test. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate. 0.1ml of the bacterial culture (TA100 or WP2uvrA-), 2ml of molten trace histidine or tryptophan supplemented, top agar, 0.1ml of the test material formulation and 0.5ml of S9 -mix or phosphate buffer were mixed, and were applied to sterile plates of Vogel Bonner Minimal agar (30ml/plate). 10 concentrations of the test material formulation and a vehicle control (acetone) were tested. 0.1ml of the maximum concentration of the test material and 2ml of molten, trace histidine or tryptophan supplemented, top agar were also overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48hrs 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.

Method: Main Test: Experiment 1:
Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels (50, 150, 500, 1500 and 5000µg/plate), in triplicate, both with and without the addition of rat liver homogenate metabolising system (10% liver S9 in standard co-factors). Measured aliquots (0.1ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1ml of the test formulation, vehicle or positive control, as well as either 0.5ml of S9 -mix or phosphate buffer.The dose range was determined in a preliminary toxicity assay and was 50 to 500µg/plate in the first experiment. The contents of each test tube were mixed and evenly distributed onto Vogel-Bonner Minimal agar plates (one tube per plate). Incubation was at 37 °C and lasted for 48hrs, after which the frequency of revertant colonies was assessed using a Domino colony counter.

Method: Main Test: Experiment 2:
The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test formulations.

Acceptance Criteria:
The reverse mutation assay may be considered to be valid if the following criteria are met:
-All tester strain cultures exhibit a characteristic noumber of spontaneous revertants per plate in the vehicle and untreated controls.
-The appropriate characteristics for each tester strain have been confirmed e.g. 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 positive control value should be at least twice the respective vehicle control for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9 -mix.
-There should be a minimum of 4 non-toxic material dose levels.
-There should be no evidence of excessive contamination.


METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: no data
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: 3 per concentration/vehicle control/positive and negative control

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range testes and/ora reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevence of the results will be considered first, statistical methods, as recommended by UKEMS can also be used to aid evaluation (not only determing factor). A testmaterial will be considered non-mutagenic (negative) if the test system if the above criteria are not met.

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:

See below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Remarks on results:

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was therefore tested up the maximum recommended dose level of 5000µg/plate. An oily precipitate was obseved at 5000µg/plate but this did not prevent the scoring of revertant colonies. no significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, 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.

Table 1: Preliminary toxicity test:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	86	145	131	121	87	112	117	132	147	126	117P
+	TA100	101	82	86	82	77	89	99	106	120	92	98P
-	WP2uvrA-	24	24	22	24	19	20	18	27	23	13	20P
+	WP2uvrA-	41	36	33	29	29	33	38	36	41	37	37P

Table 2: Main study: Spontaneuous Mutation Rates (Concurrent Negaative Controls): Experiment 1:

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA-	TA98	TA1537
71	15	21	13	2
60                (72)	11                (14)	28               (24)	21                (18)	8                   (6)
85	15	22	18	8

Table 2: Main study: Spontaneuous Mutation Rates (Concurrent Negaative Controls): Experiment 2:

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA-	TA98	TA1537
90	29	27	18	14
110            (103)	30               (28)	18               (22)	13               (18)	6                   (8)
109	24	22	22	5

Table 3: Main Study: Test Results: Experiment 1 - Without Metabolic Activation:

Test period		From: 16 August 2005			To: 19 August 2005
With or without S9-Mix	Test substance concentration (µg)	Number of revertants (mean number of colonies per plate)
		Base Pair Substitution type			Frameshift type
		TA100	TA1535	WP2uvrA-	TA98	TA1537
-	0	89	14	18	25	11
		86     (86)	10    (13)	33     (23)	16    (20)	9      (10)
		82      3.5*	15   2.6	19      8.4	18    4.7	11    1.2
-	50	102	13	19	27	9
		91     (93)	10   (14)	16     (20)	19     (24)	14     (11)
		86     8.2	18   4.0	25     4.6	25     4.2	10     2.6
-	150	97	13	31	14	8
		101   (99)	10    (15)	19     (23)	14      (13)	13     (11)
		98      2.1	23    6.8	18      7.2	11      1.7	13     2.9
-	500	81	11	18	24	8
		104   (101)	11     (12)	22    (21)	23     (22)	14      (9)
		117    18.2	15     2.3	23   2.6	18     3.2	6        4.2
-	1500	105	14	17	21	13
		110   (106)	26    (19)	25    (19)	32      (28)	15     (12)
		102    4.0	17    6.2	15    5.3	32      6.4	9        3.1
-	5000	87P	15P	28P	24P	8P
		101P (103)	10P   (15)	28P   (26)	21P     (25)	8P       (9)
		121P  17.1	19P   4.5	22P   3.5	29P      4.0	10P     1.2
Positive Controls   S9-Mix -	Name Concentration (µg/plate) No. colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		283 225   (261) 276     31.7	136 134   (138) 145      5.9	521 452   (466) 424    49.9	162 123   (147) 156    21.0	634 296   (434) 372   177.3

- ENNG = N-ethyl-N'-nitro-N-nitrosguanidine

- 4NQO = 4 -Nitroquinoline-1 -oxide

- 9AA = 9 -Aminoacridine

- P = precipitate.

- * = standard deviation

Table 4: Main Study: Test Results: Experiment 1 - With Metabolic Activation:

Test period		From: 16 August 2005			To: 19 August 2005
With or without S9-Mix	Test substance concentration (µg)	Number of revertants (mean number of colonies per plate)
		Base Pair Substitution type			Frameshift type
		TA100	TA1535	WP2uvrA-	TA98	TA1537
+	0	92	13	26	23	11
		70    (79)	13    (11)	26     (29)	26    (27)	11    (11)
		74     11.7*	8      2.9	34      4.6	31    4.0	11    0.0
+	50	64	8	20	33	13
		82     (77)	9       (9)	23     (22)	28     (27)	10     (10)
		86     11.7	9       0.6	24     2.1	19     7.1	8      2.5
+	150	67	9	18	28	11
		70     (71)	13    (13)	22     (22)	19      (22)	18     (14)
		77      5.1	17    4.0	26      4.0	20      4.9	13      3.6
+	500	96	11	25	34	10
		97     (94)	16     (12)	36    (30)	33     (33)	9        (11)
		88      4.9	8       4.0	28    5.7	33     0.6	14       2.6
+	1500	102	14	21	29	4
		85       (98)	10    (11)	28    (27)	20      (23)	5       (6)
		107    11.5	10    2.3	32    5.6	20      5.2	10     3.2
+	5000	91P	16P	36P	32P	11P
		105P  (94)	11P   (14)	25P   (31)	33P    (35)	13P    (12)
		86P   9.8	14P   2.5	31P   5.5	39P    3.8	111P     1.2
Positive Controls   S9-Mix +	Name Concentration (µg/plate) No. colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		1024 713   (951) 1117 211.6	170 176   (174) 176   3.5	237 255   (251) 260   12.1	126 118   (115) 101   12.8	258 113  (177) 160  74.0

- 2AA = 2 -Aminoanthracene

- BP = Benzo(a)pyrene

- P = Precipitate

- * = Standard Deviation

Table 5: Main Study: Test Results: Experiment 2 - Without Metabolic Activation:

Test period		From: 26 August 2005			To: 29 August 2005
With or without S9-Mix	Test substance concentration (µg)	Number of revertants (mean number of colonies per plate)
		Base Pair Substitution type			Frameshift type
		TA100	TA1535	WP2uvrA-	TA98	TA1537
-	0	104	18	32	19	14
		102   (101)	19    (19)	22     (25)	20    (20)	14    (12)
		98      3.1*	21    1.5	21      6.1	22    1.5	9      2.9
-	50	96	20	26	16	2
		95       (96)	18       (17)	24     (24)	22     (20)	11     (11)
		100    2.6	14       3.1	23     1.5	23     3.8	11     0.6
-	150	76	14	29	23	9
		106     (98)	14    (15)	22     (29)	25      (22)	10     (10)
		112     19.3	16     1.2	35      6.5	19      3.1	10      0.6
-	500	91	14	21	16	8
		95       (99)	16     (16)	25    (25)	18     (19)	9        (9)
		112     11.2	19      2.5	30    4.5	22     3.1	11       1.5
-	1500	86	16	16	22	9
		99       (94)	15    (16)	29    (22)	18      (20)	9       (9)
		98      10.8	16    0.6	21     6.6	21      2.1	10     0.6
-	5000	87P	13P	25P	19P	13P
		104P  (99)	18P   (15)	18P   (22)	16P    (19)	8P    (10)
		107P   10.8	15P   2.5	23P   3.6	23P    3.5	9P     2.6
Positive Controls   S9-Mix -	Name Concentration (µg/plate) No. colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		491 406   (437) 414    46.9	211 196   (280) 434   133.3	710 605   (664) 678    53.8	173 191   (200) 236   32.4	733 214  (404) 264   286.3

- ENNG = N-ethyl-N'-nitro-N-nitrosguanidine

- 4NQO = 4 -Nitroquinoline-1 -oxide

- 9AA = 9 -Aminoacridine

- P = precipitate.

- * = standard deviation

Table 6: Main Study: Test Results: Experiment 2 - With Metabolic Activation:

Test period		From: 26 August 2005			To: 29 August 2005
With or without S9-Mix	Test substance concentration (µg)	Number of revertants (mean number of colonies per plate)
		Base Pair Substitution type			Frameshift type
		TA100	TA1535	WP2uvrA-	TA98	TA1537
+	0	87	12	36	23	20
		77      (88)	12    (12)	38     (39)	41    (32)	21    (21)
		100   11.5*	12     0.0	44      4.2	31    9.0	23     1.5
+	50	93	12	31	24	16
		85       (96)	10      (11)	31     (35)	34     (80)	18     (17)
		87       4.2	11       1.0	42     6.4	27     5.1	18     1.2
+	150	75	13	32	34	15
		79     (82)	12    (12)	30    (30)	36      (34)	16     (17)
		92     8.9	12     0.6	29      1.5	31      2.5	19      2.1
+	500	98	12	30	37	14
		91       (95)	11     (12)	41    (35)	36     (34)	18      (16)
		95       3.5	13      1.0	35    5.5	30     3.8	15       2.1
+	1500	89	12	37	35	16
		88       (93)	10    (11)	34    (36)	30      (30)	19       (18)
		102      7.8	10     1.2	36     1.5	26      4.5	20       2.1
+	5000	98P	15P	38P	31P	20P
		87P    (95)	12P   (13)	35P   (39)	27P    (30)	22P    (21)
		99P    6.7	11P   2.1	44P   4.6	33P    3.1	21P     1.0
Positive Controls   S9-Mix +	Name Concentration (µg/plate) No. colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		1218 1061(1083) 971  125.0	246 196   (209) 186    32.1	631 611   (664) 567    53.8	187 201   (187) 174   13.5	374 306  (326) 297   42.1

- ENNG = N-ethyl-N'-nitro-N-nitrosguanidine

- 4NQO = 4 -Nitroquinoline-1 -oxide

- 9AA = 9 -Aminoacridine

- P = precipitate.

- * = standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The registered substance was determined to be non-mutagenic.

Executive summary:

A genetic study of the substance was carried out according to EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) using Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvr A.

The registered substance and the bacteria were applied to agar and after 3 days the substance was determined to be non-mutagenic.