D-tetramethrin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1,3,4,5,6,7-hexahydro-1,3-dioxo-2H-isoindol-2-yl)methyl (1R-trans)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

EC Number:

214-619-0

EC Name:

(1,3,4,5,6,7-hexahydro-1,3-dioxo-2H-isoindol-2-yl)methyl (1R-trans)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

Cas Number:

1166-46-7

Molecular formula:

C19H25NO4

IUPAC Name:

(1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1R,3R)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Constituent 2

Reference substance name:

(1,3,4,5,6,7-hexahydro-1,3-dioxo-2H-isoindol-2-yl)methyl (1R-cis)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

EC Number:

257-144-4

EC Name:

(1,3,4,5,6,7-hexahydro-1,3-dioxo-2H-isoindol-2-yl)methyl (1R-cis)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate

Cas Number:

51348-90-4

Molecular formula:

C19H25NO4

IUPAC Name:

(1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1R,3S)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Constituent 3

Reference substance name:

1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1S,3S)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Cas Number:

1166-48-9

Molecular formula:

C19H25NO4

IUPAC Name:

1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1S,3S)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Constituent 4

Reference substance name:

(1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1S,3R)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Cas Number:

51348-91-5

Molecular formula:

C19H25NO4

IUPAC Name:

(1,3-dioxo-1,3,4,5,6,7-hexahydro-2H-isoindol-2-yl)methyl (1S,3R)-2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropanecarboxylate

Test material form:

solid: crystalline

Details on test material:

the racemate is characterized by CAS-number 7696-12-0

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB,rfa,pKM101,pAQ1 as genetic markers beside histidine deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate

Test concentrations with justification for top dose:

The test item TETRAMETHRIN was found to be soluble in DMSO at a concentration of 300 mg/ml. Since 100 µl of the test item solution are used in the preparation of each plate, this permitted a maximum concentration of 5000 µl/plate to be used in the toxicity test.
No signs of toxicity were observed at any dose-level tested, in any tester strain, in the absence or presence of S9 metabolic activation. On the basis of these results a maximum concentration of 5000 µg/plate was selected for the Main Assay I with all tester strains. Precipitation of the test item was observed at 5000 µg/plate.
Two main experiments were performed.
In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate.
in Main Assay II the same dose-levels used in Main Assay I were employed. A pre-incubation step was included in all treatments with the exception of testing TA100 in the presence of S9 metabolism. With this tester strain the test item was assayed (in the presence of S9 metabolism) under the same experimental conditions employed in Main Assay I.

Vehicle / solvent:

Dimethylsulphoxide (DMSO): (Fluka AG, batch 404161/1 13400).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-Aminoanthracene, sterile distilled water

Details on test system and experimental conditions:

Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose-levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.
Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Main experiments
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
(i) Overlay agar (held at 45 °C) 2 ml
(ii) Test or control item solution 0.1 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml
(iv) Bacterial suspension 0.1 ml

The second experiment was performed using a pre-incubation method with all tester strains with the exception of TA100 in the presence of S9 metabolism: this treatment was, instead, carried out using the plate incorporation method.
In the pre-incubation method the components were added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 ml
(ii) Test item and control solution 0.05 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml

The incubate was vortexed and placed at 37 °C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

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:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In order to confirm the results obtained with TA100, in Main Assay II the test item was assayed, with this tester strain in the presence of metabolic activation, under the same experimental conditions used in Main Assay I. The other treatments of Main Assay II included a pre-incubation step and used the same dose-levels as Main Assay I. Thinning of the background lawn was observed at the highest dose-level in all tester strains in the absence of S9 metabolism.
Increases in revertant colonies were again observed in TA100 in the presence of S9. However, these increases did not reach twice the control value at any dose-level assayed and were not concentration-related over the range tested.
Precipitation of the test item was observed at 5000 and 2500 µg/plate in both experiments.
The sterility of the S9 mix and the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Remarks on result:

other: without pre-incubation

Any other information on results incl. tables

TABLE 1 - WITHOUT METABOLIC ACTIVATION

STUDY NO.: 9324

SOLVENT: DMSO

EXPERIMENT: Toxicity test

Dose-level (µg/plate}	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	TA-102 Rev/pl.
Untreated	18	19	32	131	384
0.00	15	20	25	119	376
50.0	24	22	29	149	399
158	16	19	29	130	406
500	18	14	32	155	397
1580	13	16	29	145	390
5000	18	16	36	177	358

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 2 - WITH METABOLIC ACTIVATION

STUDY NO.: 9324

SOLVENT: DMSO

EXPERIMENT: Toxicity test

Dose-level (µg/plate)	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	TA-102 Rev/pl.
Untreated	12	23	38	159	409
0.00	15	24	37	134	417
50.0	12	21	40	138	407
158	13	22	39	152	447
500	16	17	40	221	470
1580	14	18	42	214	451
5000	12	20	41	229	487

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 3 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA1535                               Titre: 232

Dose-level    Without metabolic activ.   With metabolic activation

[pg/pl]       Plate counts   Mean S. E.       Plate counts      Mean S. E.

Untreated	24	16	19	20	2.3	19	14	16	16	1.5
0.00	20	19	19	19	0.3	20	17	19	19	0.9
313	18	18	22	19	1.3	17	16	19	17	0.9
625	21	16	21	19	1.7	15	13	16	15	0.9
1250	18	25	21	21	2.0	13	13	19	15	2.0
2500	20	24	19	21	1.5	19	15	16	17	1.2
5000	24	27	21	24	1.7	17	13	10	13	2.0

Regression analysis

Points	S9	Intercept	Slope	Corr.coeff.	t	P-value
1  3	-	4.396	0.0000	-0.01667	0.0441	0.96605
1- 4		4.351	0.0002	0.32072	1.0708	0.30944
1-5	-	4.388	0.0001	0.31501	1.1967	0.25279
1-6		4.380	0.0001	0.57959	2.8449	0.01170
1-3	+	4.348	-0.0008	-0.78438	3.3457	0.01232
1- 4	+	4.249	-0.0004	-0.58151	2.2603	0.04734
1-5	+	4.112	-0.0001	-0.21169	0.7809	0.44882
1-6	+	4.132	-0.0001	-0.47471	2.1575	0.04652

Positive and negative controls Treatment

S9         Plate counts             Mean     S. E.

Untreated                                 -            24 16 19                   20         2.3

Sodium Azide 1 pg/pl              -            647 667 626             647       11.8

DMSO           100 pl/pl             +            20 17 19                    19         0.9

2-Aminoanthracene 1 pg/pl    +            126 131 114             124        5.0

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 4 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324 SOLVENT: DMSO

Strain: TA1537                               Titre: 221

Dose-level  Without metabolic activ.    With metabolic activation

[µg/pl]       Plate counts   Mean S. E.       Plate counts  Mean S. E.

Untreated	18	17	20	18	0.9	25	18	25	23	2.3
0.00	18	17	18	18	0.3	19	25	20	21	1.9
313	16	13	15	15	0.9	20	18	20	19	0.7
625	18	21	17	19	1.2	24	18	20	21	1.8
1250	15	14	15	15	0.3	25	19	18	21	2.2
2500	17	15	14	15	0.9	24	24	23	24	0.3
5000	19	15	17	17	1.2	20	21	19	20	0.6

Regression analysis

Points	S9	Intercept	Slope	Corr.        coeff.	t	P-value
1-3		4.059	0.0002	0.17859	0.4802	0.64572
1- 4		4.150	-0.0002	-0.34834	1.1751	0.26716
1-5		4.106	-0.0001	-0.34012	1.3041	0.21483
1-6		4.037	0.0000	-0.01146	0.0458	0.96400
1-3	+	4.551	-0.0001	-0.11856	0.3159	0.76128
1-4	+	4.525	0.0000	-0.01818	0.0575	0.95527
1-5	+	4.461	0.0001	0.42601	1.6978	0.11335
1-6	+	4.557	0.0000	0.04910	0.1966	0.84659

Positive and negative controls

Treatment                                 S9 Plate counts    Mean S.E.

DMSO	100	µl/p1	-	18	17	18	18	0.3
9-Aminoacridine	50	µg/p1	-	167	143	186	165	12.4
DMSO	100	Al/p1	+	19	25	20	21	1.9
2-Aminoanthracene	1	µg/p1	+	96	105	101	101	2.6

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 5 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324 SOLVENT: DMSO

Strain: TA102                                Titre: 269

Dose-level    Without metabolic activ.    With metabolic activation

[µg/pl]       Plate counts   Mean S. E.      late counts   Mean S. E.

Untreated	359	374	386	373	7.8	414	407	395	405	5.5
0.00	398	364	381	381	9.8	387	421	400	403	9.9
313	370	393	362	375	9.3	446	431	429	435	5.4
625	365	376	390	377	7.2	454	456	447	452	2.7
1250	384	392	389	388	2.3	483	472	475	477	3.3
2500	380	396	371	382	7.3	457	421	432	437	10.7
5000	396	366	360	374	11.1	484	479	457	473	8.3

Regression analysis

Points	S9	Intercept	Slope	Corr.        coeff.	t	P-value
1-3	-	19.482	-0.0002	-0.12630	0.3369	0.74609
1- 4	-	19.396	0.0002	0.28166	0.9283	0.37512
1-5	-	19.452	0.0001	0.18155	0.6657	0.51726
1-6		19.513	0.0000	-0.10362	0.4167	0.68241
1-3	+	20.129	0.0019	0.89109	5.1949	0.00126
1  4	+	20.270	0.0013	0.91902	7.3719	0.00002
1- 5	+	20.751	0.0002	0.35048	1.3493	0.20028
1  6	+	20.785	0.0002	0.53459	2.5303	0.02227

Positive and negative controls treatment		S9	Plate counts	Mean	S. E.
DMSO	100 µl/p1	-	398  364   381	381	9.8
Cumene hydroperoxide	100 µg/p1	-	1254 1219 1181	1218	21.1
DMSO	100 µl/p1	+	387 421  400	403	9.9
2-Aminoanthracene	10 µg/pl	+	1528  1496 1581	1535	24.8

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 6 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA98                                    Titre: 242

Dose-level    Without metabolic activ.    With metabolic activation

[µg/pL]       Plate counts   Mean S.E.      Plate counts   Mean S.E.

Untreated	32	31	29	31	0.9	40	37	36	38	1.2
0.00	29	31	27	29	1.2	40	37	38	38	0.9
313	28	30	31	30	0.9	36	41	39	39	1.5
625	31	31	28	30	1.0	47	38	38	41	3.0
1250	29	27	28	28	0.6	40	39	38	39	0.6
2500	30	31	27	29	1.2	36	37	37	37	0.3
5000	30	29	36	32	2.2	40	37	39	39	0.9

Regression analysis

Points	S9	Intercept	Slope	Corr.   coeff.	t	P-value
1-3	-	5.388	0.0001	0.27288	0.7505	0.47744
1-4	-	5.445	-0.0001	-0.27746	0.9132	0.38260
1 -5	-	5.413	0.0000	-0.07646	0.2765	0.78652
1-6	-	5.375	0.0000	0.35336	1.5109	0.15031
1-3	+	6.165	0.0003	0.35163	0.9938	0.35343
1-4	+	6.231	0.0001	0.12475	0.3976	0.69927
1  5	+	6.286	-0.0001	-0.30385	1.1499	0.27089
1-6	+	6.246	0.0000	-0.14440	0.5837	0.56755

Positive and negative

controls Treatment           S9   Plate counts  Mean S. E.

DMSO	100 µl/p1		-	29	31	27	29	1.2
2-Nitrofluorene	2µl/p1		-	197	198	184	193	4.5
DMSO	100   µl/p1		+	40	37	38	38	0.9
2-Aminoanthracene	1  µl/pl		+	574	621	619	605	15.3

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 7 -Experiment I      - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA100                               Titre: 226

[µg/pl]	Plate counts			Mean	S.   E.	Plate counts			Mean	S.E.
Untreated	135	139	127	134	3.5	143	134	139	139	2.6
0.00	123	133	136	131	3.9	135	140	134	136	1.9
313	149	156	143	149	3.8	198	203	213	205	4.4
625	171	176	174	174	1.5	254	272	270	265	5.7
1250	177	189	195	187	5.3	308	294	280	294	8.1
2500	143	154	141	146	4.0	286	268	261	272	7.4
5000	188	187	158	178	9.8	282	300	302	295	6.4

Regression analysis

Points	S9	Intercept	Slope	Corr.coeff.	t	P-value
1-3	-	11.400	0.0028	0.96353	9.5269	0.00003
1-4	-	11.645	0.0018	0.92647	7.7842	0.00001
1-5	-	12.352	0.0002	0.18444	0.6766	0.51051
1-6	-	12.339	0.0002	0.39015	1.6949	0.10946
1-3	+	11.782	0.0074	0.99120	19.8166	0.00000
1-4	+	12.555	0.0042	0.91682	7.2607	0.00003
1-5	+	13.706	0.0016	0.69098	3.4465	0.00434
1-6	+	14.334	0.0007	0.63087	3.2524	0.00500

Treatment	S9	Plate counts	Mean	S.   E.
Untreated	-	135   139  127	134	3.5
Sodium Azide 1 µg/pl	-	954   977  926	952	14.7
DMSO 100 µl/pl	+	135   140  134	136	1.9
2-Aminoanthracene 1 µg/pl	+	1194 1159 1165	1173	10.8

 

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 8 - Experiment II       - Preincubation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA1535, Titre: 231

Dose-level[µg/pl]	Without metabolic activation					With metabolic activation
	Plate counts			Mean	s.e.	Plate counts			Mean	s.e.
Untreated	15	18	20	18	1,5	14	16	12	14	1,2
0.00	19	18	12	16	2,2	17	15	11	14	1,8
313	17	13	12	14	1,5	10	18	19	16	2,8
625	19	17	18	18	0,6	13	19	15	16	1,8
1250	20	19	16	18	1,2	18	11	12	14	2,2
2500	18	15	14	16	1,2	19	11	10	13	2,8
5000	14	15	19 *	16	1,5	12	10	11	11	0,6

Regression analysis:

Points	S9	Intercept	Slope	Corr. Coeff	t	P-value
1 - 3	-	3,888	0,0004	0,25197	0,6889	0,51307
1 - 4	-	3,898	0,0003	0,41431	1,4395	0,18056
1 - 5	-	4,025	0,0000	0,05840	0,2109	0,83623
1 - 6	-	4,045	0,0000	-0,02969	0,1188	0,90689
1 - 3	+	3,792	0,0003	0,17173	0,4612	0,65864
1 - 4	+	3,885	-0,0001	-0,11427	0,3637	0,72362
1 - 5	+	3,887	-0,0001	-0,21189	0,7817	0,44838
1 - 6	+	3,891	-0,0001	-0,43244	1,9184	0,07308

* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

 TABLE 9 - Experiment II  - Preincubation method -

STUDY NO.: 9324

SOLVENT: DMSO

Dose-level [µg/pl]	Without metabolic activation					With metabolic activation
	Plate counts			Mean	s.e.	Plate counts			Mean	s.e.
Untreated	20	16	14	17	1,8	22	16	17	18	1,9
0,00	17	18	17	17	0,3	21	19	18	19	0,9
313	11	18	17	15	2,2	18	20	22	20	1,2
625	18	15	18	17	1,0	23	20	21	21	0,9
1250	17	11	15	14	1,8	17	19	22	19	1,5
2500	19	11	13	14	2,4	20	15	17	17	1,5
5000	11	7	12	10	1,5	16	12	19	16	2,0

Regression analysis:
Points	S9	Intercept	Slope	Corr. coeff.	t	P-value
1 - 3	-	4,081	-0,0001	-0,06265	0,1661	0,87279
1 - 4	-	4,125	-0,0003	-0,36073	1,2231	0,24935
1 - 5	-	4,078	-0,0001	-0,35658	1,3761	0,19203
1 - 6	-	4,108	-0,0002	-0,68025	3,7123	0,00189
1 - 3	+	4,382	0,0004	0,50432	1,5452	0,16621
1- 4	+	4,469	0,0000	-0,00721	0,0228	0,98227
1 - 5	+	4,523	-0,0001	-0,45447	1,8396	0,08877
1- 6	+	4,516	-0,0001	-0,62715	3,2207	0,00534

* Thinning of the background lawn

Positive and negative controls Treatment			S9	Plate counts			Mean	s.e.
DMSO	50 µl/pl		-	17	18	17	17	0,3
9-Aminoacridine	50 µg/pl		-	125	146	186	152	17,9
DMSO	50 µl/pl		+	21	19	18	19	0,9
2-Aminoanthracene	1 µg/pl		+	110	112	127	116	5,4

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium
TABLE 10 - Experiment II - Preincubation method -
STUDY NO.: 9324
SOLVENT: DMSO
Strain: TA102 Titre: 255
Dose-level [µg/pl]	Without metabolic activation						With metabolic activation
	Plate counts				Mean	s.e.	Plate counts			Mean	s.e.
Untreated	359	346	385		363	11,5	392	416	420	409	8,7
0,00	369	376	382		376	3,8	427	399	407	411	8,3
313	379	388	394		387	4,4	404	424	418	415	5,9
625	373	389	376		379	4,9	415	422	397	411	7,4
1250	370	381	368		373	4,0	417	411	420	416	2,6
2500	401	388	392		394	3,8	435	427	410	424	7,4
5000	370	393	384	*	382	6,7	427	410	431	423	6,4
Regression analysis:
Points	S9	Intercept	Slope	Corr. coeff.	t	P-value
1 - 3	-	19,462	0,0002	0,19337	0,5215	0,61814
1 - 4	-	19,530	-0,0001	-0,28647	0,9455	0,36668
1 - 5	-	19,420	0,0001	0,44488	1,7910	0,09659
1 - 6	-	19,490	0,0000	0,21205	0,8680	0,39825
1 - 3	+	20,305	0,0000	0,01375	0,0364	0,97200
1 - 4	+	20,291	0,0001	0,14742	0,4713	0,64751
1 - 5	+	20,271	0,0001	0,40922	1,6171	0,12986
1 - 6	+	20,315	0,0001	0,39399	1,7147	0,10571
Positive and negative treatment controls
		S9	Plate counts			Mean	s.e.
DMSO	50 µl/pl	-	369	376	382	376	3,8
Cumene hydroperoxide	100 µg/pl	-	1066	1192	1064	1107	42,3
DMSO	50 µl/pl	+	427	399	407	411	8,3
2-Aminoanthracene	20 µg/pl	+	1615	1596	1600	1604	5,8
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 11 - Experiment II

STUDY NO.: 9324

SOLVENT: DMSO

 - Preincubation method -

Strain: TA98 Titre: 244
Dose-level [µg/pl]	Without metabolic activation					With metabolic activation
	Plate counts			Mean	s.e.	Plate counts			mean	s.e.
Untreated	37	30	34	34	2,0	39	44	40	41	1,5
0.00	31	31	32	31	0,3	38	36	43	39	2,1
313	31	32	35	33	1,2	38	35	41	38	1,7
625	32	34	34	33	0,7	36	38	35	36	0,9
1250	31	29	34	31	1,5	38	42	36	39	1,8
2500	29	27	33	30	1,8	38	38	41	39	1,0
5000	26	25	29 *	27	1,2	43	38	37	39	1,9
Regression analysis:
Points	S9	Intercept	Slope	Corr. coeff.	t	P-value
1 - 3	-	5,607	0,0003	0,57691	1,8687	0,10388
1 - 4	-	5,680	0,0000	-0,05669	0,1796	0,86109
1 - 5	-	5,713	-0,0001	-0,44214	1,7773	0,09890
1 - 6	-	5,724	-0,0001	-0,74260	4,4351	0,00042
1 - 3	+	6,250	-0,0003	-0,42210	1,2319	0,25776
1- 4	+	6,171	0,0000	-0,04192	0,1327	0,89707
1- 5	+	6,150	0,0000	0,13195	0,4800	0,63923
1- 6	+	6,154	0,0000	0,20438	0,8351	0,41594
Positive and negative control - TREATMENTS			S9	Plate counts			Mean	s.e.
DMSO 50µl/pl			-	31	31	32	31	0,3
2-Nitrofluorene 2 µg/pl			-	191	194	154	180	12,9
DMSO 50 µl/pl			+	38	36	43	39	2,1
2-Aminoanthracene 2 µg/pl			+	698	666	649	671	14,4
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 12 - Experiment II      - Preincubation method -

STUDY NO.: 9324

SOLVENT DMSO

Titre: 224
Strain: TA100
Dose-level [µg/pl]	Without metabolic activation
	Plate counts				Mean	S.e.
Untreated	133	140	137		137	2,0
0,00	130	126	133		130	2,0
313	134	152	153		146	6,2
625	140	161	166		156	8,0
1250	147	150	155		151	2,3
2500	145	138	150		144	3,5
5000	152	157	164	*	158	3,5
Regression analysis:
Points	S9	Intercept	Slope	Corr. coeff.	t	P-value
1 - 3	-	11,441	0,0017	0,78057	3,3039	0,01305
1 - 4	-	11,707	0,0006	0,57570	2,2265	0,05014
1 - 5	-	11,932	0,0001	0,23129	0,8572	0,40689
1 - 6	-	11,930	0,0001	0,45188	2,0262	0,05976
Positive and negative controls
Treatment	S9	Plate counts			Mean	S. E.
Untreated	-	133	140	137	137	2.0
Sodium Azide 1 µg/pl	-	1075	1069	1003	1049	23.1
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 13 - Experiment II      - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA100 Titre: 224
Dose-level [µg/pl]	With metabolic activation
	Plate counts			Mean	S.E.
Untreated	138	132	138	136	2,0
0,00	143	134	145	141	3,4
313	199	212	200	204	4,2
625	254	251	225	243	9,2
1250	262	262	234	253	9,3
2500	248	247	261	252	4,5
5000	253	235	263	250	8,2
Regression analysis:
Points	S9	Intercept	Slope	Corr. coeff.	t	P-value
1 - 3	+	12,039	0,0060	0,96884	10,3485	0,00002
1 - 4	+	12,763	0,0030	0,85286	5,1652	0,00042
1 - 5	+	13,531	0,0012	0,70021	3,5363	0,00365
1 - 6	+	14,093	0,0005	0,56022	2,7053	0,01560
Positive and negative controls
Treatment	S9	Plate counts			Mean	s.e.
DMSO 100 µl/p1	+	143	134	145	141	3,4
2-Aminoanthracene 1 µl/p1	+	1246	1243	1222	1237	8

Applicant's summary and conclusion

Conclusions:

Although increases in revertant numbers were observed in the presence of S9 with TA100 tester strain (the dose response was not reproduced in a second experiment), the criteria for a clear mutagenic effect were not fully met and the outcome for the test item TETRAMETHRIN was regarded as inconclusive. All other tester strains assessed (TA1535, TA1537, TA98 and TA102) were clearly negative (with and without S9).

Executive summary:

The test item TETRAMETHRIN was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium.

The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and beta naphthoflavone.

In the toxicity test, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No signs of toxicity were observed at any dose-level tested, in any tester strain, in the absence or presence of S9 metabolic activation.

Two main experiments were performed.

In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. Increases in revertant numbers, which were at least two-fold greater than the control value, were observed at the higher dose-levels with TA100 tester strain in the presence of S9 metabolism.

On the basis of these results, in Main Assay II the same dose-levels used in Main Assay I were employed. A pre-incubation step was included in all treatments with the exception of testing TA100 in the presence of S9 metabolism. With this tester strain the test item was assayed (in the presence of S9 metabolism) under the same experimental conditions employed in Main Assay I. Increases in revertant colonies were again observed in TA100 in the presence of metabolic activation, but these increases did not reach twice the control value.

Precipitation of the test item was observed at 5000 and 2500 µg/plate.

It is concluded that, although increases in revertant numbers were observed in the presence of S9 with TA100 tester strain, the results obtained preclude making a definite judgement about the activity of the test item TETRAMETHRIN.