Tetrahydro-4-methyl-2H-pyran

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:

2009-03-27 to 2009-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

n/a

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-4-methyl-2H-pyran

EC Number:

225-207-5

EC Name:

Tetrahydro-4-methyl-2H-pyran

Cas Number:

4717-96-8

Molecular formula:

C6H12O

IUPAC Name:

4-methyltetrahydro-2H-pyran

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 4-methyltetrahydropyran (MTHP)
- CAS no.: 4717-96-8
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / MTHP204347
- Expiration date of the lot/batch: not stated
- Molecular weight: 100.16 g/mol
- Purity: 99.98%

Method

Target gene:

see below

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:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose range finder: -/+S9: 0, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 ug/plate
Main study:
-S9:
TA100; WP2uvrA; TA98: 0, 156, 313, 625, 1250, 2500, 5000 ug/plate
TA1535; TA1537: 0, 39.1, 78.1, 156, 313, 625, 1250 ug/plate

+S9:
TA100; TA1535; TA1537: 0, 39.1, 78.1, 156, 313, 625, 1250 ug/plate
WP2uvrA; TA98: 0, 156, 313, 625, 1250, 2500, 5000 ug/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9: TA1535, WP2uvrA

Controls 2

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9: TA100, TA98, TA1537

Controls 3

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

-S9: TA100, TA98, WP2uvrA

Controls 4

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9: TA1535

Controls 5

Positive controls:

yes

Positive control substance:

other: ICR-191 (6-Chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride)

Remarks:

-S9: TA1537

Details on test system and experimental conditions:

The pre-incubation methodology was used for the dose range finder and a main test. Duplicate plates were used for the dose range finder and main tests.

Test tubes containing 0.1 mL of vehicle, positive control or test article formulation were mixed with 0.5 mL S9 mix (or PBS in the absence of metabolic activation). 0.1 mL fresh bacterial culture was added and the mixture was incubated while shaking at 37°C for 20 minutes.

After pre-incubation, 2 mL of top agar (kept at 45°C) was added to each tube and this mixture was shaken and overlaid onto Vogel-Bonner agar plates (minimal glucose agar plates). E.coli containing tubes were poured onto minimal agar plates.

Agar plates were incubated at 37°C for 48 h in the dark for the bacterial colonies (his+ or typ+ revertants) counted.

The background lawns of the plates were examined for signs of toxicity. Other toxicity indicators that may have been noted included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Evaluation criteria:

The test chemical was considered positive in this assay if the following criteria were met:
- dose-related and reproducible increase in the number of revertant colonies (i.e. doubling of the spontaneous mutation rate in at least one tester strain either –S9 or +S9)

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

Statistics not warranted

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

A. Dose range finder:

No precipitation was observed at any dose level, - or + S9. Growth inhibition (evaluation by bacterial background lawn) was observed at 1250 ug/plate and above for strains TA100 (+S9), TA1535 (+/-S9); TA1537 (+/-S9) and at 5000ug/plate for strains TA100 (-S9), WP2uvrA (+/-S9), TA98 (+/-S9). MTHP was deemed not mutagenic in the dose range finder experiment following a pre-incubation methodology. For the main mutation test, as growth inhibition was the limiting factor the maximum dose level was set at 1250 ug/plate for strains TA98 (+S9), TA1535 (+/-S9) and TA1537 (+/-S9).for all other strains +/-S9 the maximum dose level was 5000 ug/plate.

 

Table 7.6.1/01-1: Bacterial (reverse) gene mutation pre-incubation data – Dose range finder

Conc. (ug/plate)	TA100		TA1535		WP2uvrA			TA98		TA1537
	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
0	92	137	13	10	27		27	32	57	7	11
1.22	96	109	12	9	29		28	26	47	4	6
4.88	98	114	9	6	21		25	29	42	7	5
19.5	103	96	14	11	28		26	32	39	7	7
78.1	108	117	12	10	26		21	42	54	2	7
313	97	118	12	13	23		28	35	43	4	8
1250	115	111B	8B	8B	28		34	36	42	5B	8B
5000	0A,B	77B	0A,B	4B	19B		21B	0A,B	18B	0A,B	5B
+ve	545	964	226	220	85		1122	478	432	1318	127
A: total growth inhibition B: reduced background lawn +ve controls: -S9 (absence of metabolic activation): TA100, WP2uvrA, TA98: furylfuramide TA1535: sodium azide TA1537: ICR-191						+S9 (presence of metabolic activation): TA100, TA98, TA1537: Benzo[a]pyrene TA1537, WP2urvA: 2-aminoanthracene

 

B. Main mutation tests

No precipitation was observed at any dose level. For main mutation test, growth inhibition (evaluation by bacterial background lawn) was observed at 1250 ug/plate, the maximum dose tested for strains  TA100 (+S9), TA1535 (+/-S9) and TA1537 (+/-S9); at 2500ug/plate and above for strains TA100 (+S9); TA98 (+S9), WP2uvrA (+/-S9), TA98 (+/-S9) and at 5000 ug/plate for strains Wp2uvrA (+/-S9)

 

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

 

Following MTHP treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control in two independent experiments. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system (refer to Table 7.6.1/01-2,).

 

Table 7.6.1/01-2: Bacterial (reverse) gene mutation pre-incubation data – Main mutation experiment

Conc. (ug/plate)	TA100		TA1535		WP2uvrA			TA98		TA1537
	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
0	106	122	11	14	30		22	22	41	7	10
39.1	NT	123	13	13	NT		NT	NT	NT	9	11
78.1	NT	119	12	14	NT		NT	NT	NT	4	13
156	100	119	12	14	28		31	24	29	9	12
313	110	123	10	12	29		22	30	38	6	7
625	107	135	13	16	23		23	29	44	5	14
1250	117	123B	12B	17B	20		29	32	41	8B	9B
2500	114B	NT	NT	NT	23		18	29	33B	NT	NT
5000	29B	NT	NT	NT	12B		8B	0B	13B	NT	NT
+ve	595	1112	317	308	72		1103	583	441	1333	140
B: reduced background lawn NT: not treated +ve controls: -S9 (absence of metabolic activation): TA100, WP2uvrA, TA98: furylfuramide TA1535: sodium azide TA1537: ICR-191						+S9 (presence of metabolic activation): TA100, TA98, TA1537: Benzo[a]pyrene TA1537, WP2urvA: 2-aminoanthracene

 

Table 7.6.1/01-3: Bacterial (reverse) gene mutation data – Historical vehicle and positive control data

Conc. (ug/plate)	TA100		TA1535		WP2uvrA			TA98		TA1537
	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9	-S9	+S9
Vehicle control
Mean ±SD	123	136	10	10	21		23	20	35	11	14
Lower limit	86	95	4	4	9		11	8	17	4	6
Upper limit	159	176	16	16	34		34	32	52	18	22
Positive control
Mean ±SD	592	1061	317	284	78		945	468	383	1383	127
Lower limit	473	849	222	199	54		756	375	307	691	89
Upper limit	710	1274	412	369	101		1134	562	460	2074	165
Lower limit: ± 2 S.D Upper limt: ± 2 S.D   -S9 (absence of metabolic activation): TA100, WP2uvrA, TA98: furylfuramide TA1535: sodium azide TA1537: ICR-191						+S9 (presence of metabolic activation): TA100, TA98, TA1537: Benzo[a]pyrene TA1537, WP2urvA: 2-aminoanthracene

 

C. Deficiencies:

Under the requirements of OECD 471 triplicate plates should be used for each dose level, although the use of duplicate plating is acceptable when scientifically justified.

This study was conducted in compliance with GLP and under the Japanese regulations, with the reproducibility was confirmed in two independent experiments. For this reason the use of duplicate plates was considered acceptable.

Applicant's summary and conclusion

Conclusions:

It was concluded that MTHP did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration in accordance with current regulatory requirements), in the absence and presence of a rat liver metabolic activation system (S9) using pre-incubation methodology.

Executive summary:

In a reverse gene mutation assay in bacteria, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were exposed to MTHP formulated in dimethyl sulphoxide (DMSO). The pre-incubation methodology was used. Following a dose range-finder experiment, a main mutation experiment was conducted using concentrations which spanned the range of 39.1 – 5000 ug/plate. Growth inhibition was observed in all strains, at differing concentrations.

 

For main mutation test, growth inhibition (evaluation by bacterial background lawn) was observed at 1250 ug/plate, the maximum concentration tested for strains  TA100 (+S9), TA1535 (+/-S9) and TA1537 (+/-S9); at 2500 ug/plate and above for strains TA100 (+S9); TA98 (+S9), WP2uvrA (+/-S9), TA98 (+/-S9) and at 5000 ug/plate for strains Wp2uvrA (+/-S9). No precipitation was observed at any concentration.

 

The positive controls induced an acceptable increase in revertant colony numbers, thereby demonstrating the sensitivity and specificity of the test system.

 

Following MTHP treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were equal to or greater than 2-fold above the concurrent vehicle control. This study was therefore considered to have provided no evidence of any mutagenic activity in this assay system.

 

It was concluded that MTHP did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/mL (the maximum recommended concentration in accordance with current regulatory requirements), in the absence and presence of a rat liver metabolic activation system (S9) using pre-incubation methodology.