Tetrahydro-4-methyl-2H-pyran

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

When tested in vitro, MTHP was negative in an Ames test, a mammalian cell gene mutation assay with mouse lymphoma L5178Y cells and a mammalian rodent cell line (Chinese hamster fibroblast cells) chromosomal aberration assy.

 

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.

 

MTHP did not induce mutation at the tk locus of L5178Y mouse lymphoma cells. These conditions included treatments up to 1002µg/mL (equivalent to 10 mM), the maximum recommended dose in accordance with current in vitro genotoxicity regulatory assays) in the absence (3 hours) or presence (3 hours at 2% (v/v) final concentration of S-9 fraction) of a rat liver metabolic activation system following a single mutation experiment.

 

It can be concluded that MTHP did not show an increase in the incidence of chromosome aberrations in cultured Chinese hamster lung fibroblast cells. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (6 hours [+ 18 hours recovery], 24 h [+0h], 48 h [+0 h]) and presence (6 h [+18 h]) of a rat liver metabolic activation system (S9).

Link to relevant study records

Reference

Reference 1

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

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

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%

Target gene:

see below

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

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

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9: TA100, TA98, TA1537

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

-S9: TA100, TA98, WP2uvrA

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9: TA1535

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

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-02 to 2009-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration assay

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 / MTHP213536
- Expiration date of the lot/batch: not stated
- Molecular weight: 100.16 g/mol
- Purity: 100%

Species / strain / cell type:

lymphocytes:

Remarks:

human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
6h -/+S9, 24h, -S9, 48h -S9: 0, 8.20, 16.4, 32.8, 65.6, 131, 263, 525, 1050 ug/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity)

Chromosome aberration experiment:
6h -/+S9, 24h, -S9, 48h -S9: 0, 263, 525, 1050 ug/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity)

Vehicle / solvent:

DMSO (dimethyl sulphoxide)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cultured CHO were exposed to the test article in the presence and absence of S9 mix (rat phenobarital / 5,6 benzoflavone). Without S9 mix cells were exposed for short-term (6 h) with an 18 h recovery or continuously for 24 or 48 h without recovery. In the presence of S9 mix exposure was limited to 6 h, with cells harvested 18 h later. On the basis of the results of the preliminary toxicity test the following concentrations (in duplicate) were selected in order that an appropriate range of toxicity was observed:

-/+S9: 0, 263, 525, 1050 µg/mL

Positive controls were included.

From 200 metaphases/dose level with 20 centromeres, chromosome number, all chromosomes normal or some aberrant, and specific types and numbers of aberrations were recorded.

Rationale for test conditions:

refer to "Any other information on materials and methods incl. tables"

Evaluation criteria:

In accordance with the criteria by Ishidate et al, the chromosome aberration inducibility of the test article was classified as follows based on the incidence of (%) cells with chromosome structural and numerical abnormalities:
- lower than 5% - negative
- lower than 10% but not lower than 5% - false positive
- greater than 10% - positive.

Statistics:

no statisitical analysis undertaken

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

6 h treatments

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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24 h treatment

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

48 h

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

48 h treatment

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

Preliminary cytotoxicity assay

Test item concentrations between 8.20 - 1050 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity following a 6 h treatment or 24 and 48 h treatments without S9. In the pre-test for toxicity, no precipitation of the test article was observed. No relevant influence on osmolality or pH value was observed. No clear cytotoxicity was observed up to the highest concentration (equivalent to 10 mM), with cell growth inhibition values of 50% estimated at concentrations of 700 ug/mL in the 6 h +S9 and at concentrations greater than 1050 ug/mL for all other treatments.

 

Cytogenetic assay:

- Short term treatment (6 h) -S9:

No clear cytotoxicity was observed up to the highest concentration (equivalent to 10 mM), with cytotoxicity values of 0%, 8%, and 0% at concentrations of 263, 525 and 1050 µg/mL, respectively.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 263, 525 and 1050 µg/mL were 1.5%, 0.0% and 0.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (0.5%) and did not meet the criteria for a positive result, as defined by Ishidate et al (i.e. aberrant cells <5% frequency deemed to be devoid of clastogenic potential).

 

Table 7.6.1/03-1: Experiment 1: 6 h [+16 h]) -S9 cytotoxicity and chromosomal aberration data

Conc		Cell growth		Mean aberrant cells (%)
(ug/mL)	(mM)	(%)	Cytotoxicity (%)
0	0	100	0	0.5
263	2.5	100	0	1.5
525	5	92	8	0.0
1050	10	102	0	0.0
MMC		105	0	26.5

 

- Short term treatment (6 h) +S9: - Experiment 1:

No overt cytotoxicity was observed (i.e. >50%) up to the highest concentration (equivalent to 10 mM), with cytotoxicity values of 29%, 39%, and 33% at concentrations of 263, 525 and 1050 µg/mL, respectively.

 

An increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 263, 525 and 1050 µg/mL were 4.0%, 4.0% and 5.0%, respectively. The frequency of aberrant cells (excluding gaps) was exceeded the concurrent control value (0.5%) and meet the criteria for a false positive result, as defined by Ishidate et al (i.e. aberrant cells <10% but not lower than 5% frequency). In order to better define this response, a further experiment was undertaken

 

Table 7.6.1/03-2: Experiment 1: 6 h [+16 h]) +S9 cytotoxicity and chromosomal aberration data

Conc		Cell growth		Mean aberrant cells (%)
(ug/mL)	(mM)	(%)	Cytotoxicity (%)
0	0	100	0	0.5
263	2.5	71	29	4.0
525	5	61	39	4.5
1050	10	67	33	5.0
CPA		87	13	81

 

- Short term treatment (6 h) +S9: - Experiment 2:

No overt cytotoxicity was observed (i.e. >50%) up to the highest concentration (equivalent to 10 mM), with cytotoxicity values ranging from 18% to 44% over a concentration range of 344 to 1050 µg/mL.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 344, 430, 538, 672, 840 and 1050 µg/mL were 1.0%, 2.5%, 3.5%, 0.5%, 1.0% and 1.0%, respectively. The frequency of aberrant cells (excluding gaps) whilst exceeding the concurrent control value (0.0%) did not meet the criteria for a positive result, as defined by Ishidate et al (i.e. aberrant cells <5% frequency deemed to be devoid of clastogenic potential).

 

Table 7.6.1/03-3: Experiment 2: 6 h [+16 h]) +S9 cytotoxicity and chromosomal aberration data

Conc		Cell growth		Mean aberrant cells (%)
(ug/mL)	(mM)	(%)	Cytotoxicity (%)
0	0	100	0	0.0
344	3.3	75	25	1.0
430	4.1	82	18	2.5
538	5.1	78	22	3.5
672	6.4	56	44	0.5
840	8	82	18	1.0
1050	10	68	32	1.0
CPA		71	29	78.0

 

- Continuous treatment (24 h) -S9: - Experiment 1:

No overt cytotoxicity was observed (i.e. >50%) up to the highest concentration (equivalent to 10 mM), with cytotoxicity values of 0%, 10%, and 10% at concentrations of 263, 525 and 1050 µg/mL, respectively.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 263, 525 and 1050 µg/mL were 1.0%, 0.0% and 0.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (0.0%) and did not meet the criteria for a positive result, as defined by Ishidate et al (i.e. aberrant cells <5% frequency deemed to be devoid of clastogenic potential).

 

Table 7.6.1/03 -4: Experiment 1: 24 h [+0 h]) -S9 cytotoxicity and chromosomal aberration data

Conc		Cell growth		Mean aberrant cells (%)
(ug/mL)	(mM)	(%)	Cytotoxicity (%)
0	0	100	0	0.0
263	2.5	105	0	1.0
525	5	90	10	0.0
1050	10	90	10	0.0
MMC		114	0	33.5

 

- Continuous treatment (48 h) -S9: - Experiment 1:

No overt cytotoxicity was observed (i.e. >50%) up to the highest concentration (equivalent to 10 mM), with cytotoxicity values of 15%, 4%, and 18% at concentrations of 263, 525 and 1050 µg/mL, respectively.

 

No biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 263, 525 and 1050 µg/mL were 0.0%, 0.0% and 0.0%, respectively. The frequency of aberrant cells (excluding gaps) was consistent with the concurrent control value (0.5%) and did not meet the criteria for a positive result, as defined by Ishidate et al (i.e. aberrant cells <5% frequency deemed to be devoid of clastogenic potential).

 

Table 7.6.1/03 -5: Experiment 1: 24 h [+0 h]) -S9 cytotoxicity and chromosomal aberration data

Conc		Cell growth		Mean aberrant cells (%)
(ug/mL)	(mM)	(%)	Cytotoxicity (%)
0	0	100	0	0.5
263	2.5	85	15	0.0
525	5	96	4	0.0
1050	10	82	18	0.0
MMC		114	0	60.5

 

Overall it can be concluded that MTHP did not show an increase in the incidence of chromosome aberrations in cultured Chinese hamster lung fibroblast cells. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (6 hours [+ 18 hours recovery], 24 h [+0h], 48 h [+0 h]) and presence (6 h [+18 h]) of a rat liver metabolic activation system (S9).

No evidence of an increase in polyploid metaphases were noticed after treatment with the test article as compared to the control cultures.

 

In all experiments, either MMC (0.05, 0.075 µg/mL) or CPA (14 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Overall it can be concluded that MTHP did not show an increase in the incidence of chromosome aberrations in cultured Chinese hamster lung fibroblast cells. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (6 hours [+ 18 hours recovery], 24 h [+0h], 48 h [+0 h]) and presence (6 h [+18 h]) of a rat liver metabolic activation system (S9).

Executive summary:

In a mammalian chromosomal aberration assay, cultured Chinese hamster lung fibroblast cells, invitro were exposed to MTHP formulated in DMSO in either the presence of metabolic activation (+S9, 6 h [+18 h recovery]) and absence of metabolic activation (-S9, 6h [+18 h], 24h [+0 h] and 48h [+0 h]).

 

In the absence of S9 (6 h, 24 h and 48 h) treatment concentrations of 263, 525 and 1050 µg/mL were examined for structural and numerical aberrations. No precipitation was observed by eye at the end of treatment. No overt cytotoxicity (>50%) was observed.

In the presence of S9, no overt cytotoxicity was observed (i.e. >50%) up to the highest concentration (equivalent to 10 mM), with cytotoxicity values of 29%, 39%, and 33% at concentrations of 263, 525 and 1050 µg/mL, respectively.

 

An increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 263, 525 and 1050 µg/mL were 4.0%, 4.0% and 5.0%, respectively. The frequency of aberrant cells (excluding gaps) was exceeded the concurrent control value (0.5%) and meet the criteria for a false positive result, as defined by Ishidate et al (i.e. aberrant cells <10% but not lower than 5% frequency). In order to better define this response, a further experiment was undertaken

 

In the confirmatory experiment, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells (excluding gaps) after treatment with the test article at concentrations of 344, 430, 538, 672, 840 and 1050 µg/mL were 1.0%, 2.5%, 3.5%, 0.5%, 1.0% and 1.0%, respectively. The frequency of aberrant cells (excluding gaps) whilst exceeding the concurrent control value (0.0%) did not meet the criteria for a positive result, as defined by Ishidate et al (i.e. aberrant cells <5% frequency deemed to be devoid of clastogenic potential).

 

Overall it can be concluded that MTHP did not show an increase in the incidence of chromosome aberrations in cultured Chinese hamster ovary cells. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (6 hours [+ 18 hours recovery], 24 h [+0h], 48 h [+0 h]) and presence (6 h [+18 h]) of a rat liver metabolic activation system (S9).

No evidence of an increase in polyploid metaphases were noticed after treatment with the test article as compared to the control cultures.

 

In all experiments, either MMC (0.05, 0.075 µg/mL) or CPA (14 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

 

Overall it can be concluded that MTHP did not show an increase in the incidence of chromosome aberrations in cultured Chinese hamster lung fibroblast cells. These conditions included treatments up to the maximum recommended concentration (10 mM) in accordance with current in vitro genotoxicity test guidelines in the absence (6 hours [+ 18 hours recovery], 24 h [+0h], 48 h [+0 h]) and presence (6 h [+18 h]) of a rat liver metabolic activation system (S9).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-24 to 2017-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward gene mutation

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 / MTHP-72715
- Expiration date of the lot/batch: not stated
- Molecular weight: 100.16 g/mol
- Purity: 100%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphthoflavone

Test concentrations with justification for top dose:

Range-finder:
3 h -/+S9: 0, 4, 31.31, 62.63, 125.3, 250.5, 501, 1002 ug/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity)

Main mutation assay:
3 h -/+S9: 0, 7.828, 15.66, 31.31, 62.63, 125.3, 250.5, 501, 1002 ug/mL (equivalent to 10 mM, the maximum recommended concentration in accordance with current regulatory guidelines for in vitro mammalian genotoxicity assays in the absence of toxicity)

Vehicle / solvent:

DMSO (dimethyl sulphoxide)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Preliminary cytotoxicity assay:
Cells were exposed to test article formulations, solvent controls for 3 h in the absence and presence of S9. Single cultures were used for the vehicle control and test article formulation cultures. At the end of treatment cultures were washed, cell culture counted, adjusted (where necessary to 3 x 10^5 cells/mL) transferred to cell culture flasks and incubated for 2 days, with cell adjustment made every 24 hours. After 2-days growth period the relative suspension growth (RSG) of the treated cell cultures was calculated to estimate toxicity.
Osmolality and pH were measured on post-treatment media. As no marked changes are observed, further measurements were not deemed necessary for the mutation experiments.

Mutation assay:
The mutation assay was conducted as detailed above for the preliminary cytotoxicity assay, with the following exceptions:
- Positive controls included.
- At the end of the 2-days of growth, cells were sub-cultured to assess cytotoxicity and to initiate the phenotypic expression.
- Mutation frequency was determined by plating ~2000 cells/well in cell culture medium containing 4 µg TFT/mL. Plates were incubated for 10-14 days. After this period the number of well without growth was counted to provide CE in TFT. Wells with growth in indicated evidence of TFT-resistance mutants. Colony sizing was performed on negative and positive controls.
- Cloning efficiency was determined by plating ~1.6 cells/well into two 96 well plates. Plates were incubated for 10-14 days. After this period the number of wells without growth of cells was counted.
Solubility of the test article in culture medium was assessed, by eye, at the beginning and end of treatment.

Rationale for test conditions:

refer to justifcation of dose levels

Evaluation criteria:

Acceptance criteria:
The assay was considered acceptable if all the following criteria (as defined by Moore et al ) were met:
- The mean mutant frequencies in the vehicle control cultures fell within the normal range (50 to 170 mutants/106 viable cells)
- At least one positive control showed either an absolute increase in mean total MF of at least 300 x 10-6 (at least 40% of this should be in the small colony MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control
- The RTG for the positive controls was greater than 10%
-The mean cloning efficiency of the vehicle controls from the Mutation Experiments were between the range 65% to 120%
The mean SG of the vehicle controls from the Mutation experiments were between the range of 8 to 32 following 3 hour treatments.

Evaluation criteria:
The test article was considered mutagenic in this assay (as defined by Moore et al1) if :
- The MF of any test concentration exceeded the sum of the vehicle control mutant frequency plus GEF
- The linear trend test was statistically significant
- Any observed response is reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.

Statistics:

not warranted

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Preliminary cytotoxicity assay:

The maximum concentration tested in the preliminary test was 1002 µg/mL (equivalent to 10 mM).

In the absence (3 h) and presence (3 h) of S9, no overt toxicity was observed such that the highest concentration analysed (1002 µg/mL) gave RSG values of 98-116%.

 

Osmolality and pH were measured on post-treatment media. As no marked changes are observed, further measurements were not deemed necessary for the mutation experiments.

 

Main mutation assay:

-S9:

The maximum concentration tested was 1002 µg/mL. Toxicity, expressed in terms of relative total growth (RTG) gave 72% RTG. In the absence of S9, no increases in mutant frequency (MF) which exceeded the sum of the Global Evaluation Factor (GEF) of 126 + the vehicle control MF, were observed in any treated cultures (refer to Table 7.6.1/02-1).

 

Table 7.6.1/02-1: Mouse lymphoma toxicity and mutant frequency data: 4 h –S9

Conc.		-S9		% of small colonies
(ug/mL)	(mM)	RTG	MF
0	0	100	135.64	45
7.828	0.0781	129	108.51	38
15.66	0.156	93	90.57	34
31.31	0.313	115	170.85	43
62.63	0.625	98	143.19	29
125.3	1.25	138	125.22	57
250.5	2.5	111	126.85	42
501.0	5	154	94.35	43
1002	10	72	109.43	46
MMS	-	71	585.07$	62

+ve controls: MMS - methyl methanesulphonate

$ Sum of the vehicle control mutant frequency (MF) + GEF (126) exceeded [262.64]

 

+S9:

The maximum concentration tested was 1002 µg/mL. Toxicity, expressed in terms of RTG gave 92% RTG.

In the presence of S9, no increases in mutant frequency (MF) which exceeded the sum of the Global Evaluation Factor (GEF) of 126 + the vehicle control MF, were observed in any treated cultures (refer to Table 7.6.1/02-2).

 

The positive controls induced an acceptable increases in mutation frequency and an acceptable increase in the number of small colony mutants under both treatment conditions, thereby demonstrating the sensitivity and specificity of the test system.

 

Table 7.6.1/02-2: Mouse lymphoma toxicity and mutant frequency data: 4 h +S9

Conc.		-S9		% of small colonies
(ug/mL)	(mM)	RTG	MF
0	0	100	79.90	36
7.828	0.0781	93	95.62	34
15.66	0.156	116	58.22	60
31.31	0.313	97	116.14	46
62.63	0.625	102	70.46	27
125.3	1.25	104	77.85	48
250.5	2.5	102	84.00	59
501.0	5	124	87.15	34
1002	10	92	87.02	39
MMS	-	13	993.54$	68

+ve controls: B[a]P – cyclophosphamide

$ Sum of the vehicle control mutant frequency (MF) + GEF (126) exceeded [205.9]

Conclusions:

It is concluded that MTHP did not induce mutation at the tk locus of L5178Y mouse lymphoma cells. These conditions included treatments up to 1002µg/mL (equivalent to 10 mM), the maximum recommended dose in accordance with current in vitro genotoxicity regulatory assays) in the absence (3 hours) or presence (3 hours at 2% (v/v) final concentration of S-9 fraction) of a rat liver metabolic activation system following a single mutation experiment.

Executive summary:

4-Methyltetrahydropyran was assayed for the ability to induce mutation at the thymidine kinase locus (5-trifluorothymidine [TFT] resistance) in cultured mouse lymphocytes (L5178Y TK+/-clone 3.7.2C) cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by one Mutation Experiment, each conducted in the absence and presence of metabolic activation by phenobarbital and 5,6-benzoflavone-induced rat liver post-mitochondrial fraction (S9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and dosed at 1% v/v.

 

A 3 hour treatment incubation period was used for all experiments performed in the presence of S9. In the absence of S9, the Range-Finder Experiment was performed using 3 and 24 hour treatment incubation periods and the Mutation Experiment was performed using a 3 hour treatment incubation.

 

In the cytotoxicity Range-Finder Experiment, 3 hour treatment, in the absence and presence of S9 and 24 hour treatment in the absence of S9, six concentrations were tested ranging from 31.31 to 1002 µg/mL (equivalent to 10 mM at the highest concentration tested). As the cytotoxicity Range-Finder Experiment was restricted to 2 days post treatment, toxicity was expressed in terms of relative suspension growth (RSG). In the 3 hour treatment in the absence and presence of S9, RSG values of 85% and 93% were obtained, respectively. In the 24 hour treatment in the absence of S9, RSG values were reduced to 50%. No significant changes in pH (more than one unit) or osmolality (more than 50 mOsm/kg) in the medium following the addition of test article concentrations  were observed compared to concurrent negative controls. It was therefore not deemed necessary to undertake further pH and osmolality measurements in the mutation assay. Precipitation in the medium (observed by eye at the end of treatment) was not observed in any of the test article treatment groups.

 

In the Mutation Experiment (3 hour treatment) eight concentrations, ranging from 7.828 to 1002 µg/mL in the absence and presence of S-9 were tested. Two days after treatment the highest concentrations analysed to determine viability and TFT resistance were 1002 µg/mL in both the absence and presence of S9, which gave 72% and 92% relative total growth (RTG), respectively.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S9) and cyclophosphamide (with S9). Therefore the study was accepted as valid.

 

The MF of the concentrations plated were all less than the sum of the mean control MF plus the global evaluation factor (GEF, 126 mutants per 106viable cells) for both the 3 hour treatments with4-Methyltetrahydropyran when tested up to an concentration equivalent to 10 mM, indicating a negative result under these treatment conditions.

 

It is concluded that MTHP did not induce mutation at the tk locus of L5178Y mouse lymphoma cells. These conditions included treatments up to 1002µg/mL (equivalent to 10 mM), the maximum recommended dose in accordance with current in vitro genotoxicity regulatory assays) in the absence (3 hours) or presence (3 hours at 2% (v/v) final concentration of S-9 fraction) of a rat liver metabolic activation system following a single mutation experiment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivio genotoxicity test is not triggered.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Comparison with the CLP criteria

There was no indication that MTHP has a mutagenic effect on somatic cells in the Ames, mammalian gene mutation or mammalian chromosomal aberration in vitro assays.

The criteria for classification for mutagenicity were not met.