Tetrahydro-4-methyl-2-phenyl-2H-pyran

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD 471, Safepharm Laboratories Limited 2006

Link to relevant study records

Reference

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:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected August 2005; signature: November 2005

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

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 ug/plate.
Main test:
- Experiment 1 (range-finding test): Salmonella strains - 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate; E.coli strain - 15, 50, 150, 500, 1500 and 500 ug/plate.
- Experiment 2 (main test): TA100 (without S9-mix): 5 to 1500 ug/plate; TA100 (with S9-mix), TA1535, TA98, TA1537: 15 to 15000 ug/plate; WP2uvrA·: 50 to 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): incubated at 37 degrees C for approximately 48 hours

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity- this will be reported as equivocal.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at 5000ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

cytotoxicity

Remarks:

observed at 500ug/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No test material precipitate was observed .on the plates at any of the doses tested in either the presence or absence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Range finding study

Range-finding test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	98	92	10	14	18	24	21	19	14	12
		73		20		21		19		10
		104		12		34		18		12
-	1.5	106	102	7	11	NT	NT	29	17	13	14
		109		10				13		15
		92		15				10		13
-	5	86	94	12	10	NT	NT	16	17	12	13
		88		8				18		14
		109		9				16		13
-	15	81	80	21	15	26	30	18	19	14	13
		80		13		21		25		13
		78		10		43		13		13
-	50	76	79	13	16	20	20	19	18	15	13
		75		12		18		12		13
		86		23		22		22		11
-	150	87	86	14	19	30	25	21	18	13	12
		81		21		21		19		11
		89		22		23		15		13
-	500	90*	92	21	21	24	20	31	22	9	10
		95*		18		18		25		9
		91*		25		19		11		12
-	1500	0*	0	0*	0	22	22	0*	0	0*	0
		0*		0*		22		0*		0*
		0*		0*		22		0*		0*
	5000	N/T		N/T		19*	14	N/T		N/T
						13*
						9*
+	0	77	75	8	10	32	33	18	23	22	23
		74		14		37		20		24
		74		7		30		30		24
+	1.5	64	70	7	8	NT		26	27	14	19
		78		10				41		24
		67		8				14		20
+	5	71	71	16	11	NT		25	23	21	19
		77		8				24		12
		66		8				19		23
+	15	81	78	13	9	37	32	26	25	15	19
		75		4		23		24		18
		79		10		35		25		23
+	50	79	72	7	11	32	30	10	15	20	16
		65		9		26		16		14
		73		16		32		20		14
+	150	82	80	19	12	46	30	21	26	18	20
		81		11		26		36		20
		78		7		19		21		21
+	500	81	77	8	8	22	25	19	20	11	15
		76		7		30		22		21
		74		8		23		19		12
+	1500	0*	0	0*	0	23	31	20*	19	13*	13
		0*		0*		34		15*		9*
		0*		0*		35		22*		18*
+	5000	NT		NT		19	21	NT		NT
						23
						22
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		674	710	1085	1096	672	955	214	214	2274	2171
		712		1110		1070		216		2205
		743		1092		1123		212		2035
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		1620	1671	302	300	647	625	312	316	651	569
		1598		291		633		316		736
		1794		306		594		320		320

Table 2. Main test

Main test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	80	84	9	13	32	30	15	16	21	12
		90		14		30		17		5
		81		15		27		16		9
-	5	75	73	N/T		NT		N/T		N/T
		68
		76
-	15	82	78	16	13	NT		25	21	17	15
		66		9				17		10
		85		15				22		19
-	50	95	85	15	12	41	32	17	21	10	12
		89		11		27		22		11
		71		9		29		24		16
-	150	95	91	20	18	22	25	25	20	16	16
		85		15		19		19		20
		94		19		34		16		13
-	500	95	95	16*	17	22	28	24	17	7	9
		99		17*		38		11		14
		92		19*		24		17		7
-	1500	0*	0	0*	0	21	20	0*	0	0*	0
		0*		0*		16		0*		0*
		0*		0*		24		0*		0*
-	5000	N/T		N/T		11*	13	N/T		N/T
						10*
						18*
+	0	74	87	11	11	47	42	15		26	25
		96		11		39		17		28
		92		10		41		22		21
+	15	56	64	7	11	N/T		18		23	19
		56		14				22		17
		79		11				13		17
+	50	68	70	9	10	37	39	14		19	17
		62		7		39		22		19
		79		13		40		17		13
+	150	61	68	5	7	30	35	19		9	15
		71		7		39		20		21
		71		9		37		9		15
+	500	73	71	7	8	29	31	24		17	19
		66		6		31		27		22
		75		10		34		15		18
+	1500	0*	0	0*	0	25	30	5*		21*	20
		0*		0*		31		13*		18*
		0*		0*		33		8*		20*
+	5000	N/T		N/T		19*	21	N/T		N/T
						19*
						25*
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		463	458	244	252	887	891	196	177	1936	1896
		473		215		900		162		1633
		437		302		887		173		2119
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		2596	2510	305	285	418	416	206	223	458	434
		2502		288		365		221		385
		2433		262		464		241		458

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance is considered to be non-mutagenic.

Executive summary:

The study was performed to the requirements of guideline OECD 471 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in both the presence and absence of S-9 mix. Salmonella typhimurium strains TA1535, TA1537, TA98 and TAIOO and Escherichiacoli strain WP2uvrK were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 1.5 and 5000 µg/plate, depending on strain type. The experiment was repeated on a separate day using a similar dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels (1.5, 5 and 15 µg/plate) were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies withinthe normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, initially at and above 500 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate or its toxic limit depending on strain type. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

OECD 471, 2006 - The study was performed to the requirements of OECD Guideline 471 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in both the presence and absence of S-9 mix. A preliminary test was performed to determine the test concentrations for main study. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, initially at and above 500 ug/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate or its toxic limit depending on strain type. In the main study, the plate incorporation method was used and the test substance was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA in the presence and absence of S-9 mix.

Justification for selection of genetic toxicity endpoint
Study selected is an in vitro study (Klimisch 1)

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for mutagenicity

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity