Reaction mass of 2,4-dimethyl-6-phenyl-3,6-dihydro-2H-pyran and 2-methyl-4-methylene-6-phenyltetrahydro-2H-pyran and 4,6-dimethyl-2-phenyl-3,6-dihydro-2H-pyran

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 January 2012 and 14 February 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Kanpoan No. 287 -- Environment Protection Agency

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Eisei No. 127 -- Ministry of Health & Welfare

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Heisei 09/10/31 Kikyoku No. 2 -- Ministry of Economy, Trade & Industry

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:

Reaction mass of 2,4-dimethyl-6-phenyl-3,6-dihydro-2H-pyran and 2-methyl-4-methylene-6-phenyltetrahydro-2H-pyran and 4,6-dimethyl-2-phenyl-3,6-dihydro-2H-pyran

Molecular formula:

C13H160

IUPAC Name:

Reaction mass of 2,4-dimethyl-6-phenyl-3,6-dihydro-2H-pyran and 2-methyl-4-methylene-6-phenyltetrahydro-2H-pyran and 4,6-dimethyl-2-phenyl-3,6-dihydro-2H-pyran

Test material form:

other: Liquid

Details on test material:

- Name of test material: Pelargene
- Physical state: liquid
- Lot no. / Batch no. NX0002642
- Purity: 99.03 %
- Expiration date of the lot/batch: 2012-08-14
- Storage condition of test material: At room temperature, light protected

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Base on the toxic effects observed eight concentrations were tested and 2500 µg/plate were chosen as maximal concentration for the Salmonella typhimurium strains and 5000 µg/plate were chosen as maximal concentration for the E. coli strain in experiment II.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
S. typhimurium strains: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
E. coli strain: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Migrated to IUCLID6: sodium azide, NaN3 used with Strains TA 1535, TA 100 @ 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD used with Strains: TA 1537, TA 98 @ 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: methyl methane sulfonate, MMS used with Strains: WP2 uvrA @ 3 µL/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA used with Strains:TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA @ 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),10 µg/plate (WP2 uvrA)

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 27.7 mg/mL (lot no. R 041111) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in experiment I with and without S9 mix and at 2500 and 5000 µg/plate in experiment II with S9 mix . No precipitation of the test item in the overlay agar was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Reduced background growth was observed at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 333 - 5000 1000 - 500 100 - 5000 100 - 5000
TA 1537 333 - 5000 333 - 5000 100 - 5000 100 - 5000
TA 98 333 - 5000 1000 - 5000 100 - 5000 100 - 5000
TA 100 333 - 5000 333 - 5000 100 - 5000 100 - 5000
WP2 uvrA / 2500, 5000 / /
/ = no reduced background growth observed


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 333 - 5000 1000 - 5000 333 - 2500 333 - 2500
TA 1537 333 - 5000 1000 - 5000 100, 2500 1000, 5000
TA 98 333 - 5000 2500, 5000 333 - 2500 1000, 5000
TA 100 333 - 5000 2500, 5000 2500 1000, 5000
WP2 uvrA / 5000 / /
/ = no toxic effects observed

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1455002	Study Code: Harlan CCR 1455002
Experiment: 1455002 VV Plate	Date Plated: 26/01/2012
Assay Conditions:	Date Counted: 01/02/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			13 ± 1	17 ± 3	32 ± 2	89 ± 4	53 ± 2
	Untreated			12 ± 5	15 ± 5	25 ± 3	91 ± 6	45 ± 7
	Pelargene	3 µg		15 ± 2	16 ± 3	25 ± 4	83 ± 3	47 ± 3
		10 µg		15 ± 1	15 ± 5	21 ± 6	77 ± 7	54 ± 2
		33 µg		14 ± 2	12 ± 4	28 ± 2	73 ± 9	55 ± 13
		100 µg		16 ± 0	13 ± 6	23 ± 4	84 ± 11	45 ± 12
		333 µg		5 ± 3^M R	5 ± 1^M R	12 ± 2^M R	38 ± 6^M R	37 ± 9
		1000 µg		0 ± 0^M R	0 ± 0^M R	10 ± 1^M R	15 ± 3^M R	37 ± 5
		2500 µg		0 ± 0^M R	0 ± 0^M R	6 ± 2^M R	0 ± 0^M R	36 ± 1
		5000 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	29 ± 4
	NaN3	10 µg		1817 ± 63			2160 ± 39
	4-NOPD	10 µg				296 ± 7
	4-NOPD	50 µg			64 ± 2
	MMS	3.0 µL						1267 ± 109
With Activation	DMSO			22 ± 7	19 ± 2	44 ± 3	113 ± 3	71 ± 8
	Untreated			27 ± 1	24 ± 4	48 ± 10	111 ± 6	87 ± 6
	Pelargene	3 µg		26 ± 3	20 ± 4	42 ± 7	107 ± 9	72 ± 15
		10 µg		23 ± 2	29 ± 1	37 ± 4	111 ± 12	67 ± 4
		33 µg		24 ± 2	23 ± 5	45 ± 7	112 ± 29	69 ± 7
		100 µg		28 ± 2	23 ± 7	40 ± 8	124 ± 4	66 ± 4
		333 µg		16 ± 1	19 ± 6^R	36 ± 9	86 ± 3^R	62 ± 2
		1000 µg		5 ± 2^M R	5 ± 2^M R	21 ± 2^M R	68 ± 8^M R	41 ± 5
		2500 µg		0 ± 0^M R	0 ± 0^M R	15 ± 1^M R	0 ± 0^M R	38 ± 5^R
		5000 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	31 ± 3^M R
	2-AA	2.5 µg		300 ± 9	270 ± 12	1720 ± 259	2127 ± 92
	2-AA	10.0 µg						264 ± 12

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	M R	Manual count Reduced background growth

1.1     Summary of Results Experiment II Study Name: 1455002 Study Code: Harlan CCR 1455002 Experiment: 1455002 HV2 Pre Date Plated: 08/02/2012 Assay Conditions: Date Counted: 14/02/2012   Metabolic Activation Test Group Dose Level (per plate)   Revertant Colony Counts (Mean ±SD)                           TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA                   Without Activation DMSO     23 ± 3 13 ± 2 26 ± 8 88 ± 2 41 ± 14 Untreated     24 ± 2 13 ± 3 24 ± 4 97 ± 5 48 ± 8 Pelargene 1 µg   24 ± 7 13 ± 2 26 ± 1 88 ± 6     3 µg   23 ± 6 12 ± 6 25 ± 3 102 ± 4 47 ± 7   10 µg   27 ± 1 15 ± 4 28 ± 6 100 ± 10 41 ± 3   33 µg   23 ± 6 10 ± 2 26 ± 8 96 ± 7 39 ± 2   100 µg   11 ± 5^R 4 ± 3^R 17 ± 8^R 67 ± 6^R 31 ± 7   333 µg   4 ± 1^M R 4 ± 2^M R 9 ± 4^R M 71 ± 10^R 25 ± 3   1000 µg   0 ± 0^M R 0 ± 0^M R 0 ± 0^M R 56 ± 9^M R 27 ± 5   2500 µg   0 ± 0^M R 0 ± 0^M R 0 ± 0^M R 7 ± 3^M R 24 ± 2   5000 µg           29 ± 6 NaN3 10 µg   1794 ± 60     2241 ± 151   4-NOPD 10 µg       348 ± 10     4-NOPD 50 µg     80 ± 7       MMS 3.0 µL           275 ± 22                                                       With Activation DMSO     32 ± 5 21 ± 4 38 ± 10 123 ± 9 53 ± 7 Untreated     30 ± 1 23 ± 5 45 ± 4 119 ± 8 60 ± 12 Pelargene 1 µg   30 ± 6 20 ± 9 40 ± 3 102 ± 9     3 µg   38 ± 12 19 ± 7 35 ± 5 134 ± 15 56 ± 5   10 µg   35 ± 5 16 ± 4 36 ± 5 128 ± 6 55 ± 4   33 µg   38 ± 7 20 ± 4 48 ± 4 108 ± 9 56 ± 6   100 µg   20 ± 1^R 17 ± 7^R 40 ± 1^R 109 ± 17^R 47 ± 7   333 µg   12 ± 6^M R 9 ± 3^M R 23 ± 4^M R 83 ± 4^R 48 ± 7   1000 µg   0 ± 0^M R 1 ± 1^M R 2 ± 3^M R 14 ± 2^M R 36 ± 5   2500 µg   0 ± 0^M R 0 ± 0^M R 0 ± 0^M R 0 ± 0^M R 40 ± 3   5000 µg           29 ± 4 2-AA 2.5 µg   263 ± 24 217 ± 18 1691 ± 63 1901 ± 33   2-AA 10.0 µg           357 ± 42                     Key to Positive Controls Key to Plate Postfix Codes     NaN3 2-AA 4-NOPD MMS sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate R M Reduced background growth Manual count

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Pelargene is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Pelargene to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II,  S. typhimurium strains: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
                         E. coli strain: 3; 10; 33; 100; 333; 1000; 2500;and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at higher concentrations with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at higher concentrations with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pelargene at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.