Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane extraction

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:

26 July - 24 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 471 without any deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

dated 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

dated August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

28 October 2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane extraction

EC Number:

946-584-9

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane extraction

Test material form:

other: Pale yellow to dark yellow paste

Details on test material:

Substance type: UVCB (Natural Complex Substance)
Name: Elemi resinoid
Test substance storage: Dry area, unopened containers, optimum temp. 11-25°C.
Purpose of testing: REACH
Date of expiration: 17 May 2021

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1003395243
- Expiration date of the lot/batch: 17 May 2021
- Purity test date: 18 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark (closed containers)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer and sonication for 10 minutes at 40°C. The test item was confirmed as a UVCB product and, therefore, no purity correction was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Method

Target gene:

histidine locus for Salmonella strains and tryptophan for E. coli strain

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in house, the test item was noted as insoluble in dimethyl sulphoxide at 50 mg/mL but fully soluble in dimethyl formamide at the same concentration and acetone at 100 mg/mL. Acetone was selected as the vehicle. Distilled water was not evaluated as a vehicle in this test system as information provided by the sponsor suggested it was unstable with the test item.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours for both Experiment

CONTROLS:
- Vehicle/solvent control: Acetone
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

NUMBER OF REPLICATIONS: Triplicate

- OTHER: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (i.e. maximum recommended dose level). The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Evaluation criteria:

Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it will be considered to exhibit mutagenic activity in this test system (Cariello and Piegorsch, 1996).

A test item 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

refer tables 7.6.1/4 and 7.6.1/5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted in both experiments in both the presence and absence of S9 from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY
- The vehicle (acetone) control plates gave counts of revertant colonies within the 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.
- Experiment 1 (Plate Incorporation)
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).
- Experiment 2 (Pre-Incubation)
Consequently, in the second mutation test (pre-incubation method), the same maximum dose level was selected as the maximum dose level. In the second experiment, the test item caused a visible reduction in all of the Salmonella bacterial lawns in the absence of S9-mix from 1500 μg/plate. In the presence of S9-mix, toxicity was noted to all of the Salmonella bacterial strains at 5000 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.

HISTORICAL CONTROL DATA [with ranges, means and standard deviation and confidence interval (e.g. 95%)]
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Any other information on results incl. tables

Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
Experiment 1
TA100		TA1535		WP2uvrA		TA98		TA1537
80		28		12		20		15
89	(84)	18	(25)	13	(16)	22	(20)	11	(14)
82		28		23		18		15
Experiment 2
TA100		TA1535		WP2uvrA		TA98		TA1537
73		31		23		16		8
80	(71)	17	(26)	25	(25)	18	(19)	16	(11)
60		30		27		22		10

 

Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation

 

Test Period		From: 07 August 2017					To: 10 August 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	80 68 68	(72) 6.9#	24 38 29	(30) 7.1	18 18 24		(20) 3.5	17 23 18	(19) 3.2	19 14 14	(16) 2.9
	1.5 µg	71 74 74	(73) 1.7	34 27 33	(31) 3.8	21 20 25		(22) 2.6	19 20 18	(19) 1.0	16 14 9	(13) 3.6
	5 µg	71 74 99	(81) 15.4	29 23 22	(25) 3.8	18 18 21		(19) 1.7	15 20 25	(20) 5.0	11 12 17	(13) 3.2
	15 µg	102 64 74	(80) 19.7	23 31 25	(26) 4.2	26 9 17		(17) 8.5	17 13 25	(18) 6.1	17 23 20	(20) 3.0
	50 µg	81 74 61	(72) 10.1	24 27 26	(26) 1.5	16 19 9		(15) 5.1	33 17 10	(20) 11.8	23 19 13	(18) 5.0
	150 µg	82 79 87	(83) 4.0	32 22 27	(27) 5.0	16 14 10		(13) 3.1	14 22 21	(19) 4.4	13 20 15	(16) 3.6
	500 µg	101 74 73	(83) 15.9	30 33 36	(33) 3.0	12 16 11		(13) 2.6	23 18 25	(22) 3.6	18 21 11	(17) 5.1
	1500 µg	79 P 83 P 76 P	(79) 3.5	25 P 28 P 24 P	(26) 2.1	20 P 16 P 22 P		(19) 3.1	22 P 38 P 20 P	(27) 9.9	7 P 11 P 11 P	(10) 2.3
	5000 µg	68 P 66 P 67 P	(67) 1.0	31 P 34 P 33 P	(33) 1.5	11 P 12 P 14 P		(12) 1.5	32 P 19 P 25 P	(25) 6.5	7 P 8 P 10 P	(8) 1.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		346 357 357	(353) 6.4	387 507 661	(518) 137.4	554 586 539		(560) 24.0	171 169 201	(180) 17.9	113 110 113	(112) 1.7

ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:       Test item precipitate

#:       Standard deviation

 

Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation

 

Test Period		From: 07 August 2017					To: 10 August 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	65 82 81	(76) 9.5#	19 22 19	(20) 1.7	24 19 17		(20) 3.6	22 16 20	(19) 3.1	14 13 12	(13) 1.0
	1.5 µg	95 69 86	(83) 13.2	11 17 29	(19) 9.2	25 24 16		(22) 4.9	17 24 22	(21) 3.6	16 7 16	(13) 5.2
	5 µg	71 70 81	(74) 6.1	21 16 26	(21) 5.0	23 22 17		(21) 3.2	37 24 15	(25) 11.1	10 13 15	(13) 2.5
	15 µg	75 72 74	(74) 1.5	10 33 19	(21) 11.6	15 29 26		(23) 7.4	22 23 21	(22) 1.0	21 19 9	(16) 6.4
	50 µg	75 60 78	(71) 9.6	21 22 24	(22) 1.5	28 22 24		(25) 3.1	35 23 23	(27) 6.9	15 14 13	(14) 1.0
	150 µg	69 83 76	(76) 7.0	31 29 23	(28) 4.2	12 26 22		(20) 7.2	26 25 26	(26) 0.6	14 23 10	(16) 6.7
	500 µg	74 94 86	(85) 10.1	21 14 23	(19) 4.7	19 22 15		(19) 3.5	21 20 27	(23) 3.8	10 17 15	(14) 3.6
	1500 µg	80 P 78 P 72 P	(77) 4.2	32 P 29 P 29 P	(30) 1.7	9 P 17 P 16 P		(14) 4.4	19 P 17 P 28 P	(21) 5.9	16 P 12 P 17 P	(15) 2.6
	5000 µg	77 P 71 P 75 P	(74) 3.1	21 P 22 P 26 P	(23) 2.6	19 P 17 P 24 P		(20) 3.6	22 P 26 P 16 P	(21) 5.0	15 P 17 P 13 P	(15) 2.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		849 1124 1178	(1050) 176.4	223 235 242	(233) 9.6	120 123 132		(125) 6.2	188 184 196	(189) 6.1	388 349 395	(377) 24.8

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

P:        Test item precipitate

#:       Standard deviation

 

 

Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation

 

Test Period		From: 21 August 2017					To: 24 August 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	61 79 71	(70) 9.0#	25 22 30	(26) 4.0	30 19 22		(24) 5.7	23 17 15	(18) 4.2	17 9 11	(12) 4.2
	15 µg	70 75 77	(74) 3.6	20 25 27	(24) 3.6	18 18 27		(21) 5.2	22 18 23	(21) 2.6	16 9 8	(11) 4.4
	50 µg	66 66 64	(65) 1.2	22 10 24	(19) 7.6	19 23 20		(21) 2.1	19 19 22	(20) 1.7	14 18 7	(13) 5.6
	150 µg	71 69 69	(70) 1.2	16 13 22	(17) 4.6	22 33 21		(25) 6.7	22 16 24	(21) 4.2	12 11 9	(11) 1.5
	500 µg	60 79 68	(69) 9.5	27 23 17	(22) 5.0	26 21 21		(23) 2.9	12 21 15	(16) 4.6	15 11 13	(13) 2.0
	1500 µg	71 PS 74 PS 71 PS	(72) 1.7	22 PS 26 PS 11 PS	(20) 7.8	35 P 19 P 20 P		(25) 9.0	21 PS 31 PS 16 PS	(23) 7.6	18 PS 7 PS 12 PS	(12) 5.5
	5000 µg	71 PS 90 PS 81 PS	(81) 9.5	0 PV 0 PV 0 PV	(0) 0.0	13 P 11 P 15 P		(13) 2.0	19 PS 15 PS 17 PS	(17) 2.0	8 PS 8 PS 7 PS	(8) 0.6
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		1138 945 1001	(1028) 99.3	877 980 960	(939) 54.6	885 856 915		(885) 29.5	234 210 244	(229) 17.5	309 405 634	(449) 167.0

ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:       Test item precipitate

V:       Very weak bacterial background lawn

S:        Sparse bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation

 

Test Period		From: 21 August 2017					To: 24 August 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	67 61 66	(65) 3.2#	23 26 26	(25) 1.7	30 31 36		(32) 3.2	26 22 24	(24) 2.0	9 17 8	(11) 4.9
	15 µg	64 67 67	(66) 1.7	24 31 16	(24) 7.5	36 31 44		(37) 6.6	20 21 23	(21) 1.5	15 22 17	(18) 3.6
	50 µg	76 76 65	(72) 6.4	19 25 23	(22) 3.1	26 34 35		(32) 4.9	29 15 36	(27) 10.7	9 8 20	(12) 6.7
	150 µg	63 60 62	(62) 1.5	23 18 29	(23) 5.5	38 25 35		(33) 6.8	25 18 16	(20) 4.7	9 14 7	(10) 3.6
	500 µg	68 66 66	(67) 1.2	21 21 18	(20) 1.7	26 26 36		(29) 5.8	22 21 28	(24) 3.8	18 13 16	(16) 2.5
	1500 µg	61 P 67 P 61 P	(63) 3.5	25 P 19 P 26 P	(23) 3.8	25 P 23 P 28 P		(25) 2.5	27 P 29 P 29 P	(28) 1.2	15 P 16 P 21 P	(17) 3.2
	5000 µg	62 PS 70 PS 76 PS	(69) 7.0	19 PS 18 PS 16 PS	(18) 1.5	33 P 39 P 14 P		(29) 13.1	15 PS 20 PS 18 PS	(18) 2.5	16 PS 24 PS 4 PS	(15) 10.1
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1102 1076 1067	(1082) 18.2	272 252 242	(255) 15.3	246 220 247		(238) 15.3	145 134 127	(135) 9.1	322 196 386	(301) 96.7

2AA:   2-Aminoanthracene

BP:      Benzo(a)pyrene

P:       Test item precipitate

S:        Sparse bacterial background lawn

#:       Standard deviation

 

 

Table 7.6.1/6:History Profile of Vehicle and Positive Control Values

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015
Strain S9-Mix		TA100				TA1535				TA102				WP2uvrA				TA98				TA1537				WP2uvrA pKM101				WP2pKM101
		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9	+S9
Values†		274		278		504		285		26		13		461		229		526		299		506		282		42		51		39	49
Min		60		61		7		7		222		278		10		12		11		10		4		6		87		98		89	93
Max		166		175		31		29		376		388		58		43		45		46		27		27		237		254		174	177
Mean		91		95		16		14		286		333		24		27		21		24		12		13		156		164		123	137
SD		19.3		19.1		4.5		4.0		48.7		37.6		5.6		5.9		6.2		6.1		3.8		3.4		42.2		35.6		23.1	21.2
POSITIVE CONTROL VALUES 2015
Strain S9-Mix	TA100				TA1535				TA102				WP2uvrA				TA98				TA1537				WP2uvrA pKM101				WP2pKM101
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9			+S9
Values	276		280		252		264		13		13		231		227		262		276		253		261		20		35		20			35
Min	222		250		79		118		953		673		116		103		100		78		164		97		430		494		745			325
Max	2266		2402		2779		457		3140		1655		2769		550		502		705		2318		823		1696		2264		3662			1174
Mean	614		927		472		246		2303		1093		792		266		222		218		911		336		761		1461		2257			569
SD	260.6		452.5		434.8		55.7		815.2		376.5		342.1		97.7		70.2		107.6		412.4		135.7		350.0		382.0		790.7			220.3
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016
Strain S9-Mix		TA100				TA1535				TA102				WP2uvrA				TA98				TA1537				WP2uvrA pKM101				WP2pKM101
		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9	+S9
Values		399		401		758		393		60		30		690		345		788		415		762		398		32		32		16	24
Min		63		66		8		8		216		221		10		13		8		12		3		4		97		104		78	52
Max		154		156		34		39		340		375		53		53		49		51		24		23		268		243		148	166
Mean		90		93		15		15		268		310		22		27		21		25		12		13		161		159		118	110
SD		14.5		14.3		4.5		5.2		26.4		31.1		5.8		6.3		4.8		5.7		3.5		3.5		39.2		32.3		17.0	29.3
POSITIVE CONTROL VALUES 2016
Strain S9-Mix	TA100				TA1535				TA102				WP2uvrA				TA98				TA1537				WP2uvrA pKM101				WP2pKM101
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9			+S9
Values	409		406		381		386		30		28		341		335		388		385		379		381		14		24		8			16
Min	221		284		84		92		897		629		107		102		100		96		95		101		445		574		1674			372
Max	2222		2863		2994		879		2326		2140		1611		637		449		4357		1413		639		1117		1855		2823			945
Mean	724		1264		854		240		1633		950		718		240		186		188		406		290		743		1271		2379			535
SD	320.4		562.9		664.9		62.1		564.5		382.7		338.6		98.2		49.8		230.8		227.0		92.7		214.6		326.5		426.2			143.3

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

The vehicle (acetone) control plates gave counts of revertant colonies within the 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.

No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). In Experiment 2 (pre incubation method), the test item caused a visible reduction in all of the Salmonella bacterial lawns in the absence of S9-mix from 1500 µg/plate. In the presence of S9-mix, toxicity was noted to all of the Salmonella bacterial strains at 5000 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.

A test item precipitate (greasy and particulate in appearance) was noted in both experiments in both the presence and absence of S9 from 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.