Absolute of Cistus ladaniferus (Cistaceae) obtained from cistus concrete by heat treatment and ethanol 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:

May - October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Absolute of Cistus ladaniferus (Cistaceae) obtained from cistus concrete by heat treatment and ethanol extraction.

Molecular formula:

not applicable for UVCB

IUPAC Name:

Absolute of Cistus ladaniferus (Cistaceae) obtained from cistus concrete by heat treatment and ethanol extraction.

Test material form:

liquid: viscous

Remarks:

brown-red viscous liquid

Details on test material:

Name CISTUS ABSOLUTE SIS
Batch no. 0317/3
Appearance brown-red, viscous liquid
CAS No. 89997-74-0
EINECS-No. 289-711-7
Purity 100% UVCB substance
Production date March 2017
Expiry date March 2019
Storage Room Temperature (20 ± 5°C)

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: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate, without S9-mix for TA100, TA1535 and TA 1537 and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98 without S9. 15, 50, 150, 500, 1500and 5000 μg/plate with S9 for all strains.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
In solubility checks performed in house the test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

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: preincubation
All testing will be performed using the pre-incubation method (20 minutes at 37 °C) except for the untreated controls.

DURATION
- Preincubation period: 20 minutes.
- Exposure duration: ca. 48 hours

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). Manual counts were performed at 5000 µg/plate because of test item precipitation. Several further manual counts were also 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 dose range used for Experiment 2 was determined by the results of Experiment 1. Up to seven 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 toxic limit of the test item.

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.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (light and globular in appearance) was noted at 5000 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.
- In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA100, TA 1535 and TA 1537) and 1500 µg/plate (TA98 and WP2uvrA) in the absence of S9 mix and to two bacterial strains (TA100 and TA1535) at 5000 µg/plate in the presence S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA1535, TA100 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to three of the bacterial tester strains at 5000 µg/plate (TA100, TA1535 and 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 1 and Experiment 2 .
- 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
87		16		31		31		9
86	(84)	19	(17)	15	(22)	15	(24)	21	(15)
80		17		19		25		16
Experiment 2
TA100		TA1535		WP2uvrA		TA98		TA1537
65		18		36		20		15
65	(64)	23	(18)	32	(34)	15	(18)	18	(16)
61		14		34		19		16

 

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

Test Period		From: 25 July 2017					To: 28 July 2018
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)	83 81 88	(84) 3.6#	26 14 24	(21) 6.4	20 28 25		(24) 4.0	26 16 11	(18) 7.6	15 21 19	(18) 3.1
	1.5 µg	90 71 84	(82) 9.7	9 16 12	(12) 3.5	25 19 24		(23) 3.2	15 21 25	(20) 5.0	18 8 16	(14) 5.3
	5 µg	76 71 85	(77) 7.1	8 21 15	(15) 6.5	16 25 20		(20) 4.5	25 16 14	(18) 5.9	8 13 8	(10) 2.9
	15 µg	96 94 111	(100) 9.3	12 16 9	(12) 3.5	18 24 32		(25) 7.0	10 21 17	(16) 5.6	9 9 14	(11) 2.9
	50 µg	84 82 93	(86) 5.9	9 9 8	(9) 0.6	28 25 27		(27) 1.5	16 16 21	(18) 2.9	15 9 21	(15) 6.0
	150 µg	86 S 79 S 80 S	(82) 3.8	8 S 6 S 20 S	(11) 7.6	25 28 12		(22) 8.5	15 13 18	(15) 2.5	3 S 3 S 3 S	(3) 0.0
	500 µg	74 S 65 S 58 S	(66) 8.0	10 S 12 S 8 S	(10) 2.0	24 26 20		(23) 3.1	27 23 17	(22) 5.0	7 S 6 S 3 S	(5) 2.1
	1500 µg	16 S 15 S 26 S	(19) 6.1	14 S 5 S 12 S	(10) 4.7	23 S 20 S 8 S		(17) 7.9	12 S 12 S 14 S	(13) 1.2	3 S 2 S 2 S	(2) 0.6
	5000 µg	0 VP 0 VP 0 VP	(0) 0.0	0 VP 0 VP 0 VP	(0) 0.0	14 SP 14 SP 13 SP		(14) 0.6	18 SP 18 SP 17 SP	(18) 0.6	0 VP 0 VP 0 VP	(0) 0.0
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
		1692 1753 1736	(1727) 31.5	596 637 489	(574) 76.4	719 710 811		(747) 55.9	311 277 286	(291) 17.6	251 111 175	(179) 70.1

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: 25 July 2017					To: 28 July 2018
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)	89 82 90	(87) 4.4#	15 17 16	(16) 1.0	32 20 41		(31) 10.5	29 40 32	(34) 5.7	15 17 20	(17) 2.5
	1.5 µg	74 85 78	(79) 5.6	10 10 18	(13) 4.6	21 20 31		(24) 6.1	34 16 29	(26) 9.3	12 11 17	(13) 3.2
	5 µg	97 76 91	(88) 10.8	18 15 14	(16) 2.1	24 23 30		(26) 3.8	31 44 32	(36) 7.2	12 10 12	(11) 1.2
	15 µg	81 82 78	(80) 2.1	14 18 24	(19) 5.0	18 21 25		(21) 3.5	13 25 30	(23) 8.7	21 21 20	(21) 0.6
	50 µg	68 90 94	(84) 14.0	20 13 12	(15) 4.4	31 31 33		(32) 1.2	25 23 31	(26) 4.2	14 12 20	(15) 4.2
	150 µg	96 95 84	(92) 6.7	12 21 14	(16) 4.7	34 26 24		(28) 5.3	35 36 27	(33) 4.9	17 13 17	(16) 2.3
	500 µg	92 72 105	(90) 16.6	9 23 15	(16) 7.0	31 33 19		(28) 7.6	25 28 23	(25) 2.5	21 20 13	(18) 4.4
	1500 µg	90 82 84	(85) 4.2	16 26 12	(18) 7.2	29 25 24		(26) 2.6	23 29 33	(28) 5.0	12 13 21	(15) 4.9
	5000 µg	64 SP 66 SP 71 SP	(67) 3.6	10 SP 14 SP 10 SP	(11) 2.3	39 P 18 P 15 P		(24) 13.1	23 P 34 P 20 P	(26) 7.4	7 P 11 P 11 P	(10) 2.3
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
		1170 1028 1286	(1161) 129.2	288 320 285	(298) 19.4	168 168 166		(167) 1.2	144 158 170	(157) 13.0	467 454 475	(465) 10.6

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: 14 August 2017					To: 17 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)	78 65 61	(68) 8.9#	19 19 20	(19) 0.6	20 23 23		(22) 1.7	20 21 22	(21) 1.0	19 16 5	(13) 7.4
	0.5 µg	64 70 83	(72) 9.7	17 19 14	(17) 2.5	N/T			N/T		19 11 14	(15) 4.0
	1.5 µg	79 78 63	(73) 9.0	13 23 19	(18) 5.0	N/T			N/T		13 17 5	(12) 6.1
	5 µg	88 65 74	(76) 11.6	22 21 16	(20) 3.2	20 20 29		(23) 5.2	13 25 21	(20) 6.1	16 9 11	(12) 3.6
	15 µg	62 71 73	(69) 5.9	22 17 16	(18) 3.2	28 33 28		(30) 2.9	20 28 29	(26) 4.9	13 18 15	(15) 2.5
	50 µg	64 68 74	(69) 5.0	18 31 18	(22) 7.5	29 18 21		(23) 5.7	17 20 25	(21) 4.0	9 9 10	(9) 0.6
	150 µg	63 S 67 S 60 S	(63) 3.5	12 S 17 S 21 S	(17) 4.5	28 19 22		(23) 4.6	29 15 17	(20) 7.6	10 S 5 S 14 S	(10) 4.5
	500 µg	58 S 41 S 51 S	(50) 8.5	14 S 15 S 13 S	(14) 1.0	21 20 26		(22) 3.2	14 16 12	(14) 2.0	5 S 7 S 6 S	(6) 1.0
	1500 µg	N/T		N/T		17 S 17 S 21 S		(18) 2.3	14 S 12 S 26 S	(17) 7.6	N/T
	5000 µg	N/T		N/T		31 SP 20 SP 28 SP		(26) 5.7	9 SP 11 SP 11 SP	(10) 1.2	N/T
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
		807 620 724	(717) 93.7	1500 1528 1598	(1542) 50.5	668 627 485		(593) 96.0	175 205 180	(187) 16.1	347 152 193	(231) 102.8

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:       Test item precipitate

#:       Standard deviation

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

Test Period		From: 14 August 2017					To: 17 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)	71 67 85	(74) 9.5#	16 20 22	(19) 3.1	35 24 33		(31) 5.9	34 24 38	(32) 7.2	13 14 7	(11) 3.8
	15 µg	61 75 89	(75) 14.0	26 22 22	(23) 2.3	40 36 31		(36) 4.5	26 32 39	(32) 6.5	13 15 13	(14) 1.2
	50 µg	74 75 76	(75) 1.0	21 24 13	(19) 5.7	29 41 33		(34) 6.1	40 36 24	(33) 8.3	17 13 14	(15) 2.1
	150 µg	86 82 75	(81) 5.6	11 18 29	(19) 9.1	24 45 31		(33) 10.7	36 36 26	(33) 5.8	20 15 15	(17) 2.9
	500 µg	79 79 75	(78) 2.3	17 17 33	(22) 9.2	27 34 31		(31) 3.5	44 13 42	(33) 17.3	11 18 21	(17) 5.1
	1500 µg	62 65 65	(64) 1.7	22 21 31	(25) 5.5	33 35 26		(31) 4.7	23 32 33	(29) 5.5	11 13 8	(11) 2.5
	5000 µg	64 SP 60 SP 55 SP	(60) 4.5	26 SP 31 SP 29 SP	(29) 2.5	19 SP 23 SP 23 SP		(22) 2.3	30 P 29 P 34 P	(31) 2.6	15 P 19 P 18 P	(17) 2.1
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
		1029 1096 1037	(1054) 36.6	233 212 200	(215) 16.7	139 117 157		(138) 20.0	137 107 70	(105) 33.6	394 453 413	(420) 30.1

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

OMBINED 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

SD: Standard deviation

Min: Minimum value

Max: Maximum value

†: No. of mean values used to create dataset

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 - Pre incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate,  without S9-mix for TA100, TA1535 and TA 1537 and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98 without S9. 15, 50, 150, 500, 1500and  5000 μg/plate with S9 for all strains.

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.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA100, TA 1535 and TA 1537) and 1500 µg/plate (TA98 and WP2uvrA) in the absence of S9 mix and to two bacterial strains (TA100 and TA1535) at 5000 µg/plate in the presence S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA1535, TA100 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted to three of the bacterial tester strains at 5000 µg/plate (TA100, TA1535 and WP2uvrA).  

A test item precipitate (light and globular in appearance) was noted at 5000 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 and Experiment 2 .

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