Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate by hexane 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:

01 November - 01 December 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:

2018

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:

August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

28 November 2017

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate by hexane extraction

EC Number:

947-288-2

IUPAC Name:

Resinoid of Myroxylon balsamum var. pereirae (Fabaceae) obtained from the exudate by hexane extraction

Test material form:

liquid

Details on test material:

UVCB substance

Specific details on test material used for the study:

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, appropriate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. No correction for purity was required. 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: 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate, with and without S9-mix; repeated in TA1535 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

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

CONTROLS:
- Vehicle/solvent control: Dimethyl sulphoxide; performed in triplicate
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate; performed in triplicate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation; performed in triplicate.
- 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). A number of manual counts were performed due to colonies spreading and/or artefacts on the plates, 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. 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 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate. Eight test item concentrations per bacterial strain (including an intermediate dose concentration of 3000 µg/plate) were selected in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test item following the change in test methodology. However, after incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in TA1535 revertant colony frequency to the extent where the bacterial strain required repeat analysis (absence of S9 only) employing an amended test item dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

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.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- 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.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

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 7.6.1/1 to 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 (light and greasy in appearance) was noted at and above 3000 µg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY
- The vehicle (dimethyl sulphoxide) 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 (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in both the absence and presence S9-mix.
- In the second mutation test (pre incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 1500 µg/plate (TA100 and TA98), 3000 µg/plate (TA1537) and at 500 µg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 3000 µg/plate (TA1535) and at 500 µg/plate (TA100). Weakened lawns were not observed to WP2uvrA, TA98 and TA1537 in the presence of S9, however a reduction in WP2uvrA revertant colonies (0.4 compared to the concurrent vehicle control) were noted at 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.
- No biologically relevant 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). Minor statistical values were noted in Experiment 1 (WP2uvrA at 5000 µg/plate in the absence of S9-mix) and in Experiment 2 (TA100 at 500 µg/plate and TA98 at 15 µg/plate in the absence of S9-mix). However, these responses were, in all cases, within the in-house historical vehicle/untreated control values for the relevant tester strains and were, therefore considered of no biological relevance.
- 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
95		18		20		16		8
82	90)	21	(18)	29	(26)	22	(19)	2	(7)
93		16		29		18		10
Experiment 2
TA100		TA1535		WP2uvrA		TA98		TA1537
120		11		14		23		15
100	(113)	13	(14)	29	(25)	19	(20)	16	(14)
119		19		31		17		12
		12
		12	(12)†
		12

† Repeated at a later date (absence of S9 only) due to an insufficient number of non-toxic concentrations in the original experiment

 

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

 

Test Period		From: 16 November 2017					To: 19 November 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 (DMSO)	100 91 91	(94) 5.2#	12 17 25	(18) 6.6	22 21 20		(21) 1.0	24 18 14	(19) 5.0	10 18 13	(14) 4.0
	1.5 µg	96 101 69	(89) 17.2	20 17 26	(21) 4.6	26 20 25		(24) 3.2	20 19 14	(18) 3.2	5 8 9	(7) 2.1
	5 µg	100 89 84	(91) 8.2	16 15 13	(15) 1.5	17 21 25		(21) 4.0	26 18 23	(22) 4.0	9 9 7	(8) 1.2
	15 µg	84 117 97	(99) 16.6	16 19 16	(17) 1.7	26 23 28		(26) 2.5	14 15 18	(16) 2.1	11 8 11	(10) 1.7
	50 µg	72 100 105	(92) 17.8	21 16 13	(17) 4.0	29 23 24		(25) 3.2	21 20 13	(18) 4.4	12 8 15	(12) 3.5
	150 µg	78 81 73	(77) 4.0	12 14 13	(13) 1.0	28 14 33		(25) 9.8	21 19 13	(18) 4.2	16 3 8	(9) 6.6
	500 µg	81 65 82	(76) 9.5	8 8 16	(11) 4.6	19 27 18		(21) 4.9	17 16 19	(17) 1.5	8 6 19	(11) 7.0
	1500 µg	85 86 71	(81) 8.4	18 10 14	(14) 4.0	31 23 19		(24) 6.1	24 28 21	(24) 3.5	12 12 11	(12) 0.6
	5000 µg	58 SP 53 SP 55 SP	(55) 2.5	8 SP 7 SP 8 SP	(8) 0.6	34 P 37 P 36 P		(36) 1.5	30 SP 10 SP 12 SP	(17) 11.0	8 SP 9 SP 7 SP	(8) 1.0
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
		466 532 571	(523) 53.1	321 330 385	(345) 34.6	562 583 598		(581) 18.1	139 139 150	(143) 6.4	409 459 455	(441) 27.8

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:        Test ítem precipitate

S:       Sparse bacterial background lawn

#:       Standard deviation

 

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

 

Test Period		From: 16 November 2017					To: 19 November 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 (DMSO)	122 119 105	(115) 9.1#	17 17 20	(18) 1.7	33 26 30		(30) 3.5	13 23 21	(19) 5.3	14 17 15	(15) 1.5
	1.5 µg	96 102 101	(100) 3.2	20 12 13	(15) 4.4	32 18 29		(26) 7.4	19 22 23	(21) 2.1	13 20 3	(12) 8.5
	5 µg	88 113 104	(102) 12.7	15 19 17	(17) 2.0	28 22 17		(22) 5.5	16 19 18	(18) 1.5	17 10 10	(12) 4.0
	15 µg	100 97 112	(103) 7.9	16 13 10	(13) 3.0	20 34 28		(27) 7.0	21 26 29	(25) 4.0	16 13 9	(13) 3.5
	50 µg	98 90 103	(97) 6.6	12 12 10	(11) 1.2	33 31 33		(32) 1.2	18 30 31	(26) 7.2	7 21 10	(13) 7.4
	150 µg	80 65 83	(76) 9.6	8 14 11	(11) 3.0	21 25 17		(21) 4.0	14 22 14	(17) 4.6	7 8 14	(10) 3.8
	500 µg	92 101 94	(96) 4.7	8 10 11	(10) 1.5	20 33 24		(26) 6.7	24 22 18	(21) 3.1	15 9 8	(11) 3.8
	1500 µg	101 101 103	(102) 1.2	11 10 8	(10) 1.5	31 25 28		(28) 3.0	20 15 26	(20) 5.5	11 8 11	(10) 1.7
	5000 µg	85 SP 53 SP 64 SP	(67) 16.3	11 SP 12 SP 9 SP	(11) 1.5	46 P 32 P 30 P		(36) 8.7	18 SP 25 SP 11 SP	(18) 7.0	4 SP 15 SP 7 SP	(9) 5.7
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
		2321 2466 2205	(2331) 130.8	315 262 312	(296) 29.8	149 173 185		(169) 18.3	191 166 191	(183) 14.4	331 347 345	(341) 8.7

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

P:        Test ítem precipitate

S:       Sparse bacterial background lawn

#:       Standard deviation

 

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

 

Test Period		From: 24 November 2017 28 November 2017†					To: 27 November 2017 01 December 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 (DMSO)	69 99 67	(78) 17.9#	18 18 21	(19) 1.7	29 27 25		(27) 2.0	13 13 12	(13) 0.6	24 16 13	(18) 5.7
	0.05 µg	N/T		14 11 12	(12) 1.5	N/T			N/T		N/T
	0.15 µg	N/T		12 16 14	(14) 2.0	N/T			N/T		N/T
	0.5 µg	N/T		16 12 9	(12) 3.5	N/T			N/T		N/T
	1.5 µg	N/T		11 25 16	(17) 7.1	N/T			N/T		N/T
	5 µg	105 98 84	(96) 10.7	14 14 10	(13) 2.3	30 29 20		(26) 5.5	17 27 19	(21) 5.3	11 16 23	(17) 6.0
	15 µg	87 82 84	(84) 2.5	9 11 8	(9) 1.5	24 30 27		(27) 3.0	23 25 20	(23) 2.5	16 17 17	(17) 0.6
	50 µg	72 87 90	(83) 9.6	8 S 8 S 7 S	(8) 0.6	26 21 20		(22) 3.2	20 20 13	(18) 4.0	19 8 13	(13) 5.5
	150 µg	86 68 115	(90) 23.7	7 S 9 S 7 S	(8) 1.2	25 26 24		(25) 1.0	13 13 12	(13) 0.6	28 21 12	(20) 8.0
	500 µg	117 97 109	(108) 10.1	N/T		25 22 21		(23) 2.1	20 9 12	(14) 5.7	21 9 5	(12) 8.3
	1500 µg	0 T 0 T 0 T	(0) 0.0	N/T		17 12 22		(17) 5.0	13 S 7 S 10 S	(10) 3.0	22 6 13	(14) 8.0
	3000 µg	0 TP 0 TP 0 TP	(0) 0.0	N/T		29 P 33 P 24 P		(29) 4.5	11 SP 14 SP 14 SP	(13) 1.7	6 SP 10 SP 9 SP	(8) 2.1
	5000 µg	0 TP 0 TP 0 TP	(0) 0.0	N/T		30 SP 19 SP 19 SP		(23) 6.4	11 SP 8 SP 19 SP	(13) 5.7	15 SP 15 SP 14 SP	(15) 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
		574 751 664	(663) 88.5	811 697 813	(774) 66.4	1025 994 939		(986) 43.6	170 206 201	(192) 19.5	304 387 405	(365) 53.9

 †:       Repeated at a later date due to an insufficient number of non-toxic concentrations in the original experiment

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

N/T:    Not tested at this dose level

P:       Precipitate

S:        Sparse bacterial background lawn

T:       Toxic, no bacterial background lawn

V:       Very weak bacterial background lawn

#:       Standard deviation

 

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

 

Test Period		From: 24 November 2017					To: 27 November 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 (DMSO)	99 84 98	(94) 8.4#	10 12 4	(9) 4.2	39 35 45		(40) 5.0	17 19 26	(21) 4.7	15 15 22	(17) 4.0
	5 µg	106 94 92	(97) 7.6	5 9 10	(8) 2.6	22 32 32		(29) 5.8	24 25 15	(21) 5.5	20 12 13	(15) 4.4
	15 µg	105 90 111	(102) 10.8	12 8 16	(12) 4.0	34 29 32		(32) 2.5	28 26 23	(26) 2.5	30 19 16	(22) 7.4
	50 µg	94 92 106	(97) 7.6	7 7 9	(8) 1.2	38 28 31		(32) 5.1	17 28 25	(23) 5.7	19 17 16	(17) 1.5
	150 µg	102 71 70	(81) 18.2	7 9 12	(9) 2.5	29 29 36		(31) 4.0	30 29 23	(27) 3.8	24 15 19	(19) 4.5
	500 µg	77 84 72	(78) 6.0	7 13 10	(10) 3.0	29 33 38		(33) 4.5	19 33 23	(25) 7.2	13 23 11	(16) 6.4
	1500 µg	85 77 75	(79) 5.3	7 9 8	(8) 1.0	35 33 34		(34) 1.0	21 28 21	(23) 4.0	5 12 17	(11) 6.0
	3000 µg	76 P 68 P 71 P	(72) 4.0	15 SP 13 SP 13 SP	(14) 1.2	24 P 29 P 25 P		(26) 2.6	33 P 20 P 19 P	(24) 7.8	17 P 15 P 10 P	(14) 3.6
	5000 µg	92 SP 91 SP 87 SP	(90) 2.6	14 SP 16 SP 6 SP	(12) 5.3	17 P 17 P 16 P		(17) 0.6	14 P 27 P 19 P	(20) 6.6	10 P 10 P 9 P	(10) 0.6
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
		1095 1314 1320	(1243) 128.2	279 284 255	(273) 15.5	174 217 227		(206) 28.2	131 135 116	(127) 10.0	342 353 216	(304) 76.1

2AA:   2-Aminoanthracene

BP:      Benzo(a)pyrene

P:        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

SD:     Standard deviation

Min:     Minimum value

Max:    Maximum value

†:        Number 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 - 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: 5, 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate, with and without S9-mix

After incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in TA1535 revertant colony frequency to the extent where the bacterial strain required repeat analysis (absence of S9 only) employing an amended test item dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

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 (dimethyl sulphoxide) 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 (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in both the absence and presence S9-mix.

In the second mutation test (pre incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 1500 µg/plate (TA100 and TA98), 3000 µg/plate (TA1537) and at 500 µg/plate (WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted from 3000 µg/plate (TA1535) and at 500 µg/plate (TA100). Weakened lawns were not observed to WP2uvrA, TA98 and TA1537 in the presence of S9, however a reduction in WP2uvrA revertant colonies (0.4 compared to the concurrent vehicle control) were noted at 5000 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  

A test item precipitate (light and greasy in appearance) was noted at and above 3000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No biologically relevant 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 in Experiment 2 (pre incubation method). Minor statistical values were noted in Experiment 1 (WP2uvrA at 5000 µg/plate in the absence of S9-mix) and in Experiment 2 (TA100 at 500 µg/plate and TA98 at 15 µg/plate in the absence of S9-mix). However, these responses were, in all cases, within the in-house historical vehicle/untreated control values for the relevant tester strains and were, therefore considered of no biological relevance.

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