Dipotassium hexacyanocobalt(II)-ferrate(II)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8.10.2014-28.10.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test, Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries) and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(EC) 440/2008 of 30 May 2008

Deviations:

yes

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is included as an attachment to the study report.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

λ²-cobalt(2+) potassium hexacyanoirondiuide

EC Number:

603-073-2

Cas Number:

12549-23-4

Molecular formula:

K2[CoFe(CN)6]

IUPAC Name:

λ²-cobalt(2+) potassium hexacyanoirondiuide

Test material form:

solid: particulate/powder

Remarks:

powder

Details on test material:

- Substance type: commercial product (pure active substance)
- Physical state: Solid powder
- Storage condition of test material: Ambient temperature, humidity and pressure. Stored in sealed containers in darkness.
- Stability under test conditions: Stable
- Purity: ca 100 %
- Particle size distribution: 1.05% <100μm
- Crystal structure: Face-centered cubic structure
- Density: 2.21 x 10^3 kg/m3
- pH value: the substance has a neutral pH value in an aqueous solution

Method

Target gene:

Type of mutations indicated: frame shift mutations, base-pair substitutions

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Genotype: trp-; uvrA-:; Type of mutations indicated: base-pair substitution

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Genotype: his C 3076; rfa-; uvrB-:/ his D 3052; rfa-; uvrB-; R-factor;/ his G 46; rfa-; uvrB-;/ his G 46; rfa-; uvrB-; R-factor ; Type of mutations indicated: frame shift mutations/base-pair substitutions

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item(1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile, distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Negative (untreated) controls were used in parallel with the test item

Negative solvent / vehicle controls:

yes

Remarks:

Culture with vehicle (sterile, distilled water)

True negative controls:

no

Positive controls:

yes

Remarks:

Positive controls were used in parallel with the test item

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

2-Aminoanthracene and benzo(a)pyrene were used in the series of plates with S9-mix

Details on test system and experimental conditions:

Experiment 1.
DOSES: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h (incubation at 37 C± 3 C)

NUMBER OF REPLICATIONS: 3

OTHER EXAMINATIONS:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


Experiment 2.
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
METHOD OF APPLICATION: preincubation
DOSES: 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the pre-incubation method.

DURATION
- Exposure duration: 20 min pre-incubation (37 C± 3 C with shaking), 48 h incubation (37 C± 3 C)

NUMBER OF REPLICATIONS: 3

OTHER EXAMINATIONS:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. 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:

The individual plate counts, the mean number of revertant colonies and the standard deviations were determined for the test item, positive and vehicle controls, both with and without metabolic activation.

The analyses were appropriate to determine the mutagenicity of the test item.

A history profile of vehicle, untreated and positive control values (reference items) are also presented.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA1537, TA98, TA1535 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water. However, the test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.
- Precipitation: A brown colouration and associated test item precipitate (greasy in appearance) was observed at and above 500 ug/plate, this observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA: See table 1

Remarks on result:

other: all strains tested

Any other information on results incl. tables

Table 1. History Profile of Vehicle and Positive Control Values COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2012 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 Mean 97 96 20 14 274 313 32 37 22 25 12 13 124 153 95 123 SD 16.9 17.9 5.1 3.3 26.8 38.2 8.0 7.8 5.8 6.7 2.7 2.8 27.0 37.4 15.9 18.0 Min 62 63 9 9 209 255 14 15 11 5 4 3 79 78 66 89 Max 162 169 39 33 321 355 58 59 48 42 24 25 216 242 161 168 Values† 780 560 561 347 20 8 668 479 898 369 880 347 64 43 56 34 POSITIVE CONTROL VALUES 2012 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 Mean 546 1097 406 287 1033 840 734 330 167 204 778 245 764 1420 2027 633 SD 223.5 394.5 378.8 143.3 413.1 248.9 223.3 139.8 54.2 76.8 444.9 90.8 393.4 357.5 522.9 308.7 Min 266 268 85 108 541 418 256 120 81 85 146 98 158 714 1065 261 Max 3151 3241 4157 1335 1961 1284 1661 1409 420 677 3110 622 1910 2070 3071 1700 Values† 221 233 212 226 13 13 189 188 345 233 340 226 20 22 21 36 COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2013 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 Total Plates 843 855 1612 798 12 6 1542 765 1655 876 1646 807 42 36 42 36 Min 68 63 9 8 191 266 15 13 10 12 5 5 110 112 105 108 Max 147 153 37 29 292 292 47 54 42 43 26 23 162 181 154 162 Mean 103 101 20 15 255 279 28 33 22 26 11 13 138 152 124 137 SD 14.4 15.6 4.4 3.5 47.3 18.4 6.6 7.1 5.0 5.1 3.1 3.5 14.4 22.2 15.4 17.1 POSITIVE CONTROL VALUES 2013 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 Total Plates 849 861 810 795 6 6 765 762 828 849 825 804 21 30 21 30 Min 240 349 91 103 668 673 129 101 102 84 113 86 501 840 302 385 Max 1429 3117 3750 1153 1016 694 1275 733 783 669 2161 1238 1124 2391 2889 1198 Mean 543 1211 644 250 842 684 611 320 207 226 813 286 698 1359 1288 665 SD 192.1 509.3 685.5 98.9 246.1 14.elo 256.3 120.9 76.9 92.6 384.2 127.7 263.1 441.9 894.7 305.7

SD Standard deviation

Min Minimum value

Max Maximum value

† Number of mean values used to create dataset

Table 2 Spontaneous Mutation Rates (Concurrent Negative Controls).

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
100	(106)	7	(10)	19	(22)	16	(18)	11	(17)
118		11		24		23		24
99		13		24		15		17

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
131	(126)	13	(17)	35	(28)	16	(19)	11	(11)
122		19		23		24		16
126		20		25		16		7

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 and Table 4 for Experiment 1 and Table 5 and Table 6 for Experiment 2.

Table 3. Test Results: Experiment 1 – Without Metabolic Activation

Test Period From: 17 October 2014 to 20 October 2014
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains									Frameshift strains
		TA100			TA1535			WP2uvrA			TA98			TA1537
	Solvent Control (WATER)	107		(98)	16.00		(15)	20		(19)	19		(19)	13		(15)
		100		10.7+	12.00		2.60	12		6.6	28		9.5	19		3.5
		86			17.00			25			9			13
	1.5 ug	92		(88)	8.00		(12)	21.00		(24)	21		(17)	20		(18)
		83		4.7	8.00		6.90	27.00		3.1	8		8.1	15		2.6
		90			20.00			23.00			23			19
	5 ug	98		(101)	9.00		(10)	24.00		(21)	17		(17)	16		(15)
		99		4.40	12.00		2.10	19.00		2.6	16		1.5	16		2.6
		106			8.00			20.00			19			12
	15 ug	76		(90)	15.00		(13)	29.00		(21)	20		(20)	12		(16)
		88		15.10	11.00		2.10	13.00		8.0	23		3.0	20		4.0
		106			12.00			20.00			17.00			17
	50 ug	94		(98)	13.00		(13)	27		(20)	23		(18)	13		(16)
		96		5.30	13.00		0.60	13		7.0	21		7.6	19		3.0
		104			12.00			21			9			16
	150 ug	96		(92)	1.00		(10)	19		(22)	25		(20)	15		(18)
		87		4.70	8.00		2.60	21		4.2	15		5.0	19		2.6
		94			9.00			27			19			20
	500 ug	112	P	(110)	12.00	P	(10)	17	P	(18)	17	P	(21)	20	P	(16)
		104	P	5.30	8.00	P	2.10	21	P	2.6	24	P	3.5	13	P	3.5
		114	P		11.00	P		16	P		21	P		16	P
	1500 ug	118	P	(107)	13.00	P	(13)	13	P	(15)	12	P	(18)	16	P	(15)
		107	P	11.50	15.00	P	1.50	13	P	3.5	25	P	6.7	13	P	2.1
		95	P		12.00	P		19	P		16	P		17	P
	5000 ug	103	P	(104)	15.00	P	(17)	19	P	(22)	21	P	(22)	16	P	(14)
		112	P	8.00	24.00	P	6.70	21	P	4.2	21	P	2.3	8	P	5.7
		96	P		11.00	P		27	P		25	P		19	P
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG			ENNG			ENNG			4NQO			9AA
		3 ug			5 ug			2 ug			0.2 ug			80 ug
		627		(637)	1205		(1258)	1465		(1581)	172		(178)	484		(482)
		644		8.90	1168		125.80	1660		102.5	191		11.3	287		194.0
		640			1402			1617			171			675

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

+ Standard deviation

Table 4. Test Results: Experiment 1 – With Metabolic Activation

Test Period From: 17 October 2014 to 20 October 2014
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains									Frameshift strains
		TA100			TA1535			WP2uvrA			TA98			TA1537
	Solvent Control (WATER)	91		(104)	20		(17)	21		(25)	25		(24)	9		(12)
		116		12.5+	15		2.5	19		8.7	27		3.1	11		3.6
		104			17			35			21			16
	1.5 ug	102		(103)	13		(12)	29		(29)	24		(25)	16		(15)
		100		3.6	9		3.1	31		2.0	27		1.5	13		2.1
		107			15			27			25			17
	5 ug	107		(95)	13		(16)	19		(24)	21		(19)	15		(15)
		91		10.2	23		6.1	21		7.0	16		2.5	20		4.5
		88			12			32			19			11
	15 ug	107		(96)	15		(13)	32		(29)	19		(20)	17		(14)
		94		10.6	16		4.4	29		3.5	25		4.6	8		4.9
		86			8			25			16			16
	50 ug	92		(96)	9		(13)	17		(17)	20		(20)	20		(16)
		94		5.3	15		3.8	17		0.0	C		0.0	11		4.5
		102			16			17			20			16
	150 ug	114		(92)	11		(15)	29		(28)	24		(24)	20		(15)
		100		5.3	16		3.2	35		7.5	28		3.5	15		4.5
		92			17			20			21			11
	500 ug	75	P	(102)	20	P	(19)	27	P	(27)	23	P	(28)	8	P	(13)
		104	P	18.7	24	P	6.1	23	P	4.0	31	P	4.2	20	P	6.1
		110	P		12	P		31	P		29	P		12	P
	1500 ug	112	P	(104)	12	P	(14)	28	P	(22)	19	P	(22)	11	P	(16)
		110	P	12.2	19	P	4.0	15	P	6.6	24	P	2.9	19	P	4.2
		90	P		12	P		23	P		24	P		17	P
	5000 ug	90	P	(104)	11	P	(10)	36	P	(25)	19	P	(21)	8	P	(11)
		96	P	19.3	9	P	1.2	19	P	9.5	24	P	2.6	11	P	2.5
		126	P		11	P		20	P		20	P		13	P
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA			2AA			2AA			BP			2AA
		1 ug			2 ug			10 ug			5 ug			2 ug
		815		(834)	238		(250)	207		(223)	234		(223)	740		(781)
		858		21.9	242		18.0	207		27.1	242		26.9	766		50.8
		829			271			254			192			838

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

C Contaminated

P Test item precipitate

+ Standard deviation

Table 5. Test Results: Experiment 2 – Without Metabolic Activation

Test Period From: 24 October 2014 to 27 October 2014
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains									Frameshift strains
		TA100			TA1535			WP2uvrA			TA98			TA1537
	Solvent Control (WATER)	108		(122)	11		(16)	29		(27)	13		(19)	5		(11)
		127		12.7+	16		4.5	24		2.6	16		7.9	15		5.1
		132			20			28			28			12
	50 ug	128		(131)	13		(15)	24		(28)	19		(15)	19		(14)
		135		3.5	15		2.0	28		4.5	12		3.5	9		5.0
		131			17			33			15			13
	150 ug	135		(125)	12		(12)	24		(27)	8		(15)	5		(11)
		124		9.5	16		4.5	24		5.2	20		6.1	17		6.0
		116			7			33			16			12
	500 ug	127	P	(133)	13	P	(15)	24	P	(24)	21	P	(22)	12	P	(14)
		136	P	5.2	13	P	3.5	20	P	4.5	20	P	2.1	12	P	4.0
		136	P		19	P		29	P		24	P		19	P
	1500 ug	135	P	(133)	16	P	(16)	25	P	(27)	23	P	(20)	8	P	(7)
		135	P	4.0	21	P	4.5	31	P	3.8	17	P	3.1	4	P	2.3
		128	P		12	P		24	P		21	P		8	P
	5000 ug	122	P	(118)	17	P	(16)	24	P	(24)	24	P	(17)	3	P	(8)
		104	P	12.1	13	P	3.1	28	P	4.5	12	P	6.2	11	P	4.6
		127	P		19	P		19	P		15	P		11	P
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG			ENNG			ENNG			4NQO			9AA
		3 ug			5 ug			2 ug			0.2 ug			80 ug
		515		(498)	2323		(2023)	1215		(1188)	279		(248)	482		(657)
		430		61.3	1942		268.4	1219		49.7	250		32.0	846		182.4
		549			1805			1131			215			643

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

+ Standard deviation

Table 6.Test Results: Experiment 2 – With Metabolic Activation

Test Period From: 24 October 2014 to 27 October 2014
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains									Frameshift strains
		TA100			TA1535			WP2uvrA			TA98			TA1537
	Solvent Control (WATER)	136		(127)	15		(12)	29		(30)	27		(25)	7		(11)
		114		11.7+	11		3.1	33		3.1	23		2.1	17		5.3
		132			9			27			24			9
	50 ug	112		(118)	8		(11)	23		(28)	19		(23)	12		(12)
		124		6.0	15		3.5	35		6.4	29		5.3	12		0.0
		118			11			25			21			12
	150 ug	144		(135)	13		(14)	32		(30)	24		(23)	12		(11)
		130		7.6	15		1.2	28		2.1	21		2.1	8		2.3
		132			15			31			25			12
	500 ug	116	P	(122)	13	P	(11)	27	P	(30)	25	P	(25)	11	P	(9)
		128	P	6.0	13	P	2.9	32	P	2.6	27	P	1.5	8	P	2.1
		122	P		8	P		31	P		24	P		7	P
	1500 ug	143	P	(127)	13	P	(10)	2	P	(28)	21	P	(24)	8	P	(8)
		123	P	14.4	9	P	3.1	29	P	4.2	28	P	3.6	4	P	3.5
		115	P		7	P		31	P		23	P		11	P
	5000 ug	103	P	(122)	15	P	(14)	16	P	(21)	29	P	(24)	8	P	(8)
		144	P	20.7	16	P	2.6	12	P	12.9	16	P	7.2	8	P	0.6
		118	P		11	P		36	P		28	P		7	P
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA			2AA			2AA			BP			2AA
		1 ug			2 ug			10 ug			5 ug			2 ug
		289		(338)	263		(236)	377		(405)	128		(121)	394		(394)
		295		79.2	230		25.0	430		26.6	107		11.8	388		6.0
		429			214			408			127			400

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item

+ Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative. Not classified according to the CLP Regulation based on the Reverse Mutation Assay 'Ames Test' using Salmonella
typhimurium and Escherichia coli. The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 ug/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 50 to 5000 μg/plate.

The vehicle (sterile distilled water) 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.

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) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of S9-mix, in the second mutation test (pre-incubation method). A brown colouration and associated test item precipitate (greasy in appearance) was observed at and above 500 ug/plate, this observation did not prevent the scoring of revertant colonies.

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 S9-mix in Experiment 1 (plate incorporation method). Similarly, 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 S9-mix in Experiment 2 (pre-incubation method).

The substance was therefore considered to be non-mutagenic under the conditions of the test.