Ferbam

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:

2001-12-19 - 2002-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ferbam

EC Number:

238-484-2

EC Name:

Ferbam

Cas Number:

14484-64-1

Molecular formula:

C9H18FeN3S6

IUPAC Name:

iron(3+) tris(dimethyldithiocarbamate)

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: black powder
- Storage conditions: room temperature in the dark

Method

Target gene:

Salmonella typhimurium LT2

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9-mix in standard co-factors

Test concentrations with justification for top dose:

Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 15, 5, 150, 500, 1500 and 5000 µg/plate
Mutation study - Experiment 1: 1.5, 5, 15, 5, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide
- Justification for choice of solvent/vehicle: Dimethyl formamide is an acceptable vehicle for use in this test system

Controls

Untreated negative controls:

yes

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

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

TESTER STRAINS:
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.
TA100
TA 1535 sensitive to agents inducing base-pair substitution
TA102 sensitive to agents inducing frame-shift mutations
TA98
The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 250427 06/06) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.


PROCEDURE
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel- Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl formamide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1
Six concentrations of the test material (15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended following observations made from Experiment 1 and was as follows:
TA100 and TA1535 (with and without S9), TA98 (with S9 only): 1.5, 5, 15, 50, 150, 500, 1500 ug/plate
TA102 (with and without S9): 5, 15, 50, 150, 500, 1500, 5000 ug/plate
TA98 (without S9 only), TA1537 (with and without S9): 15, 50,150, 500,1500 ug/plate
Additional dose levels were included to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses and, in the case of TA100 and TA1535, to determine the level at which the test material first induced mutations.

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkMlOl plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

15 - 500 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

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

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

15 - 150 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

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

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

10 - 500 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material was toxic initially at and above 1500 ug/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile

Any other information on results incl. tables

Preliminary study

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation	Strain	Dose (µg/plate)
		0	0,15	0,5	1,5	5	15	50	150	500	1500	5000
-	TA 100	94	73	72	93	112	160	231	257	310	0V	0V
+	TA100	148	104	93	126	147	283	322	300	255	249	0T
T = toxic, no bacterial background lawn V = very weak bacterial background lawn

Mutation Study

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 to Table 5.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: 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		TA102		TA98		TA1537
94	(97)	27	(23)	318	(322)	26	(23)	6	(13)
82		21		325		23		18
116		22		322		20		16
Experiment 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
112	(101)	26	(22)	302	(308)	26	(21)	13	(12)
94		16		303		24		9
96		24		318		13		15

Table 2: Test Results: Experiment 1 - Without Metabolic Activation
Test Period	From: 04 January 2002						To: 07 January 2002
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
-	0	87 81 76	(81) 5.5#	32 28 26	(29) 3.1	313 341 328	(327) 14.0	24 26 19	(23) 3.6	14 14 8	(12) 3.5
-	15	222 210 270	$$$ (234) 31.7	34 46 50	$$$ (43) 8.3	309 329 335	(324) 13.6	36 24 25	(28) 6.7	11 9 12	(11) 1.5
-	50	380 352 360	$$$ (364) 14.4	57 65 75	$$$ (66) 9,0	329 330 365	(341) 20.5	28 27 32	(29) 2.6	14 12 16	(14) 2,0
-	150	280 336 291	$$$ (302) 29.7	78 73 66	$$$ (72) 6,0	305 344 368	(339) 31.8	23 31 44	(33) 10.6	11 19 22	(17) 5.7
-	500	252 244 248	$$$ (248) 4.0	64 54 61	$$$ (60) 5.1	281 250 274	(268) 16.3	28 27 31	(29) 2.1	16 15 13	(15) 1.5
-	1500	0V 0V 0V	(0) 0.0	0V 0V 0V	(0) 0.0	94 104 92	(97) 6.4	0V 0V 0V	(0) 0.0	0T 0T 0T	(0) 0.0
-	5000	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0
Positive controls S9-Mix -	Name	ENNG		ENNG		MMC		4NQO		9AA
	Concentration (µg/plate)	3		5		0.5		0.2		80
	No. Colonies per plate	548 513 438	(500) 56.2	453 311 289	(351) 89.0	809 919 924	(884) 65.0	110 117 112	(113) 3.6	1766 1924 2215	(1968) 227.8
ENNG 4NQO 9AA MMC P T V $$$ #	N-ethyl-N'-nitro-N-nitrosoguanidine 4-Nitroquinoline-1-oxide 9-Aminoacridine Mitomycin C Precipiate Toxic, no bacterial background lawn Very weak bacterial background lawn p < = 0.005 Standard deviation

Table 3: Test results: Experiment 1 - With metabolic activation
Test Period	From: 04 January 2002						To: 07 January 2002
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
+	0	84 75 95	(85) 10.0#	18 15 8	(14) 5.1	324 381 361	(327) 14.0	29 40 34	(34) 5.5	18 19 17	(18) 1.0
+	15	196 188 200	$$$ (195) 6.1	52 37 50	$$$ (46) 8.1	491 474 479	(324) 13.6	53 44 59	$$$ (52) 7.5	26 15 21	(21) 5.5
+	50	312 220 236	$$$ (256) 49.2	53 41 54	$$$ (49) 7.2	483 454 478	(341) 20.5	50 50 48	** (49) 1.2	16 28 22	(22) 6.0
+	150	320 316 272	$$$ (303) 26.6	66 55 35	$$$ (52) 15.7	483 457 453	(339) 31.8	50 46 50	** (49) 2.3	19 27 22	(23) 4.0
+	500	276 263 274	$$$ (271) 7.0	43 46 46	$$$ (45) 1.7	346 319 326	(268) 16.3	23 34 40	(32) 8.6	13 15 25	(18) 6.4
+	1500	0T 0T 0T	(0) 0.0	0T 0T 0T	(0) 0.0	155 136 142	(97) 6.4	0V 0V 0V	(0) 0.0	0T 0T 0T	(0) 0.0
+	5000	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0	24P 28P 24P	(0) 0.0	0TP 0TP 0TP	(0) 0.0	0TP 0TP 0TP	(0) 0.0
Positive controls S9-Mix +	Name	2AA		2AA		DAN		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
	No. Colonies per plate	1469 1309 1638	(1472) 164.5	177 185 183	(182) 4.2	967 588 600	(718) 215.4	257 264 165	(229) 55.2	446 469 418	(444) 25.5
BP 2AA DAN P T V ** $$$ #	Benzo(a)pyrene 2-Aminoanthracene 1,8-Dihydroxyanthraquinone Precipiate Toxic, no bacterial bachground lawn Very weak bacterial background lawn p < = 0.01 p < = 0.005 Standard deviation

Table 4: Test results: Experiment 2 - Without metabolic activation
Test Period	From: 11 January 2002						To: 14 January 2002
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
-	0	86 76 83	(82) 5.1#	15 21 17	(18) 3.1	300 372 348	(340) 36.7	14 17 20	(17) 3.0	6 6 15	(9) 5.2
-	1.5	76 70 68	(71) 4.2	13 18 16	(16) 2.5	N/T		N/T		N/T
-	5	92 79 93	(88) 7.8	14 18 9	(14) 4.5	343 320 341	(335) 12.7	N/T		N/T
-	15	123 129 123	$$$ (125) 3.5	29 22 24	(25) 3.6	365 360 369	(365) 4.5	28 22 25	(25) 3.0	11 9 12	(11) 1.5
-	50	188 196 197	$$$ (194) 4.9	36 28 32	$$$ (32) 4.0	374 377 386	* (379) 6.2	41 29 33	** (34) 6.1	12 11 17	(13) 3.2
-	150	268 282 264	$$$ (271) 9.5	37 57 53	$$$ (49) 10.6	400 357 363	(373) 23.3	24 23 39	(29) 9.0	13 14 7	(11) 3.8
-	500	304 267 261	$$$ (277) 23.3	53 42 47	$$$ (47) 5.5	285 298 298	(294) 7.5	22 33 14	(23) 9.5	11 13 12	(12) 1.0
-	1500	0V 0V 0V	(0) 0.0	0V 0V 0V	(0) 0.0	129 C 137	(133) 5.7	0V 0V 0V	(0) 0.0	0V 0V 0V	(0) 0.0
-	5000	N/T		N/T		0TP 0TP 0TP	(0) 0.0	N/T		N/T
Positive controls S9-Mix -	Name	ENNG		ENNG		MMC		4NQO		9AA
	Concentration (µg/plate)	3		5		0.5		0.2		80
	No. Colonies per plate	473 411 438	(441) 31.1	255 255 244	(251) 6.4	852 801 912	(855) 55.6	114 104 106	(108) 173.5	2429 2132 2125	(2229) 173.5
ENNG 4NQO 9AA MMC C N/T P T V * ** $$$ #	N-ethyl-N'-nitro-N-nitrosoguanidine 4-Nitroquinoline-1-oxide 9-Aminoacridine Mitomycin C Contaminated Not tested at this dose level Precipiate Toxic, no bacterial bachground lawn Very weak bacterial background lawn p < = 0.05 p < = 0.01 p < = 0.005 Standard deviation

Table 5: Test results: Experiment 2 - With metabolic activation
Test Period	From: 11 January 2002						To: 14 January 2002
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
+	0	80 113 92	(95) 16.7#	12 14 12	(13) 1.2	391 393 370	(385) 12.7	29 44 41	(38) 7.9	18 13 16	(16) 1.5
+	1,5	83 90 98	(90) 7.5	13 9 12	(11) 2.1	N/T		34 32 27	(31) 3.6	N/T
+	5	113 106 127	(115) 10.7	19 25 14	* (19) 5.5	402 434 402	(413) 18.5	32 36 35	(34) 2.1	N/T
+	15	194 183 202	$$$ (193) 9.5	23 24 33	$$$ (27) 5.5	491 437 468	* (465) 27.1	41 35 40	(39) 3.2	17 17 20	(18) 1.7
+	50	303 252 317	$$$ (291) 34.2	38 42 42	$$$ (41) 2.3	543 529 563	$$$ (545) 17.1	38 39 27	(35) 6.7	18 24 26	* (23) 4.2
+	150	321 370 310	$$$ (334) 31.9	48 62 47	$$$ (52) 8.4	472 524 515	$$$ (504) 27.8	35 35 40	(37) 2.9	24 17 17	(19) 4.0
+	500	267 297 305	$$$ (290) 20.0	31 32 36	$$$ (33) 2.6	388 378 275	(347) 62.6	33 32 25	(30) 4.4	13 12 14	(13) 1.0
+	1500	0V 0V 0V	(0) 0.0	0V 0V 0V	(0) 0.0	196 189 195	(193) 3.8	0V 0V 0V	(0) 0.0	0V 0V 0V	(0) 0.0
+	5000	N/T		N/T		34 SP 43 SP 23 SP	(33) 10.0	N/T		N/T
Positive controls S9-Mix +	Name	2AA		2AA		DAN		BP		2AA
	Concentration (µg/plate)	1		2		10		5		2
	No. Colonies per plate	1728 1747 1746	(1740) 10.7	299 254 286	(280) 23.2	916 904 755	(858) 89.7	165 215 174	(185) 26.7	611 611 636	(619) 14.4
BP 2AA DAN P T V * $$$ #	Benzo(a)pyrene 2-Aminoanthracene 1,8-Dihydroxyanthraquinone Precipitate Toxic, no bacterial background lawn Very weak bacterial background lawn p < = 0.05 p < = 0.005 Standard deviation

Applicant's summary and conclusion

Conclusions:

The test material was considered to be mutagenic under the conditions of this test.

Executive summary:

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines and was conducted in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with suspensions of the test material using the Ames plate incorporation method at up to seven dose levels (0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following observations made in Experiment 1 and ranged between 1.5 and 5000 µg/plate depending on bacterial strain type and presence or absence of S9-mix. Additional dose levels were used in both experiments (where applicable) to allow for the toxicity of the test material ensuring there were a minimum of four non-toxic doses.

The vehicle (dimethyl formamide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the tester strains, both with and without S9-mix, at and above 1500 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial tester strain type and presence or absence of S9-mix. A black, fine, powdery precipitate was observed at and above 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Dose-related, reproducible and statistically significant increases were observed at sub-toxic dose levels of the test material. TA100 and TA1535 exhibited statistically significant increases from 15 to 500 µg/plate both with and without S9-mix. TA102 exhibited reproducible increases in the presence of S9 only from 15 to 150 µg/plate.

No reproducible increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test material, either with or without metabolic activation.

Judging by the type of Salmonella strains exhibiting dose-related increases (TA100, TA1535 and TA102), the test material was concluded to be a base-pair mutagen.

The test material was considered to be mutagenic under the conditions of this test.