Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

test item	year of the study	target/RA	purity	guidelines	results	notes
Basic Green 4 oxalate	1999	target	90%	OECD471	positive, TA98, increased in the cytotoxicity range	purity detected by TLC
Basic Green 4 oxalate	1984	target	70%	OECD471	negative	ca. 90% ( based on 70.8 %, as free active substance with MW = 329.5 g/mol of the cation)
Basic Green 4 chloride	2004	RA	>94%	OECD471	negative	by HPLC at 254 nm and LG ca. 1%

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Details on mammalian cell type (if applicable):

Salmonella typhimurium strains TA97a, TA98, TA100 and TA102, obtained from B. N. Ames, were maintained from stocks stored at -75 °C as described by Maron and Ames.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from the liver of rats

Test concentrations with justification for top dose:

0.01 , 0.05, 0.1 , 0.5 , 1.0 , 5.0 , 10.0 µg per plate

Vehicle / solvent:

All test compounds were dissolved in DMSO (100 µl per plate).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

mitomycin C

other: 2-aminoanthracene

Remarks:

(+S9) 2-AA. (-S9) 4-NQO for TA97a, TA98 and TA100 and mitomycin C for TA102.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: each dose was tested in triplicate and two independent assays were performed.

DOSE SELECTION: dose ranges were selected from preliminary results on cytotoxicity.

METABOLIC ACTIVATION SYSTEM: post-mitochondrial supernatant (S9 fraction) prepared from the liver of rats induced with methylcholantrene/phenobarbital.

Evaluation criteria:

A compound is considered positive if it induces a reproducible at least twofold increase in the number of revertant colonies over the concurrent negative control, at one or more concentrations. Another criterion for determining a positive result is a concentration-related increase over the range tested.

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the mutagenicity studies with Malachite Green confirmed its cytotoxicity at higher concentrations. In the presence of metabolic activation, however, the toxicity of Malachite Green to bacteria was slightly decreased. Malachite green had no mutagenic activity in any bacterial strains, with and without metabolic activation, for doses at or lower than 10 µg per plate.
The significantly increased number of revertants with the positive controls indicated the sensitivity of the assays.

RANGE-FINDING/SCREENING STUDIES
In a preliminary assay, concentrations of Malachite Green higher than 10 µg per plate were found to be highly toxic to Salmonella strain TA100.
For doses in the range 0.1–5 µg per plate, the viability of bacteria was 48–71 % of the control values, with a significant dose–effect relationship for doses at or higher than 0.5 µg per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutagenecity testing of Malachite Green in Salmonella typhimurium*

		TA97a	TA98	TA100	TA102
Without metabolic activation
Negative control		208, 213	36, 30	223, 245	647. 692
DMSO		256, 209	26, 18	195, 255	538, 646
Malachite green (µg/plate)	10	tox, nd	14, nd	tox, nd	tox, nd
	5	143, nd	15, nd	135, nd	255, nd
	1	149, 73	22, 19	171, 163	433, 440
	0.5	176, 125	26, 22	201, 172	446, 377
	0.1	184, 170	20, 24	202, 195	528, 434
	0.05	nd, 185	nd, 25	nd, 173	nd, 387
	0.01	nd. 140	nd. 29	nd, 183	nd. 373
Positive controls		657, 684	415, 437	1670, 1943	2615, 2480
With metabolic activation
Negative control		277, 273	40, 40	240, 275	801, 952
DMSO		250, 245	34, 44	246, 247	801, 855
Malachite green (µg/plate)	10	149, nd	27, nd	164, nd	387, nd
	5	179, nd	28, nd	194, nd	517, nd
	1	191, 183	32, 31	210, 210	577, 554
	0.5	228, 163	36, 33	190, 193	729, 562
	0.1	212, 212	45, 33	189, 219	708, 610
	0.05	nd. 183	nd. 34	nd, 233	nd, 578
	0.01	nd, 239	nd, 37	nd, 210	nd, 560
Positive controls		3537, 3397	4278, 4272	4247, 4571	2358, 1657

*Data are mean values (first assay, second assay) of three replicates. nd, not determined; tox, toxic dose.

Preliminaty assay: cytotoxicity on spontaneous revertants of malachite green to Salmonella strain TA 100*

	Malachite green (µg per plate)
	Control	DMSO	0.1	0.5	1	5	10
Number of colonies	21	19	15	15	12	10	3
(% of control)		(90)	(71)	(71)	(57)	(48)	(14)

*Data are mean values of three replicates

Conclusions:

Interpretation of results (migrated information):
negative

Malachite green had no mutagenic activity in any bacterial strains, with and without metabolic activation, for doses at or lower than 10 µg per plate.

Executive summary:

The genotoxic potential of the fungicide Malachite Green oxalate was assessed in bacteria cells. The test was conducted according to OEDC guideline

471 (1983) for the testing of chemicals using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 with and without metabolic activation. Each dose was tested in triplicate and two independent assays were performed. All test compounds were dissolved in DMSO (100 µl per plate). Negative and solvent controls were included in each assay. Malachite green was tested at concentrations ranging from 0.01 to 10 µg per plate. Dose ranges were selected from preliminary results on cytotoxicity.

The results of the mutagenicity studies with Malachite Green confirmed its cytotoxicity at higher concentrations. In the presence of metabolic activation, however, the toxicity of Malachite Green to bacteria was slightly decreased. Malachite green had no mutagenic activity in any bacterial strains, with and without metabolic activation, for doses at or lower than 10 µg per plate. The significantly increased number of revertants with the positive controls indicated the sensitivity of the assays.

 

Conclusion

Malachite green had no mutagenic activity in any bacterial strains, with and without metabolic activation, for doses at or lower than 10 µg per plate.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read across from analogue substance

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The experiment is well documented and scientifically acceptable. Read across from a similar substance which has the same main component and with a different counter ion that does not influence the characteristics related to the specific end-point.

Principles of method if other than guideline:

Testing was performed as reported by Zeiger et al. (1992).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA104, and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 0.1, 0.3, 1.0, 3.3 and 10.0 μg/plate

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

mitomycin C

other: 4-Nitro-o-phenylenediamine, 2-aminoanthracene

Remarks:

(-S9): SA TA100 and TA1535, 9-AA TA97, 4-NPD TA98, mitomycin-C TA102, MMS TA104. (+S9) 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION
Malachite green chloride was incubated with the Salmonella typhimurium tester strains TA97, TA98, TA100, TA102, TA104, and TA1535 either in buffer or S9 mix for 20 minutes at 37 °C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37 °C.
The high dose was limited by toxicity.

METABOLIC ACTIVATION SYSTEM: metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver.

NUMBER OF REPLICATIONS
Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of malachite green chloride.

Evaluation criteria:

In the assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold-increase required for a chemical to be judged positive or weakly positive.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutagenicity of Malachite Green Chloride in Salmonella typhimurium*

Strain	Dose (µg/plate)	Revertants/plate**
		-S9		+ hamster S9		+ rat S9
		Trial 1	Trial 2	10 %	30 %	10 %	30 %
TA102	0.0	272 ± 2.1	284 ± 1.8	256 ± 2.6	266 ± 2.1	259 ± 2.0	337 ± 4.6
	0.1	282 ± 2.3	280 ± 1.8	250 ± 2.9	255 ±216	257 ± 1.8	342 ± 4.5
	0.3	288 ± 1.8	290 ± 2.3	258 ± 2.7	274 ± 2.7	264 ± 2.2	336 ± 5.5
	1.0	267 ± 2.1	295 ± 2.3	259 ± 1.8	271 ± 2.4	274 ± 2.3	349 ± 4.7
	3.3	277 ± 3.1	300 ± 2.6	249 ± 2.0	282 ± 311	280 ± 1.7	357 ± 4.6
	10.0	271 ±2.3	285 ± 2.4	260 ± 1.5	258 ± 1.9	259 ± 3.2	325 ± 3.4
Trial summary		Negative	Negative	Negative	Negative	Negative	Negative
Positive control***		1416±14.2	961 ± 2.3	831 ± 4.3	759 ± 4.2	892 ± 3.6	1027 ± 12.5
TA104	0.0	279 ± 9.0	343 ± 2.3	280 ± 1.3	284 ± 213	276 ± 1.9	284 ± 2.6
	0.1	272 ± 12.0	349 ± 1.5	288 ± 2.8	277 ± 14.1	283 ± 2,1	288 ± 1.5
	0.3	289 ± 0.6	358 ± 2.0	277 ± 2.0	286 ± 1.8	281 ± 4.6	290 ± 1.8
	1.0	292 ± 1.2	364 ± 2.4	286 ± 1.3	289 ± 113	278 ± 4.1	286 ± 2.1
	3.3	289 ± 1.5	358 ± 2.6	273 ± 1.8	299 ± 1.8	282 ± 3.2	290 ± 2.6
	10.0	273 ± 1.8	348 ± 2.4	277 ± 2.2	293 ± 1.8	280 ± 1.7	289 ± 1.8
Trial summary		Negative	Negative	Negative	Negative	Negative	Negative
Positive control		818 ± 6.5	1116 ± 4.9	1343 ± 17.5	1396 ± 3.8	1463 ± 13.7	989 ± 8.0
TA100	0.0	127 ± 0.9	129 ± 1.2	126 ± 0.3	151 ± 1.5	112 ± 2.0	137 ± 1.5
	0.1	127 ± 1.2	132 ± 1.2	133 ± 2.3	150 ± 1.5	115 ± 3.2	149 ± 1.7
	0.3	127 ± 0.9	131 ± 2.7	134 ± 2.7	147 ± 115	108 ± 3.2	154 ± 1.8
	1.0	127 ± 1.7	140 ± 1.9	142 ± 1.5	152 ± 2.7	113 ± 1.8	144 ± 0.9
	3.3	128 ± 1.3	138 ± 1.5	135 ± 3.0	156 ± 1.2	113 ± 1.8	145 ± 2.3
	10.0	129 ± 0.9	132 ± 1.8	125 ± 1.5	153 ± 118	114 ± 2.1	136 ± 1.0
Trial summary		Negative	Negative	Negative	Negative	Negative	Negative
Positive control		531 ± 5.2	467 ± 15.2	734 ± 5.4	729 ± 3.5	885 ± 3.8	882 ± 4.6
TA1535	0.0	17 ± 1.3	17 ± 0.9	17 ± 0.7	14 ± 0.3	17 ± 0.9	20 ± 1.5
	0.1	17 ± 0.9	17 ± 0.7	15 ± 0.9	I6 ± 1.2	18 ± 0.9	21 ± 2.1
	0.3	17 ± 1.0	19 ± 0.6	17 ± 1.2	16 ± 0.9	15 ± 1,5	20 ± 1.2
	1.0	17 ± 1.8	18 ± 1.9	16 ± 0.3	17 ± 1.0	15 ± 1.0	18 ± 1.2
	3.3	17 ± 1.7	17 ± 1.5	16 ± 1.2	17 ± 1.5	17 ± 1.2	21 ± 1.7
	10.0	17 ± 0.9	17 ± 0.9	17 ± 1.3	16 ± 1.7	16 ± 0.7	18 ± 2.6
Trial summary		Negative	Negative	Negative	Negative	Negative	Negative
Positive control		563 ± 3.5	230 ± 4.0	221 ± 4.9	180 ± 2.6	274 ± 4.8	238 ± 9.0
TA97	0.0	118 ± 1.5	123 ± 1.5	127 ± 2.1	130 ± 1.7	153 ± 4.1	128 ± 2.1
	0.1	128 ± 0.7	121 ± 2.1	122 ± 1.2	125 ± 1.9	158 ± 315	125 ± 1.3
	0.3	126 ± 1.2	125 ± 0.9	124 ± 2,0	128 ± 1.2	146 ± 3.2	1241 1.5
	1.0	126 ± 1.0	127 ± 1.7	127 ± 1.2	123 ± 0.6	157 ± 2.0	134 ± 2.3
	3.3	124 ± 0.7	120 ± 2.4	125 12.7	125 ± 2.6	137 ± 414	134 ± 0.6
	10.0	118 ± 1.3	126 ± 1.2	128 ± 1.9	128 10.9	134 13.8	1271 1.2
Trial summary		Negative	Negative	Negative	Negative	Nsgarive	Negative
Positive control		387 ± 3.5	251 ± 5.9	472 ± 6.7	78 ± 14.6	533 ± 22.3	462 ± 7.2
TA97	0.0	46 ± 0.3	21 ± 0.6	34 ± 1.8	29 ± 0.6	37 ± 1.0	35 ± 2.0
	0.1	45 ± 2.1	25 ± 0.7	34 ± 1.2	27 ± 0.9	37 ± 3.3	32 ± 0.6
	0.3	44 ± 1.5	25 ± 2.0	36 ± 1.8	29 ± 0.3	39 ± 3.2	34 ± 1.5
	1.0	45 ± 1.0	28 ± 1.2	38 ± 1.2	31 ± 0.6	39 ± 1.2	36 ± 1.5
	3.3	47 ± 0.9	24 ± 2.1	38 ± 0.3	34 ± 1.5	41 ± 1,5	36 ± 1.8
	10.0	45 ± 1.3	20 ± 0.9	34 ± 1.5	33 ± 1.8	46 ± 0.7	36 ± 0.9
Trial summary		Negative	Negative	Negative	Negative	Negative	Negative
Positive control		285 ± 3.8	344 ± 9.6	983 ± 6.7	829 ± 2.6	360 ± 3.5	442 ± 3.8

*Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Zeiger et al. (1992).

0 μg/plate was the solvent control.

**Revertants are presented as mean ± standard error from three plates.

*** The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97), 4-nitroo-phenylenediamine (TA98), mitomycin-C (TA102), and methyl methanesulfonate (TA104). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Conclusions:

Interpretation of results (migrated information):
negative

Malachite green chloride was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, TA104, or TA1535, with or without induced rat or hamster liver S9 activation enzymes.

Executive summary:

Malachite green chloride was tested for mutagenicity in bacteria. Malachite green chloride was incubated with the Salmonella typhimurium tester strains TA97, TA98, TA100, TA102, TA104, and TA1535 either in buffer or S9 mix for 20 minutes at 37 °C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37 °C. The high dose was limited by toxicity.

Malachite green chloride (0.1-10.0 μg/plate) was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, TA104, or TA1535, with or without induced rat or hamster liver S9 activation enzymes.

Conclusion

Malachite green chloride was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, TA104, or TA1535, with or without induced rat or hamster liver S9 activation enzymes.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The experiment is scientifically acceptable, nevertheless details about test method and procedures are missing.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1981

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

0.05, 0.26, 1.28, 6.4, 32, 160 µg/plate

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-antramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard quantitative plate incorporation test.

NUMBER OF REPLICATIONS: each dose was tested in triplicate and all assays were performed twice.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

usually encountered at 1.28 µg/plate unless S-9 was added

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

usually encountered at 1.28 µg/plate unless S-9 was added

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

usually encountered at 1.28 µg/plate unless S-9 was added

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

a significant increase was seen in the three highest doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

usually encountered at 1.28 µg/plate unless S-9 was added

Positive controls validity:

valid

Additional information on results:

No significant increases in the numbers of revertants were seen at dose level 0.05, 0.26, 1.28, 6.4, 32, and 160 µg/plate in the strains TA 1535, TA 1537, and TA 100 with or without S-9 mix added.
Cell toxicity was usually encountered at 1.28 µg/plate unless S-9 was added.
The microsomal preparation extended some protection against the cytotoxic properties of malachite green. In TA 98 there was no increase without metabolic activation, but a significant increase was seen in the three highest doses after addition of S-9. A supplementary investigation in a narrower dose range showed a dose dependency in the range 20-70 µg/plate.

Conclusions:

Interpretation of results (migrated information):
ambiguous

Negative in TA 1535, TA 1537, and TA 100 with or without S-9 mix; negative in TA98 without metabolic activation. Significant increase was seen in TA98 strains at the three highest doses (from 6.4 µg/plate) after addition of S-9.

Executive summary:

Point mutations. Malachite green was tested using the standard quantitative plate incorporation test (OECD 1981; Ames et al. 1975). The Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were used with and without metabolic activation. Each dose was tested in triplicate and all assays were performed twice. Sodium azide, 2-nitrofluorene and 2-antramine were used as positive controls.

No significant increases in the numbers of revertants were seen at dose level 0.05, 0.26, 1.28, 6.4, 32, and 160 µg/plate in the strains TA 1535, TA 1537, and TA 100 with or without S-9 mix added.

Cell toxicity was usually encountered at 1.28 µg/plate unless S-9 was added.

The microsomal preparation extended some protection against the cytotoxic properties of malachite green. In TA 98 there was no increase without metabolic activation, but a significant increase was seen in the three highest doses after addition of S-9. A supplementary investigation in a narrower dose range showed a dose dependency in the range 20-70 µg/plate.

Conclusion

Negative in TA 1535, TA 1537, and TA 100 with or without S-9 mix; negative in TA98 without metabolic activation. Significant increase was seen in TA98 strains at the three highest doses (from 6.4 µg/plate) after addition of S-9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo gene mutation, OECD 489, oral, rats, negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In house bred animals
- Age at study initiation: 7 weeks for pre-study and the main studies
- Weight at study initiation:
Male – 186.24 to 225.53 g, Female – 186.04 to 220.75g (Pre study)
Male – 203.30 to 233.98 g, Female – 189.23 to 227.18g (Main Study)
Male – 181.23 to 187.51 g, Female – 181.09 to 186.97g (Follow up Main Study)

- Assigned to test groups randomly:
The animals were weighed and arranged in ascending order of their body weights. These stratified body weights were distributed to all the experimental groups using Microsoft Excel Spread sheet. In pre study, the body weight variations of animals selected for the experiment were -8.44 to +10.40 % and -6.70 to +8.31%. In main study, the body weight variations of animals selected for the experiment were -5.40 to +8.12 % and -8.73 to +8.90 % of the mean body weight in males and females, respectively. In follow up main study, the body weight variations of animals selected for the experiment were -1.76 to +2.00 % and -1.20 to +2.14 % of the mean body weight in males and females, respectively. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out the statistically significant differences between groups within each sex.

- Fasting period before study: none
- Housing: Maximum of three animals of same sex and group were housed together in a standard polypropylene cage (L 430 × B 285 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) will be provided ad libitum to the animals throughout the experimental period. The contaminant analysis test reports of feed will be included in the study report.

- Water (e.g. ad libitum):Water will be provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit will be provided in plastic water bottles with stainless steel sipper tubes.

- Acclimation period: Healthy adult animals were acclimatized for five days to laboratory conditions and were observed for clinical signs daily. Veterinary examination of all the animals was performed on the day of receipt and on the day of randomization.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 22.9°C
- Humidity (%): relative humidity 48 to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 28 July 2022 To: 14 November 2022

Route of administration:

oral: gavage

Vehicle:

Solubility/miscibility test was performed using distilled water, 0.5% carboxymethyl cellulose and corn oil. Test item formed suspension in distilled water, uniform suspension in 0.5% carboxymethyl cellulose and suspension in corn oil. Corn oil was selected as vehicle and the same was used for the formulation preparation. The substance in fact can form the carbinol form in water that increases its concentration with increasing pH. Therefore corn oil was selected as the most appropriate vehicle.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required quantity of test item was weighed as per the dose. The weighed test item was transferred to a clean mortar and ground using pestle by adding a small quantity of vehicle to get a uniform suspension. The content was transferred to a measuring cylinder. Again, a small quantity of vehicle was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item was transferred completely into the measuring cylinder. The final volume was made up with vehicle to get the desired concentration as per the dose requirement. Formulation of the test item was prepared freshly every day before dosing.

Doses selected for the dose range finding study: 0, 20, 60 and 180 mg/kg bw
Doses selected for the main study: 0, 20, 50 and 125 mg/kg bw
Doses selected for the main study follow up: 0, 10, 50 and 75 mg/kg bw

Concentration of test item in corn oil formulation: 0, 5, 15 and 45 mg/ml (for a dose volume of 4 ml/kg). For the stability and homogeneity and for method validation the concentrations at 0.5 mg/ml and 45 mg/ml were selected.

Stability of Dose Formulation
The stability of the test item in dose formulations was established under Bioneeds Study No.: BIO-ANM 1964.

Homogeneity and Dose Concentration Analysis
Homogeneity and Dose concentration analysis for concentration verification for study was done by Analytical Department of Bioneeds India Private Limited.
The analysis was done as per validated analytical methods detailed in the study plan of Study No.: BIO-ANM 1964. Sampling and analysis was performed for different dilutions/concentrations prepared for testing in study and the details was reported in the study report.
The samples prepared (top, mid and bottom layer) were transferred to Analytical Department of Bioneeds India Private Limited for dose concentration analysis. Only one set of aliquots of each concentration was analysed for concentration verification. The second aliquot was stored at suitable temperature and used for re-analysis (if required) otherwise was discarded after the confirmation of the results.

The validation of the analytical method was performed for the determination of content of Basic Green 4 Oxalate in Corn Oil using HPLC method. The validation was performed in terms of matrix effect, specificity, linearity, accuracy, precision, homogeneity and stability. The content of Basic Green 4 Oxalate was determined by using the validated method.
The method validation parameters evaluated for Basic Green 4 Oxalate meet the acceptance criteria. The results obtained are within the specified limits. See section 8 for further details. Thus, this method is suitable for the analysis of the Basic Green 4 Oxalate content in Corn Oil for dose formulation in the Genotoxicity studies

Duration of treatment / exposure:

2 days

Frequency of treatment:

once a day for two consecutive days for dose range finding study, main study and follow up study

Post exposure period:

none

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

follow up main study doses

No. of animals per sex per dose:

dose range finding: 3 animals/sex/dose
main study and follow up study: 5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

positive control: Ethyl methanesulfonate
Ethyl methanesulfonate will be dissolved in distilled water and will be administered at a dose of 250 mg/kg bw/day.

Tissues and cell types examined:

glandular stomach
duodenum
liver

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on dose range finding and first main study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were euthanized by cervical dislocation post 2 to 6 hours after the second treatment. Selected tissues (liver, glandular stomach and duodenum) were removed, dissected and a portion was collected for the comet assay. The tissues for the comet assay were placed into ice-cold mincing and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and were stored in ice-cold mincing buffer until processed.


-PREPARATION OF CELL NUCLEI OR SINGLE CELL
Single cell suspensions were prepared by chopping the tissue with scalpels in a small amount of ice-cold mincing solution. The scraped tissue solution was transferred into a homogenization tube and then gently homogenized using a homogenizer. Homogenate was filtered through a 70 micron pore nylon mesh and was then centrifuged at 800xg for 10 minutes. Single cells were suspended in DPBS. Cell suspensions were maintained at 2 to 8°C.

-PREPARATION OF SLIDES
Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g. 1/3 to 3/3) and sex of each animal using slide marker. To reduce the possibility of detachment of the agarose during the procedure, slides were pre coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of 1.0% low-melting agarose gel was mixed and rapidly pipette onto the surface of the pre coated slides and a coverslip was placed on it. Slides were placed on ice packs until the Agarose layer hardened (5 minutes). Slides were immersed in chilled lysing solution in the dark for overnight. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.


METHOD OF ANALYSIS:
The slides were placed onto a platform of submarine-type electrophoresis unit containing a chilled electrophoresis solution. Buffer reservoirs were filled with freshly made pH>13 Electrophoresis Buffer until the liquid level completely covers the slides.
Slides were allowed to sit in the alkaline buffer for 20 minutes to allow for unwinding of the DNA and the expression of alkali-labile damage. Power supply ~0.8 V/cm was turned on and current was adjusted to 300 milliamperes by raising or lowering the buffer level.
Electrophoresis solution was maintained a constant temperature usually between 2 to 10°C during electrophoresis under dimmed light. After completion of electrophoresis, power was turned off. Slides were removed from the buffer and place on a drain tray.
The slides were immersed in the neutralization buffer for 10 minutes. All slides were dehydrated by immersing the slides into absolute ethanol up to 10 minutes. Slides were stained with 80 μL 1X Ethidium. bromide for 5 minutes and then dipped in chilled distilled water to remove excess stain. Drained slides were kept in cold 100% methanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50±1°C for 30 minutes.

All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with Ethidium bromide, covered with a fresh coverslip and cells were scored under 400 X magnification.
At least 150 cells were analyzed per sample. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.
After slide observation finished, slides were drained by keeping in cold 100% methanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50±1°C for 30 minutes

Evaluation criteria:

The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.

Statistics:

Body weight were analyzed by SPSS at a 95% level of confidence (p<0.05) of significance. Inter group comparison of Body weight (day 1 and day 2) and percent tail DNA. The dose correlation was done using ‘t’ test. The statistical significances are designated by the superscripts as given below throughout the tables: *: Statistically significant (p<0.05).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Rationale for exposure: the substance was tested for dose range finding and consequently tested. High toxicity was observed for both studies up to 120 mg/kg bw. Bioavailability of the substance at the selected organs is therefore demonstrated, due to the toxic effects recorded (stomach ballooning and mortality). The highest dose of the follow up main study was selected to be in the range 60 to 125 mg/kg bw, as being the maximum tolerated dose, i.e. 75 mg/kg bw, which is in very good agreement with the highest dose selected also for the in vivo studies reported in the pubblication "Genotoxicity of Malachite Green and Leucomalachite Green in female big blue B6C3F mice" (450 ppm in diet = 67.5 mg/kg for mouse).

Dose Range Finding Study (Pre study)
-Clinical Signs of Toxicity and Mortality
No clinical signs or mortality were observed in animals treated with test item at 20 and 60 mg/kg and vehicle control in either sex.
At 180 mg/kg, on day 2 lethargy was observed in all animals. After dosing on day 2 lethargy was observed in all animals and nasal discharge was observed in one male and two females . Mortality was recorded for one female at 180 mg/kg.

-Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 20 and 60 mg/kg and vehicle control. Stomach ballooning was observed in all animals dosed at 180 mg/kg.


Main Study
Clinical Signs of Toxicity and Mortality
In main study, no clinical sign was observed in any of the animals dosed 20, 50 mg/kg and no mortality was observed in any of the animals in both sexes.
On day 2 lethargy and nasal discharge was observed in all animals dosed at 125 mg/kg. One female animal found dead and one female animal found in the moribund status; and were sacrificed humanely.

-Body Weight
There was no statistically significant change in mean body weight among the positive control dosed animals and Basic Green 4 oxalate dosed animals in comparison with vehicle dosed animals in both the sexes.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 20, 50 and 125 mg/kg, vehicle control and positive control.


Follow up Main Study
-Clinical Signs of Toxicity and Mortality
In main study, no clinical sign was observed in any of the animals dosed 10, 50 and 75 mg/kg and no mortality was observed in any of the animals in both sexes.

-Body Weight
There was no statistically significant change in mean body weight among the positive control dosed animals and Basic Green 4 oxalate dosed animals in comparison with vehicle dosed animals in both the sexes.

-Gross Pathology
No gross pathological findings were observed in any of the animals dosed at 10, 50 and 75 mg/kg, vehicle control and positive control.

DOSE CONCENTRATION ANALYSIS
In the comet assay, the percent recovery of Basic Green 4 oxalate at the tested concentrations on Day 1 (24/08/2022) ranged from 94.30 to 98.58 % and on Day 2 (25/08/2022) ranged from 91.10 to 99.12 % of the nominal concentrations. The results obtained indicate recovery was within the acceptance criteria of ±15% of the nominal concentrations .

Sex	Group & Dose (mg/kg)	Animal No.		Organ
			Liver	Glandular Stomach	Duodenum
Male	G1 & 0	Rh3765	2.085	1.837	2.047
		Rh3766	3.520	5.594	4.254
		Rh3767	4.740	5.464	4.052
		Rh3768	3.807	4.945	4.467
		Rh3769	4.461	5.43	5.233
		Mean	3.72	4.65	4.01
		±SD	1.04	1.59	1.19
	G2 & 10	Rh3770	2.527	3.033	1.971
		Rh3771	3.208	6.819	4.000
		Rh3772	4.774	4.113	4.174
		Rh3773	4.283	5.175	4.412
		Rh3774	4.477	5.94	5.344
		Mean	3.85	5.02	3.98
		±SD	0.95	1.49	1.24
	G3 &50	Rh3775	3.164	2.875	2.232
		Rh3776	3.262	6.577	3.934
		Rh3777	4.820	4.029	4.43
		Rh3778	4.283	4.965	4.135
		Rh3779	4.514	6.129	5.327
		Mean	4.01	4.92	4.01
		±SD	0.75	1.52	1.13
	G4 & 75	Rh3780	3.176	3.084	2.243
		Rh3781	3.312	6.861	3.876
		Rh3782	4.935	4.126	4.354
		Rh3783	4.382	5.275	4.518
		Rh3784	4.517	5.948	5.591
		Mean	4.06	5.06	4.12
		±SD	0.78	1.49	1.22
	G5 & 250  (EMS)	Rh3785	5.837	8.199	7.064
		Rh3786	5.336	8.281	6.334
		Rh3787	5.391	7.619	6.388
		Rh3788	5.567	8.216	6.488
		Rh3789	5.193	8.222	6.536
		Mean	5.46*	8.11*	6.56*
		±SD	0.25	0.27	0.29

 

Sex	Group & Dose (mg/kg)	Animal No.		Organ
			Liver	Glandular Stomach	Duodenum
Female	G1 & 0	Rh3790	3.111	4.016	2.635
		Rh3791	4.602	6.016	3.768
		Rh3792	4.421	4.325	5.511
		Rh3793	4.428	5.008	4.936
		Rh3794	4.063	4.921	5.372
		Mean	4.13	4.86	4.44
		±SD	0.60	0.77	1.22
	G2 & 10	Rh3795	3.144	2.881	3.007
		Rh3796	3.701	7.964	5.322
		Rh3797	5.046	4.216	5.766
		Rh3798	4.647	3.774	3.382
		Rh3799	4.608	7.707	5.035
		Mean	4.23	5.31	4.50
		±SD	0.78	2.36	1.23
	G3 & 50	Rh3800	2.752	3.960	3.256
		Rh3801	3.884	7.791	3.417
		Rh3802	5.102	4.331	5.808
		Rh3803	5.023	5.523	5.138
		Rh3804	4.620	7.596	5.483
		Mean	4.28	5.84	4.62
		±SD	0.98	1.79	1.20
	G4 & 75	Rh3805	2.926	3.861	3.175
		Rh3806	3.931	7.070	3.764
		Rh3807	5.154	4.468	5.954
		Rh3808	4.709	6.986	5.242
		Rh3809	4.633	7.476	5.623
		Mean	4.27	5.97	4.75
		±SD	0.87	1.67	1.22
	G5 & 250  (EMS)	Rh3810	6.075	8.929	6.550
		Rh3811	6.117	8.220	8.328
		Rh3812	6.826	8.613	6.129
		Rh3813	6.172	9.968	10.487
		Rh3814	6.139	10.434	6.309
		Mean	6.27*	9.23*	7.56*
		±SD	0.32	0.93	1.86

Conclusions:

The substance was tested for in vivo gene mutation following OECD489. Under the experimental conditions the substance did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 75 mg/kg.

Executive summary:

The test item was evaluated in the Comet Assay in Wistar Rats as per OECD Guideline No. 489, “In Vivo Mammalian Alkaline Comet Assay”, adopted on 29 July 2016.

This study was conducted to determine if the test item caused an increase in DNA damage in cells from specific organs. The COMET assay detects single and double stranded breaks when DNA is analyzed under alkaline conditions (>pH 13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. The pre study consisted of four groups, vehicle control, 20, 60 and 180 mg/kg; the test item was administered at a dose volume of 4 mL/kg in corn oil. In the pre study, each group of rat consisted of 3 males and 3 females. Clinical signs like lethargy, nasal discharge was observed at 180 mg/kg, mortality was observed in the animals dosed at 180 mg/kg, gross pathological findings like stomach ballooning  was observed at 180 mg/kg. For animals dosed at 20 and 60 mg/kg found normal, no body weight variations and no gross pathological findings were observed in any of the animals.

The main study consisted of 5 groups of rats and each group consisted of 5 males and 5 females. G1 group was administered with vehicle, G2 group was administered with 20 mg/kg, G3 group was administered with 50 mg/kg and G4 group was administered with 125 mg/kg of test item and G5 group was administered with positive control Ethyl methane sulfonate [EMS] at 250 mg/kg for two consecutive days by the oral route using a gavage cannula. Clinical signs like lethargy and nasal discharge at 125 mg/kg was observed for all animals; one female animal was found dead and one female animal was in the moribund status and sacrificed humanely; hence follow up main study was conducted with the dose levels of 10, 50 and 75 mg/kg.

The follow up main study consisted of 5 groups of rats and each group consisted of 5 males and 5 females. G1 group was administered with vehicle, G2 group was administered with 10 mg/kg, G3 group was  administered with 50 mg/kg, G4 group was administered with 75 mg/kg of test item and G5 group animals was administered withpositive control Ethyl methane sulfonate [EMS] at 250 mg/kg for two consecutive days by the oral route using a gavage cannula. Approximately 2 to 6 hours after the last dosing, all rats were euthanized by cervical dislocation and the designated organs (liver, glandular stomach and duodenum) were collected. Tissues were processed, single cells were isolated and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with Ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode.

The results for the assessment of the test item, to cause DNA strand breaks are provided for the doses of 10 [G2], 50 [G3] and 75 [G4] mg/kg, respectively, in male and female Wistar rats. The average % tailing for DNA from male liver cells was 3.72, 3.85, 4.01 and 4.06 and % tailing for DNA from female liver cells was 4.13, 4.23, 4.28 and 4.27. In cells from the glandular stomach, the observed average % tailing for DNA was 4.65, 5.02, 4.92 and 5.06 for males and for 4.86, 5.31, 5.84 and 5.97 females. The average % tailing of DNA observed in cells from the duodenum was 4.01, 3.98, 4.01 and 4.12 for males, and 4.44, 4.50, 4.62 and 4.75 for females. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any organ for any of the Basic Green 4 oxalate  groups when compared to the vehicle control group.

The positive control [G5], Ethyl methane sulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of Basic green 4 oxalate were fully adequate.

Dose concentration analyses conducted for dose verification were found to be within the acceptable range of ± 15% of the nominal concentration. These data support the conclusion that the nominal doses of the test item accurately represent the conditions for this COMET assay.

The data obtained under the conditions employed during this experiment support the conclusion that the test item did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 75 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genotoxic potential of Malachite Green (MG) have been investigated in many experiments conducted in vivo and in vitro and assaying genotoxicty, by different types of study; in many cases, its reduced derivative Leucomalachite Green (LG) has also been investigated.

 

IN VITRO INVESTIGATIONS OF MALACHITE GREEN

When tested in the point mutation assay on Salmonella thyphimurium strains TA97, TA98, TA100, TA104 and TA1535, MG did not have any mutagenic activity, in any bacterial strain, in the presence or in absence of metabolic activation, for doses up to 10 µg per plate (Culp, 2004).

The compound was found to be highly toxic to bacteria (TA100 strain) for doses higher than 5 µg per plate (Fessard et al., 1999).

These results partly confirm data from Clemmensen et al (1984). These authors have indicated that MG was negative in strains TA1535, TA1537 and TA100, with and without metabolic activation. The test substance was also negative in TA98 without S9 but was positive over a narrow range of doses (20–70 µg per plate) with metabolic activation and in the absence of marked cytotoxicity. The above table summarises the data collected from the three OECD471. The only positive results in one single strain TA98 was recorded for the oldest test (where the characterisations and composition of the substance was performed only by TLC) at doses close to cytoxicity; therefore it can be considered not relevant and the overall assessment of these screening tests negative.

 

MG was found to be extremely cytotoxic to mammalian cells (CHO) in culture. In the absence of metabolic activation, its mutagenic potential could be evaluated only for very low concentrations of 0.001–0.05 µg/ml medium. When the S9 fraction was added to the medium, the highest tested dose could be increased to 1 µg/ml. However, MG did not increase the number of thioguanine-resistant mutants in the CHO/HGPRT point mutation test (Fessard et al., 1999).

 

Because DNA strand breaks are generally considered to be the primary event leading to sister chromatid exchanges, chromosomal aberrations and genotoxicity, the effects of Malachite Green were evaluated in in vitro the Comet assay. Without exogenous activation, detectable DNA damage was observed only for doses (>3 µg/ml) inducing immediate cytotoxic effects (20–70 %), as measured by the Trypan blue exclusion method. There was a good relationship between DNA damage and cytotoxicity to CHO cells. When S9 fraction was added to the medium, MG had no cytotoxic effects for doses < 10 µg/ml and did not induced any DNA damage. At the two higher doses (15 and 20 µg/ml), the mean tail moment was increased significantly when only a moderate decrease in cell viability (10–20 %) was observed. The Comet assay confirmed that MG induced DNA alterations only at cytotoxic doses. After metabolic activation, however, DNA damage was observed at doses inducing only low cytotoxicity (Fessard et al., 1999).

 

IN VIVO INVESTIGATIONS OF MALACHITE GREEN

An in vivo alkaline Comet Assay (OECD489) was performed in 2022 with the target substance Malachite Green 0xalate, by oral gavage in rats, followng a decision on compliance check. The MTD was established between 75 and 125 mg/kg bw and the main study was conducted with highest dose at 75 mg/kg bw.

After exposure the animals were euthanised, gross pathology performed and slides for the  eletrophoresis analysis prepared. Under the experimental conditions the substance did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 75 mg/kg. Bioavailability of the substance is considered fulfilled since the dose was just below the lethal doses as recorded during the dose range finding and first main assay.

 

In vivo micronucleous tests performed on rat and mouse failed to demonstrate clastogenic effects of MG (Culp, 2004 and Clemmensen et al 1984).

 

Further in vivo investigations were performed in the experiment published by Mittelstaedt et al., (2004), in which transgenic female Big Blue B6C3F1 mice were fed with 450 ppm MG (the high doses used in the tumor bioassays) and evaluated genotoxicity after 4 and 16 weeks of treatment. MG did not increase the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point. Furthermore, no increases in liver cII mutant frequency were seen in the mice treated for 16 weeks with MG.

 

In general Malachite Green oxalate was tested in vitro and in vivo both for  cytogenicity and gene mutation toxicity and all results agree and confirm that the substance is not genotoxic nor mutagenic.

 

 

 

 

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

 

The test substance did not show any reasons of concern in the in vitro nor the in vivo tests performed.

 

In conclusion, the substance is not classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).