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

Biotransformation and kinetics

Administrative data

Endpoint:

biotransformation and kinetics

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Data from literature; 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.

Data source

Reference

Reference Type:

publication

Title:

Uptake, tissue distribution, and metabolism of malachite green in the channel catfish (Ictalurus punctatus).

Author:

Plakas S.M., El Said K.R. , Stehly G.R., Gingerich W.H., Allen J.L.

Year:

1996

Bibliographic source:

Canadian journal of fisheries and aquatic sciences. 53 (1996).

Report date:

1995

Materials and methods

Principles of method if other than guideline:

The disposition of malachite green was determined in channel catfish (Ictalurus punctatus) after intravascular dosing (0.8 mg/kg) or waterborne exposure (0.8 mg/l for 1 h). The effect of the pH of the exposure water on the absorption of the substance was also investigated. The determination of Malachite Green and its metabiolite Leucomalachite Green in muscle and plasma was perfromed by HPLC with visible light detection (618 nm) and quantification; total radioactive residues in tissues, tissue extracts and fluids were determined by liquid scintillation counting (LSC).

GLP compliance:

not specified

Type of medium:

aquatic

Test material

Test material information

Constituent 1

Reference substance name:

Malachite Green Chloride

IUPAC Name:

Malachite Green Chloride

Results and discussion

Transformation products:

yes

Identity of transformation products

No.:

#1

Reference

Reference substance name:

Unnamed

IUPAC name:

Leucomalachite Green

Identifier:

common name

Identity:

Leucomalachite Green

Any other information on results incl. tables

Pharmacokinetic values for Malachite Green after intravascular administration (0.8 mg/kg) in channel catfish.

Parameter^a	Unit	Value
A	µg/ml	0.926
B	µg/ml	0.498
C	µg/ml	0.142
α	/h	3.29
β	/h	0.57
γ	/h	0.111
MRT	h	5.4
Vss	ml/kg	1772
Clb	ml/h/kg	328

^aA, B, and C, coefficients of the triexponetial equation;  α,  β, and  γ, exponents of the triexponential equation; MRT, mean residence time; V_ss, apparent volume of distribution at steady state; Cl_b, total body clearance.

Concentrations of radioactive residues (in microgram equivalents of Malachite Green per gram or millilitre) in the bile and tissues of channel catfish after waterborne exposure to [¹⁴C]Malachite Green (0.8 mg/l for 1 h).

Tissue	0 h	2 h	4 h	24 h	96 h	168 h	336 h
Bile	0.59±0.04	6.83±3.54	33.8±16.2	101±41.2	62.4±17.5	98.2±74.4	78.3±37.5
Fat	1.50±0.50	16.0±7.02	11.8±2.93	19.0±4.31	24.9±6.50	30.3±6.39	26.7±8.48
Liver	33.4±7.45	13.5±2.33	13.0±2.75	4.64±0.61	2.24±0.57	3.42±0.71	2.85±1.32
Head kidney	24.6±4.86	10.2±2.36	8.61±1.06	4.55±0.73	3.05±1.50	2.55±1.31	2.01±1.13
Trunk kidney	22.0±5.38	9.04±1.65	8.36±1.81	4.68±2.34	2.51±1.03	3.10±1.21	2.11±1.57
Skin	3.99±0.68	2.81±1.01	3.36±1.37	2.89±1.67	2.20±0.52	1.40±1.05	0.92±0.51
Spleen	10.9±2.49	6.09±2.61	4.26±0.72	1.29±0.66	1.01±0.36	0.74±0.25	0.58±0.29
Muscle	3.18±0.72	1.88±0.60	1.83±0.19	1.33±0.25	1.49±0.37	1.34±0.35	0.71±0.17
Plasma	6.36±1.51	2.60±0.90	3.32±0.62	0.57±0.37	0.20±0.10	0.20±0.08	0.17±0.07

Relative distribution of Malachite Green and metabolites in extracts of catfish muscle at selected intervals after waterborne exposure to [¹⁴C]Malachite Green.

Time (h)	Peak 1[%]	Peak 2[%]	Peak 3[%]	Leucomalachite Green[%]	Malachite Green[%]
0	8.4	1.9	4.1	48.9	36.8
2	6.2	3.0	7.8	58.6	24.4
24	3.3	7.5	20.5	60.6	8.1
168	nd	5.6	16.4	77.9	nd
336	nd	3.8	16.3	80.0	nd

Mean plasma concentrations of parent Malachite Green exhibited a triphasic decline after intravascular dosing. Pharmacokinetic parameters were obtained by using a three-compartment model to fit the data. A half-life of 6.2 h was determined for the terminal phase of Malachite Green elimination.

Plasma concentrations of Malachite Green and its metabolites increased rapidly during waterborne exposure. Mean concentrations of Malachite Green and Leucomalachite Green were 2.77 and 1.56 µg/ml, respectively, at the end of the exposure period.
Leucomalachite Green levels continued to increase after dosing, achieving a maximum concentration of 2.36 µg/ml at 1 h after transfer of the fish to clean water. As with the intravascularly dosed fish, mean plasma concentrations of Malachite Green exhibited a triphasic decline after waterborne exposure.
The terminal elimination half-life was 4.7 h. At 10 h, plasma levels of Malachite Green were at the limit of determination (0.025 µg/ml) while Leucomalachite Green levels were 30 times higher.

Malachite Green and Leucomalachite Green levels in plasma collected in the tissue distribution study were similar to those of cannulated fish at the earlier sampling times. Malachite Green and Leucomalachite Green concentrations were 3.29 and 1.94 µg/ml, respectively, immediately after dosing. At 1 day after dosing, Malachite Green levels were at the limit of determination, while Leucomalachite Green levels persisted for up to 14 days (0.106 µg/ml). In muscle, Malachite Green and Leucomalachite Green concentrations were 1.18 and 1.45 µg/ml, respectively, at the end of the waterborne exposure period. At 14 days, mean Malachite Green and Leucomalachite Green concentrations in muscle were 0.012 and 0.518 µg/ml, respectively. The elimination of Malachite Green in muscle appeared biphasic, with a terminal half-life of approximately 67 h. Malachite Green was detectable, but not quantifiable, in muscle at sampling times beyond 14 days, while Leucomalachite Green was quantifiable for up to 42 days (0.019 µg/ml). The percentage of total residues composed of Leucomalachite Green increased from 49 % at 0 h to 80 % at 336 h. Although Leucomalachite Green was the major metabolite in muscle, additional unidentified metabolites eluted before Leucomalachite Green during HPLC. These individual metabolites constituted between 2 and 21 % of the total radioactive residues, depending on sampling time.
Total radioactivity in the exposure water for each group of fish decreased by approximately 15 % during the 1-h exposure period. Malachite Green constituted > 98 % of the total drug in the water throughout the dosing period, as determined by HPLC.
In intravascularly dosed fish, < 5 % of the radioactive dose was found in the gallbladder at 48 h. The mean concentration of total drug equivalents in the bile was 22.8 µg/ml. In the urine, < 0.5 % of the dose was cumulatively excreted over a 48-h period. Concentrations in the urine did not exceed 0.05 µg/ml at each collection time.
Equilibrium between the chromatic and carbinol forms of malachite green in water solutions was established within 2 h at pH 6 and 7. At pH 8, absorbance readings indicated continuous formation of the carbinol compound throughout the 5-h sampling period. Total dye content remained constant at all pH levels, as determined by spectrophotometric and HPLC analyses of acidified solutions (data not shown).
Tissue levels of Malachite Green and Leucomalachite Greenincreased with the pH of the dosing solution. The sums of the Malachite Green and Leucomalachite Green concentrations in plasma and in muscle after exposure at pH 8 were eight and five times higher, respectively, than after exposure at pH 6. At pH 7, the levels were comparable with those observed in the tissue distribution study in which the mean pH of the exposure water was 7.1.

Parent Malachite Green concentrations in the plasma of channel catfish increased rapidly during waterborne exposure, suggesting efficient uptake of the dye cation and (or) carbinol compound across the gills. After transfer of fish to clean water, Malachite Green concentrations in the plasma exhibited a triexponential decline with a terminal half-life of 4.7 h, similar to the results in intravascularly dosed fish.

The head kidney, composed primarily of interrenal and hematopoietic tissue, also was a site of residue accumulation in catfish with concentrations similar to those in the trunk kidney. In the highly perfused spleen, concentrations were consistently lower than those in the head and trunk kidneys.

Multiple factors may be involved in the distribution of residues, including blood perfusion rates and lipid contents of the tissues. In abdominal fat, residue concentrations increased with time, probably as a result of metabolism and redistribution of residues among the tissues. Residue concentrations in the fat were much higher than in any other tissue at sampling times beyond 4 h. Fat may serve as a deep storage compartment that prolongs residue elimination.

During waterborne exposure, Malachite Green concentrations in catfish plasma and muscle exceeded the initial concentration in water. Malachite Green also was rapidly and extensively metabolized to Leucomalachite Green. Leucomalachite Green comprised 49 % of the total drug equivalents in the muscle immediately after waterborne exposure and 80 % at 14 days.

Concentrations of Leucomalachite Green declined more slowly than those of Malachite Green in catfish muscle and plasma. Both Malachite Green and Leucomalachite Green were more persistent in muscle than in plasma. Metabolism has a major role in the clearance of Malachite Green. However, other factors may also influence the relative rates of elimination of Malachite Green and Leucomalachite Green, particularly differences in their lipid solubilities. In the present study, Malachite Green and Leucomalachite Green half-lives in catfish muscle were estimated at 2.8 and 10 days, respectively.

Tissue levels of Malachite Green and Leucomalachite Green increased dramatically with pH of the exposure water, suggesting that the carbinol form of malachite green is more readily absorbed than the chromatic form.

In summary, the disposition of Malachite Green in channel catfish is characterized by rapid and pH-dependent uptake during waterborne exposure, wide distribution and concentration in the tissues and extensive metabolism primarily to Leucomalachite Green. Malachite Green resulted to be persistent in tissue.

Applicant's summary and conclusion

Conclusions:

The disposition of Malachite Green in channel catfish is characterized by rapid and pH-dependent uptake during waterborne exposure, wide distribution and concentration in the tissues and extensive metabolism primarily to Leucomalachite Green. Malachite Green resulted to be persistent in tissue.

Executive summary:

The disposition of Malachite Green was determined in channel catfish (Ictalurus punctatus) after intravascular dosing (0.8 mg/kg) or waterborne exposure (0.8 mg/l for 1 h). The effect of the pH of the exposure water on the absorption of the substance was also investigated. The determination of Malachite Green and its metabiolite Leucomalachite Green in muscle and plasma was perfromed by HPLC with visible light detection (618 nm) and quantification; total radioactive residues in tissues, tissue extracts and fluids were determined by liquid scintillation counting (LSC).

After intravascular dosing, mean plasma concentrations of the parent compound exhibited a triphasic decline with a terminal elimination half-life of 6.2 h. MG was rapidly absorbed and concentrated in the tissues during waterborne exposure. The rate of accumulation was directly related to pH of the exposure water. After waterborne exposure, elimination of the parent compound from plasma also was triphasic with a terminal half-life of 4.7 h. In muscle, the half-life of the parent compound was approximately 67 h. MG and its metabolites were widely distributed in all tissues. In fish exposed to¹⁴C-labeled MG, total drug equivalent concentrations were highest in abdominal fat and lowest in plasma. MG was rapidly and extensively metabolized to its reduced form, Leucomalachite Green, which was slowly eliminated from the tissues. Leucomalachite Green is an appropriate target analyte for monitoring exposure of channel catfish to this drug.

Conclusion

The disposition of Malachite Green in channel catfish is characterized by rapid and pH-dependent uptake during waterborne exposure, wide distribution and concentration in the tissues and extensive metabolism primarily to Leucomalachite Green. Malachite Green resulted to be persistent in tissue.