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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

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:

The global 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:

Toxicity and metabolism of Malachite Green and Leucomalachite Green during short-term feeding to fischer 344 rats and B6C3F mice.

Author:

Sandra J. Culp, Lonnie R. Blankenship, Donna F. Kusewitt, Daniel R. Doerge, Louis T. Mulligan, Frederick A. Beland

Year:

1999

Bibliographic source:

Chemico-biological interactions 122 (1999) 153-170.

Materials and methods

Principles of method if other than guideline:

Mice and rats were fed for 28 days to determine the toxicity and metabolism of the dyes. N-Demethylated and N-oxidized Malachite Green and Leucomalachite Green metabolites, including primary arylamines, were detected by HPLC/MS in the livers of treated rats, 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Malachite Green Chloride

IUPAC Name:

Malachite Green Chloride

Constituent 2

Reference substance name:

Leucomalachite Green

IUPAC Name:

Leucomalachite Green

Radiolabelling:

no

Test animals

Species:

other: rats and mice

Strain:

other: Fischer 344 and B6C3F

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from the breeding colony at the National Centre for Toxicological Research, Jefferson, AR
- Age at study initiation: 6-7 weeks old.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Remarks:

not specified

Duration and frequency of treatment / exposure:

28 days

Doses / concentrations

Remarks:

Doses / Concentrations:
MG: 0, 25, 100, 300, 600, or 1200 ppm and 0, 1200 ppm
LM: 0, 290, 580, or 1160 ppm and 0, 1160 ppm
Additional groups were fed 0 or 1200 ppm of MG for and 0 or 1160 ppm LG 4 or 21 days.

No. of animals per sex per dose / concentration:

Each group was formed of eight animals per dose group per sex

Control animals:

yes, plain diet

Details on study design:

EXAMINATION
WEIGHT
Weights were measured weekly for a total of five measurements per animal and were analyzed on a per cage basis.

HEMATOLOGY MEASUREMENTS
Hematology measurements were conducted with a COBAS Minos Vet (Roche Diagnostic Systems, Branchburg, NJ) and included leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelet count, segmented neutrophils, lymphocytes, monocytes, eosinophils, and reticulocyte count.

CLINICAL CHEMISTRY
Clinical chemistry measurementswere conducted with a COBAS Mira Plus (Roche Diag- nostic Systems) and included total protein and bile acids, blood urea nitrogen, creatinine, alanine aminotransferase, and alkaline phosphatase.
In rats, aspartate amino transferase, glucose, cholesterol, triglycerides, y-glutamyl transferase, albu- min, sorbitol dehydrogenase, creatine kinase, sodium, potassium, chloride, calcium, and phosphorus were also measured.

Total T3andtotal T4were determined with a ‘Coat-A-Count’ procedure obtained from DPC, Los Angeles, CA. The procedure is a solid-phase radioim- munoassay, wherein125l-labeled T3/T4 competes with the sample for antibody sites. TSH was measured with a double antibody RIA procedure. Freshly prepared [l25I]TSH was allowed to react overnight with the specific antibody and sample. An excess of second antibody containing polyethylene glycol was then added, bound and unbound [125I]hormone were separated by centrifugation, and the radioactivity was measured in the precipitates.

Details on dosing and sampling:

SAMPLES
- Gathering: after 28 days of feeding
- Blood: to collect blood for clinical chemistry measurements animals were anesthetized with CO2
- Tissues: animals were then euthanized by CO2 exposure and a gross necropsy was performed. Tissues were fixed in neutral buffered formalin for 48 h, embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin. Selected tissues were stained by ISEL (Apoptag kit, Oncor, Gaithersburg, MD), a technique to demonstrate DNA fragmentation. For thin sections, formalin-fixed tissues were embedded in glycol methacrylate, sectioned at 2 µm, and stained with toluidine blue.

LIVER EXTRACTION AND ANALYSES
- Extraction: 1-10 g of liver was homogenized in a glass-Teflon homogenizer in a mixture of 2 ml 250 mg/ml hydroxylamine HCI in water, 3 ml 50 mM toluene sulfonic acid, 10 ml 100 mM ammonium acetate (pH 4.5), and 30 ml acetonitrile. Sodium chloride (2 g) was added and the samples were centrifuged for 10 min at 3000 rpm at 25°C. The supernatant was extracted with a mixture of 40 ml water, 2 ml diethylene glycol and 90 ml methylene chloride. The organic layer was then concentrated to approximately 1 ml. Solid phase extraction was conducted as described in Roybal et al (1995).

- Method of analyses: samples were analyzed by HPLC using a CN guard column, a 4.6 x250 mm Spherisorb CN column, and a 20 x 2.0 mm i.d.
PbO2 oxidative post-column coupled to a Waters model 996 photodiode array detector. The following elution conditions were used with a flow rate of 1 ml/min and monitoring was conducted at 618 nm. The samples were quantified by comparison to MG or LG standards.
The samples were also analyzed by HPLC in combination with APCI/MS. The separations were performed with a Dionex GP40 pump (Dionex, Sunnyvale, CA) and either a manual Rheodyne 7125 injector (Rheodyne. Cotati, CA) or a Dionex AS3500 autosampler. A Prodigy ODS-3 column (5 µm, 4,6 x 250 mm) was used for separation of leucomalachite green and metabolites. Separation of malachite green and metabolites was performed using a Spherisorb S5 Nitrile column (80A, 5 µm, 446 x250 mm) and a solvent system containing 40% acetonitrile in 50 mM ammonium acetate (pH 445) at a flow rate of 1.0 ml/min. For mass spectrometry a VG Platform 11 single quadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with
an APC1 interface was used. The total HPLC column effluent was delivered into the ion source (150°C) through a heated nebulizer probe (500°C) using nitrogen as the probe and bath gas. Positive ions were acquired in full scan mode (m/z 100—800 in 1.5 s cycle time) and a UV detector set at 260 nm was placed in-line before the mass spectrometer. Two separate scan functions with different sampling cone-skimmer potentials were used to acquire mass spectra with minimal (20 V) or significant (60 V) amounts of fragmentation through in-source co1lision—induced dissociation.

DNA
Approximately 10 µg of DNA was 32P-postlabeled using n-butanol enrichment. Adducts were separated by thin layer chromatography performed on 0.1 mm Machery Nagel 300 polyethylene imine cellulose plates (Alltech, Deerfield, IL) using the following solvent directions, D1: 0.9 M sodium phosphate (pH 6.8); D2: 3.6 M lithium formate, 8.5 M urea (pH 3.5); D3; 1.2 M lithium chloride, 0.5 M Tris-HCl, 8 M urea (pH 8.0). A iinal wash was conducted in D3 with the solvent used in D1. DNA adducts were visualized using a Storm 860 phosphor imaging system (Molecular Dynamics, Sunnyvale, CA). The adduct levels were quantified by comparison to a 10 β-(deoxyguanosin-N2-yl)-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a]-pyrene standard, obtained by reacting DNA with (±)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,l0-epoxide.

Statistics:

Analysis of variance (ANOVA) and Dunnett’s test.

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

A series of desmethyl derivatives were observed by HPLC/APCI/MS in liver extracts from both species. These results suggest a scenario in which MG undergoes a reduction to LG or cytochrome P-45O catalyzed N-demethylation to mono-and di-desmethyl MG.
LG could also undergo a similar N-demethylation by cytochrome P-450. These primary and secondary amine metabolites are similar to carcinogenic arylamines. As such, they could be oxidized to metabolites that react with DNA either directly or after esterilication.

Any other information on results incl. tables

A comparison of adverse effects suggests that exposure to LG causes a greater number of and more severe changes than exposure to MG. In the case of LG, total T4 levels were decreased, while TS1-1 levels were significantly elevated in male rats fed 1160 ppm LG, A decrease in T4 levels is consistent with primary hypothyroidism, but does not preclude other causes of low circulating T4, such as decreased pituitary function, alterations in protein binding of T4, and alterations in peripheral metabolism of T4. However, in combination with an increase in TSH levels, these findings indicate that pituitary function was normal in rats with decreased T4 levels and that primary hypothyroidism was the most likely cause of reduced T4 levels.

In LG-treated mice, histopathological data revealed that all mice in the 1160 ppm dose group had apoptosis of the transitional epithelium of the urinary bladder. The apoptotic cells were phagocytized by neighboring transitional epithelial cells and appeared to undergo dissolution in phagocytic vacuoles. This apoptotic cell death could be accompanied by a compensatory cell proliferation, which could promote the expansion of initiated cells.

³²P-Postlabeling of liver DNA indicated the formation of a DNA adduct or co-eluting adducts, that increased with increasing dose, in rats and mice fed LG or MG. In addition, a series of desmethyl derivatives were observed by HPLC/APCI/MS in liver extracts from both species. These results suggest a scenario in which MG undergoes a reduction to LG or cytochrome P-45O catalyzed N-demethylation to mono-and di-desmethyl MG. LG could also undergo a similar N-demethylation by cytochrome P-450. These primary and secondary amine metabolites are similar to carcinogenic arylamines. As such, they could be oxidized to metabolites that react with DNA either directly or after esterilication. Misreplication of these lesions may result in mutations that can lead to liver tumours.

Preliminary results from our laboratory, using the ³²P-postlabeling assay, indicate the presence of a single adduct or co-eluting adducts from thyroid DNA of rats fed LG for 28 days. This suggests that a genotoxic mechanism for thyroid tumour formation may also be possible.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
A comparison of adverse effects suggests that exposure to Leucomalachite Green causes a greater number of and more severe changes than exposure to Malachite green. Malachite Green and Leucomalachite Green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to Malachite Green.

Executive summary:

Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm Malachite Green (MG) or 1160 ppm Leucomalachite Green (LG) for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of LG. This was not observed with MG Hepatocyte vacuolization was present in rats administered MG or LG. Rats given LG also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given LG.

A comparison of adverse effects suggests that exposure of rats or mice to LG causes a greater number of and more severe changes than exposure to MG. N-Demethylated and N-oxidized MG and LG metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats, ³²P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that MG and LG are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to MG.

Conclusion

A comparison of adverse effects suggests that exposure to Leucomalachite Green causes a greater number of and more severe changes than exposure to Malachite green. Malachite Green and Leucomalachite Green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to Malachite Green.