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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

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.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

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

Author:

Culp S.J., Blankenship L.R., Kusewitt D.F., Doerge D.R., Mulligan L.T., Beland F.A.

Year:

1999

Bibliographic source:

Chemio-Biological Interactions 122 (1999) 153-170

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Principles of method if other than guideline:

NTP study report.

GLP compliance:

yes

Limit test:

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

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Center for Toxicological Research (Jefferson, AR).
- Time held before study: 2 weeks.
- Age at study initiation: 6 to 7 weeks.
- Housing: rats were housed two per cage.
- Cages: polycarbonate (Allentown Caging Equipment Co., Allentown, NJ), changed twice weekly; cages rotated weekly.
- Bedding: hardwood chips (Northeastern Products Inc., Warrensburg, NY), changed twice weekly.
- Cage Bonnets: microisolator tops (Lab Products, Inc., Maywood, NJ).
- Racks: metal animal cage racks (Allentown Caging Equipment Co., Allentown, NJ), changed weekly.
- Fasting period before study: 2 weeks.
- Diet: NIH-31 open formula meal available ad libitum.
- Water: Millipore-filtrated water via 16-oz water bottle, available ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (average): MGC rats 22 °C. Leucomalachite Green rats: 23 °C.
- Humidity: Malachite Green Chloride rats 47.5 %. Leucomalachite Green rats 51.5 %
- Air changes: at least 10/hour
- Photoperiod: room florescent light: 12 hrs/day.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

DIET PREPARATION
The dose formulations for Malachite Green chloride were prepared on 6 days by dissolving the chemical in water and then mixing it with feed. The 25 and 600 ppm dose formulations were prepared three times and the 100, 300, and 1200 ppm dose formulations were prepared twice.
The dose formulations for Leucomalachite Green were prepared by mixing the chemical with feed. A premix was prepared by hand and blended with additional feed. The 96 and 290 ppm dose formulations were prepared once and the 580 and 1160 ppm dose formulations were prepared twice.
Dose formulations for each chemical were mixed in a Patterson-Kelly twin-shell blender with the intensifier bar on for 20 minutes.
Formulations were stored in stainless steel feed cans at 4 ± 2 °C for up to 92 days (malachite green chloride) or 95 days (leucomalachite green).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability studies of the 25 ppm malachite green chloride dose formulations were performed by the study laboratory using HPLC. Homogeneity was confirmed. Stability of the malachite green chloride formulation was confirmed for 92 days for dose formulations stored protected from light at 4 °C and for 10 days
for dose formulations stored at room temperature, either protected from light or open to air and light.

Stability of the leucomalachite green formulation was confirmed for 95 days for dose formulations stored protected from light at up to 8 °C and for 32 days for dose formulations stored at room temperature either protected from light or open to air.

Periodic analyses of the dose formulations of malachite green chloride were conducted by the study laboratory using HPLC. Analyses of the dose formulations of malachite green chloride were conducted on one batch each of 25, 100, and 1200 ppm dose formulations, on both batches of the 300 ppm dose formulations, and on all three of the 600 ppm dose formulations. During the 28-day studies, seven of eight dose formulations analyzed for rats and mice were within 10 % of the target concentration, with no value greater than 103 % of the target concentration. The formulation that was not within 10 % of the target concentration was diluted with feed and remixed to provide a lower (300 ppm) concentration; the remix was analyzed and found to be within 10 % of the target concentration.

Analyses of the dose formulations of leucomalachite green were conducted by the study laboratory using HPLC. During the 28-day studies, all dose formulations were analyzed. All 6 dose formulations for rats and mice were within 10 % of the target concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Malachite green chloride: 0, 25, 100, 300, 600, or 1200 ppm (core study)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Malachite green chloride: 0, 1200 ppm (thyroid hormone assays)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Leucomalachite Green: 0, 290, 580, or 1160 ppm (core study)
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
Leucoalachite green: 0, 1160 ppm (thyroid hormone assays)
Basis:
nominal in diet

No. of animals per sex per dose:

Malachite Green chloride: 8 males and 8 females (core study); 16 males and 16 females (thyroid hormone assays).
Leucomalachite Green: 8 males (core study); 16 males (thyroid hormone assay).

Control animals:

yes, plain diet

Details on study design:

Groups of eight male and eight female were fed diets containing 0, 25, 100, 300, 600, or 1200 ppm malachite green chloride for 28 days. Additional groups of eight male and eight female rats designated for thyroid hormone assays were fed diets containing 0 or 1200 ppm malachite green chloride.

Groups of eight male rats were fed diets containing 0, 290, 580, or 1160 ppm leucomalachite green for 28 days. Additional groups of eight male rats designated for thyroid hormone assays were fed diets containing 0 or 1160 ppm leucomalachite green.

Animals were distributed randomly into groups of approximately equal initial mean body weights.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
Clinical findings were recorded weekly.

BODY WEIGHT
The animals were weighed initially, weekly, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption were recorded weekly.

HAEMATOLOGY
For hematology analyses, blood was placed in tubes containing EDTA as anticoagulant. Assessment of blood cells was determined by light microscopic examination of blood smears fixed in absolute methanol. Blood was collected by cardiac puncture at the end of the exposure period for hematology and clinical chemistry.
Parameters checked in table: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration and leukocyte count and differentials.

THYROID HORMONE ASSAY
Blood was collected by cardiac puncture from special study rats on days 4 and 21 for determination of thyroidstimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) concentrations. TSH was measured with double-antibody radioimmunoassay. Freshly prepared 125I-TSH was allowed to react overnight with the specific antibody in the presence or absence of unlabeled hormone in the sample. An excess of secondary antibody containing polyethylene glycol was added. The bound and unbound 125I-labeled hormones were separated by centrifugation and the radioactivity was measured in the precipitates. The amount of TSH was calculated from a standard curve. Total T3 and total T4 were determined with a “Coat-A-Count” procedure obtained from DPC (Los Angeles, CA). The procedure is a solid-phase radioimmunoassay, wherein 125I-labeled T3 or T4 competes with the T3 or T4 in the sample for antibody sites.

CLINICAL CHEMISTRY
For clinical chemistry analyses, blood samples were placed in tubes, allowed to clot, and then centrifuged, and the serum was collected. Parameters were measured with a Roche Diagnostic Cobas Mira-Plus analyzer (Roche Diagnostic Systems, Inc., Montclair, NJ). Reagents were obtained from the equipment manufacturer.
Parameters: urea nitrogen, creatinine, total protein, alanine aminotransferase, alkaline phosphatase, and total bile acids, glucose, sodium, potassium, chloride, calcium, phosphorus, albumin, cholesterol, triglycerides, aspartate aminotransferase, creatine kinase, sorbitol dehydrogenase, γ-glutamyltransferase, thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4).

Sacrifice and pathology:

GROSS PATHOLOGY
Clinical pathology studies were conducted on all core study animals at the end of the exposure period. The animals were anesthetized with carbon dioxide, and blood was collected by cardiac puncture.
Necropsies were performed on all core study animals. The kidneys and liver were weighed. Tissues for microscopic examination were fixed and preserved in 10 % neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 μm, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
A complete histopathologic examination was performed on core study control animals, 1200 ppm animals exposed to malachite green chloride, and 1160 ppm animals exposed to leucomalachite green.
HISTOPATHOLOGY
A complete histopathologic examination was performed on core study control animals, 1200 ppm animals exposed to malachite green chloride, and 1160 ppm animals exposed to leucomalachite green. The liver, pituitary gland, and thyroid gland from all core study animals and the urinary bladder from all core study mice were examined histopathologically.
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, coagulating gland, ear, esophagus, eye, gallbladder (mice), harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, lacrimal gland, larynx, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, muscle, nose, ovary, pancreas, parathyroid gland, peripheral nerve, pituitary gland, preputial gland, prostate, salivary gland, skin, spinal cord, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina, and Zymbal’s gland.
In addition, the liver, pituitary gland and thyroid gland of rats were examined in the lower exposure groups.

Statistics:

Fisher’s exact test (Bradley, 1968) was used to compare the proportion of lesions in the control group to that in each of the exposed groups. The Cochran-Armitage test (Thomas et al., 1977) for a dose-response trend on proportions was used when all four exposure groups were examined. An exact test for this procedure was used because there were fewer than 10 animals in each exposure group. All tests are one sided in that decreases in incidence with an increasing exposure concentration were not considered. The P values within pairwise comparisons were adjusted by Holm’s modification of the Bonferroni procedure as described by Wright (1992).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significantly less than those of the controls (MG and LG)

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver weight gain significantly greater than the control group (MG and LG)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

apoptotic follicular epithelial cells in the thyroid gland (LG)

Details on results:

CLINICAL SIGNS AND MORTALITY
All rats survived to the end of the studies. There were no clinical findings related to exposure to malachite green chloride or leucomalachite green.

BODY WEIGHT AND WEIGHT GAIN
Malachite Green chloride:the mean body weight gain of males in the 1200 ppm group was significantly less than that of the controls. The final mean body weight of females in the 1200 ppm group and the body weight gains of females in the 600 and 1200 ppm groups were significantly less than those of the controls.

Leucomalachite Green: the final mean body weight of males in the 1160 ppm group and the body weight gains of males in the 580 and 1160 ppm groups were significantly less than those of the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE
Malachite Green chloride: feed consumption by all exposed groups of males and females was generally similar to that by the control groups. Exposure concentrations of 25, 100, 300, 600, and 1200 ppm resulted in average daily doses of approximately 3, 12, 40, 70, and 175 mg malachite green chloride/kg body weight to males and 3, 12, 40, 75, and 190 mg/kg to females.

Leucomalachite Green: feed consumption by all groups of exposed rats was similar to that by the controls. Dietary concentrations of 290, 580, and 1160 ppm resulted in average daily doses of approximately 30, 60, and 115 mg leucomalachite green/kg body weight.

HAEMATOLOGY and CLINICAL CHEMISTRY
Malachite Green Chloride: hematology changes in male rats included a decrease in mean cell hemoglobin values in the 300 ppm or greater groups and a significant decrease in mean cell volumes in the 600 and 1200 ppm groups compared to controls. In female rats, the erythrocyte count, hemoglobin and mean cell hemoglobin concentrations, and hematocrit and mean cell hemoglobin values in the 1200 ppm group were significantly decreased. There were no significant changes in the other hematology parameters measured in female rats.
In male rats, there was a significant decrease in sorbitol dehydrogenase activity in the 1200 ppm group Compared to that in controls. In female rats, γ-glutamyltransferase activities were significantly increased in the 600 and 1200 ppm groups and cholesterol concentrations were significantly increased in the 300 ppm or greater groups.

Leucomalachite Green: significantly decreasing linear trends in the exposed groups were observed in erythrocyte counts, hemoglobin concentrations, and hematocrit values compared to controls (statistical analyses not presented). The hemoglobin concentration, hematocrit value, and erythrocyte count in the 1160 ppm group were significantly lower than those in the control group. The alkaline phosphatase activity was significantly decreased in the 1160 ppm group, while triglyceride, creatinine, albumin, and cholesterol concentrations and alanine aminotransferase activities were generally significantly decreased in all exposed groups. The phosphorus concentration and γ-glutamyltransferase activity were significantly increased in the 1160 ppm group.

THYROID HORMONE ASSAY
Malachite Green Chloride: additional groups of male and female rats were exposed to 0 or 1200 ppm malachite green chloride for 4 or 21 days and blood was collected for thyroid-stimulating hormone, triiodothyronine, and thyroxine concentrations. The triiodothyronine concentration was significantly increased in 1200 ppm female rats on day 21. Thyroxine concentrations were significantly decreased in 1200 ppm females on days 4 and 21, while no significant differences were observed in thyroid-stimulating hormone concentrations. The thyroid-Stimulating hormone concentration in 1200 ppm males was significantly decreased on day 4 but not on day 21. There were no significant changes in triiodothyronine or thyroxine concentrations in exposed males compared to the controls.

Leucomalachite Green: additional groups of male rats were exposed to 0 or 1160 ppm leucomalachite green for 4 or 21 days and blood was collected for thyroid-stimulating hormone, triiodothyronine, and thyroxine concentrations. There were significant decreases in thyroxine concentrations and significant increases in thyroid-stimulating hormone concentrations in the 1,160 ppm group at both time points.

ORGAN WEIGHTS
Malachite Green: the relative liver weights of 600 and 1200 ppm males and the relative and absolute liver weights of 300 ppm or greater females were significantly greater than those of the controls.

Leucomalachite Green: the absolute liver weights of 1160 ppm males and the relative liver weights of all exposed groups were significantly greater than those of the control group.

GROSS PATHOLOGY
Malachite Green: no gross lesions were observed that could be attributed to malachite green chloride exposure.

Leucomalachite Green: no gross lesions were observed that could be attributed to leucomalachite green exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
Malachite Green: microscopically, the incidences of hepatocyte cytoplasmic vacuolization in 1200 ppm males and females were significantly greater than those in the controls (males: 0 ppm, 0/8; 25 ppm, 0/8; 100 ppm, 0/8; 300 ppm, 0/8; 600 ppm, 1/8; 1,200 ppm, 4/8; females: 0/8, 0/8, 0/8, 0/8, 0/8, 7/8).

Leucomalachite Green: the incidences of hepatocyte cytoplasmic vacuolization were significantly increased in 580 and 1,160 ppm males (0 ppm, 0/8; 290 ppm, 2/8; 580 ppm, 5/8; 1,160 ppm, 7/8).

HISTOPATHOLOGY: NEOPLASTIC
Leucomalachite Green: two of eight rats exposed to 1160 ppm and two of eight rats exposed to 580 ppm leucomalachite green had apoptotic follicular epithelial cells in the thyroid gland. Morphologic changes consisted of sloughed follicular cells with condensed nuclei located within follicles. No inflammatory reaction was present. There was evidence of follicular epithelium regeneration, since even most severely affected follicles were still lined by viable epithelium.

Effect levels

1

Dose descriptor:

LOEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Malachite Green chloride: decrease of mean cell hemoglobin value in males; relative liver weight greater than the control in females.

2

Dose descriptor:

LOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Leucomalachite Green: relative liver weight greater than the control

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Malachite Green chloride rat LOEL (28d): 40 mg/kg bw/day (actual dose received) (males and females)
Leucomalachite Green rat LOEL (28d): 30 mg/kg bw/day (actual dose received) (males)

Executive summary:

Malachite Green Chloride and Leucomalachite Green were nominated for toxicity and carcinogenicity testing by the Food and Drug Administration and selected by the National Institutes of Environmental Health Sciences for carcinogenicity testing by the National Toxicology Program (NTP) due to the potential for significant worker and consumer exposure and lack of carcinogenicity data. The current 28-day studies were conducted as part of an overall effort by the NTP to determine the toxicity and carcinogenicity of Malachite Green Chloride and Leucomalachite Green.

Groups of eight male and eight female rats were fed diets containing 0, 25, 100, 300, 600, or 1200 ppm malachite green chloride for 28 days. Additional groups of eight male and eight female rats designated for thyroid hormone assays were fed diets containing 0 or 1200 ppm malachite green chloride. Groups of eight male rats were fed diets containing 0, 290, 580, or 1160 ppm leucomalachite green for 28 days. Additional groups of eight male rats designated for thyroid hormone assays were fed diets containing 0 or 1160 ppm leucomalachite green.

All rats survived to the end of the studies. In the malachite green chloride study, the body weight gain of males rats in the 1200 ppm group was significantly less than that of the controls. The final mean body weight of female rats in the 1200 ppm groups and the body weight gains of female rats in the 600 and 1200 ppm groups were significantly less than those of the controls. In the leucomalachite green study, the final mean body weight of male rats in the 1160 ppm groups and the mean body weight gains of male rats in the 580 and 1160 ppm groups were significantly less than those of the control groups.

In the malachite green chloride study, feed consumption by all exposed groups of male and female rats was generally similar to that by the control groups. Exposure concentrations of 25, 100, 300, 600, and 1200 ppm resulted in average daily doses of 3 to 190 mg malachite green chloride/kg body weight to male and female rats. In the leucomalachite green study, feed consumption by all groups of exposed male rats was similar to that by the controls. Dietary concentrations of 290, 580, and 1160 ppm resulted in average daily doses of approximately 30, 60, and 115 mg leucomalachite green/kg body weight to male rats.

In female rats exposed to malachite green chloride, there was a significant increases in γ-glutamyltransferase activities with an activity in 1200 ppm females seven times greater than that in the controls. Likewise, γ-glutamyltransferase activity in male rats exposed to 1160 ppm leucomalachite green was twice that in the controls. On days 4 and 21, the concentration of thyroxine was significantly decreased in male rats exposed to 1160 ppm leucomalachite green and the concentration of thyroid-stimulating hormone was significantly increased.

In the malachite green chloride study, the relative liver weights of 600 and 1200 ppm male rats and the relative and absolute liver weights of 300 ppm or greater female rats were generally significantly greater than those of the controls. In the leucomalachite green study, the relative liver weights of 290 ppm or greater male rats were significantly greater than those of the control group.

No gross lesions were observed that were attributed to malachite green chloride exposure. Microscopically, the incidences of hepatocyte cytoplasmic vacuolization were significantly increased in 1200 ppm male and female rats exposed to malachite green chloride.

No gross lesions were observed in rats that could be attributed to leucomalachite green exposure. Microscopically, the incidences of hepatocyte cytoplasmic vacuolization were significantly increased in 580 and 1160 ppm male rats.

Conclusion

Malachite Green chloride rat LOEL (28d): 40 mg/kg bw/day (actual dose received) (males and females)

Leucomalachite Green rat LOEL (28d): 30 mg/kg bw/day (actual dose received) (males)