[4-[α-[4-(dimethylamino)phenyl]benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate

Carcinogenicity

Description of key information

There is no evidence of carcinogenic activity of MG chloride in mice exposed to 100, 225, or 450 ppm.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

From April 13, 1999 to Mat 7,2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study report Read across from a similar substance which has the same main component and with a different counter ion that doesn't influence the characteristics related to the specific end-point

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Only females rats and mice has been tested

Principles of method if other than guideline:

NTP study report

Species:

other: Rats and Mice

Strain:

other: rats F344/N and Mice B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Centre for Toxicological Research (NCTR)
- Age at study initiation: approximately 6 weeks old at the beginning of the studies.
- Housing: animals were distributed randomly into groups of approximately equal initial mean body weights. Rats were housed two per cage, mice were housed four per cage. Cages were changed once a week and rotated every 3 weeks.
- Cage: Polycarbonate cages (Allentown Caging Equipment Co., Allentown, NJ), changed weekly and rotated every 3 weeks.
- Bedding: Hardwood chips (Northeastern Products, Inc., Warrensburg, NY),changed weekly.
- Cage Bonnets: Microisolator tops
- Racks: Metal animal cage racks, changed weekly.
- Diet: Feed and water were available ad libitum. NIH-31 open formula meal pellets were autoclaved then ground to powder (Purina Mills, Richmond, IN), available ad libitum until the day before sacrifice
- Water: ad libitum. Millipore-filtered water (Jefferson municipal supply) via 480 mL water bottles.
- Acclimation period: 2 weeks
- Health check: The health of the animals was monitored during the studies according to the protocols of the Study Laboratory’s Sentinel Animal Program

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 0.5°C
- Humidity: 49.4 - 50.9%
- Air changes: at least 10/hour
- Photoperiod: 12 hours/day

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

Preparation MG:
MGC, a triphenylmethane dye, is prepared as a double salt with zinc chloride for use as a dye. It is synthesized in a stepwise reaction that involves the condensation of benzaldehyde with N,N-dimethylaniline and oxidation of the resulting bis(p-dimethylaminophenyl)phenylmethane, followed by reaction of the product with hydrochloric acid (Nelson, 1974).

DIET PREPARATION
- Rate of preparation of diet (frequency): for MGC, the dose formulations were prepared approximately every 2 months by dissolving the chemical in water and then mixing it with feed.
- Mixing appropriate amounts with (Type of food): the solution was blended with feed in a Patterson-Kelly V-shell blender using an intensifier bar and a heater under a vacuum of at least 15 mm mercury for approximately 20 minutes.
- Analytical verification: periodic analyses of the dose formulations were conducted by the study laboratory using HPLC. The dose formulations were analyzed approximately every 7 weeks. Of the MGC dose formulations analyzed and used, 96% (65/68) of the dose formulations for rats and 97% (33/34) of the dose formulations for mice were within 10% of the target concentrations. All of the LG dose formulations analyzed and used for rats and mice were within 10% of the target concentrations.
- Storage temperature of food: dose formulations were stored in stainless steel feed cans at 4° ± 2°C for up to 92 days
- Other: for MGC, a homogeneity study of a 100 ppm dose formulation and a stability study of a 25 ppm dose formulation were performed by the study laboratory using HPLC. Homogeneity was confirmed, and stability was confirmed for at least 10 days for dose formulations stored at room temperature exposed to light and for at least 92 days for formulations stored at up to 6° C protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purity of MGC was determined by the study laboratory using HPLC by system A, gas chromatography (GC), and inductively coupled plasma (ICP) spectrometry. Gas chromatography was performed with a Hewlett-Packard gas chromatograph using a flame ionization detector with a helium carrier gas at 24 psi. A Carbopak B packed column with 5% Carbowax 20M (6 ft × 2 mm, Supelco) was used with an isocratic oven temperature of 80° C.

Duration of treatment / exposure:

102 weeks

Frequency of treatment:

Feed consumption was measured weekly for the first 12 weeks and approximately every 4 weeks thereafter.

Remarks:

Doses / Concentrations:
Rats: 0, 100, 300, 600 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
Mice: 0, 100, 225, 450 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Groups of 48 female rats were fed diets containing 0, 100, 300, or 600 ppm MGC for 2 years (equivalent to average daily doses of approximately 0, 7, 21, and 43 mg MGC/kg body weight).
Groups of 48 female mice were fed diets containing 0, 100, 225, or 450 ppm MGC for 2 years (equivalent to average daily doses of approximately 0, 15, 33, and 67 mg MGC/kg body weight).

Control animals:

yes

Sacrifice and pathology:

Sacrifice: carbon dioxide asphyxiation

Statistics:

Survival Analyses
The probability of survival was estimated by the product-limited procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or removed from the study for reasons other than morbidity were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) and Tarone’s (1975) life table test to identify dose-related trends. All P values for the survival analyses are two sided.

Calculation of Incidence
The incidences of neoplasms or nonneoplastic lesions are presented as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. For calculation of statistical significance, the incidences of most neoplasms and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of animals on which a necropsy was performed. This survival-adjusted rate (based on the Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased in female RATS

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

increased trend in female rats

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

equivocal for female rats

Details on results:

RATS
CLINICAL SIGNS AND MORTALITY
Survival of all exposed groups was similar to that of the control group.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of female rats exposed to 300 or 600 ppm malachite green chloride were generally less than those of the controls during most of the study. Feed consumption by exposed rats was generally similar to that by controls throughout the study. Dietary concentrations of 100, 300, or 600 ppm resulted in average daily doses of approximately 7, 21, or 43 mg MGC/kg body weight. No clinical findings were attributed to MGC exposure.

ORGAN WEIGHTS
The relative liver weight was significantly increased in females exposed to 600 ppm MGC.

HISTOPATHOLOGY: NON-NEOPLASTIC
Mononuclear Cell Leukaemia: a dose-related decreasing trend in the incidences of mononuclear cell leukaemia occurred in female rats exposed to MGC, with statistically significant decreased incidences in the 300 and 600 ppm groups

HISTORICAL CONTROL DATA
The concurrent control group represents the most valid comparison to the treated groups and is the only control group analyzed statistically in NTP bioassays. However, historical control data are often helpful in interpreting potential treatment-related effects, particularly for uncommon or rare neoplasm types. For meaningful comparisons, the conditions for studies in the historical database must be generally similar. The historical database for these studies included studies conducted by the NCTR using dietary or drinking water exposure.

OTHER FINDINGS
Thyroid Gland (RATS)
Follicular cell adenomas and carcinomas were observed in female rats exposed to 300 or 600 ppm MGC, and the incidences of adenoma or carcinoma (combined) in these groups exceeded the historical control range. A dose-related increasing trend (P=0.049) in the incidence of cystic follicles was observed in exposed rats. Cystic follicles consisted of very large thyroid follicles that were distended with colloid and lined by flattened follicular epithelial cells. Lesions diagnosed as follicular cell hyperplasia were also cystic, but there were small fronds and foci of follicular epithelial cells protruding into distended follicles. Although the increases were not statistically significant, thyroid follicular cell hyperplasia was only observed in rats exposed to MGC.

Liver: (RATS)
There were modest, but not statistically significant, increases in the incidences of hepatocellular adenoma in female rats exposed to MGC. However, the incidences in all groups, including the controls, exceeded the historical control range. Hepatocellular adenomas consisted of well-demarcated lesions that occupied an area greater in size than one hepatic lobule with distinct compression of adjacent parenchyma. A single hepatocellular carcinoma was found in one 300 ppm female and was a large well-demarcated lesion that consisted of anaplastic hepatocytes arranged in a trabecular pattern in some areas. Corresponding increases in the incidences of eosinophilic foci and centrilobular necrosis were observed in female rats exposed to MGC. Eosinophilic foci were characterized by distinct, variably sized foci in which hepatocytes were larger than normal due to increased amounts of eosinophilic cytoplasm. The incidence of centrilobular necrosis, in which the necrotic cells were oriented around central veins in the hepatic lobule, exhibited an increasing trend (P=0.002).

Mammary Gland (RATS)
The incidence of mammary gland carcinoma in female rats exposed to 600 ppm MGC exceeded the historical control range.

Pituitary Gland (RATS)
There was a statistically significant increase in the incidence of adenoma of the pituitary gland (pars distalis) in female rats exposed to 100 ppm MGC. Incidences of pars distalis adenoma in 100 and 300 ppm females exceeded the historical control range for pars distalis adenoma or carcinoma (combined).

MICE:
BODY WEIGHT AND WEIGHT GAIN
Mean body weights of exposed female mice were generally similar to those of the controls throughout most of the study. Feed consumption by exposed groups of females was similar to that by the controls. Dietary concentrations of 100, 225, or 450 ppm MGC resulted in average daily doses of approximately 15, 33, or 67 mg MGC/kg body weight. No clinical findings were attributed to MGC exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
Urinary Bladder:
Significantly (P < 0.05) increased incidences of intracytoplasmic inclusions were observed in all groups of female mice exposed to MGC (0 ppm, 7/47; 100 ppm, 15/46; 225 ppm, 34/45; 450 ppm, 39/48). Intracytoplasmic inclusions were found in the transitional epithelium of the urinary bladder. The inclusions were variable in size, yellow-orange, slightly refractile and generally found in the superficial epithelial cells. The inclusions were found in many mice exposed to MGC and the number of inclusions (severity) increased with increasing exposure concentration (1.1, 1.1, 1.4, 1.4). The inclusions were much less prominent than those observed in female mice exposed to LG. The pathogenesis of the intracytoplasmic inclusions is unknown, but the inclusions are thought to represent degradation products. The presence of the inclusions did not have an apparent effect on the general health or mortality of the affected mice.

OTHER FINDINGS
Kidney Weights: kidney weights of female mice exposed to MGC were generally less than those of the controls. The absolute right kidney weights were significantly decreased in the 225 and 450 ppm groups as were the relative weights in the 100 and 225 ppm groups. The absolute left kidney weight was significantly decreased in the 450 ppm group, and the relative weight was decreased in the 225 ppm group.

Relevance of carcinogenic effects / potential:

No evidence of carcinogenic activity of Malachite Green Chloride.

Dose descriptor:

NOAEL

Effect level:

> 450 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Mice B6C3F1

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

< 600 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Rats F344/N; based on the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) and marginal increases in hepatocellular adenoma and mammary gland carcinoma in exposed rats

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity (migrated information)

Conclusions:

Under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity of Malachite Green Chloride (MGC) in female F344/N rats based on the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) and marginal increases in hepatocellular adenoma and mammary gland carcinoma in exposed rats. There was no evidence of carcinogenic activity of MGC in female B6C3F1 mice exposed to 100, 225, or 450 ppm.

Executive summary:

Background

Malachite Green Chloride is a dye used to prevent fungus infections in commercial fisheries. Leucomalachite Green (LG) is formed from Malachite Green (MG) and remains in the tissues of exposed fish. We studied the effects of MG on female rats and female mice, and the effects of LG on male and female rats and female mice, to identify potential toxic or cancer-related hazards to humans.

Methods

For each study we mixed the dye into the feed of rats and mice. The doses of MGC given were 100, 300, or 600 parts per million (ppm) for female rats and 100, 225, or 450 ppm for female mice. Doses of LG were 91, 272, or 543 ppm for male and female rats and 91, 204, or 408 ppm for female mice. There were 48 animals in each dose group. Control animals received the same feed with no chemical added. The study lasted for two years. Tissues from more than 40 sites were examined for every animal.

Results

Rats, but not mice, exposed to MGC or leucomalachite green weighed less on average than the control animals. In rats exposed to the dyes, there were very slight increases in a few types of tumors: cancers of the thyroid gland, liver, and mammary gland in females exposed to MGC; of the thyroid gland and testes in males exposed to LG; and of the thyroid gland and liver of females exposed to LG. We saw no increase in cancers in female mice given MGC, but there was in increase in liver tumors in female mice given LG.

Conclusions

It was concluded that tumors of the thyroid gland, liver, or mammary gland in female rats might have been caused by MGC, but that MGC did not cause cancer in female mice. LG might have caused cancers of the thyroid gland in male and female rats, and of the testes in male rats and liver in female rats. LG caused an increase in cancer of the liver in female mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

other: Rats and mice

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to CLP regulation (EC1272/2008) Malachite Green Acetate is not classified as carcinogen

Additional information

In the female rats there was a small, but statistically significant, increase in the incidence of hepatocellular adenoma, but is not clear if the increase is biologically significant; is not clear if the Malachite Green (MG) increase incidence of adenoma and incidence of pituitary gland adenoma (Culp, 2006).

Other effects attributed to MG and that seems linked to tumour promotion were reported in literature.

MG appears to be able to produce transformation SHE cells, developing aneuploid pattern; this cells can be tumorigenic, as they could produce tumours (Rao, 2001). Carcinogenesis involves an imbalance between the regulation of cell proliferation and apoptotic death, either by inhibition of apoptosis or by stimulation of cell division, or by affecting both. The Rao study (2001) suggests also that transformed cells are much less sensitive to apoptosis than normal cells. Nevertheless the exact mechanisms by which transformed SHE cells could develop resistance to MG-induced apoptosis are not clear and further studies are required to see the link between the abrogation of G2/M checkpoint control and the development of resistance to apoptosis observed (Rao, 2001).

Furthermore static protein tyrosine specific phosphatases associated with enhanced protein tyrosine and/or serine-threonine phosphorylation seems to be linked to abnormal expression of G1/S cyclins and PCNA during rat liver tumour promotion by MG (Sundarrajan, 2000), but also in this case further studies are required.

Mechanisms behind the induction of these tumours are uncertain and also are not entirely clear the relevance of these findings.

The NTP report (Cilp, 2005) explain that under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity of MG Chloride in female F344/N rats based on the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) and marginal increases in hepatocellular adenoma and mammary gland carcinoma in exposed rats. Furthermore it was concluded that there was no evidence of carcinogenic activity of MG chloride in female B6C3F1 mice exposed to 100, 225, or 450 ppm.

Studies available don't show a clearly result, so Malachite Green Acetate can't be classified ad a carcinogenic substance.