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

Carcinogenicity

Description of key information

Exposure to malachite green chloride in feed resulted in nonneoplastic lesions in the thyroid gland and liver of female rats and the urinary bladder of female mice.

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 13th, 1999 to May 7th, 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: National Toxicology Program study report. 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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

National Toxicology Program study report.

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

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. 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 was 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 %
- Air changes: at least 10/hour
- Photoperiod: 12 hours/day

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

Dose formulations were prepared approximately every 2 months by dissolving the chemical in water and then mixing it with feed. 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.
Dose formulations were stored in stainless steel feed cans at 4 ± 2 °C for up to 92 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.
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 malachite green chloride dose formulations analyzed and used, 96 % (65/68) of the dose formulations were within 10 % of the target concentrations.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 100, 300, or 600 ppm (equivalent to average daily doses of approximately 0, 7, 21 and 43 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

48 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: to assist in selecting doses for the 2-year bioassay, a 28-day range-finding study was conducted in which male and female F344 rats were fed the test item (0, 25, 100, 300, 600, or 1200 ppm) in feed.
- Rationale for animal assignment: animals were distributed randomly into groups of approximately equal initial mean body weights.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
Observed twice daily and clinical findings recorded weekly.

BODY WEIGHT
Animals were weighed initially, weekly for the first 12 weeks, approximately every 4 weeks until week 92, weekly for the last 12 weeks, and at the end of the studies.

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption was recorded weekly for the first 12 weeks and approximately every 4 weeks thereafter.

Sacrifice and pathology:

Sacrifice: carbon dioxide asphyxiation.

GROSS PATHOLOGY
Necropsies were performed on all animals. Organs weighed were the kidneys and liver and the thyroid gland.

HISTOPATHOLOGY
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone marrow (femur, sternum), brain (cerebellum, cerebrum, stem), clitoral gland, esophagus, eye, harderian gland, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, muscle (thigh), nerve (sciatic), nose, ovaries, pancreas, parathyroid gland, pituitary gland, salivary gland, skin, spinal cord (thoracic), spleen, stomach (forestomach and glandular), thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina, and Zymbal’s gland.

Statistics:

Survival Analyses: the probability of survival was estimated by the product-limited procedure of Kaplan and Meier (1958).
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: the Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.
Analysis of Continuous Variables: for both body weights and food consumption, the mixed models approach to repeated measures ANOVA was used. Organ weights, terminal body weights, and the ratios of organ weight to terminal body weight for terminally sacrificed animals were analyzed using ANOVA procedures. Terminal body weights were also used as a covariant in an ANACOVA procedure. For each end point analyzed, Dunnett’s two-sided test (Dunnett, 1955) was used to compare the control group mean to each treatment group mean, either overall or at each point of time, whichever was appropriate.

Clinical signs:

no effects observed

Description (incidence and severity):

similar to the control group

Mortality:

no mortality observed

Description (incidence):

similar to the control group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

300 and 600 ppm groups less than the control group

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

similar to the control group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

relative liver weight increased (600 ppm)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

thyroid gland: follicle, cyst (0/46, 1/48, 1/47, 3/46); liver: eosinophilic focus (5/48, 10/48, 13/48, 14/48)

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of all exposed groups was similar to that of the control group. Survival rates: 29/48, 23/48, 32/48, 25/48.
No clinical findings were attributed to malachite green chloride exposure.

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.

FOOD CONSUMPTION AND COMPOUND INTAKE
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 malachite green chloride/kg body weight.

ORGAN WEIGHTS
The relative liver weight was significantly increased in females exposed to 600 ppm malachite green chloride

PATHOLOGY AND STATISTICAL ANALYSES
Thyroid Gland: follicular cell adenomas and carcinomas were observed in female rats exposed to 300 or 600 ppm 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 malachite green chloride.

Liver: there were modest, but not statistically significant, increases in the incidences of hepatocellular adenoma in female rats exposed to test item. 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. 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: the incidence of mammary gland carcinoma in female rats exposed to 600 ppm exceeded the historical control range.

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

Mononuclear Cell Leukemia: a dose-related decreasing trend in the incidences of mononuclear cell leukemia occurred in female rats exposed, with statistically significant decreased incidences in the 300 and 600 ppm groups.

Relevance of carcinogenic effects / potential:

Ambiguous

Dose descriptor:

NOAEL

Effect level:

7 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: incidence of eosinophilic foci in the liver was increased

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

21 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: thyroid follicular cell adenoma and carcinomas, hepatocellular ademonas , mononuclear cell leukemia

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Conclusions:

It was concluded that tumors of the thyroid gland, liver, or mammary gland in female rats might have been caused by Malachite Green chloride.

Executive summary:

Malachite Green chloride effects were studied on female rats to identify potential toxic or cancer-related hazards to humans. The dye was mixed into the feed of rats and the doses given were 100, 300, or 600 ppm (equivalent to average daily doses of approximately 0, 7, 21 and 43 mg/kg bw/day). 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.

Rats exposed to Malachite Green chloride weighed less on average than the control animals. In rats exposed to the dye, there were very slight increases in a few types of tumors: cancers of the thyroid gland, liver, and mammary gland. It was concluded that tumors of the thyroid gland, liver, or mammary gland in female rats might have been caused by Malachite Green chloride.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

7 mg/kg bw/day

Study duration:

chronic

Species:

rat

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 the CLP Regulation (EC 1272/2008), for the purpose of the classification for Carcinogenicity, section 3.6, substances can be allocated in one of two categories, as follow.

A substance is classified in Category 1, as known or presumed human carcinogens, on the basis of epidemiological and/or animal data. A substance may be classified as known to have carcinogenic potential for humans, classification largely based on human evidence or presumed to have carcinogenic potential for humans, based on animal evidence.

The placing of a substance in Category 2 is done on the basis of evidence obtained from human and/or animal studies, but which is not sufficiently convincing to place the substance in Category 1, based on strength of evidence together with additional considerations.

Based on the available information, Malachite Green has not been judged as presumed either suspected human carcinogen.

In conclusion, the substance does not meet the criteria to be classified for carcinogenicity, according to the CLP Regulation (EC 1272/2008).

Additional information

Under the conditions of the NTP (2005) 2-year feed studies, there was equivocal evidence of carcinogenic activityof malachite green 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. There wasno evidence of carcinogenic activityof malachite green chloride in female B6C3F1mice exposed to 100, 225, or 450 ppm.

Under the conditions of these 2-year feed studies, there wasequivocal evidence of carcinogenic activityof leucomalachite green in male F344/N rats based on an increase in interstitial cell adenoma of the testes and the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) in exposed rats. There wasequivocal evidence of carcinogenic activityof leucomalachite green in female F344/N rats based on a marginally increased incidence of hepatocellular adenoma and the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) in exposed rats. There wassome evidence of carcinogenic activityof leucomalachite green in female B6C3F1mice based on an increase in hepatocellular adenoma or carcinoma (combined) (NTP, 2005).

Exposure to malachite green chloride in feed resulted in nonneoplastic lesions in the thyroid gland and liver of female rats and the urinary bladder of female mice; exposure to leucomalachite green in feed resulted in nonneoplastic lesions in the thyroid gland and liver of male and female rats and the urinary bladder of female mice (NTP, 2005).

Effects attributed to Malachite Green and that seems linked to tumour promotion were reported in literature. The substance appears to be able to produce transformation SHE cells, developing aneuploid pattern; these 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 et al. study 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 Malachite Green-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 Malachite Green (Sundarrajan, 2000), but also in this case further studies are required.

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