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

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: No GLP, no guidelines followed 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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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 mice

Author:

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

Year:

1999

Bibliographic source:

Chemio-Biological Interactions 122 (1999) 153-170

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Remarks:

Not all details on OECD requirement reported

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Malachite Green Chloride and Leucomalachite Green

IUPAC Name:

Malachite Green Chloride and Leucomalachite Green

Details on test material:

MALACHITE GREEN -MG-
- Name of test material: Malachite Green
- CAS: 569-64-2
- Source: Chemsyn, Lenexa, KS
- Analytical purity: ≥9 4% by high performance liquid chromatography (HPLC) with UV detection (254 nm)
- Impurities: Leucomalachite Green 1% and demethlyated derivatives of Malachite Green 3.5%

LEUCOMALACHITE GREEN -LG-
- Name of test material: Leucomalachite Green
- CAS: 129-73-7
- Source: Chemsyn, Lenexa, KS
- Analytical purity: ≥9 8% by high performance liquid chromatography (HPLC) with UV detection (254 nm)
- Impurities: the impurities detected were ≤ 2% monodesmethyl Leucomalachite Green and Malachite Green

Test animals

Species:

other: Rat 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:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: 0, 25, 100, 300, 600, or 1200 ppm MG for 28 days; fed 0, 290, 580, or 1160 ppm of LG for 28 days. Additional groups were fed 0 or 1200 ppm of MG for and 0 or 1160 ppm LG 4 or 21 days.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4, 21 or 28 days

Frequency of treatment:

Not specified

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
MG for 28 days: 0, 25, 100, 300, 600, or 1200 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
LG for 28 days: 0, 290, 580, or 1160 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
MG for 4 or 21 days: 0 or 1200 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
LG 4 or 21 days: 0 or 1160 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Each group was formed of eight animals per dose per sex

Control animals:

yes, concurrent no treatment

Examinations

Statistics:

Analysis of variance (ANOVA) and Dunnett’s test

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

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

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

ca. 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Malachite Green

2

Dose descriptor:

NOAEL

Effect level:

ca. 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Malachite Green

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Rats fed MG: In female rats there were significant decreases in the mean body weights in the 1200 ppm dose group for weeks l-4; although the male rats fed 1200 ppm MG tended to have lower body weights as compared to the control group, the differences were not significant. In the female rats, the animals in the three highest doses (300, 600, and 1200 ppm) of MG had significantly increased ratios of liver weights to body weights. The ratio of liver weight to body weight was significantly increased in the male rats fed 600 and 1200 ppm MG. In both sexes, there was a significant linear increasing trend in the levels of ϒ-glutamyl transferase, with the value in the 1200 ppm female dose group being 4.2-fold greater (P < 0.0005). Blood hematology measurements in female rats showed slight but significant, decreases in the 1200 ppm dose group in erythrocyte count, haemoglobin, haematocrit, mean erythrocyte haemoglobin and mean erythrocyte concentration. Male rats had slight, but significant, decreases in mean erythrocyte haemoglobin in the 300, 600, and 1200 ppm dose groups.

Additional groups of female and male rats were fed 0 or 1200 ppm MG for 4 or 21 days and blood was collected for T3, T4 and TSH measurements. The T3 levels were significantly higher in female rats fed 1200 ppm MG. The T4 levels were significantly lower on both days 4 and 21 in the female rats in the 1200 ppm group. There were no significant changes in T3 or T4 levels in males or in the TSH levels in either sex. Seven out of eight female rats fed 1200 ppm MG had minimal to mild hepatocyte vacuolization (P <0.01). The same lesion, primarily midzonal in location, was observed in one of eight male rats fed 600 ppm MG and four of eight male rats fed the 1200 ppm dose.

Male rats fed LG: Male rats fed 1160 ppm LG had significantly lower body weights at weeks 2, 3, and 4. There were also significant decreases in body weights in the 580 ppm dose group at weeks 3 and 4. Linear decreasing dose trends were observed for weeks 2, 3, and 4 (P < 0.003). The ratio of liver weights to body weights was significantly increased for all three dose groups.

y-Glutamyl transferase levels were 2.2-fold higher and phosphorus levels were slightly increased in male rats fed 1160 ppm LG. In addition, erythrocyte count, haemoglobin, and haematocrit levels showed slight, but significant, decreases from the controls in the 1160 ppm dose group. There was a significant decrease in T4 and increase in TSH levels on days 4 and 21. In male rats fed LG, hepatocyte vacuolization, primarily midzonal and centrilobular in location, was seen in 7/8 rats fed 1160 ppm, 5/8 rats fed 580 ppm (P < 0.04), and 2,/8 rats fed 290 ppm, a significant dose trend (P < 0.0004). Two of the eight rats fed 1160 ppm and two of the eight rats fed 580 ppm LG had apoptotic follicular epithelial cells in the thyroid gland. Morphologic changes consisted of sloughed follicular cells with condensed nuclei located within the follicles. An inflammatory reaction was not present. There was evidence of follicular epithelium regeneration, since even the most severely affected follicles were still lined by viable epithelium.

Mice fed MG: Female mice fed 1200 ppm MG had significantly lower body weights at weeks 3 (P<0.02) and 4 (P<0.03). The body weights of male mice were not significantly affected by any of the dose levels of MG. In female mice fed 600 or 1200 ppm MG, there were slight significant decreases in the erythrocyte count and haemoglobin and haematocrit levels as compared to the control group; in male mice, significant decreases in these parameters were observed in the 1200 ppm dose group, The mean erythrocyte volume was increased 1-2% in female mice fed 300, 600, and 1200 ppm. There was also an increase in reticulocytes in these groups. Male mice fed 1200 ppm MG showed a 1.6-fold increase in reticulocytes. There were no significant histopathological changes observed in the mice fed MG.

Female mice fed LG: Female mice fed 1160 ppm LG had significantly lower body weights at week 4. A marginally significant decrease from the control group was observed in the female mice fed 580 ppm at week 4. In addition, there were statistically significant linear dose trends for week 3 and week 4. All female mice fed 1160 ppm LG had scattered dead or degenerate cells in the transitional epithelium of the urinary bladder. Many of the cells lacked nuclei and when visible, the nuclei were condensed or fragmented, suggesting apoptosis. Examination of thin sections revealed that many apparent apoptotic cells were contained within phagocytic vacuoles inside viable epithelial cells. The ISEL technique showed that the cytoplasm of apparently apoptotic cells was moderately positive for the presence of DNA fragments and condensed nuclei stained intensely for DNA fragmentation. Individual cell necrosis was not accompanied by inflammatory changes. Similar apoptosis was not seen in transitional epithelium of the bladders of female mice fed 0, 290, or 580 ppm LG.

DNA adducts and metabolites: a single adduct (or co-eluting adducts) was observed with both compounds

Applicant's summary and conclusion

Conclusions:

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. A NOAEL of 100 ppm can be derived from results.

Executive summary:

Malachite Green (MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. MG is reduced to and persists as Leucomalachite Green (LG) in the tissues offish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm MG or 1160 ppm 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. Haepatocyte 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.

A NOAEL of 100 ppm can be derived from results.