[4-[4-(diethylamino)benzhydrylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium hydrogen sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP, no guidelines followed, poor details on test conditionsRead 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

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of Malachite Green and Leucomalachite Green in female big blue B6C3F mice

Author:

Roberta A. Mittelstaedt, Nan Mei, Peggy J. Webb, Joseph G. Shaddock, Vasily N. Dobrovolsky, Lynda J. McGarrity, Suzanne M. Morris, Tao Chen, Frederick A. Beland, Kevin J. Greenlees, Robert H. Heflich

Year:

2004

Bibliographic source:

Mutation Research 561(2004) 127-138

Materials and methods

Principles of method if other than guideline:

MICRONUCLEUS: were conducted on peripheral blood using the µFlow Mouse Micronucleus Analysis Kit and the manufacturer’s protocol (Litron Laboratories, Rochester, NY USA). Flow analysis was perforrned using a FACSort flow cytometer.LYMPHOCYTE Hprt: lymphocytes were removed from crushed spleens, stimulated by treatment overnight with concanavalin A, and processed for limiting-dilution cloning in 96-well dishes. LIVER CLL: High-molecular-weight DNA was extracted from the mouse livers using the RecoverEase DNA Extraction Kit (Stratagene)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 02 and 05

IUPAC Name:

Similar substance 02 and 05

Details on test material:

- Name of test material: Malachite Green -MG-- CAS: 569-64-2- Analytical purity: 88% by HPLC-PDA- Source: Chemsyn Science Laboratories- Name of test material: Leucomalachite Green -LG- - CAS: 129-73-7- Analytical purity: 99% by HPLC-PDA- Source: Chemsyn Science Laboratories

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: purchased from Stratagene (La Jolla, CA, USA) and supplied by Taconic farm (Germantown, NY, USA)- Age at study initiation: approximately 6-week-old mice- Number of animal: 12- Diet: NIH-31; 450 ppm MG, 204 o 408 ppm LG and the remaining 12% of MG was primarily LG and the desmethyl derivatives of MG and LG. It has been used the high dose of MG and the middle and the high dose of LG used in the NTP studies.CHEMICAL ANALYSIS OF THE FEED:- Chemical analysis of the feed indicated that the actual doses were withing 3% of the target doses.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

no vehicles used

Details on exposure:

View diet details

Duration of treatment / exposure:

Six mice from each group were killed after 4 weeks and the remaining mice were killed after 16 weeks of exposure.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:450 ppmBasis:nominal in dietMG

Doses / concentrations 2

Remarks:

Doses / Concentrations:204Basis:nominal in dietLG

Doses / concentrations 3

Remarks:

Doses / Concentrations:408Basis:nominal in dietLG

No. of animals per sex per dose:

12 mices were fed control diet (NIH-31) or diet containing 450 ppm of MG or 204 or 408 ppm of LG.

Positive control(s):

MICRONUCLEUS ANALYSISPositive and negative controls for the flow analysis were prepared from peripheral blood of 9-day-old neonatal B6C3F1 mice that were treated on days 1—8 with either DMSO or 200 mg/kg dideoxycytidine. These treatments were not performed concurrently with the feeding study; however, the resulting samples were analyzed for micronucleus frequency at the same time as the test samples.

Examinations

Tissues and cell types examined:

Micronucleus, lymphocyte hprt mutant, liver cll.

Statistics:

On the lymphocyte Hprt mutant frequency data has been investigated by analysis of variance (ANOVA)

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

MICRONUCLEUS ANALYSIS

Feeding female B6C3F1 Big Blue mice with MG or LG for as long as 16 weeks had no significant effect on either reticulocyte or normochromatic erythrocyte peripheral blood micronucleus frequencies. Positive controls for the micronucleus assay were significantly elevated, and their frequencies consistent with previous observations.

LYMPHOCYTE Hprt MUTANT ANALYSIS

Analysis of variance (ANOVA) on the lymphocyte Hprt mutant frequency data for mice treated for 4 weeks indicated a signilicant difference among the groups. This appeared to be due to a relatively low mutant frequency in mice treated with 204 ppm LG, and Dunnett’s test indicated that there were no significant differences between the mutant frequencies in any of the treated groups and the control. Hprz lymphocyte mutant frequencies were not significantly different from the controls after 16 weeks of treatment with MG or LG.

Hprt lymphocyte mutant frequencies were not significantly different from controls after 28 days or 16 weeks.

LIVER CLL.

Mutant frequencies were determined in the livers of mice fed 450 ppm MG or 408 ppm LG for 16 weeks. Statistical analysis by ANOVA, followed by Dunnett’s test, indicated that LG increased the liver mutant frequency while MG did not. Sequencing of mutant clones revealed that the degree of mutant independence for the control and treated mice was similar. For this analysis, different mutations isolated from the same mouse were assumed to be induced independently; the same mutation identified more than once in mutants isolated from the same mouse was assumed to be derived from an expanded mutant clone and, therefore, not independent. When the all mutant frequencies were corrected for independence, the results indicated that LG did increase liver cll mutation frequency, while MG did not. Female Big Blue rats fed 543 ppm LG for 16 weeks in a previous study were also evaluated for liver cll mutant frequency. There was no increase in either cll mutant or mutation frequency inthe treated rats.

The liver cll mutants isolated from the mutant frequency assays were sequenced to analyze the specific mutations in the clI gene. A high proportion of mutations from control mice (49%) contained transitions. The spectrum of mutations from mice treated with MG was similar to that for control mice, while the mutations from mice fed LG had increased frequencies of transversions.

MG did not increase the cII liver mutation frequency after 16 weeks.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negativeThese results indicate that the tested substance is not an in vivo mutagen; it resulted negative in micronucleous analysis, lynphocyte HPRT mutant analysis and mutant frequencies determined in the liver cells.

Executive summary:

The tested substance is rapidly reduced in vivo to Leucomalachite Green (LG). In the present study transgenic female Big Blue B6C3F₁ mice WERE FED with 450 ppm of MG and 204 and 408 ppm of LG and evaluated genotoxicity after 4 and 16 weeks of treatment. Neither MG nor LG increased the peripheral blood micronucleus frequency or Hprt lymphocyte mutant frequency at either time point; however, the 16-week treatment with 408 ppm LG did increase the liver cll mutant trequency. Similar increases in liver cII mutant frequency were not seen in the mice treated for 16 weeks with MG.

These results indicate that MG is not an in vivo mutagen; while the evaluation of LG is not clearly, because results are more ambiguous.