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

Biological effects monitoring

Administrative data

Endpoint:

biological effects monitoring

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 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:

Cytotoxic effects of sublethal concentrations of malachite green in isolated hepatocytes from rainbow trout

Author:

Zahn t. and Braunbeck T.

Year:

1995

Bibliographic source:

Toxicology in vitro, vol 9, No. 5, pp 729-741, 1995

Materials and methods

Principles of method if other than guideline:

Isolated hepatocytes of rainbow trout were used to evaluate cytotoxicity in a subcellular system and compare in vitro effects with those of in vivo experiments.

Type of study / information:

Cytotoxic effects of sublethal concentrations of Malachite Green (MG).

Test material

Test material information

Constituent 1

Reference substance name:

Malachite Green Oxalate

IUPAC Name:

Malachite Green Oxalate

Details on test material:

- Name of test material: malachite green
- IUPAC name: N-[4-[bis]4-(dimethylamino)-phenyl[phenyl]methylene] Cl 42000
- Source: Merck, Darmstadt, Germany

Results and discussion

Any other information on results incl. tables

ACUTE TOXCITY OF MALACHITE GREEN

Viability of isolated hepatocytes as evidenced by trypan blue exclusion showed a senescence dependent decline from 90 or more to about 70% after 5 days (cf. Braunbeck and Storch, 1992) in culture for all experimental groups. Likewise. LDH release into the medium, which increased from 200 ± 35 mU ml at day 1 to approximately 240 mU/Uml at day 5 in controls. Did not show significant changes after exposure to malachite green. Thus, significant cytotoxic effects of malachite green on isolated hepatocytes could not be revealed at the concentrations tested.

ULTRASTRUCTURE OF CONTROL HEPATOCYTES

Nuclei showed heterochromatin randomly distributed in the nucleoplasm with small concentrations underneath the nuclear envelope. The organelle-rich portion of the cytoplasm comprised mitochondria. peroxisomes, tubules and cisternae of smooth endoplasmic reticulum (SER) and RER stacks with up to 12 non-fenestrated cisternae arranged in parallel array. Golgi fields consisting of 3- 5 cisternae and occasional lipid droplets. In control cells, only a modest increase of small myelinated bodies in the cytoplasm and the tendency of heterochromatin to condense in the nucleus of ageing cells could he observed from day 3 in culture.

ULTRASTUCTURE OF ISOLATED HEPATOCYTES EXPOSED TO 0.01 mg LITRE MALACHITE GREEN

After 24 h of exposure to malachite green, part of the hepatocytes showed an increasing tendency of the heterochromatin to condense in the nuclearperiphery and around the nucleolus. - Cytoplasmic effects after 3 days of exposure comprised a closer association of peroxisomes with single RER cisternae. a more intimate association o f RER cisternae with glycogen rosettes, a slight increase of lipid droplets and induction of glycogenosomes.

After 5 days of exposure, additional effects were a less regular compartmentation of hepatocytes, formation of mitochondria clusters and their more intimate association with RER cisternae and lipid droplets, an increased number of peroxisomes, RER fractionation, dilation, vesiculation and transformation into concentric membrane whorls, and elevated amounts of SER tubules. Rare observations were crystal-like inclusions in dilated RER cisternae.

ULTRASTUCTURE OF ISOLATED HEPATOCYTES EXPOSED TO 0.1 mg LITRE MALACHITE GREEN

Effects described for 0.01 mg litre malachite green were enhanced after exposure to 0.1 mg/litre. Most effects could be observed earlier. Thus, the reaction of isolated rainbow trout hepatocytes proved to be time and dose dependent. Additional effects at 0.1 mg litre comprised: - after day 1: increased amounts of lysosomes, vesiculation and decreased amounts of RER cisternae; - after day 5: increased heterogeneity of mitochondria size and morphology. Closer association of RER cisternae with mitochondria and formation of glycogen bodies.

ULTRASTUCTURE OF ISOLATED HEPATOCYTES EXPOSED TO 1 mg LITRE MALACHITE GREEN

At 1 mg litre malachite green effects were strongest. - After 1 day of exposure: the amount of RER was slightly decreased and numerous RER cisternae were transformed into concentric membrane whorls. Mitochondria were highly heterogeneous in shape and size, and the amount of peroxisomes and lysosomel elements (lysosomes, mvelinated bodies, vacuoles) was further increased. Glycogen fields were restricted to small areas and occasional glycogenosomes could be detected as early as day 1. - After prolonged exposure: increased amounts of lysosomes (day 3), myelinated bodies (day 5) and cytoplasmic vacuoles (day 5) were detected. Also, dilation and vesiculation of RER cistern were intensified after day 5

Applicant's summary and conclusion

Conclusions:

In vitro data provided evidence for nuclei and mitochondria as the major cellular targets of Malachite Green in both fish and mammals.

Executive summary:

The fish therapeutic dye malachite green was tested or sublethal cytotoxic effects it concentrations of 0, 0.01, 0.1 and 1 mg/litre. Isolated hepatocytes of rainbow trout were used to evaluate its cytotoxicity in a subcellular system and compare in vitro effects with those of in vivo experiments. Whereas conventional ability tests [trypan blue exclusion, lactate dehydrogenase (LDH) release] failed to detect any acute toxic effect by Malachite Green exposure. Electron microscopy revealed time and dose dependent responses of isolated hepatocytes with first reactions after 1 -day exposure to 0.01 mg/litre. Whereas hepatocellular nuclei took a more irregular shape, cytoplasmic changes comprised an increase in heterogeneity of mitochondrial shape. Closer association of mitochondria with cisternae of the rough endoplasmic reticulum (RER), fractionation, dilation and vesiculation of RER. Formation of cytoplasmic membrane 0whorls glycogen bodies and induction of myelinated bodies and cytoplasmic vacuoles. In agreement with conclusions drawn from in vivo experiments, in vitro data provided evidence for nuclei and mitochondria as the major cellular targets of Malachite Green in both fish and mammals.