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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:

Based on REACH regulation annex VIII column 2 (specific rules for adaptation from column 1) the study does not need to be conducted if a pre-natal developmental toxicity study (Annex IX, 8.7.2) or a two-generation reproductive toxicity study (Annex IX, section 8.7.3) is available.

Effects on developmental toxicity

Description of key information

The outcomes from the rabbit study provide evidence that Malachite Green may cause developmental toxicity and therefore classification is justified, supporting the current classification  Repr 2, H361d

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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: No GLP, no guidelines followed, poorly details on test conditions 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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Malachite Green was administrated to Rabbits by oral gavage. Thalidomide was used as positive control and untreated animals as untreated control.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

ANIMALS TESTED
- Specie: Oryctolagus cuniculus
- Source: commercial laboratory supplier Scott Rabbit Products, Langley, Washington
- Age at study initiation: time-pregnant
- Housing: all animals were housed individually
- Diet: Purina Laboratory Rabbit Chow supplemented by fresh greens
- Water: water ad libitum

CONTROL
- Group: two control group were used: one untreated and one like positive control (treated by Thalidomide, a known teratogen)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DIET
- Dose: 5, 10, 20 mg/kg
- Solution: Malachite Green was administrated as an aqueous solution.
- Administration: orally by gavage on days 6 through 18 of gestation
- N. initial animals: 20

POSITIVE CONTROL SOLUTION
- Solution: thalidomide was used as a positive control
- Preparation: Thalidomide was prepared as a suspension in corn oil
- Dose: 150 mg/kg

Duration of treatment / exposure:

18 days of gestation; orally administration by gavage on days 6 through 18 of gestation

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5, 10, 20 mg/kg
Basis:
nominal in diet
Malachite Green solution

Remarks:

Doses / Concentrations:
150 mg/kg
Basis:
nominal in diet
Positive control

No. of animals per sex per dose:

At dose 5 mg/kg 16 animals; at 10 mg/kg 21; at 20 mg/kg 16.

Control animals:

yes, concurrent vehicle

Details on study design:

PRELIMINARY TEST
Consequently, doses of 2, 50, and 75 mg/kg were selected to determine the maximum dose to be used with rabbits. Three young, nonpregnant adult New Zealand white rabbits were dosed daily for 13 days in each treatment. Data from this limited study indicated that nonpregnant female rabbits could tolerate 13 consecutive daily doses of 50 mg/kg of Malachite Green. Stanford Research Institute experience has been that pregnant rabbits are much more sensitive to drugs than nonpregnant ones so 20 mg/kg was selected as the maximum level to be used in the teratology study.

Maternal examinations:

GENERAL SIGN OF TOXICITY: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: were recorded gestation days 0, 6, 9, 12, 15 and 18 and one day 29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Examination: on day 29 the animals were sacrificed and progeny were delivered by caesarean section. During delivery, all resorptions in each doe were recorded.

OTHER:
Young were thoroughly examined at delivery, weighed and incubated for 24 hours. During incubation they were observed hourly for viability during the first 4 hours and again at 24 hours

Fetal examinations:

- External examinations: Yes
- Skeletal examinations: Yes
- Visceral examination: Yes

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Treated adult animals were consistently lower in average total body weight than the untreated controls at the end of the study, but there were no overt signs of toxicity.

Remarks on result:

other: See report below

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetal toxicity was evident in the reproductive performance of does treated with Malachite Green (MG). At all three dosage levels of MG, there were significant increases in pre-implantation losses and in the ratio of dead implants to total implants and decrease in the number of living fetuses (P = 0.05). Early resorption accounted for most of the dead implants.
At the time of caesarean section mean body weights of progeny were significantly less in the thalidomide positive control and 5 mg/kg MG groups than in the untreated controls. Weights of foetuses from the 20 mg/kg MG group were also significantly lighter, but the difference was not as extreme.
Significant effects were observed in all three types of anomalies: gross, visceral and skeletal. Groups treated with 5 and 20 mg/kg MG showed significant differences from control animals in skeletal deviations. All treated groups showed significantly more progeny with deviations than were observed in the controls. Although delayed ossification is not an anomaly, this response was observed in varying ratios among all MG treated lots.

Dose descriptor:

LOAEL

Effect level:

> 5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

During treatment with malachite green, the does showed a greatly reduced intake of dry food. This reduced food consumption is believed to have been related to the chemical treatments rather than the result of an incomplete diet.

Conclusions:

Malachite Green produced significant tetratological affects at all levels of treatment at dose as low as 5mg/kg. Abnormalities noted in rabbits were anomalies most frequently involved the skeleton, liver, heart, and kidneys.

Executive summary:

Malachite Green Oxalate caused significant developmental abnormalities when administered to pregnant New Zealand whiter rabbits Oryctolagus cuniculus. All dose levels affected foetal development of rabbits. At the dosages given, the incidence of anomalies caused by Malachite Green was about half that observed in thalidomide-treated positive controls and two to three times that in untreated controls.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

An old study on Malachite Green (MG) conducted in rabbits provides indication of developmental toxicity, evidenced by an increase in the number of resorptions at doses that did not cause significant maternal toxicity (Meyer et al., 1983). MG produced significant tetratological affects at all levels of treatment at dose (5, 10 and 20 mg/kg bw) as low as 5 mg/kg. Abnormalities noted in rabbits were most frequently involved the skeleton, liver, heart, and kidneys.

Although no evidence of developmental toxicity was found in rats in Reynolds reports (1991, 1992), which demonstrated the absence of effects of oral administration (by gavage) of MG to pregnant rats during the period of organogenesis at a dose levels of 2, 10, 50 and 100 mg/kg bw/day on litter and foetal responses, the outcomes from the rabbit study provide evidence that MG may cause developmental toxicity and therefore classification is appropriated, supporting the current classification Repr 2, H361d.

The actual classification of Malachite Green as H361d (Suspected of damaging the unborn child) can be accepted with no further investigation for Malachite Green Acetate.

Toxicity to reproduction: other studies

Additional information

Fertility

No data available. No classification proposed

 

Developmental toxicity

The developmental toxicity of Malachite Green (MG) has been investigated in two species, rat and rabbit. No evidence of developmental toxicity was evident in rats at dose levels causing maternal toxicity (increased mortality and reduced bodyweight); nevertheless the older study on rabbits provides some indication of possible developmental toxicity, evidenced by an increase in the number of resorptions at doses that did not cause significant maternal toxicity.

 

Although the inconsistent results found on rats and rabbits, the outcomes of the rabbit study provide evidence that MG may cause developmental toxicity and therefore classification is appropriated, supporting the current harmonized classification Repr. Cat. 2;

H361d

.

Justification for classification or non-classification

The findings from the rabbit study provide evidence that malachite green may cause developmental toxicity and therefore classification is justified, supporting the current harmonized classification Repr.2 H361d

The actual classification of Malachite Green as H361d (Suspected of damaging the unborn child) can be accepted with no further investigation for Malachite Green Acetate.