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

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

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:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The experiment is scientifically acceptable, nevertheless details about test method and the test substance composition are missing.

Principles of method if other than guideline:

Malachite Green oxalate was administered orally to timed-pregnant New Zealand white rabbits by gavage on days 6 through 18 of gestation at doses of 5, 10 and 20 mg/kg. Two control groups were used: thalidomide, a known teratogen, was used as a positive control agent; water, the carrier for Malachite Green, was used with the untreated control group.

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: 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, ad libitum.
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Malachite Green was administrated as an aqueous solution.

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:

On days 6 through 18 of gestation.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5, 10, 20 mg/kg
Basis:
actual ingested
Malachite Green solution

Remarks:

Doses / Concentrations:
150 mg/kg
Basis:
actual ingested
Positive control

No. of animals per sex per dose:

5 mg/kg: 16 animals
10 mg/kg: 21animals
20 mg/kg: 16 animals.

Control animals:

yes, concurrent vehicle

other: positive control (thalidomide)

Details on study design:

PRELIMINARY TEST
A range-finding study was conducted with female Sprague-Dawley rats to determine the acute oral toxicity of Malachite Green oxalate in a water carrier. Eight dosages ranging from 12.5 to 1.600 mg/kg were used; there were three rats in each treatment group. The range from 300 to 750 mg/kg was then used to determine the median lethal dose (LD50). Ten female rats were treated at each of four dosages: 500, 450, 600 and 750 mg/kg. The 24-hour LD50 for Malachite Green in fasted female rats was 520 mg/kg (95 % confidence limits, 435 to 624 mg/kg). Effects observed included depression, prostration, emaciation, coma, and death. As dosage levels increased, there was a curresponding increase in the severity of observed effects.
ln other work at Stanford Research Institute rabbits have been found to tolerate only 1/100 the chemical doses tolerated by rats.
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:

CLINICAL OBSERVATION
All animals were observed daily for external signs of toxicity.

BODY WEIGHT
Weights were recorded on gestation days 0. 6, 9. 12, 15, and 18 and on day 29, when the animals were sacrificed and progeny were delivered by caesarean section.

POST-MORTEM EXAMINATIONS
On day 29 animals were sacrificed and progeny were delivered by caesarean section.

Ovaries and uterine content:

During delivery, all resorptions in each doe were recorded.

Fetal examinations:

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.
Alter 24 hours of incubation, all progeny were killed, sexed and examined for gross developmental anomalies. About one-third ofthe young were dissected to check for visceral anomalies.
The remaining young were stained with Alizarin red S for skeletal examination. Visceral anomalies found during stripping for skeletal staining were recorded, as were skeletal anomalies observed during dissection.

Statistics:

Fisher's exact test was used to examine significance for the gross, visceral and skeletal deviations and the number of progeny with deviations. All other data were tested for significance by the t-test on actual data or data transformed by arc sine or square root.

Details on maternal toxic effects:

Details on maternal toxic effects:
CLINICAL SIGNS AND BODY WEIGHT
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.

FOOD CONSUMPTION
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.

WEIGHT GAIN
Does in the untreated control group gained an average of 230 g; thalidomide-treated animals lost an average of 50 g each.
ln the Malachite Green treated groups, those dosed at 5 mg/kg gained an average of 60 g; those dosed at 10 mg/kg lost 30 g; and those dosed at 20 mg/kg lost 60 g. Although the differences in means seem large and dose-related, the variation within groups was such that only the thalidomide and 10-mg/kg Malachite Green groups were signilicantly different from the water-placebo control group (P ≤ 0.05).

Remarks on result:

other: see results in the other sections

Remarks:

see results in the other sections

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
FOETAL TOXICITY
Foetal toxicity was evident in the reproductive performance of does treated with Malachite Green. At all three dosage levels of Malachite Green, 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).
Talidomide effects for these variables were significann at P= 0.01I. Early resurption accounted for must of rhe dead implants The percentage of does wirh one or more resorptions was 35 % for the untreated controls, 95 % for the thalidomide-treated group, and 69, 67, and 69 % for rhe groups treared with 5, 10 and 20 mg/kg Malachite Green, rcspectively.
The ratio ofmale to female fetuses was about 2:1 in all treated and untreated groups. This ratio remained constant, even among drastically reduced numbers of offspring.

POST CAESAREAN
At the time of caesarean section, mean body weights of progeny were significantly less (P = 0.01) in the thalidomide (34.2 g) and 5 mg/kg Malachite Green (34.2 g) groups than in the untreated controls (37.1 g), Weights of fetuses from the 20 mg/kg Malachite Green group (35.2 g) were also significantly lighter (F = 0.05), but the difference was not as extreme. Although the average weight in the 10-mg/kg group (35.8 g) was also less than that of the untreated controls. the difference was not statistically significant.
All fetuses were incubated and observed for 24 hours after removal from the uterus. Survival of the untreated controls was 84 % during this period. In the treated groups the rates of survival for the different groups were: thalidomide-44 %; 5 mg/kg Malachite Green-65 %; 10 mg/kg Malachite Green-87 %; 20 mg/kg Malachite Green-78 %. The reduction in survival observed in the thalidomide and 5-mg/kg Malachite Green groups was significant (P= 0.01 and P = 0.05, respectively). Survival at the two higher dosages of Malachite Green was comparable to that of the untreated controls.
A variety of developmental anomalies was observed in the treated groups.
ln general, the occurrence of developmental deviations in animals dosed with Malachite Green was twice that in the control but only half that in animals dosed with thalidomide. Teratological effects of Malachite Green were observed at all treatment levels and were not dose-related.
Significant effects were observed in all three types of anomalies (gross, visceral, and skeletal) for thalidomide. Groups treated with 5 and 20 mg/kg Malachite Green 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 ossifcation is not an anomaly, rhis response was observed in varying ratios among all Malachile Green treated lots.

Dose descriptor:

NOAEL

Effect level:

5 other: mg/kg (actual dose)

Based on:

test mat.

Basis for effect level:

skeletal malformations

Developmental effects observed:

not specified

Reproductive performance of pregnant female New-Zealand white rabbits dosed with thalidomide (used as positive control) or Malachite Green. Number in parentheses are average number per doe.

Treatment group and dose	Number of doe	Number of corpora lutea	Total number of implants	Number of resorptions	Number of fetuses
					Aborted	Dead	Live
Untreated	20	186 (9.3)	171 (8.6)	15 (0.8)	7	3	146 (7.3)
Thalidomide* 15 mg/kg	21	195 (9.3)	163 (7.8)	107 (3.1)	5	1	50** (2.4)
Malachite Green 5 mg/kg	16	149 (9.3)	121 (7.6)	34 (2.1)	0	8	79*** (4.9)
Malachite Green 10 mg/kg	21	206 (9.8)	164 (7.8)	49 (2.3)	6	0	109*** (5.2)
Malachite Green 20 mg/kg	16	173 (10.8)	144 (9.0)	60 (3.8)	0	1	83*** (5.2)

*Used as positive control

** Significantly fewer than controls at P = 0.01

*** Significantly fewer than controls at P = 0.05

Incidence of developmental anormalies observed in day-old progens of New Zealand white rabbits dosed with water, Malachite Green or thalidomide (expressed as percent of animals examined)*

Anomaly	Dosage and (in parentherses) number of animals examined
	Water	Malachite Green			Thalidomide
	1 mg/kg (146)	5 mg/kg (79)	10 mg/kg (109)	20 mg/kg (83)	150 mg/kg (50)
Visceral
Enlarged liver	0	12.7	5.5	19.3	42.0
Mottler liver	0	8.9	0	0	24.0
Hemorrhaging in bladder	13.7	8.9	19.3	9.6	24.0
Enlarged heart	0	0	5.5	6.0	10.0
Edematous abdominal cavity	2.0	8.9	0	0	34.0
Hydronephrosis	0	3.8	0	3.6	0
Skeletral
Incomplete ossification of caudal vertebrae	0	0	14.7	4.8	0
Incomplete ossification of fifth phalange	0	14.0	0.9	1.2	0
Incomplete ossification of first phalange	0	3.8	2.8	8.4	12.0
Incomplete ossification of sternebrae	6.8	1.3	64	3.6	0
Extended ossification of caudal vertebrae	0	5.1	0.9	3.6	0
Incomplete ossification of skull	2.0	0	0.9	8.4	16.0
Malformed skull	0.7	3.8	3.7	1.2	22.0
Fused caudal vertebrae	0	0	8.3	0	36.0
Flexure of wrists (one or both)	0	0	1.8	0	38.0
Twisted ankles (one or both)	0	1.3	0	0	14.0
Shortened tail	2.7	0	0	0	54.0
Extended ossification of sternebrae	0	1.3	0	3.6	0
Extended ossification of thoracic vertebrae	0	0	3.7	0	0
Malformed caudal vertebrae (cleft)	0	0	2.8	0	8.0
Malformed scapula	0	0	0	1.2	6.0
Miscellaneous
Encephalitis	0	3.8	0	0	24.0
Hemorrhaging in brain	0	8.9	0	3.6	0

*Statistical evaluation of the significance of each anomaly were not run.

Summary of teratological effects of Malachite Green in day-old progeny of New Zealand white rabbits

Treatment group and dose	Total progeny	Progeny with deviation from normal*			Progeny with deviation*	Percent with deviation	Total deviation*
		Gross	Visceral	Skeletal
Control (water 1 mg/kg)	146	0	7/44	21/103	27/146	18.5	33
Thalidomide (150 mg/kg)	50	42/50**	16/21**	33/36**	47/50**	94.0	187
Malachite Green (5 mg/kg)	79	3/79***	9/23***	20/56***	3/79**	38.0	36
Malachite Green (10 mg/kg)	109	2/109	set-36	29/77***	37/109**	33.9	52
Malachite Green (20 mg/kg)	83	8/83**	12/31***	22/58***	39/83**	47.0	44

*Some individuals had more than one anomaly, so numbers are not additive across these columns. The second number in each entry is the number of animals examined for the respective category of deviations.

** Significantly fewer than controls at P = 0.01

*** Significantly fewer than controls at P = 0.05

Conclusions:

Malachite Green oxalate caused significant developmental abnormalities when administered to pregnant New Zealand whiter rabbits. All dose levels teated (equal/higher than 5 mg/kg) affected foetal development of rabbits.

Executive summary:

Malachite Green oxalate was administered orally to timed-pregnant New Zealand white rabbits by gavage on days 6 through 18 of gestation at doses of 5, 10 and 20 mg/kg. Two control groups were used: thalidomide, a known teratogen, was used as a positive control agent; water, the carrier for Malachite Green, was used with the untreated control group.

All animals were observed daily for external signs of toxicity. The animals were sacrificed and progeny were delivered by caesarean section. During delivery, all resorptions in each doe were recorded. 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.

Alter 24 hours of incubation, all progeny were killed, sented and examined for gross developmental anomalies.

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.

Conclsuion

Malachite Green oxalate caused significant developmental abnormalities when administered to pregnant New Zealand whiter rabbits. All dose levels teated (equal/higher than 5 mg/kg) affected foetal development of rabbits.

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

The developmental toxicity of malachite green has been investigated in two species, rat and rabbit

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. 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 (Meyer and Jogenson., 1983). The poor quality of the study and concerns relating to its conduct cast some doubt on the reliability of the findings.

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.

An harmonized classification and labelling (C&L) has already been approved for Malachite Green chloride (EC: 209-322-8) and oxalate (EC: 219-441-7) salts, based on the data here reported. The classification has been discussed by Commission of the European Communities and the related information was presented in ECBI/54/02 report, 18th July, 2002 (Health and Safety Executive, UK. 2005). The Commision concluded that "the findings from the rabbit study provide evidence that Malachite Green may cause developmental toxicity and therefore classification is justified, supporting the current classification Repr. Cat. 3; R63".

Although, there are inconsistent findings in the rat and rabbit studies, a classification and labelling agree with harmonized C&L is proposed.

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

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. 3; R63.

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.