Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested,while LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day.When male and female Fischer 344 rats were treated with gentian violet (548-62-9) orally.

 

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data from handbook or collection of data
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
The three-generation reproductive toxicity study of Gentian violet was performed in Fischer 344 rats.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available
Specific details on test material used for the study:
- Name of test material (as cited in study report): Gentian violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
No data available
Route of administration:
oral: feed
Vehicle:
other: Feed
Details on exposure:
No data available
Details on mating procedure:
- M/F ratio per cage:No data available
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:No data available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)]No data available
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol:No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
total:280-294
F0 generation :at least 80days
F1b generation:100-140 days
F2b generation:100-140 days
F3a generation:up to weaning
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
After at lest 80 daysfrom start of study, In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation


- Selection of parents from F1 generation when pups were [...] days of age.:
At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2a generation. Same procedure was used to produce F2b generation

- Age at mating of the mated animals in the study: [...] weeks
After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation.
Remarks:
0, 5,15 and 30 mg /kg bw /day
No. of animals per sex per dose:
Total :450
Control group:180
Male :90
female 90
treated group:270
5mg/kg bw/day:45male and 45 female
15mg/kg bw/day:45male and 45 female
30mg/kg bw/day:45male and 45 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:No data
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]: No data available
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); for F1b and F2a generation one male and one female from each litter were seleted and 2 male and 2 female per litter for F3a generation were selected excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:yes
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, were observed

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]No data available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]No data available
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.] No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.: yes in F3a generations were observed
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. yes in F3a generation
Statistics:
No data available
Reproductive indices:
No data available
Offspring viability indices:
No data available
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No dose related effect on mortality was observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .
Clinical signs:No data available
Mortality : not dose related mortality was observed,

Body weight and food consumption: The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group.

Test substance intake:No data available

Reproductive function: estrous cycle:No effect observed

Reproductive function: sperm measures:No data available

Reproductive performance:No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .

Organ weights:No data available

Gross pathology:No data available

Histopathology:No data available
other findings:No data available
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect observed on reproductive performance
Critical effects observed:
not specified
System:
other: not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
not dose related mortality was observed,
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .
Clinical signs:No data available

Mortality : not dose related mortality was observed,

Body weight and food consumption: The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group

Test substance intake:No data available

Reproductive function: estrous cycle:No effect observed

Reproductive function: sperm measures:No data available

Reproductive performance:No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .

Organ weights:No data available

Gross pathology:No data available

Histopathology:No data available
other findings:No data available
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect observed on reproductive performace
Critical effects observed:
not specified
System:
other: not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose related effects on incidence of gross deformities were noted
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
No dose related effects on incidence of gross deformities were noted
Dose descriptor:
NOAEL
Generation:
other: F2b
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: gross deformities
Remarks on result:
other: No effect observed at dose 30mg/kg bw
Critical effects observed:
not specified
System:
other: not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Histopatological finding: Dose related trend for focal dilatation of the renal cortex and tubules ,for necrosis of the thymus and inverse dose-response relationship for red pulp haematopoietic cell proliferation of the spleen
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Histopatological finding: Dose related trend for focal dilatation of the renal cortex and tubules ,for necrosis of the thymus and inverse dose-response relationship for red pulp haematopoietic cell proliferation of the spleen
Dose descriptor:
LOAEL
Generation:
other: F3a
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Dose related effect observed
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Organ:
kidney
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
no
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
Treatment related:
no
Conclusions:
The NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested,while LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day.When male and female Fischer 344 rats were treated with gentian violet (548-62-9) orally.
 
Executive summary:

In three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. gentian violet (548-62-9) was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation.

 

In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1ageneration used for separate study while F0 generation animals were mating second times in same dose group following birth of F1bgeneration. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2bgeneration .After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues.

 

 

The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group.Also No dose related effects were observed on gross deformities in each generation.

 

The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female).

 

 Hence the NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested. While LOAEL for offspring in F3a generation w as considered at 5mg/kg bw/day when male and female Fischer 344 rats were treated with gentian violet (548-62-9) orally.

 

 

 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Data is K4 and from secondary source
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity

In different studies,N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9)has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments i.e. most commonly in rats for Gentian violet (548-62-9).

In experimental study given by FAO/WHO Expert Committee on Food Additives (JECFA)( FAO/WHO Expert Committee on Food Additives (JECFA),Page no.3-34,2004) IIn three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. gentian violet (548-62-9) was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation.In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1ageneration used for separate study while F0 generation animals were mating second times in same dose group following birth of F1bgeneration. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2bgeneration .After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues.The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group.Also No dose related effects were observed on gross deformities in each generation.The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female). Hence the NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested.While LOAEL for offspringin F3a generationw as considered at 5mg/kg bw/day when male and female Fischer 344 rats were treated with gentian violet (548-62-9) orally.

 

It is supported by experimental study given by NTP (National Centre for Toxicological Research, TER82080 (study conducted in 1981-82).The reproductive and developmental toxicity study of Gentian violet (548-62-9) was performed in female New Zealand white rabbits. Total 133 animals used for study in which 30 female animals in control group. The doses were selected base upon preliminary toxicity study. All the animals provided with food and water ad libitum. Gentian violet was dissolved in distilled water in concentrations of 0, 0.5,1. 0 and 2.0 mg/ml for the conventional teratology study. Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer and analytical verification done using spectrophotometry.

 Artificially inseminated rabbits were used .Timed-pregnant CD rats were given gentian violet at dose levels of 0.0, 0.5, 1.0 or 2.0 mg/kg/day in distilled water. Dams were weighed on gestational days 0, 6-19 (prior to daily dosing) and 30 (immediately prior to sacrifice), and were also observed for clinical signs of toxicity. At sacrifice on gestational day 30, dams were evaluated for body weight, liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses). Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method. Half of the fetuses were decapitated immediately after dissection and the heads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique). All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

 The maternal mortality in the present study was 22.6% (7/31 does) in the 2mg/kg bw dose group, 15.4% (4/26) in the 1mg/kg bw dose group, 7.4% (2/27) in the0. 5mg/kg bw dose group and 0.0 (0/27) in the control group. A significant trend was seen toward reduction in maternal body weight on gestational day 19 (end of dosing), and in maternal weight gain (gestational period and treatment period). For maternal weight gain, all GV-exposed groups were significantly lower than for controls for both treatment and gestation period. Clinical signs, seen in a dose-related manner, included wheezing, diarrhea, congestion, wet nose, dyspnea, lacrimation, and anorexia and cyanosis (the latter two in those does which died). All GV-exposed groups exhibited a significant increase in the number of implantation sites per litter versus controls. Percentage of resorptions per litter and number of litters with resorptions, as well as the percentage of non-live (dead plus resorbed) and affected (non- live plus malformed) per litter exhibited a dose-related upward trend, but no significant pairwise comparisons. For live litters, the number of fetuses (male and/or female) per litter did not differ among dose groups. Average fetal body weight per litter exhibited a significant downward trend with all dose group values significantly lower than controls. When separated by sex, only female fetal body weight per litter exhibited a significant downward trend and pairwise comparisons. There were no significant dose-related effects on the incidence of gross, visceral or skeletal malformations per litter, or in the number (or percent) of fetuses, males or females, malformed per litter nor in the number or percent of litters with malformed fetuses. Examination of malformation incidence by category indicated no malformations unique to or with a higher incidence in any of the GV-exposed groups relative to control. No evidence of reproductive and developmental toxicity of gentian violet was seen when administered by gavage to pregnant NZW rabbits during organogenesis at doses which produced evidence of maternal and fetal mortality and toxicity. Hence NOAEL for maternal toxicity and fetal toxicity was considered to be 2mg/kg bw /day i.e highest tested dose. WhenNew Zealand white rabbits were treated with Gentian violet (548-62-9) orally.

Also In another experimental study given by National Center for Toxicological Research,( NTP: TER82079 (1981)) The reproductive and developmental toxicity study of Gentian violet (548-62-9) was performed in female Charles River CD rat. Total 125 animals used for study in which 32 female animals in control group. The doses were selected base upon preliminary toxicity study. All the animals provided with food and water ad libitum. Gentian violet was dissolved in distilled water in concentrations of 0, 0.5,1. 0 and 2.0 mg/ml for the conventional teratology study. Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer and analytical verification done using spectrophotometry.

 Timed-pregnant CD rats were given gentian violet at dose levels of 0.0, 2.5, 5.0 or 10.0 mg/kg/day in distilled water by gavage on gestational days 6 through 15.For proof ofpregnancyexamined vaginal smear for the presence of sperm and it considered as GD 0.Dams were weighed on gestational days 0, 6-15 (prior to daily dosing) and 20 (immediately following sacrifice), and were also observed for clinical signs of toxicity. At sacrifice on gestational day 20, dams were evaluated for body weight, liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses). Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method. Half of the fetuses were decapitated prior to dissection and the beads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique). All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

 The maternal mortality rate in the present study was 9.4% (3/32dams) in the 10 mg/kg bw dose group; all other dams from all dose groups survived to terminal sacrifice on gestational day (gd) 20. A significant trend for reduced maternal body weight was found on gd 11 and 15 with the value for the 10 mg/kg bw dose group significantly different from controls on gd 11. A significant trend toward reductions in maternal weight gain for the gestation period, treatment period and absolute weight gain (weight gain during gestation minus gravid uterine weight) were found with the values for these three parameters in the 10mg/kg bw dose group significantly below those of control. Weight gain during the treatment period was also significantly reduced in the 10mg/kg bw dose group versus controls.

 Clinical signs of toxicity presented a dose-response pattern, and included the weight loss of more than 5 grams in 24 hours ,wheezing, lethargy, weakness, diarrhea, lacrimation and rough coat. There were no dose-related differences observed in the reproductive measures as number of implantation sites per litter; number or percent of resorptions, fetal deaths or non-live (dead plus resorptions) per litter. Among live litters, the number of live fetuses per litter, number or percent males per litter, average fetal body weight, average male or female fetal body weight per litter were also unaffected by treatment. There was a significant trend toward an increased number and percent affected (non-live plus malformed) per litter with dose; the number of litters with affected fetuses was significantly elevated in the 10mg/kg bw dose group versus controls.

 

 There were no gross malformations observed in any dose group in the study. Major visceral malformations observed included hydronephrosis (left or right) and hydroureter (unilaterally or bilaterally) with some fetuses exhibiting both malformations. Skeletal defects observed consisted mainly of short rib involving the thirteenth rib uni- or bilaterally. When incidences of all malformations were analyzed, there was a significant trend across dose groups toward increased number and percentage of fetuses, males, and females malformed per litter. In pairwise comparisons, the number and percentage of fetuses malformed per litter was significantly elevated in the 10mg/kg bw dose group versus controls as was the number of litters with malformed fetuses.

 Gentian violet caused an increase in hydroureter, hydronephrosis and short ribs, the incidence being significant only in the 10mg/kg bw dose group. These malformations were accompanied by signs of maternal toxicity i.e. reduction in maternal weight on gd 11, maternal weight gain during gestation and treatment periods, absolute weight gain and clinical signs. There was no significant incidence of malformations in the lower dose groups, in the absence of maternal toxicity (2.5mg/kg bw ) or in the presence of limited maternal toxicity (5mg/kg bw ). Hence, NOAEL for maternal toxicity was considered to be 2.5 mg/kg bw/day and for embryo or fatal toxicity was considered to be 5mg/kg bwday based on fetal effect seen in conjunction with maternal toxicity at 10mg/kg bw/day

 

 Based on the above study on ,N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9)it can be concluded that The NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested. When male and female Fischer 344 rats were treated with gentian violet (548-62-9) orally.

Effects on developmental toxicity

Description of key information

The NOAEL value of Gentian violet in parent New Zealand white rabbits were considered to be 0.5mg/kg/day and for F1 generation was 2 mg/kg/day as no maternal toxicity and Foetal malformations were observed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data from experimental study report
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
Teratogenicity of Gentian Violet (CAS No. 548-62-9) in New Zealand White Rabbits was evaluated following maternal exposure
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Name of test material (as cited in study report): Gentian violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Details on test animal & Environmental conditions

TEST ANIMALS
- Source:Dutchland Laboratory Animals, Inc. (Denver, PA)
- Age at study initiation:approximately six months 14 days.
- Weight at study initiation:Female : 3.5-4.6 kg
- Housing:stainless steel cages with mesh flooring Minimum cage dimensions were 18" x 24" x 18" and maximum cage dimensions were 24" x 24" x
20".
- Diet (e.g. ad libitum):Purina Certified Rabbit Chow ad libitum
- Water (e.g. ad libitum):deionized/filtered water ad libitum
- Acclimation period:14days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):16.66-22.22°C
- Humidity (%):57.13 ± 2.86%
- Air changes (per hr):No data avaliable
- Photoperiod (hrs dark / hrs light):12 hr

IN-LIFE DATES: From: To:


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Gentian violet was dissolved in distilled water in concentrations of 0, 0.5, 1.0 and 2.0 mg/mlmg/ml for the conventional teratology study.
Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer
Analytical verification of doses or concentrations:
yes
Remarks:
by spectrophotometry
Details on analytical verification of doses or concentrations:
yes,by spectrophotometry
Details on mating procedure:
Artificially inseminated rabbits
Duration of treatment / exposure:
13 days i.e. Gestational days 6 through 19
Frequency of treatment:
Daily
Duration of test:
30 days
Remarks:
0.0, 0.5, 1.0 or 2.0 mg/kg/day
No. of animals per sex per dose:
Total:133
0.0mg/kg : 30
0.5mg/kg: 32
1.0mg/kg: 31
2.0mg/kg: 40
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the pilot study
Maternal examinations:
CAGE SIDE OBSERVATIONS:No data available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on gestational days 0, 6-19

BODY WEIGHT: Yes
- Time schedule for examinations:Dams were weighed on gestational days 0, 6-19 (prior to daily dosing) and 30 (immediatelyprior to sacrifice)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # :sacrifice on gestational day 30,
- Organs examined: liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses).

OTHER:
Ovaries and uterine content:
No data available The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes:Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.

- Soft tissue examinations: Yes: All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method.

- Skeletal examinations: Yes: All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

- Head examinations: Yes: half per litter :Half of the fetuses were decapitated prior to dissection and theheads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique).
Statistics:
ANOVA was performed on selected response variables 30 employing General Linear Hodel (GLH) procedures for the Statistical Analysis System (SAS) Library .Prior to GLM analysis, the arcsine-square root transformation was performed on all maternal or litter-derived percentage data (Snedecor and Cochran, 1967), and Bartlett's test for homogeneity of variance (alpha level =0.001) (Winer, 1962) was performed on all data to be analyzed by GLM.The statistical tests that were performed on each parameter are described in Appendix II (protocol), Amendment II, Table 2 and Amendment1. Two dependent variables (i.e. average fetal body weight per litter, and percent malformed fetuses per litter), were analyzed in a three-way(dose x replicate x sex) ANOVA design with sex as a repeated measure within each litter. When either of these variables showed a significant (p<0.05) interaction between sex and dose as tested against the general error term, the data for that variable were examined separately for eachsex in a two-way (dose x replicate) design. When either variable showed a nonsignificant (p>0.05) dose x sex interaction.
Indices:
No data available
Historical control data:
Yes. -- Historical data on 1142 New Zealand White rabbit control matings have been summarized across 75 teratology studies from pharmaceutical companies or biological research organizations by the Middle Atlantic Reproduction and Teratology Association
(Woo and Hoar, 1982)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs were taken daily during the dosing period (gd 6 through 19), and at necropsy (gd 30). The following symptoms were seen in does in a dose-related manner: wheezing, diarrhea, congestion, wet nose, dyspnea, lacrimation, and anorexia and cyanosis (the latter two in those which died).

Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
Maternal mortality followed a clear dose-response trend with seven deaths (out of 31 retained in the study, 22.6%) in the 2.0 mg/kg/day dose group, four deaths (out of 26 retained in the study, 15.4%) in the 1.0 mg/k/day dose group and two deaths (out of 27 retained in the study, 7.4%) in the 0.5 mg/kg/day dose group. No deaths occurred in the control group (out of 27 does retained in the study).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A number of weight related parameters also indicated maternal toxicity. There was a significant downward trend for maternal body weight on gd 19 (at the end of the dosing period), and maternal weight gain (gestation period and treatment period).Significant pairwise comparisons were present only for maternal weight gain, where all gentian violet-exposed groups were significantly lower than controls for this parameter for the gestation period and treatment period.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment- related effects on gravid uterine weight, liver weight or relative liver weight.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality
Clinical signs: in a dose-related manner, included wheezing, diarrhea, congestion, wet nose, dyspnea, lacrimation, and anorexia and cyanosis (the latter two in those does which died).

Mortality: The maternal mortality in the present study was 22.6% (7/31 does) in the 2 mg/kg bw dose group, 15.4% (4/26) in the 1 mg/kg bw dose group, 7.4% (2/27) in the 0.5 mg/kg bw dose group and 0.0 (0/27) in the control group.

Dermal irritation: No data

Body weight and weight gain: There was a significant downward trend for maternal body weight on gd 19

Food consumption and compound intake: No data

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis: No data available

Neurobehaviour: No data

Organ weights: There were no treatment- related effects on gravid uterine weight, liver weight or relative liver weight.

Gross pathology: No data available
Histopathology: No data available
Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment related effects on the number or percent of dead conceptuses per litter. There was a significant upward trend in the percentage of non-live (dead plus resorbed) and affected (non-live plus malformed) conceptuses per liter but no significant pairwise comparisons
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Examination of reproductive parameters indicated a significant increase in the number of implantation sites per litter in all compound-exposed groups versus controls. Percent resorptions per litter and number of litters with resorptions exhibited a dose-related upward trend but no significant pairwise comparisons
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Percent resorptions per litter and number of litters with resorptions exhibited a dose-related upward trend but no significant pairwise comparisons
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
For litters with one or more live fetuses (i.e “live litters") the number of fetuses (male and/or female) did not differ among dose groups.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Dose descriptor:
NOEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
maternal abnormalities
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Average fetal body weight per litter exhibited a significant dose-related downward trend with values from all gentian violet exposed groups significantly lower than from controls. When separated by sex, only the female foetuses exhibited the significant pairwise comparisons
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Average fetal body weight per litter exhibited a significant dose-related downward trend with values from all gentian violet exposed groups significantly lower than from controls. When separated by sex, only the female foetuses exhibited the significant pairwise comparisons
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
For litters with one or more live fetuses (i.e “live litters") the number of fetuses (male and/or female) did not differ among dose groups
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
There were no significant dose-related effects on incidence of gross malformations per litter, nor in the number (or percent) of males, females or foetuses malformed per litter, nor in the number or percent of litters with malformed fetuses
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no significant dose-related effects on incidence of skeletal malformations per litter, nor in the number (or percent) of males, females or foetuses malformed per litter, nor in the number or percent of litters with malformed fetuses
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no significant dose-related effects on incidence of visceral malformations per litter, nor in the number (or percent) of males, females or foetuses malformed per litter, nor in the number or percent of litters with malformed fetuses
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Fetal body weight changes: When separated by sex, only female fetal body weight per litter exhibited a significant downward trend and pairwise comparisons. When separated by sex, only female fetal body weight per litter exhibited a significant downward trend and pairwise comparisons.
Reduction in no of live offspring: The number of fetuses (male and/or female) per litter did not differ among dose groups
Changes in sex ratio: no effects observed
Fetal/pup body weight changes: Average fetal body weight per litter exhibited a significant downward trend with all dose group values significantly lower than controls.
Changes in litter size and weight: no effects observed
Changes in postnatal survival: no effects observed
External malformation: no effects observed
Skeletal malformation: no effects observed
Visceral malformation: no effects observed
Other effect: No data available

Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Developmental effects observed:
no
Lowest effective dose / conc.:
2 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified


Assignment of CD Rats in Replicate and Dose Groups in the Teratological Evaluation of Gentian Violet

 

Gentian Violet (mg/kg/day, po)

Replicate

0.0

2.5

5

10

I

14

13

14

14

II

18

17

17

18

Total Treated

32

30

31

32

Summary of Analysis of Teratological Defects Observed in CD Rat Fetuses Following Maternal Exposure to Various Dose Levels of Gentian Violet by Oral Gavage on Gestational Days 6 Through 15

 

Gentian Violet (mg/kg/day, po)

 

0

2.5

5

10

 fetuses examineda

264

299

264

235

 litter examinedb

25

26

24

22

Gross malformations per litterc

0

0

0

0

Visceral malformations

 

 

 

0

Foetusesc

0

2

1

5f

Litterd

0

1

1

4

Skeletal malformations

 

 

 

 

Foetusesc

1

2

5

5

Litterd

1

2

3

5

Fetuses malformed per litterc

0.04

±0.04

0.15

±0.09

0.25

±0.14

0.45**

±0.14

Percent fetuses malformed per litterc

0.31

±0.31

1.50

±0.86

1.91

±1.02

6.70**

±2.70

No. d fetuses malformed

1

4

6

10

Percent fetuses malformedd

0.4

1.3

2.3

4.3

No. litters with malformationse

1

3

4

8

Percent litters with malformationse

4.0

11.5

18.7

36.4

Males malformed per litterc

0.04h

±0.04

0.08h

±0.06

0.09h

±0.06

0.29h

±0.12

Females malformed per litterc

0.0

1.41

0.98

2.56

1.53

8.30

5.00

Percent males malformed per litterc

0.52

±0.52

1.20

±0.88

1.24

±0.86

4.54

±1.95

Percent females malformed per litterC

0.0

1.41

±0.98

2.56

±1.53

8.30

±5.00

aOnly live fetuses were examined for malformations.

blncludes only litters with live feluses.

cfetuses with one or more malformations.

dLitters with one or more malformed fetuses.

eReported as mean + S.E.H. for all live litters.

fOne litter ofhead(7) was lost.

gOne fetus was lost during staining process.

hOne litter had no male fetuses:

iOne litter had no female fetuses.

jTwolitter. had no female fetuse

§p<0.05 Jonckheere's Test.

§§p<0.01Jonckheere's Test.

*p<0.05 Kruskal-Wallis.

**p<0.01 Mann-Whitney U.

***p<0.05 Fisher's Exact Test.

Specific Teratological Defects Observed Following Maternal Exposure to Various Dose Levels of Gentian Violet by Oral Gavage on Gestational Days 6 Through 15

 

Gentian Violet (mg/kg/day, po)

 

00

2.5

5.0

10

Total fetuses examinedb

264

299

264

235

Total litters examinedc

25

26

24

22

GROSS MALFORMATIONS f

0

0

0

0

VISCERAL MALFORMATIONSg

 

 

 

 

Hydronephrosis: left

 

1

 

 

right

 

 

 

2

Hydroureter: bilateral

 

 

 

1

right

 

 

 

2

left

 

2

1

1

Extra liver lobe

 

 

 

1

SKELETAL Malformations

 

 

 

 

Lumbar centra misaligned

 

 

1

 

Thnracic centra off center

 

1

1

 

Short rib

1

1

3

5

Missing ribf

 

 

1

 

VARIATIONSf,g,h

 

 

 

 

Hematoma: jaw

 

1

 

 

neck

1

 

 

 

lower limb

1

 

 

 

back

 

1

 

 

No. innominate

 

1

1

1

wavy rib

3

 

 

 

Misaligned sternebrae

2

2

2

3

Extra ossification sites -

 

6

 

 

spinal cord and tail

 

 

 

 

Doubled centra

 

5

3

1

Clubbed limb w/o bone change

 

 

1

 

Bilateral papilla 1/4 normalsize or less

1

 

 

1

Left papilla 1/4 normalsize or less

 

 

 

1

Right papilla 1/4 normalsize or less

2

 

1

1

Very soft tissue - kidney

 

1

 

 

Incomplete ossification

 

1

1

 

Distended ureter(s)

21

18

8

15

aA single fetus may have more than one malformation or variation,

bAll live fetuses were examined for gross malformations.

cAll live fetuses were examined for visceral malformations of the trunk. Fifty percent of the heads were examined for soft tissue malformations.

dFifty percent decapitated carcasses and50%fetal preparations with heads intact were examined for skeletal malformations.

eThirteenth rib was two-thirds or less normal length, unilaterally or bilaterally.

fone fetus had both twelfth and thirteenth ribs missing.

 

Reproductive Parameters after Exposure of Pregnant CD Rats to Various Dose Levels of Gentian Violet by Oral Gavage on Gestational Days 6 Through 15

 

 

Gentian Violet (mg/kg/day, po)

 

0.0

2.5

5.0

10

ALL LITTERSa

 

 

 

 

(Pregnant Dams)

25

26

24

25

Implantation sites per 1itterb

11.48

±0.70

12.35

±0.53

11.50

±0.86

11.52

±0.77

Resorptions per litterb

0.92

±0.38

0.85

±0.51

0.50

±0.17

2.12

±0.93

Percent Resorptions per litterb

9.3

±3.8

5.6

±3.1

5.4

±2.1

17.5

±6.6

No. Litters with resorptions

8

7

8

11

Percent litters with resorptions

32.0

26.9

33.3

44.0f

Dead per litterb

0

0

0

0

Percent dead per litterb

0

0

0

0

No. Litters with dead

0

0

0

0

Percent Litters with dead

0

0

0

0

0Non-live per litterb,c

0.92

±0.38

0.85

±0.51

0.50

±0.17

 

2.12

±0.93

Percent non-live per litterb, c

9.3

±3.8

5.8

±3.1

 

5.4

±2.1

17.5

±6.6

No. litters with non-live

8

7

8

11

Percent litters with non-live

32.0

26.9

33.3

44.0

Affected per 1itterb ,d

0.96

±0.39

1.00

±0.51

0.75

±0.21

 

2.52

±0.91

Percent affected per litterb

9.5

±3.8

7.2

±3.2

7.2

±2.3

 

22.3

±6.7

No. litters with affected

8

9

11

17

Percent litters with affected

32.0

34.6

45.8

68.0

LIVE LITTERSe

 

 

 

 

(No. litters with live fetuses)

25

26

24

22

Live fetuses per litterb

10.56

±0.85

11.50

±0.59

11.00

±0.89

10.68

±0.82

Males per litterb

5.56

±0.57

6.04

±0.53

5.25

±0.49

 

5.18

±0.52

Percent males per litterb

53.1

±4.2

52.0

±3.9

 

50.2

±4.5

48.2

±3.9

Average fetal body weight (g) per litter

3.686

±0.077

3.642

±0.059

3.486

±0.072

3.606

±0.090

Average male fetal body weight (g) per litter

3.741

±0.078

3.688

±0.067

3.587

±0.080

3.767

±0.061

Average female fetal body weight (g) per litter

3.560

±0.069

3.553

±0.073

3.422

±0.073

3.510

±0.088

 

aincludes all dams pregnant at sacrifice; litter size = no. implantation sites per dam.

bReported as mean! S.E.M.

cNon-live =dead plus resorbed.

dAffected =non-live plus malformed.

eincludes only dams with live fetuses; litter size =no. live fetuses per dam.

fTwo dams had all resorptions.

gOne litter had no males.

hOne litter had no females.

iOne litter had no females.

ip,0.05 Jonckhecre'. Test.

'p<0.05Fisher exact test

Maternal Parameters after Exposure to Pregnant CD Rats to Various Dose Levels of Gentian Violet by Oral Gavage on Gestational Days 6 Through 15

 

 

Gentian Violet (mg/kg/day, po)

 

0.0

2.5

5

10

SUBJECTS (Dams)

 

 

 

 

Total treated

32

30

31

32

Removed

0

0

0

0

Deaths

0

0

0

0

Non-pregnant at sacrifice

7

4

7

4

Pregnant (%) at sacrifice

25 (78.1)

26(86.7)

24(77.4)

25(86.2)

MATERNAL BODY WEIGHT(g)

 

 

 

 

Gestational day 0a

228.55

±3.11

232.87

±3.51

227.90

±2.97

234.17

±4.14

Gestational day 6a

250.20

±3.51

 

251.83

±3.96

248.47

±3.10

255.58

±3.73

Gestational day 11

272.25

±3.52

272.08

±3.86

262.70

±3.81

250.72

±5.69

Gestational day 15

290.12

±4.26

288.99

±3.97

277.00

±4.39

268.39

±6.93

Gestational day 20

345.00

±6.03

347.83

±6.52

334.89

±6.53

319.01

±9.21

Maternal Weight Gain (g)

 

 

 

 

Gestation period

116.45

±4.38

114.96

±5.85

106.98

±5.34

84.84

±9.35

Treatment period

39.92

±1.36

37.15

±2.16

27.29

±3.27

12.81

±2.13

Absolute weight gain

54.08

±3.36

47.74

±4.38

46.01

±3.30

30.09

±6.13

Gravid Uterine Weight (g)a

62.37

±4.38

67.22

±3.23

60.98

±4.64

54.75

±5.49

Maternal Liver Weight (g)a

14.15

±0.26

14.40

±0.29

13.74

±0.35

13.46

±0.47

Relative Maternal Liver Weight

4.10

±0.08

4.10

±0.09

4.11

±0.07

4.18

±0.08


aincludes alldams pregnant at sacrifice;

bweight gain during gestation minus gravid uterine weight.

CThree dams hadall resorptions.

dOne liver weight had not recorded.

eOne liver weight was incorrectly recorded.

ip<0.05Jonckheeres̔ Test.

iip<0.01Jonckheeres̔ Test.

iiip0.001Jonckheeres̔ Test.

tp<0.05 Kruskal-wallis.

ttp<0.01 Kruskal-Wallis.

tttp<0.001 Kruskal-walli&

 p<0.01Mann-whintlleyU

 p<0.01Mann-whintlleyU.

 

 

 

 

 

 

Conclusions:
NOAEL for maternal toxicity and fetal toxicity was considered to be 2mg/kg bw /day i.e highest tested dose. When New Zealand white rabbits were treated with Gentian violet (548-62-9) orally.
Executive summary:

The teratogenicity study of Gentian violet (548-62-9) was performed in femaleNew Zealand white rabbits. Total 133 animals used for study in which 30 female animals in control group. The doses were selected base upon preliminary toxicity study. All the animals provided with food and water adlibitum.Gentian violet was dissolved in distilled water in concentrations of 0, 0.5,1. 0 and 2.0 mg/ml for the conventional teratology study. Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer and analytical verification done using spectrophotometry.

 

Artificially inseminated rabbits were used .Timed-pregnant CD rats were given gentian violet at dose levels of 0.0, 0.5, 1.0 or 2.0 mg/kg/day in distilled water. Dams were weighed on gestational days 0, 6-19 (prior to daily dosing) and 30 (immediately prior to sacrifice), and were also observed for clinical signs of toxicity. At sacrifice on gestational day 30, dams were evaluated for body weight, liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses). Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method. Half of the fetuses were decapitated immediately after dissection and the heads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique). All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

 

The maternal mortality in the present study was 22.6% (7/31 does) in the 2mg/kg bw dose group, 15.4% (4/26) in the 1mg/kg bw dose group, 7.4% (2/27) in the0. 5mg/kg bw dose group and 0.0 (0/27) in the control group. A significant trend was seen toward reduction in maternal body weight on gestational day 19 (end of dosing), and in maternal weight gain (gestational period and treatment period). For maternal weight gain, all GV-exposed groups were significantly lower than for controls for both treatment and gestation period. Clinical signs, seen in a dose-related manner, included wheezing, diarrhea, congestion, wet nose, dyspnea, lacrimation, and anorexia and cyanosis (the latter two in those does which died). All GV-exposed groups exhibited a significant increase in the number of implantation sites per litter versus controls. Percentage of resorptions per litter and number of litters with resorptions, as well as the percentage of non-live (dead plus resorbed) and affected (non- live plus malformed) per litter exhibited a dose-related upward trend, but no significant pairwise comparisons. For live litters, the number of fetuses (male and/or female) per litter did not differ among dose groups. Average fetal body weight per litter exhibited a significant downward trend with all dose group values significantly lower than controls. When separated by sex, only female fetal body weight per litter exhibited a significant downward trend and pairwise comparisons.

There were no significant dose-related effects on the incidence of gross, visceral or skeletal malformations per litter, nor in the number (or percent) of fetuses, males or females, malformed per litter nor in the number or percent of litters with malformed fetuses. Examination of malformation incidence by category indicated no malformations unique to or with a higher incidence in any of the GV-exposed groups relative to control. No evidence of teratogenicity of gentian violet was seen when administered by gavage to pregnant NZW rabbits during organogenesis at doses which produced evidence of maternal and fetal mortality and toxicity. Hence NOAEL for maternal toxicity and fetal toxicity was considered to be 2mg/kg bw /day i.e highest tested dose. When New Zealand white rabbits were treated with Gentian violet (548-62-9) orally.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
0.5 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Data is K4 and from secondary source
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

Developmental toxicity

In different studies,N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9)has been investigated for developmental toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments i.e. most commonly in rats and rabbits for Gentian violet (548-62-9).

In experimental study given by NTP (National Center for Toxicological Research, TER82080 (study conducted in 1981-82).The teratogenicity study of Gentian violet (548-62-9) was performed in female New Zealand white rabbits. Total 133 animals used for study in which 30 female animals in control group. The doses were selected base upon preliminary toxicity study. All the animals provided with food and water ad libitum. Gentian violet was dissolved in distilled water in concentrations of 0, 0.5,1. 0 and 2.0 mg/ml for the conventional teratology study. Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer and analytical verification done using spectrophotometry.

Artificially inseminated rabbits were used .Timed-pregnant CD rats were given gentian violet at dose levels of 0.0, 0.5, 1.0 or 2.0 mg/kg/day in distilled water. Dams were weighed on gestational days 0, 6-19 (prior to daily dosing) and 30 (immediately prior to sacrifice), and were also observed for clinical signs of toxicity. At sacrifice on gestational day 30, dams were evaluated for body weight, liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses). Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method. Half of the fetuses were decapitated immediately after dissection and the heads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique). All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

  The maternal mortality in the present study was 22.6% (7/31 does) in the 2mg/kg bw dose group, 15.4% (4/26) in the 1mg/kg bw dose group, 7.4% (2/27) in the0. 5mg/kg bw dose group and 0.0 (0/27) in the control group. A significant trend was seen toward reduction in maternal body weight on gestational day 19 (end of dosing), and in maternal weight gain (gestational period and treatment period). For maternal weight gain, all GV-exposed groups were significantly lower than for controls for both treatment and gestation period. Clinical signs, seen in a dose-related manner, included wheezing, diarrhea, congestion, wet nose, dyspnea, lacrimation, and anorexia and cyanosis (the latter two in those does which died). All GV-exposed groups exhibited a significant increase in the number of implantation sites per litter versus controls. Percentage of resorptions per litter and number of litters with resorptions, as well as the percentage of non-live (dead plus resorbed) and affected (non- live plus malformed) per litter exhibited a dose-related upward trend, but no significant pairwise comparisons. For live litters, the number of fetuses (male and/or female) per litter did not differ among dose groups. Average fetal body weight per litter exhibited a significant downward trend with all dose group values significantly lower than controls. When separated by sex, only female fetal body weight per litter exhibited a significant downward trend and pairwise comparisons.

There were no significant dose-related effects on the incidence of gross, visceral or skeletal malformations per litter, nor in the number (or percent) of fetuses, males or females, malformed per litter nor in the number or percent of litters with malformed fetuses. Examination of malformation incidence by category indicated no malformations unique to or with a higher incidence in any of the GV-exposed groups relative to control. No evidence of teratogenicity of gentian violet was seen when administered by gavage to pregnant NZW rabbits during organogenesis at doses which produced evidence of maternal and fetal mortality and toxicity. Hence NOAEL for maternal toxicity and fetal toxicity was considered to be 2mg/kg bw /day i.e highest tested dose. When New Zealand white rabbits were treated with Gentian violet (548-62-9) orally.

 In another experimental study given by National Centre for Toxicological Research,( NTP: TER82079 (1981)) The teratogenicity study of Gentian violet (548-62-9) was performed in female Charles River CD rat. Total 125 animals used for study in which 32 female animals in control group. The doses were selected base upon preliminary toxicity study. All the animals provided with food and water ad libitum. Gentian violet was dissolved in distilled water in concentrations of 0, 0.5,1. 0 and 2.0 mg/ml for the conventional teratology study. Dosing solutions were stored at 5°C except at the time of dosing when they were warmed to room temperature, and stirred continuously with a magnetic stirrer and analytical verification done using spectrophotometry.

 

Timed-pregnant CD rats were given gentian violet at dose levels of 0.0, 2.5, 5.0 or 10.0 mg/kg/day in distilled water by gavage on gestational days 6 through 15.For proof of pregnancy examined vaginal smear for the presence of sperm and it considered as GD 0.Dams were weighed on gestational days 0, 6-15 (prior to daily dosing) and 20 (immediately following sacrifice), and were also observed for clinical signs of toxicity. At sacrifice on gestational day 20, dams were evaluated for body weight, liver weight, gravid uterine weight and status of uterine implantation sites (i.e., implantation sites, resorptions, dead fetuses, live fetuses). Live fetuses were dissected from the uterus and evaluated for live litter size, body weights, sex ratios and gross morphological abnormalities.All live fetuses were examined for visceral malformations employing the Staples' fresh tissue dissection method. Half of the fetuses were decapitated prior to dissection and the beads were fixed in Bouin's solution for free hand sectioning and examination (Wilson's Technique). All fetal carcasses were cleared and stained with Alizarin Red S and examined for skeletal malformations.

The maternal mortality rate in the present study was 9.4% (3/32dams) in the 10 mg/kg bw dose group; all other dams from all dose groups survived to terminal sacrifice on gestational day (gd) 20. A significant trend for reduced maternal body weight was found on gd 11 and 15 with the value for the 10 mg/kg bw dose group significantly different from controls on gd 11. A significant trend toward reductions in maternal weight gain for the gestation period, treatment period and absolute weight gain (weight gain during gestation minus gravid uterine weight) were found with the values for these three parameters in the 10mg/kg bw dose group significantly below those of control. Weight gain during the treatment period was also significantly reduced in the 10mg/kg bw dose group versus controls.

  Clinical signs of toxicity presented a dose-response pattern, and included the following: weight loss of more than 5 grams in 24 hours, wheezing, lethargy, weakness, diarrhea, lacrimation and rough coat. There were no dose-related differences observed in the following reproductive measures: number of implantation sites per litter; number or percent of resorptions, fetal deaths or non-live (dead plus resorptions) per litter. Among live litters, the number of live fetuses per litter, number or percent males per litter, average fetal body weight, average male or female fetal body weight per litter were also unaffected by treatment. There was a significant trend toward an increased number and percent affected (non-live plus malformed) per litter with dose; the number of litters with affected fetuses was significantly elevated in the 10mg/kg bw dose group versus controls.

There were no gross malformations observed in any dose group in the study. Major visceral malformations observed included hydronephrosis (left or right) and hydroureter (unilaterally or bilaterally) with some fetuses exhibiting both malformations. Skeletal defects observed consisted mainly of short rib involving the thirteenth rib uni- or bilaterally. When incidences of all malformations were analyzed, there was a significant trend across dose groups toward increased number and percentage of fetuses, males, and females malformed per litter. In pairwise comparisons, the number and percentage of fetuses malformed per litter was significantly elevated in the 10mg/kg bw dose group versus controls as was the number of litters with malformed fetuses.

Gentian violetcaused an increase in hydroureter, hydronephrosis and short ribs, the incidence being significant only in the 10mg/kg bw dose group. These malformations were accompanied by signs of maternal toxicity i.e. reduction in maternal weight on gd 11, maternal weight gain during gestation and treatment periods, absolute weight gain and clinical signs. There was no significant incidence of malformations in the lower dose groups, in the absence of maternal toxicity (2.5mg/kg bw ) or in the presence of limited maternal toxicity (5mg/kg bw ). Hence, NOAEL for maternal toxicity was considered to be 2.5 mg/kg bw/day and for embryo or fatal toxicity was considered to be 5mg/kg bw day based on fetal effect seen in conjunction with maternal toxicity at 10mg/kg bw/day

Based on the above study on ,N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9)it can be concluded that NOAEL value is 30mg/kg bw in male and female Fischer 344 rats for reproductive toxicity and NOAEL value in parent New Zealand white rabbits were considered to be 0.5mg/kg/day, whereas for F1 generation was 2 mg/kg/day for developmental toxicity.

Justification for classification or non-classification

Based on the above study on ,N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9)it can be concluded that NOAEL value is 30mg/kg bw in male and female Fischer 344 rats for reproductive toxicity and NOAEL value in parent New Zealand white rabbits were considered to be 0.5mg/kg/day, whereas 2 mg/kg/day for developmental toxicity in F1 generation.