[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

review article or handbook

Title:

Teratologic Evaluation of Gentian Violet (CAS No. 548-62-9) in New Zealand White Rabbits

Author:

NTP

Year:

1982

Bibliographic source:

National Center for Toxicological Research, TER82080 (study conducted in 1981-82)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

EC Number:

208-953-6

EC Name:

[4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

Cas Number:

548-62-9

Molecular formula:

C25H30ClN3

IUPAC Name:

4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride

Test material form:

solid

Details on test material:

- 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

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

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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:

Administration / exposure

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

Doses / concentrations

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

Examinations

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)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

maternal abnormalities

mortality

Results (fetuses)

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

Effect levels (fetuses)

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

Overall developmental toxicity

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

Any other information on results incl. tables

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 examined^a	264	299	264	235
litter examined^b	25	26	24	22
Gross malformations per litter^c	0	0	0	0
Visceral malformations				0
Foetuses^c	0	2	1	5^f
Litter^d	0	1	1	4
Skeletal malformations
Foetuses^c	1	2	5	5
Litter^d	1	2	3	5
Fetuses malformed per litter^c	0.04 ±0.04	0.15 ±0.09	0.25 ±0.14	0.45** ±0.14
Percent fetuses malformed per litter^c	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 malformations^e	1	3	4	8
Percent litters with malformationse	4.0	11.5	18.7	36.4
Males malformed per litter^c	0.04^h ±0.04	0.08^h ±0.06	0.09^h ±0.06	0.29^h ±0.12
Females malformed per litter^c	0.0	1.41 0.98	2.56 1.53	8.30 5.00
Percent males malformed per litter^c	0.52 ±0.52	1.20 ±0.88	1.24 ±0.86	4.54 ±1.95
Percent females malformed per litter^C	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 examined^b	264	299	264	235
Total litters examined^c	25	26	24	22
GROSS MALFORMATIONS f	0	0	0	0
VISCERAL MALFORMATIONS^g
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 rib^f			1
VARIATIONS^f,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 LITTERS^a
(Pregnant Dams)	25	26	24	25
Implantation sites per 1itter^b	11.48 ±0.70	12.35 ±0.53	11.50 ±0.86	11.52 ±0.77
Resorptions per litter^b	0.92 ±0.38	0.85 ±0.51	0.50 ±0.17	2.12 ±0.93
Percent Resorptions per litter^b	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	44.0^f
Dead per litter^b	0	0	0	0
Percent dead per litter^b	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 litter^b, 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 litter^b	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 LITTERS^e
(No. litters with live fetuses)	25	26	24	22
Live fetuses per litter^b	10.56 ±0.85	11.50 ±0.59	11.00 ±0.89	10.68 ±0.82
Males per litter^b	5.56 ±0.57	6.04 ±0.53	5.25 ±0.49	5.18 ±0.52
Percent males per litter^b	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 0^a	228.55 ±3.11	232.87 ±3.51	227.90 ±2.97	234.17 ±4.14
Gestational day 6^a	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.

 

 

 

 

 

 

Applicant's summary and conclusion

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.