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

Endpoint summary

Description of key information

Repeated dose toxicity Oral:

Combined repeated dose & carcinogenicity study was performed to determine the mutagenic nature of gentian violet. The study was performed using male and female Fischer F344 rats. Male and female weanling animals (F0) were randomly divided into four groups under barrier conditions and administered 0 (control), 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and females: 0, 40, 100 or 200 mg/Kg bw) GV in their feed for at least 80 days. All rats had access to feed and drinking-water ad lib. While receiving dosed feed, the females were mated with males (one male/ female) of the same dose level. Brother/sister matings were avoided. Two males and two females were selected randomly from each litter (F1a generation) and allocated three animals per cage as weanlings to the chronic study. Litter mates were not assigned to the same cage. The F1a animals continued on the same dose levels as their respective parents for 12, 18 or 24 months. The animals were observed for changes in body weight, food consumption, mortality and morbundity and the presence of lesions.

 

In females, the body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups. In males, at 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk.

 

Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for the females and 25-30 g food/kg body weight for the males.

 

253 rats were found to be in the moribund stage throughout the study period. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw. The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels.

 

No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the 160 mg/Kg bw group. The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls. The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered GV in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months. Incidences of leukemia were high in all groups of female rats fed GV for 24 months and statistical analysis showed no significant differences overall or in the dose groups. In the males fed GV for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed GV for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively. No incidence of mononuclear cell leukaemia show a dose response in male rats fed GV for either 18 or 24 months. No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. Innon-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes.

 

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for Gentian violet in male and female rats is considered to be 30 and 40 mg/Kg bw respectively.

Repeated dose toxicity Inhalation:

Gentian violet has a very low vapor pressure (2.57 X 10-12 Pa), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point was considered for waiver.

Repeated dose toxicity Dermal:

Repeated dose dermal toxicity study was performed to determine the dermal toxic nature of Gentian violet. The study was performed using female Charles River CD rats. 2 mL/Kg dye formulation P-23 containing 0.002% ( 0.00942 mg/Kg) test compound was applied to the dorso-scapular area. The application was made during the gestation days 1, 4, 7, 10, 13, 16 and 19. The animals were observed for clinical signs, body weight changes, dermal irritation if any and food consumption. No dye formulation related toxicity was noted.Changes in female body weights and food consumption were similar for rats in the untreated controls and all dye-treated groups. No irritation or other changes in appearance were noted except for changes in skin and hair color at the site of topical application of the dye formulation. Based on the observations made,the No Observed Adverse Effect Level (NOAEL) for gentian violet in female Charles River CD rats is considered to be 0.00942 mg/Kg.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose & carcinogenicity

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 is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Combined repeated dose & carcinogenicity study was performed to determine the chronic toxic nature of gentian violet

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material : Gentian violet
- Molecular formula : C25H30ClN3
- Molecular weight : 407.986 g/mol
- Substance type: Organic
- Physical state: 99%
- Impurities (identity and concentrations) : 1% methyl violet

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Center for Toxicological Research
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were housed in barrier housed conditions containing hardwood chips as cage bedding in filter topped cages
- Diet (e.g. ad libitum): Feed ad libitum
- Water (e.g. ad libitum): Drinkng water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± -16.6 °C
- Humidity (%): 50 ±5 %
- Air changes (per hr): 14-16 changes of air/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark cycle

IN-LIFE DATES: From: To: No data

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Gentian violet was dissolved in ethanol and sprayed directly into the feed in a sanitized chamber at dose levels of 0, 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and Females: 0, 40, 100 or 200 mg/Kg bw). Ethanol was subsequently removed from the prepared feed during a 30 min blending process using a vacuum

DIET PREPARATION
- Rate of preparation of diet (frequency): Mixing of GV into the feed was done on a weekly basis and feed was used in less than 30 days after mixing.
- Mixing appropriate amounts with (Type of food): Purina 5010M autoclavable, Purina MIlles, Inc. Richmong IN, USA
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: Males: 0, 30, 80 or 160 mg/Kg bw
Females: 0, 40, 100 or 200 mg/Kg bw
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

80 days

Frequency of treatment:

Daily

Remarks:

Males: 0, 30, 80 or 160 mg/Kg bw
Females: 0, 40, 100 or 200 mg/Kg bw

No. of animals per sex per dose:

Total: 1140
0 mg/Kg bw: 210 males and 210 females
30 (males) and 40 (females) mg/Kg bw: 120 males and 120 females
100 (males) and 80 (females) mg/Kg bw: 120 males and 120 females
200 (males) and 160 (females) mg/Kg bw: 120 males and 120 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): F0 animals were randomly divided into dose groups
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly
- Cage side observations checked in table [No.?] were included. Mortality and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 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: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data - Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the animals were subjected to complete necropsy
HISTOPATHOLOGY: Yes

Other examinations:

No data

Statistics:

The statistical procedure
(CHRONIC) used in the analysis of the data are described by Kodell et al. This procedure is a
computer program for statistical analysis of carcinogenesis data that was developed into a Statistical Analysis System Procedure. This program follows a unified approach for the estimation and testing of the
time to onset, prevalence and mortality distribution functions. The onset and mortality functions represent "net" rather than 'crude" probabilities in that they are adjusted for mortality from causes of death other than the tumour of interest. The prevalence function represents a probability filrther adjusted tbr mortality caused by the tumour. Specifically, the mortality
function characterizes the mortality rate due to a tumour, the prevalence function characterizes the incidental (non-fatal) tumour rate and the time-to-onset function characterizes the distribution of time to histological appearance of the tumour (disease of interest). CHRONIC performs age-adjusted comparisons of tumour rates between each dose group and the controls and also provides an overall test for a dose response as described by Peto et al. In reporting statistical significance, the Bonferroni correction was applied to the nominal 0.05 significance level to adjust the false positive error rate for multiple comparisons with the controls, in this case
requiring a P-value of less than 0.053 – 0.0167 for statistical significance with a false positive rate less than 0.05.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Other effects:

not specified

Details on results:

Clinical signs and mortality:
Clinical signs: 253 rats were found to be in the moribund stage throughout the study period.

Mortality: At the end of 24 months, mortality was approximately 33% for both males and females in the control group and approximately 66% for females and 48% for males in the highest 200 or 160 mg/Kg bw dosed group for males and females respectively. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw.

The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels.

Body weight and weight gain:
Female: The body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups.

Males: At 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk.

Food consumption and compound intake: Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for
the females and 25-30 g food/kg body weight for the males.

Based on the consumption rates, the dosage of GV remained relatively constant following the rapid growth stage in the first 20 wk.

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology : No data

Clinical chemistry: No data

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No data

Gross pathology: No data

Histopathology: No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the
160 mg/Kg bw group.

The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls.

The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered GV in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months.

Incidences of leukemia were high in all groups of female rats fed GV for 24 months and statistical analysis showed no significant differences overall or in the dose groups.

In the males fed GV for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed GV for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively.

No incidence of mononuclear cell leukaemia show a dose response in male rats fed GV for either 18 or 24 months.

No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. In non-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes.

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Changes noted in mortality, body weight, Gross pathology effects

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Changes noted in mortality, body weight, Gross pathology effects

Critical effects observed:

not specified

Table: Incidence of neoplastic lesions in male Fischer F344 rats fed GV in the diet for 18 and 24 months

Site and type of neoplastic lesion	Incidence according to dose (ppm)*
	18 months				24 months
	0	100	300	600	0	100	300	600
Liver
Hepatocellular adenoma	0/15 (0)	1/15 (7)	0/15 (0)	0/14 (0)	1/179 (0/5)	1/90 (1)	3/88 (3)	4/89 (4)
Testes
Malignant mesothalioma	0/15 (0)	0/15 (0)	1/15 (7)	1/15 (7)	0/177 (0)	0/90 (0)	0/87 (0)	1/90 (1)
Thyroid
Follicular cell adenocarcinoma	0/15 (0)	0/15 (0)	0/14 (0)	0/13 (0)	1/163 (1)	4/84 (5)	2/74 (3)	5/79 (6)
Follicular cell adenoma	0/15 (0)	0/15 (0)	1/15 (7)	1/15 (7)	1/163 (1)	0/84 (0)	0/74 (0)	2/79 (3)
Follicular cell adenoma and adenocarcinoma	0/15 (0)	0/15 (0)	1/15 (7)	1/15 (7)	2/163 (1)	4/84 (5)	2/74 (3)	2/78 (9)
Multiple organs
Mononuclear cell leukemia	6/15 (40)	1/15 (7)	3/15 (20)	4/15 (27)	104/180 (58)	66/90 (73)	69/90 (77)	51/90 (57)

*Significant trend at 0.05 level for overall; 0.05.3 for control v. dose comparison (Bonferroni corrected). Significant trend at 0.01 level for overall: 0.001/3 for control dose comparison (Bonferrom corrected). Significant trend at 0.001 level for overall: 0.001/3 for control dose comparison (Bonferroni corrected).

This significance value arises from the small number of tumours; the result was determined using Fisher's exacl test

Table: Incidence of neoplastic lesions in female Fischer F344 rats fed GV in the diet for 18 and 24 months

 

Site and type of neoplastic lesion	Incidence according to dose (ppm)
	18 months				24 months
	0	100	300	600	0	100	300	600
Liver
Hepatocellular adenoma	0/15 (0)	0/11 (0)	0/10 (0)	0/14 (0)	1/170 (0)	1/90 (1)	2/84 (2)	1/87 (1)
Heart
Mononuclear cell leukemia	0/15 (0)	0/11 (0)	0/10 (0)	2/14 (14)	27/169 (16)	16/90 (18)	19/83 (23)	22/87 (25)
Thyroid
Follicular cell adenocarcinoma	0/15 (0)	0/11(9)	0/10 (0)	0/14 (0)	1/159 (1)	1/83 (1)	4/76 (5)	6/77 (8)
Follicular cell adenoma	0/15 (0)	0/11 (0)	0/10 (0)	0/14 (0)	1/159 (1)	2/83 (2)	3/76 (4)	3/77 (4)
Follicular cell adenoma and adenocarcinoma	0/15 (0)	1/11 (9)	0/10 (0)	0/14 (0)	2/159 (1)	3/83 (4)	7/76 (9)	9/77 (12)
Multiple organs
Mononuclear cell leukemia	0/15 (0)	2/11(18)	2/10 (20)	6/14 (43)	77/171 (45)	38/90 (42)	45/87 (52)	40/87 (46)

 

*Significant trend at 0.05 level for overall; 0.05.3 for control v. dose comparison (Bonferroni corrected). Significant trend at 0.01 level for overall: 0.001/3 for control dose comparison (Bonferrom corrected). Significant trend at 0.001 level for overall: 0.001/3 for control dose comparison (Bonferroni corrected).

This significance value arises from the small number of tumours; the result was determined using Fisher's exacl test

 

Table. Incidence of non-neoplastic lesions in Fischer 344 rats fed GV in the diet for 24 months

Site and type of non- neoplastic lesion	Incidence according to dose (ppm)
	24 months
	0	100	300	600
Liver
Clear cell foci	6/179 (3)	5/60 (6)	5/88 (6)	8/89 (9)
Eosinophilic foci	7/179 (4)	5/90 (6)	20/88 (23)	33/89 (37)
Mixed cell foci	32/179 (18)	26/90 (29)	28/88 (24)	47/89 (53)
Regeneration	7/179 (4)	11/90 (12)	21/88 (24)	15/89 (17)
Centrilobular necrosis	5/179 (3)	4/90 (4)	8/88 (9)	11/89 (12)
Thyroid gland
Follicular cysts	18/163 (11)	7/84 (8)	9/74 (12)	17/79 (22)
Spleen
Red pulp hyperplasia	11/175 (6)	7/88 (8)	9/74 (12)	17/79 (2)
Lymph node
Mesenteric hyperplasia	8/168 (5)	9/86 (10)	5/84 (6)	11/81 (14)
Females
Liver
Clear cell foci	1/170 (1)	1/90 (1)	3/84 (4)	1/87 (1)
Eosinophilic foci	0/170 (0)	0/90 (0)	6/84 (7)	10/87 (11)
Mixed cell foci	29/170 (17)	23/90 (36)	39/84 (46)	30/87 (34)
Regeneration	4/170 (2)	9/90 (10)	20/84 (24)	18/87 (21)
Centrilobular necrosis	7/170 (4)	8/90 (9)	6/84 (7)	20/87 (23)
Thyroid gland
Follicular cysts	8/159 (5)	9.83 (11)	8/76 (11)	7/77 (9)

*Incidence is expressed as the no. animals with the specified non-neoplastic lesion/no. animals at risk. Values in parentheses represent the incidence of the non-neoplastic lesion expressed as percentage of the no. animals surviving

 

Table: Incidence of non-neoplastic lesions expressed as levels of significance [P-values] in Fischer 344 rats fed GV in the diet for 24 months

Lesion	Significance level (p-values)*
	Overall	100 ppm dose level	300 ppm dose level	600 ppm dose level
Males
Liver
Clear cell foci	0.00023	0.189	0.074	0.00044
Eosinophilic foci	0.00005	0.136	0.00005	0.00005
Mixed cell foci	0.00005	0.0061	0.00005	0.00005
Regeneration	0.0001	0.004	0.00005	0.00016
Centrilobular necrosis	0.165	0.0335	0.001	0.183
Thyroid gland
Follicular cysts	0.003	0.582	0.207	0.007
Spleen
Red pulp hyperplasia	0.0004	0.276	0.556	0.0006
Lymph node
Mesenteric hyperplasia	00.005	0.037	0.277	0.001
Females
Liver
Clear cell foci	0.128	0.266	0.005	0.149
Eosinophilic foci	0.00005		0.00005	0.00005
Mixed cell foci	0.00009		0.080	0.0009
Regeneration	0.00005	0.0001	0.00005	0.00005
Centrilobular necrosis	0.00005	0.003	0.00005	0.00005
Thyroid gland
Follicular cysts	0.00005	0.070	0.1448	0.00005

*Significant trend at 0.05 level for overall: 005/3 for control dose comparison (Bonferroni corrected)

Significant trend at 0 01 level for overall: 0.01/3 for control dose comparison (Bonferroni corrected)

Significant trend at 0 001 level lot overall: 0001/3 for control dose comparison (Bonferroni corrected]

 

Conclusions:

The No Observed Adverse Effect level (NOAEL) for Gentian violet in male and female rats is considered to be 40 and 30 mg/Kg bw respectively.

Executive summary:

Combined repeated dose & carcinogenicity study was performed to determine the mutagenic nature of gentian violet. The study was performed using male and female Fischer F344 rats. Male and female weanling animals (F0) were randomly divided into four groups under barrier conditions and administered 0 (control), 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and females: 0, 40, 100 or 200 mg/Kg bw) GV in their feed for at least 80 days. All rats had access to feed and drinking-water ad lib. While receiving dosed feed, the females were mated with males (one male/ female) of the same dose level. Brother/sister matings were avoided. Two males and two females were selected randomly from each litter (F1a generation) and allocated three animals per cage as weanlings to the chronic study. Litter mates were not assigned to the same cage. The F1a animals continued on the same dose levels as their respective parents for 12, 18 or 24 months. The animals were observed for changes in body weight, food consumption, mortality and morbundity and the presence of lesions.

 

In females, the body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups. In males, at 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk.

 

Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for the females and 25-30 g food/kg body weight for the males.

 

253 rats were found to be in the moribund stage throughout the study period. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw. The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels.

 

No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the 160 mg/Kg bw group. The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls. The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered GV in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months. Incidences of leukemia were high in all groups of female rats fed GV for 24 months and statistical analysis showed no significant differences overall or in the dose groups. In the males fed GV for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed GV for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively. No incidence of mononuclear cell leukaemia show a dose response in male rats fed GV for either 18 or 24 months. No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. Innon-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes.

 

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for Gentian violet in male and female rats is considered to be 30 and 40 mg/Kg bw respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is from K2 peer reviewed publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because the substance is unreactive, insoluble and not inhalable and there is no evidence of absorption and no evidence of toxicity in a 28-day 'limit test' and human exposure is limited

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

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 is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Repeated dose dermal toxicity study was performed to determine the dermal toxic nature of Gentian violet using Charles River CD female rats

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material : Basic violet 3
- Molecular formula : C25H30ClN3
- Molecular weight : 407.986 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): No data

Species:

rat

Strain:

other: Charles River CD

Details on species / strain selection:

No data

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: the mated female animals were housed individually in temperature- and
humidity-controlled rooms
- Diet (e.g. ad libitum): Ralston Purina Laboratory Chow ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Controlled environment
- Humidity (%):Controlled environment
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Type of coverage:

not specified

Vehicle:

not specified

Details on exposure:

TEST SITE
- Area of exposure: The dorso-scapular area
- % coverage: No data
- Type of wrap if used: No data
- Time intervals for shavings or clipplings: The hair at the site of application on the dorso-scapular area was shaved closely the day prior to application

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No data
- Time after start of exposure: No data

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg of the P-23 dye formulation
- Concentration (if solution): 0.002% (2.0 mg/Kg)
- Constant volume or concentration used: No data
- For solids, paste formed: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Amount(s) applied (volume or weight with unit): No data
- Concentration (if solution): No data
- Lot/batch no. (if required): No data
- Purity: No data

USE OF RESTRAINERS FOR PREVENTING INGESTION: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Duration of treatment: 19 days
Duration of exposure: 1, 4, 7, 10, 13, 16 and 19 day of gestation

Frequency of treatment:

Once on days 1, 4, 7, 10, 13, 16 and 19 of gestation

Remarks:

0.002% (0.00942 mg/Kg)

No. of animals per sex per dose:

Total: 100
0 mg/Kg: 60 female rats
2.0 mg/Kg: 20 female rats
Positive control: 20 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

Acetylsalicylic acid

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: No data

BODY WEIGHT: Yes
- Time schedule for examinations: 1, 4, 7, 10, 13, 16 and 19 day of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY:
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data - Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: No data sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

No data

Other examinations:

No data

Statistics:

All statistical analyses compared the treatment groups with the control groups. Statistically significant differences between groups were judged valid only when there were significant differences between any one of the dye treated groups and each of the three untreated control groups.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Details on results:

Clinical signs and mortality
Clinical signs: No signs of toxicity were noted during the study period
Mortality: No data

Dermal irritation: No irritation or other changes in appearance were noted except for changes in skin and hair color at the site of topical application of the dye formulation

Body weight and weight gain: Changes in female body weights were similar for rats in the untreated controls and all dye-treated groups at 0.00942 mg/Kg.

Food consumption and compound intake: Mean food consumption for all groups throughout gestation was similar

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: No data

Clinical chemistry: No data

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No data

Gross pathology: No data

Histopathology: No data

Dose descriptor:

NOAEL

Effect level:

0.009 other: mg/Kg

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No evidence of compound-induced toxicity was observed

Remarks on result:

other: No adverse effects were observed

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for gentian violet in female Charles River CD rats is considered to be 0.00942 mg/Kg.

Executive summary:

Repeated dose dermal toxicity study was performed to determine the dermal toxic nature of Gentian violet. The study was performed using female Charles River CD rats. 2 mL/Kg dye formulation P-23 containing 0.002% ( 0.00942 mg/Kg) test compound was applied to the dorso-scapular area. The application was made during the gestation days 1, 4, 7, 10, 13, 16 and 19. The animals were observed for clinical signs, body weight changes, dermal irritation if any and food consumption. No dye formulation related toxicity was noted. Changes in female body weights and food consumption were similar for rats in the untreated controls and all dye-treated groups. No irritation or other changes in appearance were noted except for changes in skin and hair color at the site of topical application of the dye formulation. Based on the observations made, the No Observed Adverse Effect Level (NOAEL) for gentian violet in female Charles River CD rats is considered to be 0.00942 mg/Kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

0.009 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from K2 peer revewed publication

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the target chemical was reviewed to determine the toxic nature of Gentian violet upon repeated exposure by oral, inhalation and dermal route. The summary is as mentioned below:

Repeated dose toxicity Oral:

Combined repeated dose & carcinogenicity study was performed to determine the mutagenic nature of gentian violet. The study was performed using male and female Fischer F344 rats. Male and female weanling animals (F0) were randomly divided into four groups under barrier conditions and administered 0 (control), 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and females: 0, 40, 100 or 200 mg/Kg bw) GV in their feed for at least 80 days. All rats had access to feed and drinking-water ad lib. While receiving dosed feed, the females were mated with males (one male/ female) of the same dose level. Brother/sister matings were avoided. Two males and two females were selected randomly from each litter (F1a generation) and allocated three animals per cage as weanlings to the chronic study. Litter mates were not assigned to the same cage. The F1a animals continued on the same dose levels as their respective parents for 12, 18 or 24 months. The animals were observed for changes in body weight, food consumption, mortality and morbundity and the presence of lesions.

 

In females, the body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups. In males, at 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk.

 

Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for the females and 25-30 g food/kg body weight for the males.

 

253 rats were found to be in the moribund stage throughout the study period. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw. The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels.

 

No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the 160 mg/Kg bw group. The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls. The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered GV in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months. Incidences of leukemia were high in all groups of female rats fed GV for 24 months and statistical analysis showed no significant differences overall or in the dose groups. In the males fed GV for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed GV for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively. No incidence of mononuclear cell leukaemia show a dose response in male rats fed GV for either 18 or 24 months. No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. Innon-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes.

 

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for Gentian violet in male and female rats is considered to be 30 and 40 mg/Kg bw respectively.

In another study by Littlefield (Fundamental and Applied Toxicology, 1985), Combined repeated dose & carcinogenicity study was performed to determine the chronic nature of gentian violet. The study was performed using 720 male and 720 female B6C3F1 mice for 12, 18, and 24 months of continuous dosing. The test chemical was mixed with feed and given by oral gavage route at dose levels of 0, 10, 300 or 600 ppm (0, 75-100, 225-250, 450-475 mg/Kg bw/week for males and 0, 100, 250-275 or 500 mg/Kg bw/week for females). The animals were observed for Clinical signs, body weight changes, food consumption, clinical chemistry and were subjected to gross and histopathology. There was no effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates. Mortality in the controls of both sexes was less than 15% at 24 months, but was approximately 64% in the females and 23% in the males given the high dose . Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in males at 24 months and in females at 18 and 24 months. Statistical tests for dose-related trends with respect to (1) mortality due to liver neoplasms, (2) prevalence of liver neoplasms, and (3) time to onset of liver neoplasms showed positive trends in both males and females.

 

Histopathological examination revealed several liver neoplasms (hepatocellular carcinoma). A slight dose response at 18 months for the liver neoplasms was noted in females and no other dose responses were noted in the 12- or 18- month sacrifice groups. Essentially, all dose-related lesions were noted in the 24-month sacrifice groups. Malignant liver neoplasms occurred with an incidence of 4, 5, 32, and 77% in the female controls, 100, 250-275, and 500- mg/Kg bw/week-dose groups, respectively, by 24 months. A background incidence of 15% was noted in the control males by 24 months compared to an incidence of 17, 18, and 35% in the 75-100, 225-250, and 450-475 mg/Kg bw/week-dose groups, respectively. The incidence in the females by 18 months was 2, 0, 4, and 13% for the controls, 100, 250-275, and 500 mg/Kg bw/week dose groups, respectively. The incidence of liver neoplasms in males by 18 months did not rise above the background levels of 10% observed in the controls. The males demonstrated a lower susceptibility than females to liver carcinogenicity from gentian violet. While the overall comparisons for both malignant tumors alone and malignant plus benign tumors showed a significant positive trend for mortality, prevalence, and onset in both sexes, the pairwise comparisons of doses to controls showed less positive trends in the males. For malignant liver neoplasms, positive increases from control were noted only in prevalence and onset at 500 mg/Kg in females and 450-475 mg/Kg in males. The results for malignant plus benign lesions were essentially the same as malignant only except positive increases from control were noted for prevalence and onset in the 250-275 mg/Kg in females and 225-250 mg/Kg in males also. Dose responses were noted in females for erythropoiesis in the spleen with 7, 16, 20, and 44% responding for the 0-, 100-, 250-275-, and 500-mg/Kg bw/week-dose levels, respectively, and for atrophy of the ovaries with the incidence of 6, 15, 28, and 42%. Significant dose responses were also noted for the occurrence of reticulum cell sarcomas (Type A) in the uterus, vagina, bladder, and ovaries. These lesions were absent from the controls, except for 1/182 for RCS in the vagina. The following incidences were noted for the respective dose levels of 0, 100, 250-275, and 500 mg/Kg bw/week: RCS of the uterus (Type A), 0, 2, 7, and 13%; RCS of the vagina (Type A), 0.5, 1, 5, and 9%; RCS of the bladder (Type A), 0, 2, 3, and 6%; RCS of the ovaries (Type A) 0, 1, 3, and 6%. Adenoma of the Harderian gland was noted in 4, 12, 20, and 16% of the females and in 4, 7, 11, and 10% of the males in the respectively, by 24 months.

 

Lymphoreticular tissue and the liver were the target tissues for tissue morphology. Benign hepatocellular neoplasms (adenomas) were usually small, and they compressed adjacent parenchyma focally. The adenomas were composed of well-differentiated cells in which the cytoplasm was either basophilic, eosinophilic, clear or vacuolated. Little pleomorphism of nuclei was present. The cells were of uniform size and formed either regular cords of not more than two cell layers thick or solid masses. The hepatocellular carcinomas observed were of the trabecular pattern. Hemorrhage and necrosis occurred in some of the cases. The 4 non hepatocellular metastases in the lung were from an undifferentiated sarcoma of muscle and subcutis of the thigh, from a mammary gland tumor, from a Harderian gland tumor, and from a granulosa cell tumor of the right ovary. The other tumor cellular pattern also found was a solid pattern which was composed of either small immature neoplastic hepatocytes or extremely large anaplastic hepatocytes. The cytoplasm was either acidophilic or vacuolated and the nuclei were large with prominent nucleoli. Mitotic figures were not numerous. The RCS (Type A) of the female genital organs was composed of sheets of elongated spindled cells with basophilic ovoid nuclei and scanty acidophilic cytoplasm, involving the wall of the vagina, cervix, and uterus.

 

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for Gentian violet in male and female < 100 mg/L in male and female B6C3F1 mice.

Repeated dose toxicity Inhalation:

Gentian violet has a very low vapor pressure (2.57 X 10-12 Pa), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point was considered for waiver.

Repeated dose toxicity Dermal:

Repeated dose dermal toxicity study was performed to determine the dermal toxic nature of Gentian violet. The study was performed using female Charles River CD rats. 2 mL/Kg dye formulation P-23 containing 0.002% ( 0.00942 mg/Kg) test compound was applied to the dorso-scapular area. The application was made during the gestation days 1, 4, 7, 10, 13, 16 and 19. The animals were observed for clinical signs, body weight changes, dermal irritation if any and food consumption. No dye formulation related toxicity was noted.Changes in female body weights and food consumption were similar for rats in the untreated controls and all dye-treated groups. No irritation or other changes in appearance were noted except for changes in skin and hair color at the site of topical application of the dye formulation. Based on the observations made,the No Observed Adverse Effect Level (NOAEL) for gentian violet in female Charles River CD rats is considered to be 0.00942 mg/Kg.

In the same mentioned study by Burnett et al, Repeated dose dermal toxicity study was performed to determine the dermal toxic nature of Gentian violet. The study was performed using male and femaleNew Zealand White rabbits. 1 mL/Kg dye formulation P-23 containing 0.002% ( 0.00471 mg/Kg) test compound was applied to the back and dorso-lateral aspects of the thoracic-lumbar area (one on each side of the midline). The application was made twice weekly for 13 weeks exposure duration. The animals were observed for mortality, body weight changes, hematological, clinical chemistry and urine analysis, gross and histopathology. No dye formulation related toxicity was noted.Body weight gain of all test groups was at least equal to that of the controls. Scattered statistically significant differences in the hematologic and clinical chemistry values were noted between test and control groups at the various sampling intervals. However, these differences were not considered to be of any toxicologic significance because of either the direction or continuity of the differences or the fact that they fell within the range of historical control values. No remarkable urinanalysis data was noted. No dye discoloration of the urine was seen at any time during the test. Statistically significant differences in relative organ weights between a test group and the combined controls were observed where the differences were not significant when the group was compared with each control group separately. No gross and microscopic lesions were noted that were judged to be due to the administration of the hair dye formulations containing the test compound. Based on the observations made, the No Observed Adverse Effect Level (NOAEL) for gentian violet in New Zealand White rabbits is found to be 0.00471 mg/Kg during the twice dermal application done in 13 weeks study period.

Based on the data available for the target chemical, Gentian violet in reported doses is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route as per the criteria mentioned in CLP regulation. However doses higher than NOAEL dose for chronic administration orally should be avoided.

Justification for classification or non-classification

Based on the data available for the target chemical, Gentian violet is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route as per the criteria mentioned in CLP regulation. However doses higher than NOAEL dose for chronic administration orally should be avoided.