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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Chronic Toxicity/Carcinogenicity Studies Of Gentian Violet In Fischer 344 Rats: Two-Generation Exposure
Author:
N. A. Littlefield, D. W. Gaylor, B.-N. Blackwell And R. R. Allen
Year:
1989
Bibliographic source:
Fd Chem. Toxic. Vol. 27, No. 4, pp. 239-247, 1989

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

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

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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]

 

Applicant's summary and conclusion

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.