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Carcinogenicity

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Description of key information

The NOAEL was considered to be for female mice 100 mg/kg bw  for 12 month and for male mice 225-250mg/kg bw for 18 month. When B6C3F mice were treated with gentian violet orally for 24 month to determine its carcinogenicity.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
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 peer reviewed journal
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
The carcinogenicity study of gentian violet was performed in mice.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report):Gentian violet
- IUPAC name: N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride
- Molecular formula : C25H30N3.Cl
Molecular weight : 407.986 g/mol
- Smiles notation: C(\c1ccc(N(C)C)cc1)(c1ccc(N(C)C)cc1)=C1\C=C\C(=[N+](/C)C)C=C1.[ClH-]
- InChl:1S/C25H30N3.ClH/c1-26(2)22-13-7-19(8-14-22)25(20-9-15-23(16-10-20)27(3)4)21-11-17-24(18-12-21)28(5)6;/h7-18H,1-6H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
Species:
mouse
Strain:
B6C3F1
Remarks:
(C57BL/6 x C3H)
Details on species / strain selection:
no data available
Sex:
male/female
Details on test animals and environmental conditions:
- Source: National Center for Toxicological Research
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 8-15g
- Fasting period before study: no data available
- Housing:The animals were housed four animals/cage in barrier-type animal holding room with hardwood chips as the bedding material and filter top cages
- Diet (e.g. ad libitum): feed (Purina Mills, Richmond, IN), ad libitum
- Water (e.g. ad libitum): Drinking water -ad libitum
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22.22 ± 2 °C
- Humidity (%):50±5 %
- Air changes (per hr):14-16
- Photoperiod (hrs dark / hrs light):12hr

Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The gentian violet was dissolved in ethyl alcohol and sprayed directly into the feed (Purina 5010M, autoclavable) at dose levels of 0, 100, 300, and 600 ppm. The ethyl alcohol was subsequently removed from the prepared feed during a 30-min blending process by a vacuum.
DIET PREPARATION
- Rate of preparation of diet (frequency):weekly
- Mixing appropriate amounts with (Type of food): no data

VEHICLE
- Justification for use and choice of vehicle (if other than water):feed
- Concentration in vehicle: for female : 500, 250-275, and 100 mg gentian violet/kg body wt/week
for male: 450-475, 225-250, and 75-100 mg gentian violet, respectively

- Amount of vehicle (if gavage):no data
- Lot/batch no. (if required):no data
- Purity: 99%gentian violet and 1% methyl violet.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data available
Duration of treatment / exposure:
12,18 or 24 month
Frequency of treatment:
daily
Post exposure period:
no data available
Remarks:
For female : 500, 250-275, and 100 mg
For male : 450-475, 225-250, and 75-100 mg
No. of animals per sex per dose:
Total:1440
Male:720
Control :288
75-100mg /kg :144
225-250mg/kg:144
450-475mg/kg:144
Female :720
Control:288
100mg/kg :144
250-275mg/kg:144
500mg/kg:144
Control animals:
yes
yes, concurrent vehicle
Details on study design:
no data available
Positive control:
no data available
Observations and examinations performed and frequency:
Observation and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included. Mortality or moribund condition


DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: weekly

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

BODY WEIGHT: Yes
- Time schedule for examinations: daily for1-4 week then for 4week interval after 4week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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 AND COMPOUND INTAKE (if drinking water study): / No data
- Time schedule for examinations:

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

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

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

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

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

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
no data available
Statistics:
Statistical procedure Cochran-Armitage and Fischer’s exact test was used for analysis of RCS.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
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:
effects observed, treatment-related
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:
effects observed, treatment-related
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: Mortality (adjusted for sacrifices) 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 i.e. for male475mg/kg bw and for female 500mg/kg bw.
BODY WEIGHT AND WEIGHT GAIN: No dose related effect on body weight and weight gain
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No dose related effect on Food consumption & compound intake in control and treated group
FOOD EFFICIENCY: no data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) no data available

OPHTHALMOSCOPIC EXAMINATION: no data available

HAEMATOLOGY: no data available

CLINICAL CHEMISTRY: There were no observed effects at 12 months. At 18 months there was a small increase in direct bilirubin; however, this was probably caused by interference of the blue pigment in the serum.At 24 months, positive effects were noted for AST-GOT, ALT-GPT, serum cholesterol, α1 serum protein, α2 serum protein, and triglycerides. All of these effects related to abnormalities of the liver. In addition, most of the statistically positive trends were in the females at 24 months and at 500 mg/Kg bw/week

URINALYSIS: no data available

NEUROBEHAVIOUR: no data available

ORGAN WEIGHTS: no data available

GROSS PATHOLOGY: performed on all animals. All were dose related response greatest response in liver neoplasm in 24 month

HISTOPATHOLOGY: NON-NEOPLASTIC: No non neoplastic lesions observed in male and female rats treated at 12 and 18 month necropsies whereas erythropoiesis in the spleen was observed in female.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): 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. Mortality at this dose level showed a borderline p value of 0.02. 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.



OTHER FINDINGS: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 nonhepatocellular 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 similar to those described by Dunn (1954). The tumor 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.

Relevance of carcinogenic effects / potential:
A positive dose response for hepatocellular carcinoma was noted in male at 24 months and in females at 18 and 24 months. The incidence of atrophy of the ovaries, adenoma of the harderian gland, and the presence of type A reticulum cell sarcomas in the urinary bladder, uterus, ovaries, and vagina observed in female.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: 12 month
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: for 18 month
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
other: hepatocellular
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
275 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
bladder
ovary
uterus
vagina
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table: Dunnett's / (Comparison to Control) Applied In Connection With the Unweighted Means Analysis (Significant Two-Tailed P Values At 24-Month Sacrifice)

 

Females

Males

100

300

600

100

300

600

AST-GOT

NS°

.05 P

.01 P

NS

NS

.01 P

ALT-GPT

NS

.01 P

.01 P

NS

NS

.01 P

ALP

QNS

QNS

QNS

NS

NS

NS

Triglycerides

NS

NS

.01 N

NS

NS

NS

Cholesterol

NS

NS

.01 P

NS

.05 N

NS

αP2-%

NS

.01 P

.01 P

NS

NS

NS

αl-CONC

NS

NS

.01 P

NS

NS

NS

QNS, quantity (of serum) not sufficient; NS, not significant at p = 0.05; N, change in negative direction; P, change in positive direction.

Table: Microscopic Histopathology Summary of Female Mice

Site (Lesion)

18 Month sacrificeb(Dose, ppm)

24 Month sacrificeb(Dose, ppm)

0

100

300

600

0

100

300

600

Liver, neoplasm, benign

3/47

0/22

3/24

8/24

8/185

8/93

36/93

20/95

6C

0

13

33

4

9

39

21

Liver, neoplasm, malignant

1/47

0/22

1/24

3/24

7/185

5/93

30/93

73/95

2

0

4

13

4

5

32

77

Uterus, RCS Type A

0/47

0/22

1/24

1/24

0/188

2/95

6/90

12/93

0

0

4

4

0

2

7

13

Bladder, RCS Type A

0/47

1/22

1/24

0/23

0/188

2/92

3/89

5/91

0

0

4

0

0

2

3

6

Spleen erythropoiesis

2/47

1/21

1/24

0/23

13/190

15/96

18/92

42/95

4

5

4

0

7

16

20

44

Ovaries, Atrophy

0/45

0/21

0/22

1/21

11/178

13/90

25/89

37/89

0

0

0

5

6

15

28

42

Harderian gland, adenoma

2/46

2/21

3/23

1/23

8/186

11/93

18/89

15/94

4

10

13

4

4

12

20

16

Ovaries, RCS Type A

0/45

0/21

0/22

0/21

0/178

1/90

3/89

5/89

0

0

0

0

0

1

3

6

Vagina, RCS Type A

0/46

0/22

1/23

0/22

1/182

1/90

4/88

8/87

0

0

4

0

0.5

1

5

9

aAll incidences in the 12-month sacrifice group were 0% except a 4% incidence in Harderian gland adenoma at 0

and 300 ppm, vagina RCS (Type A) at 100 ppm, and vagina RCS (total) at 100 ppm.

bIncludes dead or moribund animals that were removed from the study prior to the scheduled sacrifice dates.

cNo. positive for lesion/total No., and percentage.

Table: Microscopic Histopathology Summary of male Mice

Site (Lesion)

18 Month sacrificeb(Dose, ppm)

24 Month sacrificeb(Dose, ppm)

0

100

300

600

0

100

300

600

Liver, neoplasm, benign

3/48

0/24

2/24

2/22

17/183

14/92

20/93

37/93

6C

0

8

9

10

15

22

38

Liver, neoplasm, malignant

5/48

1/24

2/24

2/22

27/183

15/92

17/93

33/93

0

4

8

9

15

17

18

35

Harderian gland, adenoma

2/47

2/24

2/23

0/21

7/187

7/92

10/94

9/89

4

8

9

0

4

7

11

10

° All incidences in the 12-month sacrifice group were 0% except an 8% incidence in liver neoplasm (benign) at 100 ppm.

* Includes dead or moribund animals that were removed from the study prior to the scheduled sacrifice dates.

cNo. positive for lesion/total No., and percentage

Table: Metastatic lesions

Dose

Males

Females

Controls

3

0

100 ppm

1

0

300 ppm

1

3

600 ppm

3

13

Conclusions:
The NOAEL was considered to be for female mice 100 mg/kg bw for 12 month and for male mice 225-250mg/kg bw for 18 month. When B6C3F mice were treated with gentian violet orally for 24 month to determine its carcinogenicity.
Executive summary:

The gentian violet (548-62-9) was studied in the diet of 720 males and 720 females of B6C3F,mice(C57BL/6 x C3H) at dose levels For female 500, 250-275, and 100 mg and for male :450-475, 225-250, and 75-100 mg was done to determine its toxicity and carcinogenicity. Sacrifices were conducted after 12, 18, and 24monthsof continuous dosing.

There were effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates. Mortality (adjusted for sacrifices) in the control of both sexes was less than15% at 24 months, but was approximately64% in the females and 23% in the males given the high dose i.e 500mg/kg bw .Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in male at 24 months and in females at l8 and 24months.Statistical tests for dose-related trends with respect to (1) mortality due to liver neoplasms,(2)prevalence of liver neoplasms, and(3)time to on set of liver neoplasms showed positive trends in both males and females. Other dose-related toxicological responses, particularly in the female mice, included erythropoiesis in the spleen ,atrophy of the ovaries, adenoma of the harderian gland, and the presence of type A reticulum cell sarcomas in the urinary bladder, uterus, ovaries, and vagina.

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 tumours alone and malignant plus benign tumours 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.

Mortality at this dose level showed a borderline p value of 0.02. 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. The estimation of risk of 10-6over background for malignant liver neoplasms using linear extrapolations showed a lower bound on the virtually safe dose (VSD) to be 2 ppb for the female mice and I ppb for the male mice. For benign and malignant liver tumors together, the lower bound on the VSD was essentially the same as for malignant liver neoplasm alone. Under the conditions of the experiment described above, gentian violet appears to be a carcinogen in mice several different organ sites. Hence NOAEL was considered to be for female mice 100 mg/kg bw for 12 month and For male mice 225 -250mg/kg bw for 18 month. WhenB6C3F mice were treated with gentian violet orally for 24 month.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Data from peer reviewed journal
System:
hepatobiliary
Organ:
liver

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Carcinogenicity by oral route

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

In experimental study given by Nei A. Littlefield, Boon-Nam Blackwell, Cynthia C. Hzwim, and David W. Gaylor(FUNDAMENTAL AND APPLIED TOXICOLOGY 5, 902-9 12 ,1983) The gentian violet (548-62-9) was studied in the diet of 720 males and 720 females of B6C3F,mice(C57BL/6 x C3H) at dose levels For female 500, 250-275, and 100 mg and for male :450-475, 225-250, and 75-100 mg was done to determine its toxicity and carcinogenicity. Sacrifices were conducted after 12, 18, and 24months of continuous dosing.There were effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates.Mortality(adjustedforsacrifices) in the control of both sexes was less than15% at 24 months, but was approximately 64% in the females and 23% in the males given the high dose i.e 500mg/kg bw .Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in male at 24 months and in females at l8 and 24months.Statistical tests for dose-related trends with respect to (1) mortality due to liver neoplasms,(2)prevalence of liver neoplasms, and(3)time to on set of liver neoplasms showed positive trends in both males and females. Other dose-related toxicological responses, particularly in the female mice, included erythropoiesis in the spleen ,atrophy of the ovaries, adenoma of the harderian gland, and the presence of type A reticulum cell sarcomas in the urinary bladder, uterus, ovaries, and vagina. 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 tumours alone and malignant plus benign tumours 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. Mortality at this dose level showed a borderline p value of 0.02. 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. The estimation of risk of 10-6over background for malignant liver neoplasms using linear extrapolations showed a lower bound on the virtually safe dose (VSD) to be 2 ppb for the female mice and I ppb for the male mice. For benign and malignant liver tumors together, the lower bound on the VSD was essentially the same as for malignant liver neoplasm alone. Under the conditions of the experiment described above, gentian violet appears to be a carcinogen in mice several different organ sites. Hence NOAEL was considered to be for female mice 100 mg/kg bw for 12 month and For male mice 225 -250mg/kg bw for 18 month. WhenB6C3F mice were treated with gentian violet orally for 24 month.

The another experimental study given by N.A Littlefield and D. W. Gaylor (Fd. chem toxic, Vol.27,no:4, pp239-247,1989) The carcinogenicity of gentian violet was studied in male and female Fischer 344 rats for 24 month. The gentian violet in dose concentration for Males: 0, 30, 80 or 160 mg/Kg bw and for Females: 0, 40, 100 or 200 mg/Kg bw was administered to the animals in feed by oral route. 570male and 570 female were used for study. All the animals provide with feed and water ad libitum. All the animals were examined for clinical sing, body weight, food consumption. The rats were killed and necropsied after 12,18 and 24 months of continuous dosing.Female body weight increases for all dose group while male in higher dose group had lower average body weight than those for any other dose group while food consumption in week 1-20 showed rapid decrease then stable after 90 weeks it’s increased. The female had higher mortality rate than the dosed male. Mortality first appeared in female at about 258 days and in male at about 177days .At end of dosing period mortality rate in female were 33, 38, 60 and 66% for 0, 40, 100 or 200 mg/Kg bw dose group respectively. While mortality rate in male were 33, 33, 48 and 39 % for0, 30, 80 or 160 mg/Kg bw dose group respectively. The incidence of mononuclear cell leukemia not observed on male rats fed with GV for 18 or 24 months but in female it appeared in time related response while the follicular cell adenocarcinoma of thyroid gland in female rats at 24 months necropsy was 1, 1, 5 and 8%in0, 40, 100 or 200 mg/Kg bw dose group respectively. No non neoplastic lesions observed in male and female rats treated at 12 and 18 month necropsies whereas non neoplastic lesions observed in liver at 24 month in female rats. Hence The Gentian violet considered to be carcinogenic as there was equivocal evidence of carcinogenic activity of gentian violet in F344 rats based on the occurrence of thyroid gland follicular cell adenoma or carcinoma (combined) in exposed rats for 24 month.

 

 

 Based on the above study on N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9) it can be concluded that NOAEL value for mice and rats was considered to be 100 mg/kg bw for 18 month. Thus, comparing this value with the criteria of CLP regulation, N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9) can be not classified for Carcinogenicity at 100mg/kg bw for 18 months, in higher dose it may be carcinogenic.

 

 

Justification for classification or non-classification

Based on the above study on N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (548-62-9) it can be concluded that NOAEL was considered to be 100 mg/kg bw in both the species i.e B6C3F,mice and Fischer 344 rats when treated orally above NOAEL dose it should be avoided as it may be carcinogenic in higher dose for chronic used.