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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from SCCS report

Data source

Reference
Reference Type:
secondary source
Title:
Opinion on Basic Violet 2 Colipa N° B115
Author:
Scientific Committee on Consumer Safety
Year:
2011
Bibliographic source:
European Commission, SCCS/1340/10, 2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Repeated dose oral toxicity study was performed to determine the oral toxic nature of Basic Violet 2 upon repeated exposure for 13 weeks to rats
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Basic Violet 2
- IUPAC name: 4,4'-[(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methylene]bis(2-methylaniline) hydrochloride
- Molecular formula: C22H23N3ClH
- Molecular weight: 365.906 g/mol
- Substance type: Organic
- Physical state: No data
Specific details on test material used for the study:
- Name of test material: New Fuchsin
- Molecular formula: C22H23N3ClH
- Molecular weight: 365.906 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): 6%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd: SD
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
No data

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in water to give the appropriate dose range of 0, 3, 10 or 30 mg/kg bw/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 3, 10 or 30 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/Kg bw/day
- 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:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
0, 3, 10 or 30 mg/kg bw/day
No. of animals per sex per dose:
Total: 40 males and 40 females
0 mg/kg bw: 10 males and 10 females
3 mg/kg bw: 10 males and 10 females
10 mg/kg bw: 10 males and 10 females
30 mg/kg bw: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

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

HAEMATOLOGY: Yes
- 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. Hematocrit, RBC, MCH, MCV, hemoglobin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. NA. Ca, K, Cholesterol, glucose, ASAT and ALP activity

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: Neurotoxicity was noted
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, relative kidney, heart and liver weight was measured

HISTOPATHOLOGY: Yes, histological examination was performed
Other examinations:
No data
Statistics:
No data

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Staining of the faeces in all test groups. Occasional hunched posture and dyspnoea in a few 30 mg/kg bw/day rats was noted
Mortality:
mortality observed, non-treatment-related
Description (incidence):
On day 57, 2 females from the 30 mg/Kg bw/day died due to misdosing
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females of the 30 mg/Kg bw/day showed reduced body weight from day 8 of treatment.

With regard to body weight a statistically significant reduction was observed in females at 30 mg/kg/bw and at 10 mg/kg bw/d also a reduction was noted. In males only a slight reduction was observed at 30 mg/kg/bw. This was accompanied by reduced body weight gains in all these groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was lowered in the 30 mg/Kg bw/day females in the last week.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Dose related effects were noted in the hematological parameters.

Haematological observations were reduction in red blood cells counts in both males and females which were statistically significant at 10 and 30 mg/kg bw/d for males and at 30 mg/kg bw/d for females. Furthermore, haematocrit values were slightly reduced in all dose groups in females. Further, the values for mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were slightly increased in males and females of all dose groups. However, all changes were in the range of the historical controls
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Dose related effects were noted in the clinical parameters.

Some differences were observed with regard to Na, Ca, K, glucose, cholesterol as well as in the levels of alkaline phosphatase and aspartate aminotransferase. It was argued that all values were well within the range of historical controls.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motility impairment in a few 30 mg/Kg/day rats
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative organ weights were statistically significantly increased in xmales and females at 30 m/kg bw/d for liver, kidneys and heart, the latter being additionally elevated (statistically significant) in males at 10 mg/kg bw/d. In addition, at 30 mg/kg bw slight increases were observed in relative testes weights in males and relative brain weights in females (both statistically significant). Despite the results of the statistical analyses of differences to controls, the differences were slight and all mean values remained well within the range of historical control data. The only organ in which a histopathological correlate was observed was the liver (centrilobular hepatocytic hypertrophy in 5 10 mg/Kg bw/day males and in most high-dose males and females. Hepatocytic necrosis in one 30 mg/Kg bw/day male).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dark and firm areas in the liver in three 30 mg/Kg bw/day males.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hepatocytic hypertrophy in five 10 mg/Kg bw/day males and in most 30 mg/Kg bw/day males and females. Hepatocytic necrosis in one 30 mg/Kg bw/day male. Nephropathy (tubular basophilia / dilatation and /or chronic inflammation) in five 10 mg/Kg bw/day males and six 30 mg/kg bw/day males.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
other: Significant changes were noted at doses above 3 mg/Kg bw/day

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
other: Hepatic and renal system
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table: Details on hematological and clinical chemistry parameters

Dose (mg/Kg bw/day)

3

10

30

Dose related

Male

Female

Male

Female

Male

Female

Hematology

 

 

 

 

 

 

 

Hematocrit

Dc

-

Dc

-

Dc

-

yes

RBC count

D

D

Dc

D

Dc

Dc

Yes

Hemoglobin

-

-

-

Dc

-

Dc

Yes

MCH

-

-

Ic

-

Ic

-

Yes

MCV

-

-

-

-

Ic

-

 

 

 

 

 

 

 

 

 

Clinical chemistry

 

 

 

 

 

 

 

Na

I

Ic

Ic

Ic

Ic

Ic

Yes (males)

Ca

Ic

Ic

Ic

Ic

Ic

Ic

Yes (males)

K

Ic

-

Ic

-

Ic

-

 

Cholesterol

-

-

-

-

Ic

Ic

 

Glucose

-

Ic

-

I

-

Ic

 

ASAT activity

-

-

-

Dc

Dc

Dc

Yes

ALP activity

-

-

-

Dc

-

Dc

Yes

 

 

 

 

 

 

 

 

Organ weight

 

 

 

 

 

 

 

Relative liver

-

-

-

-

Ic

Ic

 

Relative kidney

-

-

-

-

Ic

Ic

 

Relative Heart

-

-

Ic

-

Ic

Ic

 

Dc/Ic: Statistically significant decreased/increased compared to the controls

D/I: Decreased/increased, but not statistically significantly compared to the controls

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for the test compound Basic violet 2 is considered to be 3 mg/Kg bw/day.
Executive summary:

Repeated dose oral toxicity study was performed to determine the oral toxic nature ofBasic Violet 2upon repeated exposure for 13 weeks to rats. The test chemical dissolved in water as vehicle at dose levels of 3, 10 or 30 mg/kg bw/day was administered by the gavage route to 10 male and 10 female Sprague Dawley Hsd: SD strain rats for 13 weeks. The animals were observed for mortality, clinical signs, ophthalmology, neurotoxicity, body weight and organ weight changes, food intake, hematological and clinical chemistry changes. Staining of the faeces in all test groups. Occasional hunched posture and dyspnoea in a few 30 mg/kg bw/day rats was noted. On day 57, 2 females from the 30 mg/Kg bw/day died due to misdosing. Females of the 30 mg/Kg bw/day showed reduced body weight from day 8 of treatment.With regard to body weight a statistically significant reduction was observed in females at 30 mg/kg/bw and at 10 mg/kg bw/d also a reduction was noted. In males only a slight reduction was observed at 30 mg/kg/bw. This was accompanied by reduced body weight gains in all these groups. In females at 30 mg/kg/bw also food consumption was reduced. Food intake was lowered in the 30 mg/Kg bw/day females in the last week. Dose related effects were noted in the hematological parameters.Haematological observations were reduction in red blood cells counts in both males and females which were statistically significant at 10 and 30 mg/kg bw/d for males and at 30 mg/kg bw/d for females. Furthermore, haematocrit values were slightly reduced in all dose groups in females. Further, the values for mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) were slightly increased in males and females of all dose groups. However, all changes were in the range of the historical controls.Dose related effects were noted in the clinical parameters.Some differences were observed with regard to Na, Ca, K, glucose, cholesterol as well as in the levels of alkaline phosphatase and aspartate aminotransferase. It was argued that all values were well within the range of historical controls. Relative organ weights were statistically significantly increased in xmales and females at 30 m/kg bw/d for liver, kidneys and heart, the latter being additionally elevated (statistically significant) in males at 10 mg/kg bw/d. In addition, at 30 mg/kg bw slight increases were observed in relative testes weights in males and relative brain weights in females (both statistically significant). Despite the results of the statistical analyses of differences to controls, the differences were slight and all mean values remained well within the range of historical control data. The only organ in which a histopathological correlate was observed was the liver (centrilobular hepatocytic hypertrophy in five 10 mg/Kg bw/day males and in most high-dose males and females. Hepatocytic necrosis in one 30 mg/Kg bw/day male).Dark and firm areas in the liver in three 30 mg/Kg bw/day males. Centrilobular hepatocytic hypertrophy in five 10 mg/Kg bw/day males and in most 30 mg/Kg bw/day males and females. Hepatocytic necrosis in one 30 mg/Kg bw/day male. Nephropathy (tubular basophilia / dilatation and /or chronic inflammation) in five 10 mg/Kg bw/day males and six 30 mg/kg bw/day males.

 

At the doses 30 and/or 10 mg/kg bw/day histopathological findings in liver and kidney indicate organ toxicity which is supported by changes in body and organ weights as well as in biochemical parameters (cholesterol, aspartate aminotransferase, alkaline phosphatase). Based on the observations made, the No Observed Adverse Effect Level (NOAEL) for the test compound Basic violet 2 is considered to be 3 mg/Kg bw/day