[4-[bis[4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadien-1-ylidene]dimethylammonium acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 journal

Data source

Reference

Reference Type:

publication

Title:

Acute (Mouse and Rat) and Short-term (Rat) Toxicity Studies on Blue VRS

Author:

D. E. Hall and I. F. Gaunt, Madge Farmer and P. Grasso

Year:

1967

Bibliographic source:

Fd Cosmet. Toxicol Vol. 5, pp. 165-170, 1967

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Chronic repeated dose toxicity was studied was performed to determine the toxic nature of Blue VRS in rats orally.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

EC Number:

204-934-1

EC Name:

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Cas Number:

129-17-9

Molecular formula:

C27H32N2O6S2.Na

IUPAC Name:

hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Details on test material:

- Name of test material: Blue VRS
- Molecular formula: C27H31N2O6S2.Na
- Molecular weight: 566.672g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities: 5.5 %

Specific details on test material used for the study:

- Name of test material: Blue VRS
- Molecular formula: C27H31N2O6S2.Na
- Molecular weight: 566.672g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities: 5.5 %

Test animals

Species:

rat

Strain:

other: SPF Carworth Farm E

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: No data available
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: 5/cage
- Diet (e.g. ad libitum): Spillers Small Laboratory Animal diet, ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data available


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

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Spillers Small Laboratory Animal diet

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: The test chemical was made to be available in Spillers Small Laboratory Animal food at dose levels of 0.0, 0.3, 0.75, 1.5 and 3.0% (0, 150, 375, 750 and 1500 mg/kg/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): Spillers Small Laboratory Animal food
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Spillers Small Laboratory Animal diet
- Concentration in vehicle: 0.0, 0.3, 0.75, 1.5 and 3.0% (0, 150, 375, 750 and 1500 mg/kg/day)
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0.0, 0.3, 0.75, 1.5 and 3.0 % (0, 150, 375, 750 and 1500 mg/kg/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Total : 180
0 mg/Kg/day : 15 male and 15 female
0 mg/Kg/day: 15 male and 15 female
150 mg/Kg/day : 15 male and 15 female
375 mg/Kg/day : 15 male and 15 female
750 mg/Kg/day: 15 male and 15 female
1500 mg/Kg/day: 15 male and 15 female

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: Daily
- Cage side observations checked in table [No.?] were included: Mortality were observed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

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: 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 available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available
- Time schedule for examinations: No data available

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 6 week and on terminally
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data
- How many animals: All the treated animals were examined.
- Parameters checked in table [No.?] were examined: Erythrocytes, Packed cell volume, Hemoglobin concentration in all teeated animals and total and Dtfferentlal Leucocytes count in 0, 750 and 1500 mg/Kg/day dose groups were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At week 12
- Animals fasted: No data available
- How many animals: All the treated animals were examined.
- Parameters checked in table [No.?] were examined: Serum urea was examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at week 12, a 6-hr period of water deprivation and during a 4-hr period beginning 16 hr after a water load of 25 ml/kg.
- Metabolism cages used for collection of urine: : No data available
- Animals fasted: : No data available
- Parameters checked in table [No.?] were examined: pH, Specific gravity and Volume were examined.

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

OTHER:
Absolute and relative organ weights were examined.

Organ examined.
Liver, Left and Right Kidneys, Brain, Heart, Spleen, Adrenals and Gonads were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Various organs were weighed and gross abnormalities were examined.

HISTOPATHOLOGY: Yes
Organ examined. Liver, Left and Right Kidneys, Brain, Heart, Spleen, Adrenals and Gonads were examined.

Other examinations:

No data available

Statistics:

No data available

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):

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality: All treated animals beared normal health during the study period. No mortality was observed but one male in the 750 mg/Kg/day group was killed on day 82 since it had a large mass in the neck which appeared to be an enlarged salivary gland.

Body weight and weight gain: Impairment of growth was observed during the first 4 weeks and was attributed to reduced food intake

Food consumption and compound intake: Reduced food intake was observed during the first 4 weeks

Food efficiency: Reduced
Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No departure from normality was observed; red cell morphology was also unaffected.

Clinical chemistry: No effect on kidney function investigated during the last week of treatment or on serum urea nitrogen determined terminally

Urinanalysis: The urine of all the animals receiving Blue VRS in the diet was bluish-green and slightly more acid than that of the controls.

Proteinurea was comparable in test and control groups and no reducing substances were detected in the urine of any group.

Neurobehaviour: No data available

Organ weights: No effect on the absolute or relative (g/100 g body weight) organ weights was observed

Gross pathology

Histopathology: Histopathological changes included occasional fatty changes in the liver, especially in the females and an increase in the number of active acini in the thyroids of all the animals at 750 and 1500 mg/Kg/day. In addition the kidneys of the males at 1500 mg/Kg/day contained blue material in the lumen of the convoluted tubules associated with a coloured line observed at autopsy at the cortico-medullary junction.

Other pathological changes were similar in incidence and severity in the test and control groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, clinical sign, body weight, food consumption, compound intake, haematology, clinical chemistry, organ weights, gross pathology and histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect-level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats were treated with Blue VRS.

Executive summary:

In a subchronic repeated dose toxicity study, SPF Carworth Farm E male and female rats were treated with Blue VRS at a concentration of 0.0, 0.3, 0.75, 1.5 and 3.0 % (0, 150, 375, 750 and 1500 mg/kg/day) orally. Impairment of growth, Reduction in food consumption, compound intake and bluish-green and slightly more acid urine were observed in 750 and 1500 mg/Kg/day (1.5 and 3.0 %) treated male and female as compared to control. In addition, Fatty changes in the liver of female rat and increase number of active acini in the thyroids of male rat of 750 and 1500 mg/Kg/day (1.5 and 3.0 %) treated animals. Blue material in the lumen of convoluted tubules associated with a coloured line observed at the cortcomedullary junction of kidney in male of 1500 mg/Kg/day (3.0 %) treated dose groups but this was not necessarily attributable to Blue VRS. Histological changes associated with acinal activity occur in response to stress of various kinds were noted. Therefore, the no observed adverse effect level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats were treated with Blue VRS orally for 90 days.