Registration Dossier

Administrative data

Description of key information

Repeated dose subacute toxicity study was performed to determine the toxic nature of Direct blue 86 (EC name: Disulfo copper phthalocyanine amine salt). The chemical was administered by oral gavage route at dose levels of 0 or 1000 mg/Kg bw/day to 20 male and 20 females rats in a standard volume of 10 mL/kg. The treatment period was followed by a 15 day recovery period. 22 daily doses were given over a period of 30 days i.e 5 times/week, 1 dose/day for 4 weeks plus Monday and Tuesday of the last week. The doses were selectedwere selected on the basis of acute oral LD50 study. The oral LD50 value in rats was in excess of 5000 mg/Kg bw/day and no signs of toxicity were noted and hence the dose was subacute study was selected to be 1000 mg/Kg bw/day. Each day, clinical signs and body weights were recorded. Food intake was determined on a weekly basis. At the end of the treatment and the recovery period, hematology, blood chemistry, and urinalysis measurements were carried out by standard methods on animals from control and test groups. At the end of the treatment period or after the recovery period, each rat was examined externally and by dissection for macroscopic abnormalities. Each rat was autopsied and selected organs, including liver, kidneys, adrenals, and spleen were submitted for histopathological examination, following determination of the organ weights. Other organ tissues were fixed in formalin and are held for reference in the archives. Appropriate statistical analyses were performed on the different measurements. No mortality or signs of toxicity were observed during the study period and food and water consumption were within the normal range following treatment with the eight selected colorants. There was evidence of absorption, based on the coloration of urine and/or tissues following treatment with Direct Blue 86. Comparison of the urinalysis of test and control animals, and all bilirubin and urobilinogen tests were negative. The clinical examinations of liver function revealed no signs of toxicity following treatment and recovery. No overall effects were observed on the hematological profiles of the test animals compared with the controls. The statistical analyses did not reveal evidence for any treatment-related effects on liver or adrenals weights. The kidney weights were increased at termination of treatment with Direct Blue 86, and Direct Blue 15, but were normal after the 2-week recovery. This effect may relate to the morphological changes of the tubules of the kidneys, which were seen at the end of the treatment period, but not after the recovery period. Other histopathological examinations of selected tissues showed no significant effects related to the treatment.

 

The No Observed Adverse Effect Level (NOAEL) for Direct blue 86 in male and female rats is 1000 mg/Kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: 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 is from peer reviewed publication
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Subacute toxicity study was performed to determine the toxic nature of Direct Blue 86 upon repeated exposure by oral route
GLP compliance:
not specified
Limit test:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of the test material: Disulfo copper phthalocyanine amine salt
- EC name: Disulfo copper phthalocyanine amine salt
- Molecular formula: C32H14CuN8O6S2.2Na
- Molecular weight: 780.1706 g/mol
- Substance type: Organic
- Purity: No data
Species:
rat
Strain:
not specified
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals 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: Rats were housed under standardized conditions
- Diet (e.g. ad libitum): pelleted standard diet ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

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

IN-LIFE DATES: From: To: No data
Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
other: Vehicle was used. Details are not mentioned
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Direct Blue 86 was dissolved in appropriate vehicle to give a final dose range of 0 or 1000 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): No data
- Concentration in vehicle: 1000 mg/Kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg
- 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:
30 days
Frequency of treatment:
22 Daily doses over a period of 30 days i.e 5 times/week, 1 dose/day for 4 weeks plus Monday and Tuesday of the last week
Remarks:
0 or 1000 mg/Kg bw/day
No. of animals per sex per dose:
Total: 80
0 mg/Kg bw/day: 20 males and 20 females
1000 mg/Kg bw/day: 20 males and 20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected on the basis of acute oral LD50 study. The oral LD50 value in rats was in excess of 5000 mg/Kg bw/day and no signs of toxicity were noted and hence the dose was subacute study was selected to be 1000 mg/Kg bw/day
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: After termination of treatment 10 females and 10 males of each group were given a 2-week recovery period, if effects had been observed or suspected in the main study.
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No data
Positive control:
No data
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: Every day

BODY WEIGHT: Yes
- Time schedule for examinations: Every day

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: Yes
- Time schedule for collection of blood: At the end of treatment and recovery period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Control and test group animals
- Parameters checked in table [No.?] were examined. Hematocrit, hemoglobin, erythrocytes count, total and differential leucocyte counts, median cell volume, median
cell hemoglobin, platelet count and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of treatment and recovery period
- Animals fasted: No data
- How many animals: Control and test group animals
- Parameters checked in table [No.?] were examined. Serum alkaline phosphatase (SAP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT).


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of treatment and recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. Volume, color, pH, specific gravity, bilirubin, quantitative glucose, quantitative protein, urobilinogen, and urea as well as analysis of the sediment.

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, At the end of the treatment period or after the recovery period, each rat was examined externally and by dissection for macroscopic abnormalities. Each rat was autopsied and selected organs, including liver, kidneys, adrenals, and spleen were weighed to determine the organ weight.

HISTOPATHOLOGY: Yes, At the end of the treatment period or after the recovery period selected organs, including liver, kidneys, adrenals, and spleen were submitted for histopathological examination.
Other examinations:
No data
Statistics:
Statistical analyses were performed. Organ weights (liver, kidneys, adrenals) were subject to analysis of variance and analysis of covariance on final body weight
Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
Clinical signs and mortality: No clinical signs of toxicity and mortality were observed.

Feces were found to be blue in colour

Body weight and weight gain: The body weight gain observed in test animals was in the normal range as compared to controls following treatment

Food consumption and compound intake: The food consumption in test animals was in the normal range as compared to controls following treatment

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: Any hematological effects observed were in the normal range as compared to controls

Clinical chemistry: The clinical examinations of liver function revealed no signs of toxicity following treatment and recovery and were in the normal range
Urinanalysis Urinanalysis effects were found to be in the normal range. Urine was found to be green-blue in colour. Urinalysis of test and control animals and all bilirubin and urobilinogen tests were negative.

Neurobehaviour: No data

Organ weights: Increased kidney weights was noted but were normal after the 2-week recovery. This effect may relate to the morphological changes of the tubules of the kidneys, which were seen at the end of the treatment period, but not after the recovery period.

Gross pathology: Kidneys and intestine showed blue colouration

Histopathology: Microscopic examination revealed normal changes observed if any. Other histopathological examinations of selected tissues showed no significant effects related to the treatment with these compounds.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant alterations were noted at the mentioned dose level
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) for Direct blue 86 in male and female rats is 1000 mg/Kg bw/day.
Executive summary:

Repeated dose subacute toxicity study was performed to determine the toxic nature of Direct blue 86 (EC name: Disulfo copper phthalocyanine amine salt). The chemical was administered by oral gavage route at dose levels of 0 or 1000 mg/Kg bw/day to 20 male and 20 females rats in a standard volume of 10 mL/kg. The treatment period was followed by a 15 day recovery period. 22 daily doses were given over a period of 30 days i.e 5 times/week, 1 dose/day for 4 weeks plus Monday and Tuesday of the last week. The doses were selectedwere selected on the basis of acute oral LD50 study. The oral LD50 value in rats was in excess of 5000 mg/Kg bw/day and no signs of toxicity were noted and hence the dose was subacute study was selected to be 1000 mg/Kg bw/day. Each day, clinical signs and body weights were recorded. Food intake was determined on a weekly basis. At the end of the treatment and the recovery period, hematology, blood chemistry, and urinalysis measurements were carried out by standard methods on animals from control and test groups. At the end of the treatment period or after the recovery period, each rat was examined externally and by dissection for macroscopic abnormalities. Each rat was autopsied and selected organs, including liver, kidneys, adrenals, and spleen were submitted for histopathological examination, following determination of the organ weights. Other organ tissues were fixed in formalin and are held for reference in the archives. Appropriate statistical analyses were performed on the different measurements. No mortality or signs of toxicity were observed during the study period and food and water consumption were within the normal range following treatment with the eight selected colorants. There was evidence of absorption, based on the coloration of urine and/or tissues following treatment with Direct Blue 86. Comparison of the urinalysis of test and control animals, and all bilirubin and urobilinogen tests were negative. The clinical examinations of liver function revealed no signs of toxicity following treatment and recovery. No overall effects were observed on the hematological profiles of the test animals compared with the controls. The statistical analyses did not reveal evidence for any treatment-related effects on liver or adrenals weights. The kidney weights were increased at termination of treatment with Direct Blue 86, and Direct Blue 15, but were normal after the 2-week recovery. This effect may relate to the morphological changes of the tubules of the kidneys, which were seen at the end of the treatment period, but not after the recovery period. Other histopathological examinations of selected tissues showed no significant effects related to the treatment.

 

The No Observed Adverse Effect Level (NOAEL) for Direct blue 86 in male and female rats is 1000 mg/Kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from K2 reliable publication

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose toxicity: Oral

Peer reviewed publication was reviewed to determine the toxic nature of Disulfo copper phthalocyanine amine salt. The study was performed on rats for a duration of 30 days. The details of the study are mentioned as below:

Leist ( Ecotoxicology And Environmental Safety, 1982) performed repeated dose subacute toxicity study to determine the toxic nature of Direct blue 86. The chemical was administered by oral gavage route at dose levels of 0 or 1000 mg/Kg bw/day to 20 male and 20 females rats in a standard volume of 10 mL/kg. The treatment period was followed by a 15 day recovery period. 22 daily doses were given over a period of 30 days i.e 5 times/week, 1 dose/day for 4 weeks plus Monday and Tuesday of the last week. The doses were selectedwere selected on the basis of acute oral LD50 study. The oral LD50 value in rats was in excess of 5000 mg/Kg bw/day and no signs of toxicity were noted and hence the dose was subacute study was selected to be 1000 mg/Kg bw/day. Each day, clinical signs and body weights were recorded. Food intake was determined on a weekly basis. At the end of the treatment and the recovery period, hematology, blood chemistry, and urinalysis measurements were carried out by standard methods on animals from control and test groups. At the end of the treatment period or after the recovery period, each rat was examined externally and by dissection for macroscopic abnormalities. Each rat was autopsied and selected organs, including liver, kidneys, adrenals, and spleen were submitted for histopathological examination, following determination of the organ weights. Other organ tissues were fixed in formalin and are held for reference in the archives. Appropriate statistical analyses were performed on the different measurements. No mortality or signs of toxicity were observed during the study period and food and water consumption were within the normal range following treatment with the eight selected colorants. There was evidence of absorption, based on the coloration of urine and/or tissues following treatment with Direct Blue 86. Comparison of the urinalysis of test and control animals, and all bilirubin and urobilinogen tests were negative. The clinical examinations of liver function revealed no signs of toxicity following treatment and recovery. No overall effects were observed on the hematological profiles of the test animals compared with the controls. The statistical analyses did not reveal evidence for any treatment-related effects on liver or adrenals weights. The kidney weights were increased at termination of treatment with Direct Blue 86, and Direct Blue 15, but were normal after the 2-week recovery. This effect may relate to the morphological changes of the tubules of the kidneys, which were seen at the end of the treatment period, but not after the recovery period. Other histopathological examinations of selected tissues showed no significant effects related to the treatment.

 

The No Observed Adverse Effect Level (NOAEL) for Direct blue 86 in male and female rats is 1000 mg/Kg bw/day.

Based on the key study summarized, Disulfo copper phthalocyanine amine salt is not likely to be toxic upon repeated exposure by oral route.

Justification for classification or non-classification

Based on the key study summarized, Disulfo copper phthalocyanine amine salt (CAS no 1328 -51 -4) is not likely to be toxic upon repeated exposure by oral route.