Tetrasodium 2,2'-[1,4-phenylenebis[carbonylimino[2-acetamido-4,1-phenylene]azo]]bis[5-[(4-sulphonatophenyl)azo]benzenesulphonate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

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 publication

Data source

Reference

Reference Type:

publication

Title:

Long-Term Toxicity Study Of Black Pn In Mice

Author:

J.-P. Drake, K. R. Butterworth, I. F. Gaunt And P. Grasso

Year:

1977

Bibliographic source:

Food and cosmetics toxicology; Vol 15, pp. 503-508, 1977

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

A repeated dose study was performed investigating the effect of Brilliant Black PN in CFW strain mice when administered orally for 80 wk.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

EC Number:

219-746-5

EC Name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

Cas Number:

2519-30-4

Molecular formula:

C28H21N5O14S4.4Na

IUPAC Name:

tetrasodium 4-acetamido-5-hydroxy-6-({7-sulfonato-4-[(4-sulfonatophenyl)diazenyl]-1-naphthyl}diazenyl)naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Brilliant Black PN
- Molecular formula (if other than submission substance): C28H17N5Na4O14S4
- Molecular weight (if other than submission substance):867.66716 g\mol
- Substance type: Organic
- Physical state: Solid

Specific details on test material used for the study:

- Name of test material: Brilliant Black PN
- Molecular formula: C28H17N5Na4O14S4
- Molecular weight: 867.66716 g\mol
- Substance type: Organic
- Physical state: Solid
- Purity: tetrasodium 8-acetamido-2-(7- sulpho-4-p- sulphophenylazo- 1-naphthylazo)- 1-naphthol-3,5-disulphonate; dye content*, min. 82%; subsidiary dyes*, max 4%; matter volatile at 135°C *, max 10%; matter insoluble in water*, max 0.1%; matter soluble in diisopropyl ether*, max 0.2%; chloride and sulphate (as sodium salts)*, max 8.0%; copper*, max 10ppm; arsenic*, max 1 ppm; lead*, max 10 ppm; heavy metals (as sulphides)*

Test animals

Species:

mouse

Strain:

other: CFW strain

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: From a specified-pathogen-free colony
- Age at study initiation: No data available
- Weight at study initiation: The mice were weighed at the start of the experiment (exact weight not mentioned)
- Fasting period before study: No
- Housing: They were caged in groups of 15 in a room
- Diet (e.g. ad libitum): Oxoid pasteurized breeding diet,ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±1°C
- Humidity (%):50-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

not specified

Details on oral exposure:

No dataPREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose level of 0, 0.1, 0.25,0.5 or 1.0% (0, 130, 325, 650 or 1300 mg/kg bw/d)

DIET PREPARATION
- Rate of preparation of diet (frequency): Oxoid pastuerized feeding diet
- 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): Oxoid pastuerized feeding diet
- Concentration in vehicle: 0, 0.1, 0.25,0.5 or 1.0% (0, 130, 325, 650 or 1300 mg/kg bw/d)
- 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

Duration of treatment / exposure:

80 wk

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.1, 0.25,0.5 or 1.0% (0, 130, 325, 650 or 1300 mg/kg bw/d)

No. of animals per sex per dose:

Total:
0 mg/Kg bw/day: 30 male and 30 female mice
130 mg/Kg bw/day: 30 male and 30 female mice
325 mg/Kg bw/day: 30 male and 30 female mice
650 mg/Kg bw/day: 30 male and 30 female mice
1300 mg/Kg bw/day: 30 male and 30 female mice

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Frequently
- Cage side observations checked in table [No.?] were included.: General condition and behaviour

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At wk 28 and 55 from the caudal vein of ten males and ten females from the control group and from the groups of 0.5 and 1.0% dietary levels.
At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 20 animals (10 male and 10 female)
- Parameters were examined: haemoglobin concentration and packed cell volume, as well as for counts of erythrocytes and leucocytes. In addition, the methaemoglobin concentrations were determined in the samples collected at 80 wk. Preparations for counting the reticuiocytes and the different types of leucocytes were made but, in the absence of consistent effects on the other measurements, these counts were not carried out.

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters were examined: No data

URINALYSIS: Yes
- Time schedule for collection of urine: At 28 wks at 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels (0.5 and 1.0%) of Black PN.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters were examined: protein, reducing substances, bile salts and blood as well as for colour, pH and microscopic constituents

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The animals were killed by exsanguination from the aorta under sodium pentobarbitone anaesthesia following an overnight period without food. At autopsy, macroscopic abnormalities were recorded and the brain, heart, liver, spleen, kidneys, adrenal glands and gonads were weighed.

HISTOPATHOLOGY: Yes, the brain, heart, liver, spleen, kidneys, adrenal glands and gonads alongwith salivary glands, pituitary, thyroid, thymus, various lymph nodes, pancreas, urinary bladder, lungs, stomach, duodenum, ileum, colon, caecum, rectum, striped muscle (hind limb), spinal cord, uterus, aortic arch and any other tissue that appeared abnormal were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin. All tissues from the control mice and from those fed diet containing 1% Black PN were examined histologically. At the lower dose levels, the examination was confined to the liver, kidney
and any tissues seen to be abnormal at autopsy.

Other examinations:

Carcinogenic examination

Statistics:

Chi-square test, Student's t test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The ingestion of Black PN had no effect on the condition or behaviour of the animals

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were deaths in all groups during the course of the study but at no time were there any statistically significant differences between the number of deaths in the control mice and those given Black PN

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the study the body weights of mice of both sexes were similar in all groups

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing 0.5 or 1.0% Black PN than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% Black PN in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the
females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0% Black PN were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80.

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormal constituents were detected in the urine from the control or treated mice.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1% Black PN. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight. By contrast, the relative brain weight of females fed 0.25% Black PN was higher than the control figure. Liver weights of female but not of male mice fed 0.25% Black PN were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels
of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25% Black PN.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The incidence of histological findings was similar in all groups of mice, including the controls. Similarly, most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels of Black PN, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (in a female on 0.1%), a uterine fibromyoma (0.1%) and a squamous-cell carcinoma of the skin (female, 0.5%).

The only tumour found at the highest dietary level without comparable control findings was a squamous cell carcinoma of the skin in a male mouse fed 1% Black PN.

However, the frequency of histopathological findings in the treated animals did not differ significantly from those in the controls. Hence, no relationship was obvious between these findings and treatment with Black PN.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 300 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effect were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The long term No observed adverse effect level (NOAEL) for Black PN in mice is considered to be 1300 mg/Kg bw/day.

Executive summary:

Repeated dose toxicity test were performed on mice with different concentrations from 0.1, 0.25,0.5 or 1.0% (130,325,650,1300 mg/kg bw/d)Black PN for 80 wk. 30 males and 30 females was used for the treatment and group of 60 mice of each sex as control.

The general condition and behaviour of the animals were observed frequently and any mouse that showed signs of ill-health was isolated, to be returned to its cage on recovery or to be killed if its condition deteriorated. The mice were weighed at the start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment. Blood sample were taken at wk 28 and 55.At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy. For haematology blood samples were collected and for urine sample from 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels of Black PN. Histopathology was also conducted.

The ingestion of Black PN had no effect on the condition or behaviour of the animals. There were deaths in all groups during the course of the study but at no time were there any statistically significant differences between the number of deaths in the control mice and those given Black PN. Throughout the study the body weights of mice of both sexes were similar in all groups. The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing 0.5 or 1.0% Black PN than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% Black PN in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0% Black PN were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80. No abnormal constituents were detected in the urine from the control or treated mice. There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1% Black PN. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight. By contrast, the relative brain weight of females fed 0.25% Black PN was higher than the control figure. Liver weights of female but not of male mice fed 0.25% Black PN were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25% Black PN. The incidence of histological findings was similar in all groups of mice, including the controls. Similarly, most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels of Black PN, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (in a female on 0.1%), a uterine fibromyoma (0.1%) and a squamous-cell carcinoma of the skin (female, 0.5%). The only tumour found at the highest dietary level without comparable control findings was a squamous cell carcinoma of the skin in a male mouse fed 1% Black PN. However, the frequency of histopathological findings in the treated animals did not differ significantly from those in the controls. Hence, no relationship was obvious between these findings and treatment with Black PN. Based on the considerations, the long term No observed adverse effect level (NOAEL) for Black PN in mice is considered to be 1300 mg/Kg bw/day.