Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting 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 NTP report

Data source

Reference

Reference Type:

review article or handbook

Title:

Toxicology And Carcinogenesis Studies Of Direct Blue 218 (Cas No. 28407-37-6) In F344/N Rats And B6C3F1 Mice (Feed Studies)

Author:

U.S. Department Of Health And Human Services

Year:

1994

Bibliographic source:

National Toxicology Program, NTP TR 430, NIH Publication No. 94-3161, 1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

14 days repeated dose toxicity study was conducted to determine the toxic nature of C.I. Direct blue 218

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

EC Number:

249-008-8

EC Name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Cas Number:

28407-37-6

Molecular formula:

C32H16Cu2N6O16S4.4Na

IUPAC Name:

Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)

Test material form:

other: amorphous powder

Details on test material:

Name of the test chemical: Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)
Common Name: C.I Direct Blue 218
IUPAC name: tetrasodium (3E)-5-amino-3-{2-[4-(4-{2-[(2E)-8-amino-1-oxo-3,6-disulfonato-1,2-dihydronaphthalen-2-ylidene]hydrazin-1-yl}-3- hydroxyphenyl)-2-hydroxyphenyl]hydrazin-1-ylidene}-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate dicopper
Molecular Formula: C32H20Cu2N6Na4O16S4
Molecular Weight: 1087.84 g/mol
SMILES Notation: c12c3c(c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O])cc2N)N=Nc1ccc(cc1O[Cu]O3)c1cc2c(N=Nc3c(cc4c(c3O[Cu]O2)c(cc(c4)S(=O) (=O)[O-])N)S(=O)(=O)[O-])cc1.[Na+].[Na+].[Na+].[Na+]
InChI: 1S/C32H24N6O16S4.2Cu.4Na/c33-19-11-17(55(43,44)45)5-15-9-25(57(49,50)51)29(31(41)27(15)19)37-35-21-3-1-13(7-23(21)39) 14-2-4-22(24(40)8-14)36-38-30-26(58(52,53)54)10-16-6-18(56(46,47)48)12-20(34)28(16)32(30)42;;;;;;/h1-12,39-42H,33-34H2,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;;;/q;2*+2;4*+1/p-8/b37-35-,38-36-;;;;;;
Substance Type: Organic
Physical State: Solid Deep purple to dark blue amorphous powder

Specific details on test material used for the study:

- Name of the test chemical: Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)
- Common Name: C.I Direct Blue 218
- IUPAC name: tetrasodium (3E)-5-amino-3-{2-[4-(4-{2-[(2E)-8-amino-1-oxo-3,6-disulfonato-1,2-dihydronaphthalen-2-ylidene]hydrazin-1-yl}-3- hydroxyphenyl)-2-hydroxyphenyl]hydrazin-1-ylidene}-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate dicopper
- Molecular Formula: C32H20Cu2N6Na4O16S4
- Molecular Weight: 1087.84 g/mol
- SMILES Notation: c12c3c(c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O])cc2N)N=Nc1ccc(cc1O[Cu]O3)c1cc2c(N=Nc3c(cc4c(c3O[Cu]O2)c(cc(c4)S(=O) (=O)[O-])N)S(=O)(=O)[O-])cc1.[Na+].[Na+].[Na+].[Na+]
InChI: 1S/C32H24N6O16S4.2Cu.4Na/c33-19-11-17(55(43,44)45)5-15-9-25(57(49,50)51)29(31(41)27(15)19)37-35-21-3-1-13(7-23(21)39) 14-2-4-22(24(40)8-14)36-38-30-26(58(52,53)54)10-16-6-18(56(46,47)48)12-20(34)28(16)32(30)42;;;;;;/h1-12,39-42H,33-34H2,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;;;/q;2*+2;4*+1/p-8/b37-35-,38-36-;;;;;;
- Substance Type: Organic
- Physical State: Dark blue solid
- Impurities (identity and concentrations): More than 9 minor components (>1%) were present in the dye and although it was not possible 10 determine the definite structure of these impurities, the reducible azo bond appeared 10 be present in the majoriIy of the impurities.

Test animals

Species:

mouse

Strain:

B6C3F1

Remarks:

F344/N

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 50 to 57 days old
- Weight at study initiation:
- Fasting period before study: No data
- Housing: Animals were housed 5/cage in polycarbonate cages (changed twice weekly) with heat-treated hardwood chips bedding (changes twice weekly) and reemay spun bonded polyester cage filters (changed once every 2 weeks) having Stainless steel racks (changed once/2 weeks)
- Diet (e.g. ad libitum): NIH-D7 open-formula mash diet ad libitum
- Water (e.g. ad libitum): Water was provided through Automatic watering system ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22˚C
- Humidity (%): 68%
- Air changes (per hr): 12 changes/hour
- Photoperiod (hrs dark / hrs light): Fluorescent light: 12 hours/day

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Because of limited solubility in water, the dye was administered to animals in feed.

Vehicle:

other: Feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed to give dose levels of 0, 1,000, 3000, 7000, 15000, or 30000 ppm (0, 200, 600, 1400, 3000 or 6000 mg/Kg/day). Dose formulations were prepared prior to the initiation and at the midpoint of the 14-day studies

DIET PREPARATION
- Rate of preparation of diet (frequency): Prior to the initiation and at the midpoint of the 14-day studies
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: 25˚C

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0 , 1,000, 3000, 7000, 15000, or 30000 ppm (0, 200, 600, 1400, 3000 or 6000 mg/Kg/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was confirmed and the stability of the dose formulations was established for 3 weeks when stored in the dark at temperatures up to 25° C and for 1 week when stored open to air and light. Periodic analyses of the dose formulations of C.I. Direct Blue 218 were conducted at the study laboratory and at the analytical chemistry laboratory using visible spectroscopy. For the 14-day studies, the dose formulations were analyzed prior to study
initiation

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0 , 1,000, 3000, 7000, 15000, or 30000 ppm (0, 200, 600, 1400, 3000 or 6000 mg/Kg/day)

No. of animals per sex per dose:

Total: 30 males and 30 females
0 mg/Kg/day: 5 males and 5 females
200 mg/Kg/day: 5 males and 5 females
600 mg/Kg/day: 5 males and 5 females
1400 mg/Kg/day: 5 males and 5 females
3000 mg/Kg/day: 5 males and 5 females
6000 mg/Kg/day: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Distributed using a table of random numbers.
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included. Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, weekly, and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, once a week
- 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: No data
- 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. No data

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

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: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The brain, heart, right kidney, liver, lung, right testis, and thymus were weighed.

HISTOPATHOLOGY: Yes, Complete histopathologic examinations were performed on 30,000 ppm (3000 mg/Kg/day) animals. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, epididymis, esophagus, gall bladder (mice), large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum) heart, kidney, liver, lung, mammary gland, mandibular lymph node, mesenteric lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, sternebrae, stomach (forestomach and glandular), testes, thyroid gland, trachea, thymus, urinary bladder, and uterus. In addition, the liver, thymus and gallbladder of all mice was also examined.

Other examinations:

No data

Statistics:

No data

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Blue discoloration of the feces was observed in all exposed groups of male and female mice. Male and female mice of the 30000 ppm (6000 mg/Kg/day) appeared emaciated and hyperactive during the last week of the study

Mortality:

no mortality observed

Description (incidence):

No death was noted during the study period

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice exposed to 30,000 ppm (6000 mg/Kg/day) lost weight. The final mean body weights of other exposed groups were similar to those of the controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption by exposed groups was similar to that by controls, except for the last week of the study, when the apparent amount of feed consumed by the 15,000 and 30,000 ppm groups was greater than that consumed by the controls. This increased feed consumption was related to increased feed spillage in these exposure groups.

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male mice that received 15,000 ppm (3000 mg/Kg/day), the absolute and relative liver weights were significantly greater than those of the controls, and in males exposed to 30,000 ppm (6000 mg/Kg/day) and females exposed to 15,000 and 30,000 ppm (3000 and 6000 mg/Kg/day) the relative liver weights were significantly greater than those of controls. In the 30,000 ppm groups (6000 mg/Kg/day), there were some significantly lower absolute organ weights which were attributed to the lower body weights. The relative heart weight of 30,000 ppm (6000 mg/Kg/day) females and the relative thymus weights of 30,000 ppm (6000 mg/Kg/day) males and females were significantly lower than those of the controls

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the skin and gastrointestinal tracts of exposed male and female mice had blue discoloration

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No data

Effect levels

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant toxic effects were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Survival, Mean Body Weights, and Feed Consumption of Mice in the 14-Day Feed Study of C.I. Direct Blue 218

Dose (ppm)	Survival	Mean Body weight			Final weight relative to controls (%)	Feed consumption
		Initial	Final	Change		Week 1	Week 2
Male
0	5/5	22.3±0.9	24.0±0.9	17±0.1		2.4	2.6
1000	5/5	22.3±0.7	24.5±0.9	2.2±0.2	102	2.8	2.8
3000	5/5	22.1±0.7	24.2±0.7	2.1±0.2	101	2.6	2.5
7000	5/5	22.0±1.0	23.9±0.8	1.9±0.5*	100	2.6	2.9
15000	5/5	22.0±0.9	22.3±1.4	0.3±0.6**	93	3.4	5.0^d
30000	5/5	22.1±0.9	17.9±0.8**	-4.2±0.3**	75	4.0	5.6^d
Females
0	5/5	17.5±0.4	18.8±0.4	1.2±0.2		2.2	1.8
1000	5/5	17.4±0.7	18.3±0.5	0.9±0.3	98	2.1	2.4
3000	5/5	17.8±0.4	18.8±0.2	1.0±0.3	100	2.5	2.2
7000	5/5	17.6±0.8	18.7±0.8	1.1±0.2	100	2.7	2.7
15000	5/5	17.1±0.4	18.0±0.3	0.9±0.4	96	3.1	3.9^d
30000	5/5	17.9±1.0	15.1±0.8**	-2.8±0.4**	80	3.6	4.7^d

 

* Significantly different (P≤O.05) from the control group by Williams' or Dunnett's test

** P≤0.01

Applicant's summary and conclusion

Conclusions:

The No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 3000 mg/Kg/day for males and females B6C3F1 mice.

Executive summary:

14 days repeated dose toxicity study was conducted to determine the toxic nature of C.I. Direct blue 218. The study was performed using male and female B6C3F1 mice and dosing the test chemical in feed at dose levels of0 , 1,000, 3000, 7000, 15000, or 30000 ppm (0, 200, 600, 1400, 3000 or 6000 mg/Kg/day). The animals were observed for clinical signs, body weight, changes in body weight and food consumption and were subjected to gross pathology and histopathology. No animals died during the study period.Blue discoloration of the feces was observed in all exposed groups of male and female mice. Male and female mice of the 30000 ppm (6000 mg/Kg/day) appeared emaciated and hyperactive during the last week of the study. Male and female mice exposed to 30,000 ppm (6000 mg/Kg/day) lost weight. The final mean body weights of other exposed groups were similar to those of the controls. Feed consumption by exposed groups was similar to that by controls, except for the last week of the study, when the apparent amount of feed consumed by the 15,000 and 30,000 ppm groups was greater than that consumed by the controls. This increased feed consumption was related to increased feed spillage in these exposure groups. In male mice that received 15,000 ppm (3000 mg/Kg/day), the absolute and relative liver weights were significantly greater than those of the controls, and in males exposed to 30,000 ppm (6000 mg/Kg/day) and females exposed to 15,000 and 30,000 ppm (3000 and 6000 mg/Kg/day) the relative liver weights were significantly greater than those of controls. In the 30,000 ppm groups (6000 mg/Kg/day), there were some significantly lower absolute organ weights which were attributed to the lower body weights. The relative heart weight of 30,000 ppm (6000 mg/Kg/day) females and the relative thymus weights of 30,000 ppm (6000 mg/Kg/day) males and females were significantly lower than those of the controls. At necropsy, the skin and gastrointestinal tracts of exposed male and female mice had blue discoloration. Based on the observations made, the No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 3000 mg/Kg/day for males and females B6C3F1 mice.