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-)

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

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.

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:

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

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

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.

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

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

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

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

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

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

Critical effects observed:

not specified

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 000 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

Data is from NTP report

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

Additional information

Repeated dose toxicity oral:

Data available for the target chemical was reviewed to determine the toxic nature of C.I. Direct blue 218 upon repeated exposure by oral route. The studies are as mentioned below:

 

14 days repeated dose toxicity study (NTP, 1994) in mice 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.

 

Another 14 days repeated dose toxicity study (NTP, 1994) was conducted in rats to determine the toxic nature of C.I. Direct blue 218. The study was performed using male and female F344/N rats and dosing the test chemical in feed at dose levels of0 , 1,000, 3000, 7000, 15000, or 30000 ppm (0, 100, 300, 700, 1500 or 3000 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.Rats exposed to 30,000 ppm had a blue stain around the mouth, eye, and nose, and rats exposed to 15,000 (1500 mg/Kg/day) and 30,000 ppm (3000 mg/Kg/day) excreted blue feces. Male and female rats exposed to30,000 ppm (3000 mg/Kg/day) lost weight during the study. Final mean body weights of other exposed groups of male and female rats were similar to those of the controls, although the mean body weight gain of male rats receiving 15,000 ppm (1500 mg/Kg/day) was lower than that of the controls. Feed consumption by male and female rats that received 30,000 ppm was less than that by controls and feed spillage was observed at this exposure level, apparently as a result of reduced palatability of the diet. Lower absolute organ weights in the 30,000 ppm (3000 mg/Kg/day) groups were attributed to lower body weights. The relative heart weights of 30,000 ppm males and females and the relative thymus weight of 30,000 ppm (3000 mg/Kg/day) males were significantly lower than those of the controls. At necropsy 15,000 and 30,000 ppm (1500 and 3000 mg/Kg/day) rats had mild to moderate blue discoloration of the skin and intestine. Based on the observations made, the No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 1500 mg/Kg/day for females and 700 mg/Kg/day for males.

 

In the same NTP report (1994), 13 weeks repeated dose toxicity study was conducted in mice 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 of 0 , 3000, 10000 or 20000 ppm (Males- 0, 400, 1500 or 3600 mg/Kg bw/day and females- 0, 400, 1800 or 4000 mg/Kg bw/day). The animals were observed for clinical signs, body weight, changes in body weight and food consumption, meatology and clinical chemistry and were subjected to gross pathology and histopathology. No animals died during the study period.No signs of clinical toxicity were noted. The fur, skin, and feces of alI exposed groups of mice were discolored blue.The final mean body weights and mean body weight gains of males and females that received 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) and males that received 10,000 ppm (1500 mg/Kg bw/day) were significantly lower than those of the controls. The final mean body weight of males that received 20,000 ppm (3600 mg/Kg bw/day) was 24% lower than that of the controls and the final mean body weight of females receiving 20,000 ppm (4000 mg/Kg bw/day) was 14% lower than that of the controls. Feed consumption by exposed mice was similar to that by the controls except in the 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) groups, where feed spillage was noted and slightly higher levels of feed consumption were recorded. Feed consumption by exposed mice was similar to that by the controls except in the 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) groups, where feed spillage was noted and slightly higher levels of feed consumption were recorded. The hematocrit, hemoglobin, mean erythrocyte volume, and mean erythrocyte hemoglobin values in males receiving 10,000 and 20,000 ppm (1500 and 3600 mg/Kg bw/day) were significantly lower than controls. These findings are indicative of a microcytic normochromic anemia and are suggestive of sequestration or a deficiency of iron, possibly secondary to extravascular hemolysis. Mean erythrocyte volume and mean erythrocyte hemoglobin values in female mice exposed to 10,000and 20,000 ppm (1800 or 4000 mg/Kg bw/day) were also significantly lower than those of the controls. Serum levels of alanine aminotransferase and sorbitol dehydrogenase in male and female mice receiving 10,000 and 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) were significantly higher than those of controls and are consistent with the hepatic injury observed microscopically. Most absolute and relative organ weight differences in the 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) groups were attributed to lower body weights. However, despite the lower body weights of the 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) groups, the absolute liver weights of mice in these groups were slightly greater and the relative liver weights were significantly greater than controls. This effect was attributed to the ingestion of C.I. Direct Blue 218. At necropsy, there was blue discoloration of the liver and kidneys of mice receiving 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females). The principal histologic lesions associated with the administration of C. I. Direct Blue 218 to mice occurred in the liver and spleen. Liver lesions including centrilobular hepatocyte hypertrophy, karyomegaly, multifocal individual hepatocyte necrosis, and oval cell proliferation were observed in most mice receiving 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females). The hepatic lesions in the 10,000 ppm (1500 and 1800 mg/Kg bw/day) groups were minimal and consisted only of hepatocyte hypertrophy and karyomegaly. Hypertrophy of hepatocytes was primarily centrilobular in distribution, but in some mice the change was more extensive and involved midzonal and, infrequently, periportal hepatocytes as well. The affected hepatocytes were enlarged with an increased amount of eosinophilic cytoplasm and contained enlarged nuclei (karyomegaly). Some nuclei were irregular rather than uniformly round and contained inclusions of invaginated cytoplasm. The individual cell necrosis was generally minimal in severity and involved a few individual hepatocytes or small groups of hepatocytes randomly distributed in the liver lobules. The macrophages or Kupffer cells containing intracytoplasmic yellow-green pigment were observed in the periportal areas and to a lesser extent in the hepatic sinusoids. Proliferation of oval cells was observed in male and female mice. The oval cells radiated from the portal areas of the lobule, separating and distorting the periportal hepatic cords. The oval cells had scant cytoplasm and small, dense, oval nuclei. While the spleens of all mice in each of the exposed and control groups contained hemosiderin-laden macrophages in the red pulp, the number of pigmented macrophages was increased in males and females receiving 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females). The difference between the exposed and control groups was easily discernable, but generally slight. The increased accumulation of hemosiderin in the spleen of 20,000 ppm (3600 and 4000 mg/Kg bw/day respectively for males and females) mice is consistent with an increased rate of destruction or lysis of erythrocytes and the hematology findings. Based on the observations made, the No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 1500 mg/Kg/day for males and 1800 mg/Kg bw females during the 13 week study period.

 

Also, 13 weeks repeated dose toxicity study (NTP, 1994) was conducted using rats to determine the toxic nature of C.I. Direct blue 218. The study was performed using male and female F344/N rats and dosing the test chemical in feed at dose levels of0 , 3000, 10000 or 20000 ppm (Males- 0, 200, 600 or 1300 mg/Kg bw/day and Females: 0, 200, 800 or 1400 mg/Kg bw/day). The animals were observed for clinical signs, body weight, changes in body weight and food consumption, meatology and clinical chemistry and were subjected to gross pathology and histopathology. No animals died during the study period.No signs of clinical toxicity were noted. Blue discoloration of the fur and skin occurred in all exposed rats. Male and female rats receiving 10,000 (600 and 800 mg/Kg bw/day for males and females respectively) and 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) excreted blue feces.The final mean body weights and the mean body weight gains of male and female rats receiving 20,000 ppm (1300 and 1400 mg/Kg bw/day respectively) were significantly lower than those of the controls. The final mean body weight of male rats that received 20,000 ppm (1300 mg/Kg bw/day) was 24% lower than that of the controls and the final mean body weight of female rats that received 20,000 ppm (1400 mg/Kg bw/day) was 15% lower than that of the controls. The final mean body weights of males and females receiving 10,000 ppm (600 and 800 mg/Kg bw/day respectively) were 4% lower than those of the controls. Feed consumption by rats in exposed groups was similar to that by the controls except in the 20,000 ppm (1300 mg/Kg/day) groups, where feed spillage was noted. The hematocrit, hemoglobin, mean erythrocyte volumes, and mean erythrocyte hemoglobin in male and female rats that received 10,000 (600 and 800 mg/Kg bw/day for males and females respectively) and 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) were significantly lower than those of controls. These findings are indicative of a microcytic normochromic anemia and are suggestive of sequestration or a deficiency of iron, possibly secondary to extravascular hemolysis. Serum levels of alanine aminotransferase and sorbitol dehydrogenase in male and female rats receiving 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) were significantly higher than those of controls, which is consistent with hepatocellular injury. The absolute and relative kidney weights of male and female rats exposed to 10,000 (600 and 800 mg/Kg bw/day for males and females respectively) and 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) were significantly greater than those of the controls. The lower absolute liver, lung, thymus, and testis weights of 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) rats were probably related to the lower body weights that occurred in these groups. The principal histologic lesions associated with the administration of C.I. Direct Blue 218 for 13 weeks occurred in the liver of males and the liver and kidney of females. The hepatic lesions occurred primarily in males and females receiving 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) and in males receiving 10,000 ppm (600 and 800 mg/Kg bw/day for males and females respectively). The hepatic lesions were also slightly more frequent and severe in males than in females. The overall severity of the lesions was generally minimal. The most consistent finding in the liver of male and female rats was the presence of macrophages, presumably Kupffer cells, containing pale yellow, green intracytoplasmic pigment. The distribution of the pigmented macrophages was primarily periportal, but scattered pigmented macrophages were also randomly distributed along the sinusoids. The location and staining characteristics of the pigment are consistent with iron porphyrin-containing pigment possibly resulting from the lysis of erythrocytes. The principal lesions involving hepatocytes consisted of minimal individual cell necrosis and increased numbers of binucleated or multinucleated cells. The individual cell necrosis usually involved a single or, less frequently, small groups of hepatocytes scattered randomly throughout the liver lobules. Infrequently the necrotic cells were surrounded by a few mononuclear inflammatory cells. The binucleated and multinucleated hepatocytes were also randomly distributed within the lobules. The multinucleated cells were greatly enlarged and contained four to eight nuclei per cell. Minimal bile duct hyperplasia and periportal chronic inflammation were also observed in some 20,000 ppm male and female rats (1300 and 1400 mg/Kg bw/day for males and females respectively). The affected portal areas were generally infrequent and randomly distributed. They contained increased profiles of small ductules and infiltrates of mononuclear inflammatory cells. The kidneys of male and female rats recieving 20,000 ppm (1300 and 1400 mg/Kg bw/day for males and females respectively) also contained pale, yellow-green, slightly granular pigment within the cytoplasm of the proximal convoluted tubule epithelium. Hemoglobin resulting from intravascular hemolysisis known to be excreted by the renal glomerulus and absorbed by the proximal convoluted tubule epithelium. Microconcretions, consisting of intratubular, lamellated concretions of mineral at the corticomedullary junction were observed in most control and 20,000 ppm (1400 mg/Kg bw/day) female rats, but the number of micro concretions in the kidney sections was increased in females in the 20,000 ppm (1400 mg/Kg/day) group. The mineral concretions were not associated with necrosis, inflammation, or other evidence of a host response. The distribution, histologic appearance, and sex difference observed in this study are similar to that reported to occur spontaneously. Minimal to mild degeneration of the seminiferous epithelium was observed in the testes of seven of the 10 male rats receiving 20,000 ppm (1300 mg/Kg bw/day). The minimal lesions involved one or a few tubules while the mild lesions involved up to 25% of the tubules present. The affected seminiferous tubules exhibited decreased cellularity due to necrosis and loss of the germinal epithelium, and some contained small amounts of amorphous eosinophilic cellular debris or small concretions of mineral. Occasional multinucleated cells, representing fused spermatids, were seen in tubule lumens. Based on these considerations, No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 200 mg/Kg/day for males and females during the 13 week study period.

 

2 years repeated dose toxicity study (NTP, 1994) was conducted using rats to determine the toxic nature of C.I. Direct blue 218. The study was performed using male and female F344/N rats and dosing the test chemical in feed at dose levels of0 , 1000, 3000 or 10000 ppm (Males- 0, 40, 120 or 440 mg/Kg bw/day and Females: 0, 50, 140 or 470 mg/Kg bw/day) for 103 weeks. The animals were observed for clinical signs, body weight, changes in body weight and food consumption, hematology and clinical chemistry and were subjected to gross pathology and histopathology.Survival of female rats receiving 10,000 ppm (470 mg/Kg bw/day) was slightly, but not significantly, lower than that of controls. Survival of males that received 10,000 ppm (440 mg/Kg bw/day) and males and females that received 1,000 or 3,000 ppm (Males: 40 or 120 mg/Kg bw/day for males and 50 or 140 mg/Kg bw/day for females) was similar to that of controls. No treatment related clinical signs were observed. Mean body weights of male and female rats in the 10,000 ppm (440 mg/Kg bw/day for males and 470 mg/kg bw/day for females) groups were approximately 5% to 14% lower than controls after week 15, and the final mean body weights of male and female rats receiving 10,000 ppm (440 mg/Kg bw/day for males and 470 mg/kg bw/day for females) were 11% and 9% lower than those of the controls. Feed consumption was similar for male and female rats as compared to controls. The neutrophil counts of males that received 3,000 and 10,000 (120 or 440 mg/Kg bw/day) ppm were significantly lower than those of the controls. The hematocrit, hemoglobin, mean erythrocyte volume, and mean erythrocyte hemoglobin values of females receiving 10,000 ppm were significantly lower than those of the controls. In males that received 10,000 ppm (440 mg/Kg bw/day), only the mean erythrocyte hemoglobin value was significantly lower than that of the controls. Serum levels of alanine aminotransferase and sorbitol dehydrogenase in males and females receiving 10,000 ppm (440 and 470 mg/Kg bw for males and females respectively) were slightly but significantly higher than those of the controls. While these hematology and clinical chemistry differences were considered chemical related, they were slight and not clinically important. Squamous cell papilloma occurred with a significantly increased incidence in pharynx of male rats that received 10,000 ppm (440 mg/Kg bw/day) and exceeded the NTP historical incidence for this neoplasm in control male rats (10/1,253, 0.8%; range 0%-4%). The incidence of squamous cell papilloma in exposed female rats was not significantly increased. In addition, two male rats receiving 10,000 ppm (140 mg/Kg bw/day) had focal squamous epithelial hyperplasia of the mucosa in the posterior pharynx. A squamous cell carcinoma occurred in one male rat that received 10,000 ppm (440 mg/Kg bw/day), while another had a asosquamous tumor. Squamous cell papillomas were observed in the foresetomach of two males receiving 3,000 ppm (120 mg/Kg bw/day) and in one male and one female receiving 10,000 ppm (440 or 470 mg/Kg bw/day). No papillomas were observed in control rats. The only malignant epithelial neoplasm of the forestomach, a squamous cell carcinoma, was seen in one 3,000 ppm (120 mg/Kg bw/day) male. While the basal cell hyperplasia was clearly associated with the ingestion of C.I. Direct Blue 218, it is not certain if the other proliferative lesions were chemical related. Although the incidences of focal squamous hyperplasia, squamous cell papilloma, or squamous cell carcinoma were not significantly greater than those of the controls, they occurred only in rats receiving C.I. Direct Blue 218. Further, squamous cell neoplasms of the forestomach are relatively uncommon, and the incidence of three papillomas or carcinomas in the 3,000 ppm males (120 mg/Kg bw/day) exceeded the range in historical controls (4/1,253, 0.3%; range 0%-2%). Therefore, the low incidences of squamous hyperplasia or squamous cell papilloma in the exposed males may have been chemical related. The incidences of uterine endometrial stromal polyps were significantly increased in exposed female groups (0 ppm, 1/50; 1,000 ppm, 12/50; 3,000 ppm, 10/50; 10,000 ppm, 0/50;). However, the control incidence was abnormally low, the incidences did not increase with dose, and the incidences were within the NTP historical range for this neoplasm (205/1, 251,) 6%; range 2%-30%). Based on these considerations, the No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 140 mg/Kg bw/day in females and 40 mg/kg bw for male rats during the 2 years study period.

 

Another 2 years repeated dose toxicity study in mice 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, 1000, 3000 or 10000 ppm (Males- 0, 120, 360 or 1520 mg/Kg bw/day and Females: 0, 140, 470 or 2050 mg/Kg bw/day) for 103 weeks. The animals were observed for clinical signs, body weight, changes in body weight and food consumption, hematology and clinical chemistry and were subjected to gross pathology and histopathology. No treatment related clinical signs were observed. Survival of exposed male and female mice was similar to that of the controls. Mean body weights of male and female mice receiving 10,000 ppm (1520 mg/Kg bw/day and 2050 mg/Kg bw/day) were 10% to 29% lower than those of the controls during most of the study, and the final mean body weights in these groups were 19% lower than that of the controls for males and 27% lower than that of the controls for females. Feed consumption by exposed mice was similar to that by controls. Hematocrit, hemoglobin, and mean erythrocyte volume values in males and females receiving 10,000 ppm (1520 mg/kg bw/day) was significantly lower than those of the controls. Serum levels of alanine aminotransferase and/or sorbitol dehydrogenase values in male and female mice that received 10,000 ppm (1520 and 2050 mg/Kg bw/day for males and females respectively) were significantly higher than those of controls, which is consistent with hepatocellular damage. The administration of C.I. Direct Blue 218 to mice produced significantly increased incidences of hepatocellular adenoma (0 ppm, 16/50; 1,000 ppm, 19/50; 3,000 ppm, 17/50; 10,000 ppm, 40/50) and hepatocellular carcinoma (7/50, 3/50, 8/50, 17/50) in males receiving 10,000 ppm, and a significantly increased incidence of hepatocellular adenoma in females receiving 3,000 or 10,000 ppm (7/49, 12/50, 17/49, 41149). In females that received 10,000 ppm, the incidence of hepatocellular carcinoma was marginally increased. Consistent with these findings, the incidence of hepatocellular foci of cytologic alteration, a preneoplastic lesion, was also increased in males and females in the 10,000 ppm groups. The increased incidences of hepatocellular foci, adenomas, and carcinomas were considered chemical related. Uncommon renal tubule neoplasms also occurred at low incidences in male mice receiving C.I. Direct Blue 218, but not in controls. Renal tubule adenomas were seen in two males receiving 1,000 ppm, one male receiving 3,000 ppm, and one male receiving 10,000 ppm. A renal tubule carcinoma was also seen in one male that received 1,000 ppm. Because renal tubule neoplasms are uncommon in male mice (4/1,366,0.3%; range 0%-2%), these neoplasms may have been chemical related. Carcinomas of the small intestine occurred in four male mice receiving 10,000 ppm. One was observed at the 15-month interim evaluation, while the other three were observed in mice at the end of the study. One control male mouse also had a carcinoma of the small intestine. Because of the uncommon occurrence of small intestine neoplasms in untreated male mice (12/1,374, 0.9%; range 0%-4%), the slightly higher incidence of these neoplasms in males receiving 10,000 ppm may have been chemical related. Carcinomas of the small intestine also occurred in one 3,000 ppm and one 10,000 ppm female, but the low incidences precluded drawing an association with chemical administration. Based on the observations made, the No observed adverse effect level (NOAEL) for C.I. Direct blue 218 is considered to be 140 mg/Kg bw/day in females and the low observed adverse effect level is 120 mg/kg bw for male mice during the 2 years study period.

 

Based on the majority of the data available for the target chemical, C. I. Direct blue 218 is not likely to be a toxicant upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the majority of the data available for the target chemical, C. I. Direct blue 218 (CAS no 28407 -37 -6) is not likely to be a toxicant upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.