p,p',p''-tris(diethylamino)trityl alcohol

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:

other: Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Lifetime toxicity/carcinogenicity studies of FD & C BLUE No. 1 (BRILLIANT BLUE FCF) in rats and mice

Author:

J. F. Borzelleca, K. Depukat and J. B. Hallagan

Year:

1990

Bibliographic source:

Food Chem. Toxicol. Vol. 28, No. 4, pp. 221-234, 1990 ;

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Combined Repeated dose carcinogenicity study was performed to evaluate the toxic nature of Brilliant Blue FCF.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

EC Number:

223-339-8

EC Name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Cas Number:

3844-45-9

IUPAC Name:

disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)benzenesulfonate

Details on test material:

- Name of test material: FD & C BLUE No. 1 (Brilliant blue FCF)
- Molecular formula: C37H36N2O9S3.2Na
- Molecular weight: 792.8586 g/mol
- Smiles notation: C(=C1\C=C\C(=[N+](\Cc2cc(ccc2)S(=O)(=O)[O-])CC)C=C1)(/c1c(cccc1)S(=O)(=O)[O-])c1ccc(cc1)N(Cc1cc(ccc1)S(=O)(=O)[O-])CC.[Na+].[Na+]
- Substance type: Organic
- Physical state: No data
- Analytical purity: 90%
- Impurities: 10% consisted of subsidiary colourings, volatile chlorides and sulphates, and uncombined intermediates.

Test animals

Species:

mouse

Strain:

other: Charles River CD-l, COBS (ICR-derived)

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: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Mice were housed individually in hanging wiremesh cages.
- Diet (e.g. ad libitum): basal diet of certified Purina Laboratory Rodent Chow No. 5001 (Ralston Purina Company, Inc., St Louis, MO) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): 40-60%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Basal diet(Purina Laboratory Rodent Chow No. 5001 (Ralston Purina Company, Inc., St Louis, MO))

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender and were prepared and presented weekly. Assays were performed to assess the homogeneity and stability of FD & C Blue No. l in the prepared diets before study initiation.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): basal diet of certified Purina Laboratory Rodent Chow No. 5001 (Ralsto Purina Company, Inc., St Louis, MO)
- Storage temperature of food: 20-21°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: No data
- 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:

Analyses of compound concentration were conducted weekly during the first 13 wk of study, and then monthly thereafter. Analyses of the basal feed
for heavy metals, chlorinated hydrocarbons, and aflatoxin were conducted on all lots of feed used during the study. These analyses demonstrated that the basal feed contained acceptably low levels of contaminants, that the diets were prepared properly, and that the dietary content of the test material was stable.

Duration of treatment / exposure:

24 months (104 weeks)

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0%, 0%, 0.5%, 1.5% or 5.0% (0, 0, 714.28, 2142.85 or 7142.85 mg/Kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Total: 600
0 mg/Kg bw/day: 60 males and 60 females
0 mg/Kg bw/day: 60 males and 60 females
714.28 mg/Kg bw/day: 60 males and 60 females
2142.85 mg/Kg bw/day: 60 males and 60 females
7142.85 mg/Kg bw/day: 60 males and 60 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): No data
- 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: at least twice daily (with a minimum of 5 hr between observations)
- Cage side observations checked in table [No.?] were included. morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily (with a minimum of 5 hr between observations); Detailed physical examinations for signs of toxicity and palpation for masses were conducted weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the first 14 wk, biweekly for wk 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly through the first 14 wk, biweekly for wk 16-26 and monthly thereafter
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

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: after 3, 6, 12, 18 and 24 months of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10/sex of each dose level
- Parameters checked in table [No.?] were examined. haemoglobin, haematocrit, total erythrocyte count, total and differential leucocyte counts, and erythrocyte morphology.

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, Autopsies were conducted on all animals that died spontaneously, were killed in a moribund condition, or were killed on schedule. Organ weights were recorded for the brain, gonads, kidneys, liver, spleen and thyroid, and relative organ weights were calculated.

HISTOPATHOLOGY: Yes, Histology was conducted on all animals from the two control groups and from the 7142.85 mg/Kg bw/day group. The
following tissues were examined histologically: Gall bladder, adrenal (two), aorta (abdominal), bone and marrow (femur), blood smear, brain (three sections: frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), oesophagus, eye (two, with optic nerve), heart (with coronary vessels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver, lung and mainstem bronchi (lungs inflated with formalin), lymph nodes (mesenteric and mediastinal), mammary gland (inguinal), nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivary gland (mandibular), seminal vesicles (two), skeletal muscle (biceps femoris), skin, spinal cord (cervical), spleen, stomach, testes with epididymides, thymus, thyroid with parathyroid, trachea, urinary bladder (inflated with formalin), uterus, any tissue with gross changes of an uncertain nature together with an apparently normal section of the same tissue, and any tissue masses or suspect tumours together with regional lymph nodes.

Other examinations:

No data

Results and discussion

Results of examinations

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY:
Clinical signs: A blue staining of the hair, exposed skin and faeces was observed at all treatment levels. Other physical observations were seen in similar incidence among control and treated groups and were considered common for this strain.

Mortality: Mortality and survival of rats was not affected by treatment

BODY WEIGHT AND WEIGHT GAIN: Group mean body weights for treated mice were slightly lower when compared with either control group at various intervals. Statistically significant decreases in body weight (P < 0.01) occurred at some intervals for males and females in the 2142.85 and 7142.85 mg/Kg bw/day groups. Statistical significance was mainly seen when values were compared with control group IB or with the combined control means.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Mean food consumption throughout the study remained similar for control and treated mice

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Haematological values were similar among control and treated mice throughout the study. Occasional statistically significant decreases in mean haemoglobin, haematocrit and leucocyte values of treated mice were noted, but were not considered toxicologically significant.

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOUR: No data

ORGAN WEIGHTS: No data

GROSS PATHOLOGY: No data

HISTOPATHOLOGY: NON-NEOPLASTIC A variety of microscopic lesions was observed at similar incidence in control and treated mice. The 7142.85 mg/Kg bw/day females exhibited an increased incidence of haemangioma of the spleen, but the increase was statistically significant (P < 0.01) only for the unadjusted trend analysis.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS: No data

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

7 354 other: mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No mortality; no effect on hematology, behavior, and morbidity; no carcinogenicity. at 5 % concentration (7354 mg/kg bw/day)

2

Dose descriptor:

NOAEL

Effect level:

8 966 other: mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No mortality; no effect on hematology, behavior, and morbidity; no carcinogenicity. at 5 % concentration (8966 mg/kg bw/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Survival and group mean body weights

Dietary concentration (mg/Kg bw/day)	No surviving at the end of the study (Total: 60)		Final body weight (Mean±SD)
	Males	Females	Males	Females
0	25	24	38±5	32 ± 5
0	23	31	38±3	33±4
714.28	25	31	36±5	31 ± 3
2142.85	28	28	35 ± 3	31 ± 4
7142.85	33	31	36 ± 4	29 ± 4

 

Table: Mean food and compound consumption

Dietary concentration (mg/Kg bw/day)	Food consumption (g/Kg/day)		Compound consumption (g/Kg/day)
	Males	Females	Males	Females
0	133±16	169±22	-	-
0	132±15	163±22	-	-
714.28	132±16	164±21	661±79	819±105
2142.85	138±17	171±24	2064±251	2562±357
7142.85	147±17	179±22	7354±836	8966±1115

Applicant's summary and conclusion

Conclusions:

In repeated dose chronic toxicity study dietary concentration of 5.0% (7354 mg/kg/day and 8966 mg/kg/day for male and female mice, respectively) FD & C Blue No. 1 to mouse for 104 weeks did not demonstrate consistent biologically significant, compound-related adverse effects on behavior, morbidity, mortality, haematology, general physical observations. Thus, the No Observed Adverse Effect Level (NOAEL) for chronic repeated dose toxicity by oral route was considered to be 7354 mg/kg/day and 8966 mg/kg/day for male and female mice, respectively.

Executive summary:

Combined Repeated dose carcinogenicity study was performed to evaluate the toxic nature of Brilliant Blue FCF. FD & C Blue No. 1 was fed to CD-1 mice as a dietary admixture in lifetime toxicity/carcinogenicity studies at dose levels of 0.0%, 0.0%, 0.5%, 1.5% or 5.0% (0, 0, 714.28, 2142.85 or 7142.85 mg/Kg bw/day) in a lifetime toxicity/ carcinogenicity study. The maximum exposure time was 104 wk for both males and females. Dietary concentration of 5.0% (7354 mg/kg/day and 8966 mg/kg/day for male and female mice, respectively) FD & C Blue No. 1 to mouse for 104 weeks did not demonstrate consistent biologically significant, compound-related adverse effects on behavior, morbidity, mortality, haematology, general physical observations. Thus, the no-observed-adverse-effect level (NOAEL) established in this study is a dietary concentration of 5.0% (7354 mg/kg/day and 8966 mg/kg/day for male and female mice, respectively).