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

Endpoint summary

Description of key information

CAS 3844 -45 -9 has been investigated in several studies, most of them being done prior to the introduction of GLP and harmonized testing guidelines. The European Food Safety Agency (EFSA) re-evaluated all available experimental data in 2010 ( EFSA Journal 2010;8(11):)1853) and for the use as a colorant of food stuff (E133) issued an acceptable daily intake of 6 mg/kg bw derived from a NOEL of 631 mg/kg bw reported in a chronic feeding study in rats (Borzelleca 1990). The EFSA evaluation contains all repeated-dose toxicity hazard information that was available for the EU REACH registration in 2017.

The high molecular weight and the octanol-water-partition coefficient indicate that the substance is unlikely to penetrate the skin.

An assessment of the inhalation route is not performed as the substance is not volatile and handled in a non-inhalable form (granules / water-based solutions).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Exposure started in-utero

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): FD&C Blue No. 1 (CAS 3844-45-9)
- Supplier: Hilton Davis Co., Cincinnati, Ohio, USA
- Analytical purity: 90%
- Impurities (identity): subsidiary colourings, volatile chlorides and suiphates, and uncombined intermnediates
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data, but storage conditions not considered critical
- Storage condition of test material: no data
- Other: The compound was certified by the US FDA

Species:

rat

Strain:

other: Charles-River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories,Wilmington, MA, USA
- Age at study initiation: 38 days at beginning of F0-phase
- Weight at study initiation: F1 generation started exposure at weaning
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): 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): 12/12

Other: For F1-generation a maximum of two rats per sex from each litter were selected.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.

Duration of treatment / exposure:

30 months F1 (plus in-utero phase) (with 10 animals per sex and dose for interim sacrifice after 12 months)
2 months (F0-generation)

Frequency of treatment:

daily

Dose / conc.:

1 000 ppm

Remarks:

0.1% in the diet

Dose / conc.:

10 000 ppm

Remarks:

1% (w/w) in the diet

Dose / conc.:

20 000 ppm

Remarks:

2% (w/w) in the diet

Remarks:

Doses / Concentrations:
0, 50, 514, 1072 mg/kg bw (calculated for males)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0, 62, 631, 1319 mg/kg bw (calculated for females)
Basis:
actual ingested

No. of animals per sex per dose:

60 (F0)
70 (F1)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: indicated to be based on existing studies

Two identical controi groups were used to account for random biological variation.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were morbidity, mortality and gross clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) and mean daily diet consumption calculated as g food/kg body weight/day: Yes:
- Determined weekly through die first 14 weeks, biweekly for week 16-26 and monthly thereafter

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

FOOD EFFICIENCY:
No data


OPHTHALMOSCOPIC EXAMINATION: Yes (after 3, 6, 12, 18 and 24 months af the chronic phase.)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months and before termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts. and erythrocyte morphology

CLINICAL CHEMISTRY: Yes (3, 6, 12, 18 and 24 months and before termination)
aspartate aminatransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein and creatinine

URINALYSIS: Yes (3, 6, 12, 18 and 24 months and before termination)
specific gravity, pH and presence of protein, glucose, ketones, bilirubin and occult blood, appearance (gross and microscopic)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

- Organ weights: brain, gonads, kidneys, liver, spleen and thyroid

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenal (twa), aorta (abdominal), bone and marrow (femur). blood smear, brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus. eye, (two. with optic nerve), heart (with coronar vesels), intestine (caecum, colon, duodenum and iLeum), kidneys (two). liver, lung and mainstem, subbronchi (lungs inflated with formalin), lymph nodes, mesenteric and mediastinal), mammary gland, (inguinal), nerve (sciatic). ovaries pancreas, pituitary, prostate, salivar gland (mandibular), seminal vesicles (two), skeietal muscle (biceps femoris), skin.spinal cord (cervical), spieen stomach, testes, with epididymides, tbymus. thyroid with parathyraid, trachea, urinary bladder (inflated with formalin), uterus, any tissue with gross changes of an uncertain nature together with an apparentdy normal section of thc same tissue, and any tissue masses or suspect tumours rogether with regional lymph nodes.

Other examinations:

F0-examinations:
Female rats were weighed an gestation days 0, 4, 14 and 21

Statistics:

The variances of thde two groups were tested tor equality using the F test (Gull 1978). lt the variances were equal, a standard independent two-sample test was used to -determiüne equality ot mneans. lt the variances differed, Welch's t-test was used to determine equal ity of means, using the Smith-Satterthwaite correction for unequal variances (Gill 1978). All tests were conducted at the 1.0% two-sided risk level. More detailed information is provided in Fd. Chem. Toxicol. 28, 221-234 (1990)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose group females starting week 102.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly increased at the high dose group

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Survival was decreased (P < 0.01) in die 2.0% females compared to controls.

There was blue staining of fur, feces and exposed skin.

Dose descriptor:

NOAEL

Effect level:

>= 2 other: % in the diet

Based on:

test mat.

Sex:

male

Basis for effect level:

other: ca 1000 mg/kg bw

Dose descriptor:

NOAEL

Effect level:

1 other: % in the diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: ca 630 mg/kg bw; 15% reduction in body weight (body weight affected after week 102).

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

other: presumably food efficiency

Organ:

other: no target organ identified

Treatment related:

yes

Dose response relationship:

yes

 After week 90, mean body weights of the 2.0% females began a steady downward trend that was statistically significant (P < 0.01) from week 102 until the end of the study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

631 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Toxicokinetic studies suggest that the substance is poorly taken up after ingestion. The colorant has a high molecular weight and is highly soluble in water.

Lack of significant uptake is consistent with the absence of a long-term human health hazard. High doses are osmolytically active which in oral dosing settings may result in diarrhea.

Additional information

The key study for chronic hazard assessment is a life-time feeding study in rats with dietary concentrations of 0.1, 1 and 2% (w/w) published by Borzelleca et al (1990). The study design includes the requirements of OECD testing guideline 453, and includes additional fertility and developmental toxicity elements. The chronic toxicity study started with in-utero exposure. Parental animals (each 60 males and females) were exposed via the diet during pre-mating, mating and pregnancy for a total period of 2 months. Then the offspring (70 males and 70 females) was weaned onto the test-material containing diet and kept on this diet for life-long exposure (30 months). For each ten male and female rats, an interim sacrifice was performed after 12 months. Calculated from the body weights, the doses for males were 0, 50, 514, 1072 mg/kg bw and those for females were 0, 62, 631, 1319 mg/kg bw.

 

During the study, there was blue staining of fur, feces and exposed skin which is caused by the blue color of the test substance. There were no adverse observations regarding clinical signs, mortality, urinalysis, haematology, clinical chemistry, ophthalmoscopy, organ weights and histopathology. High dose animals showed a slightly higher food consumption. After week 90, mean body weights of the 2.0% females began a steady downward trend that was statistically significant (P < 0.01) from week 102 until the end of the study; terminal body weights were 15% lower compared to control group animals. High dose females showed a reduced survival rate (10/70 versus 28/70 and 24/70 in two control groups) compared to the control group.

 

The same authors also published the results of a life-long feeding study in mice with 0.5, 1.5, and 5% (w/w) in the diet, but this study only included parameters related to carcinogenic properties. No adverse findings were noted in that study. Calculated for daily uptake per kg body weight, doses were 0, 661, 2064, 7354 mg/kg bw for males and 0, 819, 2562, 8966 mg/kg bw for females.

 

Brilliant Blue caused severe growth retardation in Wistar rats when added to a low fiber diet at a

level of 5% for 21 days (Tsujita et al.,1979). The authors showed that concurrent addition of dietary fiber from the roots of edible burdock completely protected against this toxic effect. That study was performed with five male rats per group and only investigated a few parameters. The growth retardation is considered to be the result of malnutrition as the ionic and non-absorbable substance has osmolytic activity and caused chronic diarrhea. The dose of 5% in the diet is excessive and above the limit doses for regulatory testing.

 

Limited investigations with beagle dogs were published by Hansen et al (1966). Five and three beagle dogs received a dietary level of 2% and 1% for one year, respectively. One dog at the top dose died after 17 days and another died after 46 weeks at the low dose, as a result of - in the view of the authors - intercurrent virus infections. No clinical signs, gross lesions or histological abnormalities observed were observed (Hansen et al.,1966). The doses correspond to ca 500 and 250 mg/kg bw per day.

The evaluation of the effects is consistent with the SCCS opinion by EFSA (2010). No additional experimental data on repeated dose toxicity could be found during the data search for the EU REACH registration.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

The NOAEL for chronic oral exposure in rats is 631 mg/kg bw.

Accordingly, there are no significant toxic effects at doses of less than 100 mg/kg bw upon subchronic oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.