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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1973

Report date:

1973

Materials and methods

Test guideline

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

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

Molecular formula:

C37 H34 N2 Na2 O9 S3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

further impurities not specified in detail

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

0.1% in the diet

Doses / concentrations 2

Dose / conc.:

10 000 ppm

Remarks:

1% (w/w) in the diet

Doses / concentrations 3

Dose / conc.:

20 000 ppm

Remarks:

2% (w/w) in the diet

Doses / concentrations 4

Remarks:

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

Doses / concentrations 5

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.

Examinations

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)

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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).

Target system / organ toxicity

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

Any other information on results incl. tables

 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.