Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to a guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
not applicable
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-diaminostilbene-2,2'-disulphonate
EC Number:
230-847-3
EC Name:
Disodium 4,4'-diaminostilbene-2,2'-disulphonate
Cas Number:
7336-20-1
Molecular formula:
C14H14N2O6S2.2Na
IUPAC Name:
disodium 2,2'-ethene-1,2-diylbis(5-aminobenzenesulfonate)
Details on test material:
- Name of test material (as cited in study report): no data
- Substance type: organic
- Physical state: solid
- Analytical purity: 76%
- Impurities (identity and concentrations): water (approximately 14%), sodium chloride (approximately 6%), and 4,4’ -ethylene-2-dianaline sulfonic acid (approximately 4%)
- Composition of test material, percentage of components: main component (76%), water (approximately 14%), sodium chloride (approximately 6%), and 4,4’ -ethylene-2-dianaline sulfonic acid (approximately 4%).
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: Stability studies indicated that when stored protected from light, the bulk chemical was stable for 2 weeks at temperatures up to 60 degrees C.
- Storage condition of test material: Test material was stored in the dark, under refrigeration.

Test animals

Species:
mouse
Strain:
other: B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 6 weeks old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24°C
- Humidity (%): 50 +/- 15.2 %
- Air changes (per hr): 6-12
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): premix with test material and feed was prepared using a mortar and pestle. The premix and rest of the feed were layered into a Patterson-Kelly twin-shell blender and mixed for 20 minutes with an intensifier bar.
- Storage temperature of food: Test material was stored in the dark, under refrigeration


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the diet was tested by extracting feed with a buffered solution of methanol (pH 10.0 +/- 0.1), spinning the mixture in a centrifuge, and further diluting the extract with methanol. The absorbance of the samples was measured at 342 nm. For the stability studies, feed samples
were extracted with the same solution used in the homogeneity analysis. The extracts were mixed with methanol and spun in a centrifuge. An aliquot of the supernatant was injected into an HPLC system equipped with a uBondapak C18 column and a 340 nm detector. The mobile phase was 0.005 M tetrabutylammonium hydroxide in methanol (adjusted to pH 7.4 with phosphoric acid) and 0.005 M tetrabutylammonium hydroxide in water (with the same amount of phosphoric acid used to adjust the pH of the methanol solution. The ratio of the two solvents was 14:86 and the flow rate was 1.5 ml/min. Dose formulations were analyzed at least once every eight weeks to confirm stability.
Duration of treatment / exposure:
2 years
Frequency of treatment:
continious
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
6250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
12500 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment
Details on study design:
no data
The average amount of test material ingested by males from weeks 1-13 was 836 mg/kg/day for 6250 ppm and 1738 mg/kg/day for 12500 ppm. During weeks 14-104, the average amount of test material ingested by males was 763 mg/kg/day for 6250 ppm and 1565 mg/kg/day for 12500 ppm.
The average amount of test material ingested by females from weeks 1-13 was 997 mg/kg/day for 6250 ppm and 2081 mg/kg/day for 12500 ppm.
During weeks 14-104, the average amount of test material ingested by females was 666 mg/kg/day for 6250 ppm and 1444 mg/kg/day for 12500
ppm.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for 13 weeks, then monthly (or as necessary)


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at the beginning of the study, weekly for 13 weeks, monthly through week 90, and every 2 weeks thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: 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:


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsies were performed on all animals. All organs and tissues were examined for gross lesions and all major tissues were fixed and preserved
in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic
examination. Histopathologic examinations were performed on all 15- month interim animals, all tissues with grossly visible lesions, all animals that died or were killed moribund prior to 21 months, and all control and high dose animals that survived to study termination. Tissues examined included the adrenal gland, bone (costochondrial junction), bone marrow (femur), brain, esophagus, gallbladder, heart, kidney, large intestine, liver, lung, mammary gland, mesenteric lymph node, nasal cavity, ovary, pancreas, parathyroid, pituitary gland, prostate, salivary gland, seminal vesicle, skin, small intestine, spleen, stomach, testis (with epididymis), thymus, thyroid gland, trachea, urinary bladder, and uterus were fixed and examined microscopically. Tissues examined from low dose animals that died or were killed moribund after 21 months or at study termination were gross lesions, liver (males) and lung.
Other examinations:
After 15 months, 10 animals per sex from each group were euthanized for interim examinations. Blood was withdrawn from the orbital sinus plexus of all surviving animals for hematological (hemoglobin, hematocrit, erythrocyte count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, total and differential leukocyte counts) and clinical pathology analyses (blood urea nitrogen, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase). The brain, liver and right kidney of each animal were weighed.
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier. The possibility of a doserelated effect on survival was assessed using the methods of Cox and Tarone. Tumor incidence data were analyzed using a logistic regression analysis which assumed that the tumors were discovered as the result of death from an unrelated cause. The Fisher exact test and the Cochran-Armitage trend test were used to analyze the overall proportion of tumorbearing animals. Organ and body weight data were analyzed using the multiple comparison procedures of Williams and Dunnett. Clinical chemistry and hematology data were analyzed using the multiple comparison methods of Shirley and Dunn. Jonckheere’s test was used to assess the significance of dose-response trends.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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:
not specified
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
15 month examination: “There were no biologically significant absolute or relative organ weight, clinical pathology, or histopathology findings in mice administered disodium 4,4’-diamino-2,2’-stilbene disulfonate in feed for 15 months. “
Body weight, food consumption, survival and clinical findings: “Mean body weights were marginally decreased for high dose female mice. Food consumption by dosed mice was similar to food consumption by thecontrols throughout the studies. Survival was similar among control and treated grou ps of mice. No clinical findings related to chemical
administration were observed in mice.”
on-neoplastic and Neoplastic Effects: “There were no chemical-related increased incidences of neoplasm, non-neoplastic lesions, or other toxic effects in mice.”

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 12 500 ppm
Sex:
male/female
Basis for effect level:
other: Mean body weights were marginally decreased for high dose female mice.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There was no evidence of carcinogenic activity in rats receiving 6250 or 12500 ppm test material.

Categories Display