Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from NTRL report

Data source

Reference
Reference Type:
other: Secondary Literature
Title:
Repeated dose oral toxicity study of the test chemical
Author:
NTRL report
Year:
1992
Bibliographic source:
NTRL Report

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
90 days oral toxicity study was performed to determine the toxic nature of the test chemical
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)
EC Number:
255-217-5
EC Name:
Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)
Cas Number:
41098-56-0
Molecular formula:
C40H44N12O18S6.6Na
IUPAC Name:
hexasodium 2,2'-(ethene-1,2-diylbis{(3-sulfonato-4,1-phenylene)imino[6-(diethylamino)-1,3,5-triazine-4,2-diyl]imino})dibenzene-1,4-disulfonate
Test material form:
liquid
Details on test material:
- Name of the test chemical: Hexasodium 2,2'-(vinylenebis((3-sulphonato-4,1-phenylene)imino(6-(diethylamino)-1,3,5-triazine-4,2-diyl)imino))bis(benzene-1,4-disulphonate)
- Molceular formula: C40H38N12O18S6.6Na
- Molecular weight: 1305.1422 g/mol
- Smiles: CCN(CC)c1nc(nc(n1)Nc2cc(ccc2S(=O)(=O)[O-])S(=O)(=O)[O-])Nc3ccc(c(c3)S(=O)(=O)[O-])/C=C/c4ccc(cc4S(=O)(=O)[O-])Nc5nc(nc(n5)N(CC)CC)Nc6cc(ccc6S(=O)(=O)[O-])S(=O)(=O)[O-].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+]
- Substance Type: Organic
- Physical State: Liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initation : Males (40 - 61 g) and females (37 - 59 g)
- Housing:the animals were housed in metal screen bottom cages five to a cage
- Diet : Stock diet ad libitum
- Water : ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):ca. 24°c

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: stock diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with stock diet at levels of 0, 0.2, 1.0 aud 5.0 % (corresponding to 0, 138, 762 or 4824 for males and 0, 135, 778 or 4471 mg/Kg/day for females).

DIET PREPARATION
- Rate of preparation of diet (frequency):The test chemical was thoroughly mixed into stock diet by means of a mechanical blender (type Lodige, Paderborn). The diets were freshly prepared once a fortnight and stored
- Mixing appropriate amounts with (Type of food): soybean oil meal 10, brewers' yeast 3, yellow maize 26.6, whey powder 2, Whole Wheat 36, calcium phosphate 0.5, fish meal 8
trace mineralized salt 0.5, meat scraps 4, vitamin preparation 3.4, grass meal 3, soybean oil 3
- Storage temperature of food: at room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Food
- Concentration in vehicle: 0, 0.2, 1.0 aud 5.0 % corresponding to 0, 138, 762 or 4824 for males and 0, 135, 778 or 4471 mg/Kg/day for females
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
No data
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.2 ,1.0 and 5.0 % (corresponding to 0, 138, 762 or 4824 for males and 0, 135, 778 or 4471 mg/Kg/day for females)

Basis:
nominal in diet
No. of animals per sex per dose:
0%: 10 males and 10 females
0.2%: 10 males and 10 females
1.0%: 10 males and 10 females
5.0 %: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the main study were based on dose range study conducted at dose levels of 0, 200, 1000 or 5000 mg/Kg/day for 17 days
- Rationale for animal assignment (if not random): The animals were divided according to body weight
- Fasting period before blood sampling for clinical biochemistry: No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
- Other: No data available
Positive control:
No data available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. General observations and mortality

BODY WEIGHT: Yes
- Time schedule for examinations:Body weights of the individual rats were recorded once a week

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, at weekly intervals
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

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: Yes / No / No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:The water consmnption of each group was determined only during the first week of the experiment

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 13
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: All animals
- Parameters checked in table [No.?] were examined. haemoglobin content, packed cell volume, red blood cell counts and total and differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data available
- Animals fasted: No data available
- How many animals: 5 males and 5 females of each group
- Parameters checked in table [No.?] were examined. SGPT, SGOTand SAP, G6Pase and G6PD

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: In the 14th week all surviving animals were killed by decapitation and examined microscopically for pathological changes. Heart, kidney, liver, spleen, brain, testicle/ovary, thymus, pituitary, thyroid and adrenal were weighed

HISTOPATHOLOGY: Detailed microscopic examination was carried out on all male and female rats. Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lungs, trachea. spinal cord, salivary glands, exorbital lacrimal gland, prostate, epididymis, seminal vesicle. coagulating gland, uterus, urinary bladder, skeletal muscle, femoral nerve, thoracic aorta, esophagus, stomach, duodenum, ileum, caecum, colon, pancreas, skin, axillary and mesenteric lymph nodes, preputial glands and bone marrow (sternum).

A number of sections of testicles were stained according to Von Gieson's method for connective tissue. In addition a number of selected ltidney paraffin sections were stained with PAS. Frozen kidney sections were stained with Sudan III.
Other examinations:
No data
Statistics:
No data available

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the 5% dose level all animals looked sick in the second week of the experiment. At week five, all animals of this group were dead, except for one female which succumbed in the 9th week.
Mortality:
mortality observed, treatment-related
Description (incidence):
At week five in 5% dose level, all animals of this group were dead, except for one female which succumbed in the 9th week. At lower feeding levels mortality did not occur and no abnormalities in appearance or behaviour were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 1% dose level body weights was slightly lower than those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was considerably diminished at the highest feeding level in either sex. At the 0.2 and 1% food intake was generally slightly lower than in controls. This effect was more pronounced at 1% than at 0.2 %.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency was considerably diminished at the highest feeding level in either sex. Food efficiency was slightly diminished at 1%, but not at 0.2%.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption for each unit food consumed was distinctly increased at the 5% feeding level in either sex
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the 1 % feeding level in males the percentage of' neutrophils was significantly increased, whereas the percentage of lymphocytes was significantly decreased. White blood cell counts were relatively high at 1 % but not significantly different from the controls. Packed cell volume was slightly, though significantly, increased at the 0.2% level in males, but no dose-relationship was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
SGOT activity of females yas increased and SAP activity of males was decreased at 1% dose level. A decreased SGOT activity in males fed on 0.2% test chemical was not observed at the higher feeding level and is therefore considered incidental.
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):
The relative weights of the kidney and the liver and of several other organs were considerably increased at 1% in both sexes. Testicle weight was. however, decreased. at the 0.2% dose level the organ-to-body-weight ratios of' all organs were normal in males and females as well.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Large and pale kidneys were observed at 1% in both sexes. Most of the male rats in this group had strikingly small testicles. Other lesions noted at autopsy, such as unilateral hydronephrocis, early signs of murine pnuemonia, and proteinaceous plugs in urinary bladder, are frequently encountered in the strain of rats used. They may, therefore, not be related to the ingestion of the test compound
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At microscopic examination treatment related pathological. changes were noticed in the kidney and the testicle.
KIDNEY
Necrosis, swelling, irregular vacuolization (Sudan III negative) and granular deterioration (eosinophylic, PAS-negative granules) of tubular epithelial cells were seen in the renal cortex of all male and female rats at the 1% dose level. Sloughed necrotic tubular material was often found in tubular lumens. The nuclei of viable tubulur cells varied considerably in size and chromatin content. Dilated tubules lined by flattened epithelium and filled with proteinaceous casts were seen in most animals. At the 0.2% level no signs of toxic renal damage were noticed.
Testis:
A moderate degree of bilateral testicular atrophy vas present in all males at 1% test chemical. A number of seminiferous tubules only contained
Sertoli cells and occasional spermatogonia containing an irregularly vacuolated cytoplasm, Partial inhibition of spermatogenosis occurred in other tubules in which many polynucleated giant cells vere seen. Varying numbers of seminiferous tubules still showed a normal development sperm cells. In the epididymic tubtlies cellular debris, scattered spermatozoa and a few giant cells were found. Testicles of rats at the 0.2% level were quite normal and indistinguishable from those of the controls.
Other histopathological changes, as signs of chronic respiratory disease, slight focal myocarditis, proteinaceous plugs and Trichosomoides parasites in the urinary bladder, colloid cysts in the anterior lobe of the Pituitary, slight prostatitis, slight pyelitis and unilateral hydronephrosis, nephrocalcinosis, focal accumulations of RES-cells and slight periportal lymphocytic infiltrates in the liver, were found in a single animal or were about equally distributed between the control group and the test groups. Moreover, these abnormalities are comnon findings in rats and therefore may be considred as non treatment related.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Liver enzymology: There was a slight decrease in G6Pase activity with increasing levels of the test chemical in females only. When the enzyme activity is expressed per unit body weight the figures show a slight tendency to increase with increasing levels of the test chemical in males, while in females there are no distinct differences between the groups.

G6PD activity determinations showed almost the same picture: no distinct enzyme activity changes in males and a very slight decrease in enzyme activity with increasing levels of Tinopal in females. Here, too, this decrease disappeared when the enzyme activity was expressed per unit body weight (as a result of the increase in relative liver weight).

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
138 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
135 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
haematology
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL (no observed adverse effect level ) for the test chemical was considered to be at dose level of 0.2% (138 mg/Kg/day for males and 135 mg/Kg/day for females) when rats were exposed to the test chemical for 90 days.
Executive summary:

Subchronic repeated dose oral toxicity study was performed to determine the toxic nature of the test chemical. The study was performed using male and female Wistar albino rats for 90 days. The chemical was given by diet to 10 newly weaned rats per sex per dose level at 0, 0.2, 1.0 and 5% for 90 days. Body weights were recorded once a week and food intake bi-weekly. Haematological data were collected from all animals at the 13thweek of study. The hematologic parameters included haemoglobin content, packed cell volume, red blood cell counts and total and differential white blood cell counts. At the 14thweek of study, all surviving animals were sacrificed and examined for gross pathological effects. Serums samples from selected animals (5/sex/dose) were prepared on the 14thweek of study to determine enzyme activity levels. Each animal was subjected to a detailed microscopic examination. Tissue samples from the following organs were examined grossly and microscopically: lung, trachea, spinal cord, salivary glands, exorbital lacrimal gland, prostate, epididymides, seminal vesicles, coagulating gland, uterus, urinary bladder, skeletal muscle, femoral nerve, thoracic aorta, oesophagus, stomach, duodenum, ileum, caecum, colon, pancreas, skin, axillary and mesenteric lymph nodes, preputial gland and bone marrow. All animals treated at 5.0% died before the 9thweek of study. No deaths were observed at 0, 0.2 or 1.0%. No abnormal behaviour was observed. The average terminal body weight of male rats treated at 1.0% was 32% lower compared to the controls. The average terminal body weight of female rats treated at 1.0% was 27% lower compared to the controls. The terminal body weights of male and female rats treated at 0.2% were slightly (but not statistically) lower compared to the control data. Reduced food intake was observed at≥0.2% and reduced food intake at 1.0%. Noteworthy changes in haematology included lower lymphocyte and higher neutrophils counts in male rats treated at 1.0%. Noteworthy changes in clinical chemistry included increased SGOT activity in female rats treated at 1.0%. The relative weights of several organs were significantly increased at 1.0% compared to the control data. Male rats treated at 1.0% showed considerably lower (relative) testicular weights compared to the control group. Female rats treated at 1.0% showed somewhat lower (relative) ovary weights compared to the control group. No organ weight changes were observed at 0.2%. Gross examination revealed enlarge, pale kidneys in all male and female rats treated at 1.0%. In addition, male rats treated at 1.0% showed markedly small testicles compared to the controls. Microscopic findings were limited to histopathological effects on the kidneys and testes at 1.0%. Histopathological changes of the kidneys included (but were not restricted to) necrosis swelling, irregular vacuolization and granular deterioration of tubular cells in the renal cortex. These findings were present in all male and female rats treated at 1.0%. In the testes, a moderate degree of bilateral testicular atrophy was observed in all males treated at 1.0%. A number of seminiferous tubules only contained Sertoli cells and occasional spermatogonia containing an irregular vacuolated cytoplasm. Some tubules displayed partial inhibition of spermatogenesis with many polynucleated giant cells. Varying numbers of seminiferous tubules still showed normal sperm cell development. Cellular debris, scattered spermatozoa and a few giant cells were also seen in the epididymic tubules. Testicles of rats treated at 0.2% were indistinguishable from the controls.It should be stressed that testicular effects at the 1.0% dose level were observed in a group of rats that had lost a substantial amount of weight at terminal sacrifice (>30%) compared to the controls. This effect was accompanied by a number of other effects such as decreased food intake and reduced food efficiency. The current understanding from the literature is that decreased body weight gain and reduced food intake in rats is expected to result in decreased testosterone levels due to decreased GnRH release. In turn, decreased testosterone levels in rats is associated with a number of morphological changes of the testes and epididymides. Such morphological changes include degeneration/apoptosis of germ cells, spermatid retention, atrophy of Leydig cells, and apoptosis of epithelial cells. Considering the marked difference in terminal body weight between the 1.0% dose group and the controls, together with the effects on food intake and food efficiency, it can be suspected that the testicular effects at 1.0% were secondary to systemic toxicity.No remarkable effects on prostate, seminal vesicles or uterus were found in the study at any dose level.

Categories Display