Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-08-23 to 1996-10-11
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Revised Japanese Chemical Substance Law (1987) according to the notification of Dec. 9, 1986 by EA, Environmental Agency (No. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014).
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
EC Number:
421-440-3
EC Name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
Cas Number:
778583-04-3
Molecular formula:
Hill formula: C47 H66 N12 O19 S2 CAS formula: C29 H21 N9 O10 S2 x 3 C6 H15 NO3
IUPAC Name:
tris(tris(2-hydroxyethyl)azanium) 2-(cyanoamino)-5-{2-[4-({4-[2-(6,8-disulfonaphthalen-2-yl)diazen-1-yl]-2-methoxyphenyl}carbamoyl)phenyl]diazen-1-yl}-6-hydroxypyrimidin-4-olate
Test material form:
solid: compact

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 125-181 g, Females: 98-129 g
- Housing: Groups of five in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba no. 343 rat maintenance diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1996-08-23 (delivery of the animals) To: 1996-09-27 (satellite groups: 1996-10-11)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: bi-distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a glass rod and a magnetic stirrer. Frequency of preparation: Daily, prior to each application.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test item is well soluble in water
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test article/vehicle mixtures were determined in samples taken during acclimatization and during week 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG by HPLC according to a method supplied by the sponsor.

STANDARD SOLUTIONS
Stock solutions of the test article in bi-distilled water/acetonitrile (1+1 v/v) with a concentration of 200 µg/mL were prepared. Standard solutions were prepared by respective dilution of these stock solutions with bi-distilled water/acetonitrile (1+1 v/v) to yield concentrations in the range from 20 to 100 µg/mL. These standard solutions as well as the stock solution were used to calibrate the HPLC.

ANALYSIS OF SAMPLES
The delivered samples were dissolved with about 70 mL of water/acetonitrile (1+1 v/v) by means of an ultrasonic bath. Then, the 100-mL volumetric flasks were filled to the mark with bi-distilled water/acetonitrile (1+1 v/v). Depending on the dose group, the latter sample solutions were further diluted with bi-distilled water/acetonitrile (1+1 v/v) to yield concentrations within the calibration range. Finally, a 10-µL aliquot was quantified by HPLC.

HPLC DETERMINATION
Typical Operating Conditions:
Apparatus: Merck L-6200 pump, Merck L-4000 UV detector, Merck D-2500 integrator, Merck AS-2000 sampling unit
Column: LiChrospher RP 18; 5 pm; 125 mm x 4.0 mm (i.d.)
Temperature: Room temperature
Eluent: Bi-distilled water 62%, Acetonitrile 38%, 1 g/L TBAP
Flow: 1.0 mL/min
Wave length: 230 nm
Injection volume: 10 µL

EVALUATION OF RESULTS
Injected samples were quantified by peak areas of the test article in counts with reference to the calibration curve. The latter was obtained by correlation of the peak areas of the test article with their corresponding concentrations of test article injected, using the following equation 1:
Y=a+b•X (1)
(where Y = µg/mL the substance in injected sample; a = y-axis intercept; b = slope; X = peak areas of injected sample in counts)

The concentrations of the substance in vehicle were calculated according to:
C = (Y•V•D•Q) / (W•1000)
(where C = Concentration of the substance in vehicle (mg/mL); Y = µg/mL of injected sample calculated by equation 1; V = Final volume (100 mL); D = Dilution factor; Q = Density of the test article/vehicle mixtures (assumed to be 1 g/L); W = Weight of sample (about 2 g, weighed to the third decimal place)).
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
TREATMENT GROUPS:
0 mg/kg bw/day (Control): 5 Males and 5 Females
50 mg/kg bw/day (Control): 5 Males and 5 Females
200 mg/kg bw/day (Control): 5 Males and 5 Females
1000 mg/kg bw/day (Control): 5 Males and 5 Females

RECOVERY GROUPS:
0 mg/kg bw/day (Control): 5 Males and 5 Females
1000 mg/kg bw/day (Control): 5 Males and 5 Females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon data from an acute toxicity study (RCC Project 632136) and the results of a 5-day dose-range-finding study (RCC Project 632193) in which the substance was administered by gavage at 0, 200 and 1000 mg/kg to 3 rats per group and sex. Relative liver weight of males at 200 mg/kg and animals of both sexes at 1000 mg/kg were statistically significant higher when compared with the controls (group 2 males 15%, group 3 males 15%, group 3 females 19%).
- Rationale for animal assignment (if not random): Computer-generated random algorithm.
- Rationale for selecting satellite groups: To assess the reversibility of treatment-related changes.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The food consumption was recorded weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the last day of the treatment and the recovery period, respectively
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks (recovery groups: after 6 weeks)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, for about 18h with free access to drinking water
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks (recovery groups: after 6 weeks)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, for about 18h with free access to drinking water
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks (recovery groups: after 6 weeks)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for about 18h with free access to drinking water
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Statistics:
The following statistical methods were used to analyze the body weights, organ weights all ratios as well as clinical laboratory data :
When the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the ophthalmoscopy data.

References:
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc. 50, 1096-1121 (1955).
R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
, but not related to treatment with test item
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
, but not related to treatment with test item
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
, but not related to treatment with test item
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
, but considered not of toxicological relevance
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
, but considered not of toxicological relevance
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
All animals survived their assigned study period.
The only test article-related clinical signs were orange faeces in animals of group 3 and 4 (200 and 1000 mg/kg). In one control female alopecia, necrosis of skin, sore hemorrhagic spot and scabbed wound on the right shoulder was noted. This single finding was considered to be incidental.

BODY WEIGHT AND BODY WEIGHT GAIN
No differences from controls of statistical or biological significance were noted.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption values were similar for treated groups and controls.

OPHTALMOSCOPIC EXAMINATION
Ophthalmologic findings were noted in a small proportion of animals from all groups. They included persistent pupillary membranes, persistent hyaloid vessel in vitreous body and corneal opacity. These findings occurred at similar incidences in the control and treated groups at the end of the treatment period. Therefore, they are considered to be unrelated to treatment with test article.

HAEMATOLOGY
There were no treatment-related effects on hematology data neither at termination of the treatment nor at the end of the treatment free recovery period which could be considered of toxicological significance.
However, a few minor findings with statistical significance were recorded between control and treated rats of group 4: Decreased erythrocyte count, hemoglobin concentration and hematocrit in females and increased platelet count in males.
The relation of these findings to the treatment, if any, remains unclear. In addition, there were no morphological findings or other supporting evidence which would suggest a treatment-related effect. Moreover, the findings were considered to be well within the normal range of biological variation for rats of this strain and age.

CLINICAL CHEMISTRY
There were no treatment-related effects on clinical biochemistry data neither at termination of the treatment nor at the end of the treatment free recovery period which could be considered of toxicological significance.
However, a few minor findings with statistical significance were recorded between control and treated rats of group 4: increased uric acid level in both sexes, increased total bilirubin level in males, increased triglyceride level in females, and increased sodium, potassium and chloride level in females.
The findings noted suggest metabolic adaptive changes. In addition, the findings were considered to be within the normal range of biological variation for rats of this strain and age. No toxicological relevance is therefore considered to be associated with these findings.

URINALYSIS
There were no treatment-related effects on urinalysis data neither at termination of the treatment nor at the end of the treatment free recovery period which could be considered of toxicological significance.
However, a few minor findings with statistical significance were recorded between control and treated rats of group 4: higher score for bilirubin in both sexes. This finding is considered to be related to the yellow nature of the test article which may have contributed to a somewhat deeper yellow urine pigmentation in the high-dose rats. In addition, "Ictotest" used to confirm positive bilirubin reactions was found to be negative in all cases. No toxicological relevance is therefore associated with this finding. Deep yellow coloration of the urine was noted in some males and females of group 4 (1000 mg/kg) and 2 males of group 3 (200 mg/kg).

ORGAN WEIGHTS
After 4 weeks of treatment kidney weight and/or relative kidney weight ratios were statistically significantly higher in animals of both sexes at 1000 mg/kg (absolute kidney weight: +19% in males) and kidney to body weight ratio was higher in males at 200 mg/kg.
At the end of the recovery period kidney weight and/or relative kidney weight was still statistically significantly higher in animals at 1000 mg/kg (absolute kidney weight: +17% in females) when compared with the controls.
The effects on organ weight in group 4 animals were in accordance with the results of the pathologic examination. In males at 200 mg/kg no microscopical correlates were noted, therefore the higher kidney to body weight ratio was considered to be of no toxicological relevance.
Liver to body weight ratio was statistically significantly higher in females at 1000 mg/kg after the treatment and recovery period.
Relative spleen weights were statistically significantly higher in males at 1000 mg/kg after the recovery period.
Microscopic findings in liver and spleen did not distinguish between control and treated animals.

GROSS PATHOLOGY
Yellowish discoloration of some segments of the gastrointestinal tract were noted in some group 4 rats at the end of the main treatment period and was due to the passive coloration by the test article.

HISTOPATHOLOGY
The only treatment related finding was a minimal to moderate degree of vacuolation of the outer stripe tubular epithelium in the kidneys of all group 4 rats at the end of the treatment period. This finding was no longer present following the recovery period.
All other microscopic findings recorded were within the range of background pathology encountered in rats of this age and strain and occurred at similar incidences and severity in both control and treated rats at both sacrifices.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absolute and relative kidney weight, vacuolation of the outer stripe tubular epithelium in the kidneys

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 1000 mg/kg of the substance was established as the lowest-observed-adverse-effect-level (LOAEL), 200 mg/kg as the no-observed-adverse-effect-level (NOAEL) and 50 mg/kg as the no-observed-effect-level (NOEL).
Executive summary:

In this subacute toxicity study according to Directive 92/69/EEC, B. 7. "Subacute Toxicity Oral", July 31, 1992 and OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 407, May 12, 1981 “Repeated Dose Oral Toxicity -

Rodent: 28-day or 14-day Study", the substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

The study comprised 5 animals per group and sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during the treatment and recovery periods. Ophthalmoscopic examinations were performed at the end of the treatment and recovery period. At the end of the dosing period and the treatment-free period, blood samples were withdrawn for hematology and plasma chemistry analyses, and urine samples were collected for biochemical and microscopic analyses. All animals were killed, examined post mortem and necropsied. Samples of major organs from all group 1 (0 mg/kg) and group 4 (1000 mg/kg) animals, as well as kidneys and gross lesions from all animals were processed as hematoxylin and eosin stained slides and examined by light microscopy.

The results of the study are summarized as follows:

All animals survived their assigned study period.

The only test article-related clinical signs were orange faeces in animals of group 3 and 4 (200 and 1000 mg/kg).

There was no effect on body weight.

There were no effects on food consumption or relative food consumption.

There were no treatment-related changes on the ophthalmological findings.

No morphological findings or other supporting evidence for a treatment related effect were noted for the hematological findings. The clinical biochemistry findings are considered to be of metabolic adaptive nature. Urinalysis findings (increased bilirubin level) were considered to be due to the yellow color of the test article.

After 4 weeks of treatment and at the end of the recovery period kidney weight and/or relative kidney weight ratios were higher in group 4 (1000 mg/kg) animals. The higher kidney weights in group 4 are considered to be test article related and in accordance with the microscopical findings. Yellowish discoloration of some segments of the gastrointestinal tract were noted in some group 4 rats at the end of the main treatment period and was due to the passive coloration by the test article.

The only treatment related histopathological finding was minimal to moderate degree of vacuolation of the outer stripe tubular epithelium in the kidneys, of all group 4 (1000 mg/kg) rats at the end of the treatment period. This finding was no longer present following the recovery period.

Based on the results of this study, 1000 mg/kg of the substance was established as the lowest-observed-adverse-effect-level (LOAEL), 200 mg/kg as the no-observed-adverse-effect-level (NOAEL) and 50 mg/kg as the no-observed-effect-level (NOEL).