Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 18, 1990 to January 14, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 12, 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate
EC Number:
407-240-9
EC Name:
(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate
Cas Number:
159604-94-1
Molecular formula:
C66H88Cu2N20O10S2.C3H5O3.C2H3O2
IUPAC Name:
7,7'-bis[4-(3-diethylaminopropylamino)-6-(3-diethylammoniopropylamino)-1,3,5-triazin-2-ylamino]-{μ-4,4'-dihydroxy-1:2k2O4:O4'-3,3'-[3,3'-dihydroxy-1:2k2O3:O3'-biphenyl-bisazo-1:2(N3,N4-η:N3',N4'-η)]dinaphthalene-2-sulphonato(6-)}dicuprate(2-), mixed (1:1) acetic/lactic acid salts
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar rat, outbred, SPF quality recognised by international guidelines as a recommended test system
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel Switzerland
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: ca. 170 g
- Housing: animals were housed 5 to a cage (same sex) on stainless steel suspended cages with wire mesh floors
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days. Veterinary examinations was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method: The test substance was weighted into a glass flask on an analytical balance and the vehicle (w/w) added.
Frequency of test substance formulation: Daily immediately prior to dosing
Homogeneity of test substance in vehicle: Test susbtance formulations were homogenised using an electric shaker and magnetic stirrer. Homogeneity during treatment was manteined using a magnetic stirrer. From chemical analysis the test substance appeared to be homogeneous in water at all concentrations used in this study.
Storage instructions for test substance formulation: At ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared during week 1 were analysed to check the accuracy of preparation. Actual concentrations of preparations were in agreement with the treatment levels as per protocol.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Slightly low body weight gain was noted among females receiving 1000 mg/kg/day over the 5 days of treatment. Based on these observations, a high treatment level of 1000 mg/kg/ day was selected for a study of 28 days duration.
- Rationale for animal assignment (if not random): computer-generated random algorithm according to body weight with all animals within ± 20% of the sex mean
- Fasting period before blood sampling for clinical biochemistry: the animals were fasted overnight before blood sampling, but water was provided.
- Dose range finding studies: A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration.
Slightly low body weight gain was noted among females receiving 1000 mg/kg/day over the 5 days of treatment.
No differences of biological significance were observed in clinical appearance, food consumption, macroscopic appearance or liver weights between the treated groups.
Based on these observations, a high treatment level of 1000 mg/kg/ day was selected for a study of 28 days duration.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding termination, prior to overnight fasting.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before commencement of treatment and during the last week of treatment
- Dose groups that were examined: control, 50 mg/kg bw, 200 mg/kg bw and 1000 mg/kg bw

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all rats/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Animals fasted: Yes
- How many animals: all rats/sex/group
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Slides of adrenals, heart, kidneys, liver, spleen and stomach, collected at termination from all animals of the control and high dose group as well as from all gross lesions of all animals were examined by a pathologist.
Statistics:
The fol lowing statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If 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 of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity or behavioural changes over the 29 day observation period that were considered to be related to treatment.
Short periods of excessive salivation were noted in all males and females receiving 1000 mg/kg/ day. On 1 occasion, excessive salivation was also noted in 1/5 males receiving 200 mg/kg/day. Since this may be attributed to a possible irritant effect or bad taste of the test substance, it was considered not to represent a clear sign of toxicity. In addition, all treated animals showed dark appearance of the faeces from day 5 of treatment until termination. Considering the physico-chemical properties of CARTASOL BLUE PE 3562, colour changes were considered to be due to test substance that had not been absorbed in the gastro-intestinal tract.
Regurgitation of test substance was noted intermittently in al l animals receiving 1000 mg/kg/ day and incidentally in 2/5 males receiving 50 mg/kg/day and 1/5 females receiving 200 mg/kg/day. Since this phenomenon is very rare in rats and possibly may be associated with the above mentioned irritant effect of the test substance, it was considered not to be of toxicological significance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no differences in food consumption before or after allowance for body weight between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no differences between the pretest examination and the examination at week 4 that could be attributed to treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were considered not to have been affected by treatment.
A minor statistically significantly increased platelet value, for males receiving 50 mg/kg/day in comparison with control males, remained within the range normally expected for rats of this age and strain and was considered not to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences noted between control and treated rats that were related to treatment.
Statistically significantly low total protein and albumin values, noted among males receiving 1000 mg/kg/day when compared to control values, were within the range of normal biological variation for rats of this age and strain and considered to be of no toxicological significance.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no behavioural changes over the 29 day observation period that were considered to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights and relative organ weights of treated animals were indistinguishable from those of control animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The majority of animals receiving 50 or 200 mg/kg/day and all animals receiving 1000 mg/kg/day were noted with black appearance of the contents of small parts or the entire gastro-intestinal tract. There was no black appearance of gastro-intestinal tissue. This observation was not unexpected given the dark faeces noted clinically from day 5.
All other macroscopic observations at necropsy were within the range of normal background alterations and considered not to have arisen as an effect of treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOEL (28 days): 1000 mg/kg/day
Executive summary:

RATIONALE FOR DOSE LEVEL SELECTION: A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration.


Slightly low body weight gain was noted among females receiving 1000 mg/kg/day over the 5 days of treatment.


No differences of biological significance were observed in clinical appearance, food consumption, macroscopic appearance or l iver weights between the treated groups.


Based on these observations, a high treatment level of 1000 mg/kg/day was selected for a study of 28 days duration.


METHODOLOGY: In this subacute 28-day toxicity study, the test item was administered daily by gavage to SPF-bred Wistar rats. The study comprised of four groups.


The number of rats assigned to toxicity testing per group as well as the dose levels administered were as follows:






































GroupDose level
mg/kg		No. of rats
malesfemales
101-521-25
2506-1026-30
320011-1531-35
4100016-2036-40

FINDINGS:


At 50 mg/kg/day: No treatment-related changes detected.


At 200 mg/kg/day: No treatment-related changes detected.


At 1000 mg/kg/ day: No treatment-related changes detected.


CONCLUSION: From the results presented in this report a definitive No Observed Effect Level (NOEL) of 1000 mg/kg/ day was established.