Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Remarks:

source study on the analogous substance

Adequacy of study:

key study

Study period:

From January 10 to February 21, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 12, 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, West-Germany.
- Age at study initiation: approximately 6 weeks.
- Weight at study initiation: 192 - 233 g males; 141 - 171 g females.
- Housing: animals were housed 5 to a cage (same sex) in stainless suspended cages with wire mesh floors.
- Diet: standard pelleted laboratory animal diet (RMH-B from Hope Farms, Woerden, The Netherlands), ad libitum.
- Water: tap-water, diluted with decalcified water, ad libitum.
- Acclimation period: 14 days. An examination was perfromed prior to commencement of treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C (actual range 17 - 20 °C).
- Relative dumidity: 30 - 70 % (actual range 45 - 70 %).
- Air changes: 7.5-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark per day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

water prepared by reverse osmosis

Details on oral exposure:

TREATMENT
- Dose volume: 10 ml/kg bw

TEST SOLUTION
- Method: the test article was weighed into a glass flask on an analytical balance and the vehicle (w/w) added.
- Frequency of test article formulation: daily immediately prior to dosing.
- Homogeneity of test article in the vehicle: determination of homogeneitywas not considered necessary as the test substance formed a solution with the vehicle.
- Appearance of test article in vehicle: orange solution.
- Storage of test article formulation: at ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared prior to commencement of he study and during weeks 1 and 3 were analysed to check the accuracy of mixing.
Concentration of the test articie in vehicie was determined pretreatment and on days 1 and 15.
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 / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary test results.

Examinations

Observations and examinations performed and frequency:

MORTALITY
Twice daily.

DETAILED CLINICAL OBSERVATIONS
At least once daily.

BODY WEIGHT
Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION
Weekly.

OPHTHALMOSCOPIC EXAMINATION
Prior to termination in week 4.

CLINICAL CHEMISTRY
Blood samples were collected under Iight ether anaesthesia immediately prior to post mortem examination, between 8.00 and 10.00 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into untreated tubes for clinical biochemistry parameters (> 2.0 ml).
Parameters: glucose, urea, creatinine, bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, sodium, potassium, chloride, calcium, phosphnrus, protein total, protein albumin.

HAEMATOLOGY
Blood samples were collected under Iight ether anaesthesia immediately prior to post mortem examination, between 8.00 and 10.00 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA (0.5 ml) for haematological parameters.
The following haematology parameters were determined from blood containing EDTA as an anti-coagulant.
Parameters: erythrocyte count, haemogiobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width, platelet count, total ieucocyte count, differential leucocyte count.

Sacrifice and pathology:

GROSS PATHOLOGY
All animals were necropsied on day 29 and descriptions of all macroscopic abnormalities recorded. All animals were anaesthetised with pentobarbital and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: adrenal glands, aorta, brain, cecum, clitoral gland, colon, duodenum, epididymides, exorbital lacrimal gland, eyes, femur including joint, head, heart, ileum, jejunum, kidneys, larynx/pharynx, liver (all lobes), lung (including mainstem bronchi), lymph nodes - cervical mesenteric, mammary gland, oesophagus, ovaries, pancreas, pituitary gland, preputial gland, prostate gland, rectum, salivary glands (sub-maxillary), sciatic nerve, seminal vesicles, skeletal muscle (gastrocnemius), skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach (glandular and non glandular, including pylorus), testes, thymus, thyroid (including parathyroid), tongue, trachea, urinary bladder, uterus, vagina.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded at termination on the scheduled date of necropsy listed: adrenal glands, kidneys, liver, spleen, testes.

HISTOPATHOLOGY
All organ and tissue samples for histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and azophloxine. All slides for histopathological examination were dispatched to testing laboratory.
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from animals of the control and high dose group were examined by a pathologist. All abnormalities were described and included in the report.

Statistics:

The following statistical methods were used to analyse the body weight, food consumption, organ weights and ciinicai iaboratory 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 pooied 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).
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 a reaction to treatment or behavioral change by treated rats in comparison with the controls.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain by males and females treated with test item was noted as marginally increased compared to controls, but this difference did not achieve a consistent level of statistical significance and was considered the result of normal biological variation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake of treated rats was similar to that of the controls at all times.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities noted in either treated or control rats at the ophthalmoscopic examination prior to termination.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Among males receiving 200 mg/kg, significantly low values for sodium and calcium were noted, however in the absence of corroborative changes in other electrolyte values or a treatment-related distribution, it was considered that these decreases had arisen fortuitously.
Treated females showed significantly decreased glucose levels when compared to controls. However, when the control values were examined, it was considered that they were at the upper end of the normal biological range and the values obtained for treated females were within the normal biological range. These statistically significant differences were therefore considered the result of high control values and not the result of treatment.
Females receiving 200 mg/kg had significantly decreased levels of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), however, as there was no evidence of a significant decrease in females receiving 1000 mg/kg and it was considered that these decreases could be associated with the lighter liver weights noted for this group, the decrease of these enzyme levels was therefore considered not of toxicological significance.
Other differences noted between control and treated rats that achieved a level of statistical significance were considered to have arisen fortuitously and not to be as a result of treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences that were considered to have arisen as a result of treatment. Minor statistically significant changes from control values for red blood cell numbers in males receiving 50 mg/kg/day and for white blood cell numbers in females receiving 50 or 100 mg/kg/day were not, in the absence of a treatment-related distribution, considered to be of toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Females receiving 200 mg/kg had statistically significantly lighter livers when compared to control values. However, in the absence of a treatment related distribution, this difference was considered to have arisen fortuitously and not to be of toxicologicai significance.
There were no differences in the organ weights of treated males or females receiving 50 or 1000 mg/kg when compared to control values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observation showed haemorrhages in the glandular stomach of one female receiving 200 mg/kg/day and in the cervical lymph nodes of one female receiving 1000 mg/kg. In the absence of a treatment-related distribution these findings, which may have occurred as a result of terminal procedures, were considered not to be toxicologically significant.
Any other macroscopically noted abnormalities were within the range of those normally seen for rats of this age and strain and were not treatment-related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No microscopic observations were noted that were related to treatment with the test item. Those that were observed were among those commonly found in rats of this age and strain.

Effect levels

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOEL (28d) (rat males/females) ≥ 1000 mg/kg bw day

Executive summary:

In the subacute 28-day toxicity study, the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats.

A 10-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 the 28-day study. No differences of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups of the dose range finding study.

The main study was comprised of four groups. The 5 males and 5 females were assigned to each group. The dose levels used were 0, 50, 200 and 1000 mg/kg bw/day.

No evidence of a toxic reaction to test item was noted at any of the three treatment levels.

From the results obtained, a No Observed Effect Level of 1000 mg/kg/day was established.

Conclusion

NOEL (28d) (rat males/females) ≥ 1000 mg/kg bw day