Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

the study is a DRF of a sub-chronic toxicity study

Type of information:

experimental study

Adequacy of study:

key study

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:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

the study is a DRF of a sub-chronic toxicity study performed acoording to guideline 408

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

EC Number:

248-420-5

EC Name:

Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Cas Number:

27344-06-5

Molecular formula:

C42H46N14O10S2.2Na

IUPAC Name:

disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Age at study initiation: 6 – 7 weeks
Housing conditions:
- Grouping: 2 animals of the same sex in one cage
- Food: complete pelleted diet for rats
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law
- Light cycle: 12 hour light / 12 hour dark
Microclimate: 22 + 3°C, relative humidity 30-70 %, air changes: 15 per hour
- Bedding: sterilised Lignocel or sterilized shavings of soft wood
Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight
Identification of animals: the animals will be identified by the colour marks on their fur; each cage will be marked with the number of animals, sex, number of cage, name and dose level of the test item

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqua pro iniectione

Details on oral exposure:

The application form of the test item will be administered to the stomach by gavage.
Animals will be fasted 2 hours before to test item administration. After the test item will be administered, the animals will not get food for next 2 hours.
The test item concentration at single dose level will be adjusted so that the administered volume will be constant at all dose levels – 1 ml/100 g body weight. The administration form of the test item will be prepared daily before administration and during administration it will be mixed by the magnetic stirrer.

Duration of treatment / exposure:

21 days

Frequency of treatment:

once at day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

300 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:

6

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Health condition control: performed once a day during animal handling before the application of the test item.

Body weight: recorded on automatic balances with group average computing module.
First weighing was performed before the first application and then weekly. Weight increment was computed as an average per group per week (in grams).

Clinical observations: in natural conditions in animals' cages.

Haematological examination:
Before necropsy of animals the blood samples will be collected from the orbital plexus by glass micropipette under the light diethyl ether narcosis into the PVC test tubes containing anticoagulation systems. Basic blood parameters (total erythrocyte count, total leucocyte count, mean corpuscule volume, haematocrit, haemoglobin concentration, total platelet count) was determined on haematology analyser.

Pathological examination:
During the necropsy (on the 21st day of study) a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities was carried out.

Sacrifice and pathology:

GROSS PATHOLOGY: During the necropsy (on the 21st day of study), a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities was carried out.

HISTOPATHOLOGY: Not performed

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males
Mild decreasing of body weight and body weight increment at the dose level 300 mg/kg/day was recorded compared to the control group.

Females
Mild decreasing of body weight and body weight increment at the dose level 300 mg/kg/day was recorded compared to the control group.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Males
Haematological examination showed decreasing of total count of white blood cells in males at the dose levels 100 and 1000 mg/kg/day (this value was under historical control range).
Other parameters did not show significant differences among dose levels. Other mean values are comparable with our historical control range.

Females
Value of total leucocyte count was increased in females of all dose levels compared to control group. This modulation is still in historical control range.
Other haematological parameters were comparable and in range of our historical control.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

dose level: dose for selection of maximum tested dose in the main study

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table of results

	dose (mg/kg)				notes
	0	100	300	1000
Body Weight before application ± SD, males	327.35 ± 11.13	325.98 ± 8.60	326.47 ± 16.48	326.42 ± 13.38
Body Weight on 21st day ± SD, males	431.02 ± 12.91	420.40 ± 21.95	404.43 ± 21.29	416.28 ± 21.73
Body Weight before application ± SD, females	202.85 ± 11.49	202.52 ± 10.04	203.27 ± 6.29	202.10 ± 7.88
Body Weight on 21st day ± SD, females	252.67 ± 33.62	247.30 ± 7.90	240.22 ± 13.26	246.92 ± 11.12
Body weight changes	no effects	no effects	no effects	no effects	Slight changes of body weight and body weight increment were adequate to species, sex and age of animals in experiment
Food consumption	not examined	not examined	not examined	not examined
Water consumption	not examined	not examined	not examined	not examined
Signs of toxicity	no effects	no effects	no effects	no effects
Nature, severity and duration of clinical observations (whether reversible or not)	no effects	no effects	no effects	no effects
Results of ophthalmological examination	not examined	not examined	not examined	not examined
Haematological tests with relevant baseline values, males	no effects	effects	no effects	effects	Haematological examination showed decreasing of total count of white blood cells in males at the dose levels 100 and 1000 mg/kg/day (this value was under historical control range).
WBC -Total leucocyte count (103/μL), males	11.47 ±1.27	9.02 ±1.75	12.45 ±4.94	9.83 ±2.79
Haematological tests with relevant baseline values, females	no effects	no effects	no effects	no effects
Clinical biochemistry tests with relevant baseline values	not examined	not examined	not examined	not examined
Circulating thyroid hormones (T4, T3, TSH)	not examined	not examined	not examined	not examined
Organ weights	not examined	not examined	not examined	not examined
Organ/body weight ratios	not examined	not examined	not examined	not examined
Necropsy findings	no effects	no effects	no effects	no effects
Histopathological findings	not examined	not examined	not examined	not examined
Total cauda epididymal sperm number, percent progressively motile sperm, percent morphologically normal sperm, and percent of sperm with each identified abnormality	not examined	not examined	not examined	not examined

Applicant's summary and conclusion

Conclusions:

At the dose level 1000 mg/kg/day, mean values of different parameters analyzed are comparable with historical control range. Based on these findings, the highest tested dose for the main study was selected.

Executive summary:

In the dose-range finding experiment with test item, no animal died during the application period.

Clinical observation did not detect any impact of the test item on the health condition of animals at all dose levels.

Slight changes of body weight and body weight increment were reported and were found to be adequate to species, sex and age of animals in experiment.

The administration of the test item did not induce treatment-related toxicologically significant changes of red and white blood component in both sexes.

No macroscopical changes were observed during necropsy of treated animals.