Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-(diethylamino)-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:4)

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Cas Number:

42355-78-2

Molecular formula:

C40H40N12Na4O12S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[diethylamino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

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
acclimatisation: 5 days
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:

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters did not show significant differences among dose levels, both for males and females. All mean values are comparable with control range.

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:

no

Any other information on results incl. tables

Result of the study in tabular form:

	dose (mg/kg)				notes
	0	100	300	1000
Body weight (grams, at 1st day and necropsy 21st days), males	330.08 ± 10.90 424.00 ± 23.19	329.95 ± 12.67 420.78 ± 16.96	329.77 ± 12.42 432.27 ± 11.70	329.77 ± 12.42 428.27 ± 17.86	The mean values of body weights and weight increments were similar to control group. Weight increments were adequate to species, sex and age of animals.
Body weight (grams, at 1st day and necropsy 21st day), females	241.53 ± 23.67 205.83 ± 14.51	237.78 ± 11.50 205.07 ± 10.01	245.25 ± 10.94 205.65 ± 8.81	250.75 ± 20.95 206.40 ± 11.92	The mean values of body weights were similar to control group. The mean values of body weight increments in treated and control groups were variable during the whole application period. This is probably related to the adaptation process of organism to the test item administration.
Body weight increment (grams, at 1st, 2nd and 3rd week), males	39.62/27.93/26.39	36.19/31.85/22.75	40.95/35.07/26.46	39.27/33.46/26.88
Body weight increment (grams, at at 1st, 2nd and 3rd week), females	18.06/5.6/12.04	12.25/8.33/12.11	8.40/14.98/16.24	17.64/8.05/18.62
Signs of toxicity (health condition), males, females	no effects	no effects	no effects	no effects	No changes of animal health status and clinical symptoms of intoxication were observed in treated animals
Haematological tests with relevant baseline values, M and F	no effects	no effects	no effects	no effects	Haematological parameters did not show significant differences among dose levels. All mean values are comparable with historical control range
Necropsy findings, M and F	no effects	no effects	no effects	no effects	No macroscopical changes were observed during necropsy of treated males/females and control males/females

 

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

The administration of the test item did not induce treatment-related toxicologically significant changes.

No macroscopical changes were observed during necropsy of treated animals.