5,5'-{ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-butyl-1H-pyrazole-4,1-diyl)]}diisophthalic acid

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 14 July 2011 and 04 August 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

EC Number:

700-616-6

Cas Number:

849608-59-9

Molecular formula:

C36H32K4N14O8S4

IUPAC Name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry

Female Wistar (RccHan-:WIST) strain rats were supplied by a reputable supplier. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The bodyweights fell within an interval of ±20% of the mean initial bodyweight of the first treated group.

The animals were housed in groups of three in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Procedure
Using available information on the toxicity of the test material, 2000 mg/kg was chosen as the starting dose.

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (mg/ml) (ml/kg) Female
2000 200 10 3
2000 200 10 3

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.
The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.

Individual bodyweights were recorded prior to dosing and seven and fourteen days after treatment.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Doses:

A group of three fasted females was treated with the test item at a dose level of 2000 mg/kg bodyweight. This was followed by a further group of three fasted females at the same dose level. The test item was administered orally as a suspension in distilled water.

No. of animals per sex per dose:

3 female at 2000 mg/kg
3 female at 2000 mg/kg

Control animals:

no

Details on study design:

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.

The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.

Individual bodyweights were recorded prior to dosing and seven and fourteen days after treatment.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects. Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made as shown in the schematic diagram in Appendix 1 (see attached background material section).

The results were evaluated according to Commission Directive 2001/59/EC for classification and labelling of dangerous substances.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Individual clinical observations and mortality data are given in Table 1 (see "any other information on results incl. tables").
There were no deaths.

Clinical signs:

No signs of systemic toxicity were noted.

Body weight:

Individual bodyweights and weekly bodyweight changes are given in Table 2 (see "any other information on results incl. tables").
All animals showed expected gains in bodyweight over the study period.

Gross pathology:

Individual necropsy findings are given in Table 3 (see "any other information on results incl. tables").
No abnormalities were noted at necropsy.

Other findings:

- Organ weights:
Not recorded

- Histopathology:
Not recorded

- Potential target organs:
Not recorded

- Other observations:
None

Any other information on results incl. tables

Table 1              Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0= No signs of systemic toxicity

Table 2              Individual Bodyweights and Weekly Bodyweight Changes

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight Gain (g) During Week
		0	7	14	1	2
2000	1-0 Female	165	174	181	9	7
	1-1 Female	174	198	210	24	12
	1-2 Female	172	188	208	16	20
	2-0 Female	185	203	220	18	17
	2-1 Female	180	196	217	16	21
	2-2 Female	181	199	209	18	10

Table 3              Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Female	Killed Day 14	No abnormalities detected
	1-1 Female	Killed Day 14	No abnormalities detected
	1-2 Female	Killed Day 14	No abnormalities detected
	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Killed Day 14	No abnormalities detected
	2-2 Female	Killed Day 14	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2500 mg/kg bodyweight (Globally Harmonised Classification System - Unclassified).

The test item does not meet the criteria for classification according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous substances.

Executive summary:

Introduction.

The study was performed to assess the acute oral toxicity of the test item following a single oral administration in the Wistar strain rat. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 423 “Acute Oral Toxicity – Acute Toxic Class Method” (adopted17 December 2001)

Method B1 tris (Acute Toxicity (Oral) of Commission Regulation (EC) No. 440/2008)

Method. 

A group of three fasted females was treated with the test item at a dose level of 2000 mg/kg bodyweight. This was followed by a further group of three fasted females at the same dose level. The test item was administered orally as asuspensionindistilled water. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

Mortality. 

There were no deaths.

Clinical Observations. 

There were no signs of systemic toxicity.

Bodyweight. 

All animals showed expected gains in bodyweight over the study period.

Necropsy. 

No abnormalities were noted at necropsy.

Conclusion. 

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was estimated to be greater than 2500 mg/kg bodyweight (Globally Harmonised Classification System - Unclassified).

The test item does not meet the criteria for classification according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous substances.