Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 10 May 2016 and 26 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

EC Number:

276-423-1

EC Name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Cas Number:

72152-71-7

Molecular formula:

C16H8CuN2O8S2.2Na

IUPAC Name:

Cuprate(2-), [6-(hydroxy-kO)-5-[2-[2-(hydroxy-kO)-5-sulfophenyl]diazenyl-kN1]-2-naphthalenesulfonato(4-)]-, sodium

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animal Information
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight of any previously dosed animals.
Animal Care and Husbandry
The animals were housed in groups three in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed 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. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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:

All animals were dosed once only by gavage

Doses:

2000 mg/kg

No. of animals per sex per dose:

3 animals per dose, 2 dose groups

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.
Groups of fasted animals were treated as follows:
Dose Level(mg/kg) Concentration(mg/mL) Dose Volume(mL/kg) Number of Rats(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 body weight 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 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for up to 14 days.
Individual body weights were recorded prior to dosing and 7 and 14 days after treatment or at death.
At the end of the observation period the surviving 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.

Data Evaluation
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 behavioral and clinical observations, gross lesions, body weight 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 Annex 2.

Statistics:

None recorded

Results and discussion

Preliminary study:

Not applicable.

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 - 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 95% CL not reported

Mortality:

Individual mortality data are given in Appendix 1.

One animal was killed for humane reasons, approximately 3 hours after dosing, due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence.

Clinical signs:

other: Individual clinical observations are given in Appendix 1. Signs of systemic toxicity noted in the animal that was humanely killed were hunched posture, pilo erection and tiptoe gait. This animal was also prostrate and unresponsive prior to humanely kill

Gross pathology:

Individual necropsy findings are given in Appendix 3.
Abnormalities noted at necropsy of the animal that was humanely killed during the study were dark liver, dark kidneys, gaseous stomach and dark red colored liquid present in the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Other findings:

None

Any other information on results incl. tables

Appendix 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	4F	1FU	2FU	3FU	4FU	5FU	6FU	7FU	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
		Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1U	2U	4U	1F°U	2F°U	3F°U	4F°U	5F°U	6	7	8	9	10	11	12	13	14
	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	HPWt Å·X*
	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                                 H = Hunched posture       P = Pilo-erection                 Wt = Tiptoe gait

Å= Animal prostrate and unresponsive                        * = Dark red colored staining of feces and anus          F = Dark red colored staining of the feces

U = Dark red colored staining of the urine                     F° = Pink colored staining of the feces                           ·= Dark pink colored extremities

X* = Animal killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

Appendix 2     Individual Body Weights and Body Weight Changes

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight (g) at Death	Body Weight Gain (g) During Week
		0	7	14		1	2
2000	1-0 Female	188	204	219		16	15
	1-1 Female	184	190	212		6	22
	1-2 Female	198	218	230		20	12
	2-0 Female	185	189	191		4	2
	2-1 Female	179	-	-	176	-	-
	2-2 Female	203	205	206		2	1

Appendix 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	Humanely killed Day 0	Liver: dark Kidneys: dark Stomach:gaseous                   red colored fluid present
	2-2 Female	Killed Day 14	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

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 body weight (Globally Harmonized Classification System - Category 5, >2000 – 5000 mg/kg body weight).

Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

Methods

A group of three fasted females was treated with the test item at a dose level of 2000 mg/kg body weight. This was followed by a further group of three fasted females at the same dose level. Dosing was performed sequentially.

The test item was administered orally as a suspension in distilled water. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality. One animal was killed for humane reasons, approximately 3 hours after dosing, due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence. 

Clinical Observations. Signs of systemic toxicity noted in the animal that was humanely killed were hunched posture, pilo‑erection and tiptoe gait. This animal was also prostrate and unresponsive prior to being humanely killed. There were no signs of systemic toxicity noted in the surviving animals. Dark red or pink colored staining of the feces and urine was noted in the cages of all animals.

Body Weight. Surviving animals showed expected gains in body weight.

Necropsy. Abnormalities noted at necropsy of the animal that was humanely killed during the study were dark liver, dark kidneys, gaseous stomach and dark red colored liquid present in the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

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 body weight (Globally Harmonized Classification System - Category 5, >2000 – 5000 mg/kg body weight).