Tetracopper hexahydroxide sulphate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetracopper hexahydroxide sulphate

EC Number:

215-582-3

EC Name:

Tetracopper hexahydroxide sulphate

Cas Number:

1333-22-8

Molecular formula:

Cu4H6O10S

IUPAC Name:

tetracopper(2+) hexahydroxide sulfate

Details on test material:

Batch 356 SDC

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Species: Rat (Rattus norvegicus)
Strain: Wistar
Age/Weight at Dosing: 10 to 12 weeks
Weight (g): Male: Minimum: 292.2, Maximum: 359.4 ; Female: Minimum: 184.9, Maximum: 225.1
Source: Animal Breeding Facility, Jai Research Foundation
Total Number of: Thirty (15 males and 15 females)
Animals Used Female rats were nulliparous and non-pregnant
The study was undertaken in compliance with the guidelines of the “Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International” and “Guidelines for Laboratory Animals Facility” issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.

ENVIRONMENTAL CONDITIONS
Animal Room: BMR 29, Department of Toxicology
Temperature: 19 to 23 °C
Relative Humidity: 56 to 66%
Air Changes: Minimum 15 air changes/hour
Photoperiod: The photoperiod was 12 hours artificial light and 12 hours darkness, light hours being 06:00 h – 18:00 h which was maintained through automatic timer.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Details on inhalation exposure:

- Experimental Design
The 4 hour acute inhalation exposure was carried out using nose-only inhalation exposure equipment supplied by CH Technologies, USA. This dynamic inhalation exposure system consists of a flow-past nose-only inhalation exposure chamber with Perspex rat exposure/restraint tubes, air compressor, flow meter, rotating brush generator (dry powder), cascade impactor, temperature and humidity probe (thermo-hygrometer), oxygen monitor, carbon dioxide monitor, and open face gravimetric filter sampler.

- Inhalation Equipment
The rats were exposed using a nose-only inhalation exposure system with a dynamic air flow rate of 727 to 728 air changes per hour, ensuring an adequate oxygen content of at least 19% and a homogeneous, evenly distributed, respirable test aerosol with a mass median aerodynamic diameter (MMAD) particle size ranged between 1-4 microns. An exposure atmosphere CO2 level was less than 1%.

- Description of Inhalation Chamber
The dynamic inhalation equipment has 2 main parts namely outer plenum and inner plenum. Inner plenum volume is 862 cm3 and outer plenum volume is 1,200 cm3 so the total volume = 2062 cm3 = 2.062 Liters. The exposure unit (outer plenum) is made of stainless steel with 12 portholes to accommodate transparent perspex rat exposure tubes. These exposure tubes are accommodated in the portholes of the inhalation chamber. The adjustable unit of the exposure tube is set in such a way that rats breathe the test item aerosol through the window panel of the exposure tube. Observations during the inhalation experiment are made through transparent perspex rat exposure tubes. The outlet unit connected to a suction pump. The outgoing air from the chamber passes through an impinger containing 1.0% sodium hydroxide solutions and moisture traps.

- Description of Dust Generator System
The dust generator system is manufactured by PALAS GmbH and supplied by CH Technologies (USA), Inc. The dust generator system is a long assembly of tubing having several tube fittings attached. The feed system consists of a feed stock reservoir, generating system consists of a brush housing with integrated brush, spacer holder, up/down schalter, inlet of air for diserpsion, connection of compressed air with reducer, switch for manual or external controls (air, brush speed, schalter speed).

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

The nominal concentration of aerosolized substance was calculated to be 10.277, 6.646 and 3.446 mg/L air for group I, II and III, respectively, based on the mass of test item which used during exposure and the total chamber air flow during exposure.

No. of animals per sex per dose:

In the main test, 5 males and 5 females were tested per dose.

Control animals:

no

Details on study design:

OBSERVATIONS
- Toxic Signs
All rats were observed for any signs of toxicity and mortality hourly, during the 4 h exposure period and at 1 and 2 h after the exposure on the day of exposure. Subsequently, surviving rats were observed twice a day for morbidity and mortality for a period of 14 days following exposure. Clinical signs were recorded once a day.
- Body Weight
Body weights were recorded prior to exposure on day 0 and on days 1, 3, 7, and 14 and at death.
- Necropsy
At the end of the study, all survived rats were sacrificed by intraperitoneal administration of thiopentone sodium and subjected to gross pathological examination. All found dead rats were also subjected to gross pathological examination. This consisted of an external examination and the opening of the nasal passage, abdominal, and thoracic cavities. The appearance of any macroscopic abnormality was recorded.

Statistics:

As this study was conducted as a full study at three different breathing zone concentrations, the acute inhalation median lethal concentration (LC50) of the test item was calculated from the observed mortality data by the Probit analysis (Finney, 1971) method.

Results and discussion

Effect levels

Effect levels 1

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

ca. 2.091 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Effect levels 2

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

ca. 2.314 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Percent mortalities observed (both sexes combined) were 90, 30, and 10 at the breathing zone concentrations 3.038, 2.069, and 1.194 mg/L air of the test item, respectively which indicates dose dependent mortality (see table1).

Clinical signs:

other:

Body weight:

Group I: A decrease in the body weight, was observed in surviving male rat on days 1 and 3 which increased on days 7 and 14 when compared with day 0 body weight.
Group II: A decrease in the mean body weight, was observed in surviving rats on days 1, 3 and 7 which increased on day 14 when compared with day 0 mean body weight in both the sexes.
Group III: A decrease in the mean body weight, was observed on days 1 and 3 which increased on days 7 and 14 when compared with day 0 body weight in male rats. In case of female rats a decrease in the mean body weight, was observed on days 1, 3 and 7 which increased on day 14 when compared with day 0 mean body weight.

Gross pathology:

Necropsy (Macroscopic Findings):
- External: External examination of the found dead and terminally sacrificed rats did not reveal any abnormality of pathological significance.
- Internal: Visceral examination of the found dead rats revealed liver: reddish discolouration (Rat Nº 1, 3, 4 and 9) and lungs: multiple whitish foci (Rat Nº 13, 17, 19 and 24) whereas other found dead and terminally sacrificed rats did not reveal any lesion of pathological significance. Lesions observed in the found dead rats could be correlated with the test item used in the present study.

Other findings:

External examination of the found dead and terminally sacrificed rats did not reveal any abnormality of pathological significance. Visceral examination of the found dead rats revealed liver: reddish discolouration (Rat N º 1, 3, 4 and 9) and lungs: multiple whitish foci (Rat N º 13, 17, 19 and 24) whereas other found dead and terminally sacrificed rats did not reveal any lesion of pathological significance.

Interpretation of results :
The calculated acute inhalation median lethal concentration (LC50) value of the test item for combined sex was 2.091 mg/L air with 95% lower fiducial limit of 1.717 mg/L air and upper fiducial limit of 2.548 mg/L air. The regression equation is y = -15.264 + 6.103 x.
The calculated acute inhalation median lethal concentration (LC50) value of the test item for male rats was 2.314 mg/L air with 95% lower fiducial limit of 1.524 mg/L air and upper fiducial limit of 3.515 mg/L air. The regression equation is y = -8.001 + 3.864 x.

Any other information on results incl. tables

Text Table: 1 Concentration, Mortality/Rats Treated

Breathing ZoneConcentration (mg/L air)	Males (mortality/total)	Females (mortality/total)
3.038	4/5	5/5
2.069	1/5	2/5
1.194	1/5	0/5

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The calculated acute inhalation median lethal concentration (LC50) value of test item for combined sex was 2.091 mg/L air with 95% lower fiducial limit of 1.717 mg/L air and upper fiducial limit of 2.548 mg/L air. The regression equation is y = -15.264 + 6.103 x.
The calculated acute inhalation median lethal concentration (LC50) value of the test item for male rats was 2.314 mg/L air with 95% lower fiducial limit of 1.524 mg/L air and upper fiducial limit of 3.515 mg/L air. The regression equation is y = -8.001 + 3.864 x.

Executive summary:

Threegroups of rats, consisting of five male and five female rats per group were used for the main study. Rats from groups I, II, and III were exposed to breathing zone concentration levels 3.038, 2.069 and 1.194 mg test item/L of air, respectively. Rats from all groups were exposed for 4 h and surviving rats were observed for a period of 14 days.

Sign of toxicity lethargy was observed in rats exposed with the test item.

Percent mortalities observed (both sexes combined) were 90, 30, and 10 at the breathing zone concentration levels 3.038, 2.069, and 1.194 mg/L air of test item, respectively which indicates dose dependent mortality.

Group I: A decrease in the body weight, was observed in surviving male rat on days 1 and 3 which increased on days 7 and 14 when compared with day 0 body weight.

Group II:A decrease in the mean body weight, was observed in surviving rats on days 1, 3 and 7 which increased on day 14 when compared with day 0 mean body weight in both the sexes.

Group III: A decrease in the mean body weight, was observed on days 1 and 3 which increased on days 7 and 14 when compared with day 0 body weight in male rats. While, in case of female rats a decrease in the mean body weight, was observed on days 1, 3 and 7 which increased on day 14 when compared with day 0 mean body weight.

External examination of the found dead and terminally sacrificed rats did not reveal any abnormality of pathological significance. Visceral examination of the found dead rats revealed liver: reddish discolouration (Rat N º 1, 3, 4 and 9) and lungs: multiple whitish foci (Rat N º 13, 17, 19 and 24) whereas other found dead and terminally sacrificed rats did not reveal any lesion of pathological significance.

Lesions observed in the found dead rats could be correlated with the test item used in the present study.

Breathing Zone Concentration (mg/L Air)	N° of Animals Used	Mortalities/Sex		Mortalities %
		Male	Female
3.038	5 Males + 5 Females	04	05	90
2.069	5 Males + 5 Females	01	02	30
1.194	5 Males + 5 Females	01	00	10

The calculated acute inhalation median lethal concentration (LC₅₀) value of the test item for combined sex was 2.091 mg/L air with 95% lower fiducial limit of 1.717 mg/L air and upper fiducial limit of 2.548 mg/L air. The regression equation is y = -15.264 + 6.103 x.

The calculated acute inhalation median lethal concentration (LC₅₀) value of test item for male rats was 2.314 mg/L air with 95% lower fiducial limit of 1.524 mg/L air and upper fiducial limit of 3.515 mg/L air. The regression equation is y = -8.001 + 3.864 x.