Reaction mass of copper digluconate, sodium sulphate and copper sulphate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.Nov.2008 - 14.Jul.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, Number 403: “Acute Inhalation Toxicity”, adopted May 12, 1981.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

European Communities, Directive 92/69/EEC, Part B.2 “Acute Toxicity (Inhalation)”, published December 29, 1992 and European Communities Directive 93/21/EEC, April 27,1993 amending the aforementioned Directive

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

U.S. Environmental Protection Agency, Health Effects Test Guidelines OPPTS 870.1300, Acute Inhalation Toxicity, August 1998.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper di-D-gluconate

EC Number:

208-408-2

EC Name:

Copper di-D-gluconate

Cas Number:

527-09-3

Molecular formula:

C12H22CuO14

IUPAC Name:

copper(2+) bis(2,3,4,5,6-pentahydroxyhexanoate)

Constituent 2

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

H2O4S.2Na

IUPAC Name:

disodium sulfate

Constituent 3

Reference substance name:

Copper sulphate

EC Number:

231-847-6

EC Name:

Copper sulphate

Cas Number:

7758-98-7

Molecular formula:

CuSO4

IUPAC Name:

copper(2+) ion sulfate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: HanRcc:WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 4 4414 Füllinsdorf / Switzerland
- Number of Animals per Group: 5 males and 5 females
- Number of Groups: 2
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: Group 1 and 2: Males 10 weeks, Females 11 weeks
- Weight at study initiation:
Group 1: Males: 251.7 to 267.3 g, Females: 210.2 to 228.7 g
Group 2: Males: 257.3 to 287.0 g, Females: 205.9 to 213.1 g
The weight variation did not exceed ± 7.5% of the mean weight of the corresponding sex.
- Identification: By unique cage numbers and individual unique tail numbers written with an indeligible felt-tip pen.
- Diet (e.g. ad libitum): Animals had ad libitum access to a pelleted standard Kliba-Nafag 3433 rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst, Switzerland) batch nos. 76/08 and 31/08 except during the period when they were restrained in exposure tubes. Results of the analyses for contaminants and their limits of acceptability are archived at Harlan Laboratories Ltd.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum in water bottles, except during the period when they were restrained in exposure tubes. Results of
representative analyses for contaminants are archived at Harlan Laboratories Ltd.
- Acclimation period: Performed under Harlan Laboratories Study B68308 for at least five days under laboratory conditions, after clinical health examination. Only animals without any visible signs of illness were used for the study. A further observation of clinical signs was performed on each day of exposure, before exposure start.

ENVIRONMENTAL CONDITIONS
- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environment with target temperature of 22 ± 3 °C, a target relative humidity of 30 - 70% and a 12 hour fluorescent light / 12 hour dark cycle. Values for humidity and temperature out of the target range occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. Therefore these data are not reported but retained at Harlan Laboratories Ltd. A radio program was played during the most of the light period.
- Accommodation: Animals were housed in groups of 5 of the same sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Remarks:

flow-past exposure

Vehicle:

not specified

Mass median aerodynamic diameter (MMAD):

>= 2.27 - <= 2.65 µm

Geometric standard deviation (GSD):

>= 1.98 - <= 2.41

Remark on MMAD/GSD:

Particle Size Distribution
The Mass Median Aerodynamic Diameters (MMAD) obtained from two gravimetric measurements of particle size distribution during each exposure were similar (Group 1: MMAD = 2.27 and 2.84 μm, GSD = 1.98 and 2.41; Group 2: 2.37 and 2.65 μm, GSD = 2.23 and 2.30). This led to the conclusion that the particle size of the generated aerosol was fairly stable during the whole exposure periods. The MMADs for both groups were within the target range of 1 to 4 μm, thus deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. Hence, the particle size distribution and MMADs obtained were considered to be appropriate for acute inhalation toxicity testing.
Data on particle size distribution are presented in the tables in page 24 from the study report.

Details on inhalation exposure:

TREATMENT
- Room: Inhalation laboratory no. 222, Harlan Laboratories Ltd., Füllinsdorf
- Method: Inhalation by nose-only, flow-past exposure
- Rationale for Method: Inhalation is a possible route of human exposure.
- Frequency of Administration: Single, 4-hour exposure period. Exposure of Group 1 was interrupted twice for a total of 5 minutes for changing of the nebulizer or a connection.
Nevertheless, the animals were exposed for a period of 4 hours as those interruptions were accounted for.
- Rationale for Aerosol Concentration:
--> Group 1: The target concentration of approximately 5 mg/L air (actual concentration of respirable substances) is the recommended concentration (OECD 403, “Acute Inhalation Toxicity”).
--> Group 2:
The target concentration of 1 mg/L air (actual concentration of respirable substances) for 4 hours is the limit concentration for Category 4 (GHS ST/SG/AC.10/30 2003).

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

Single, 4-hour exposure period. Exposure of Group 1 was interrupted twice for a total of 5 minutes for changing of the nebulizer or a connection. Nevertheless, the animals were exposed for a period of 4 hours as those interruptions were accounted for.

Concentrations:

The chemical aerosol concentrations determined were 6.1 mg/L air (Group 1) and 1.3 mg/L air (Group 2) as targeted.

No. of animals per sex per dose:

Group 1
Males 1-5 slightly above 5 (mg/L air)
Females 6-10 slightly above 5 (mg/L air)

Group 2
Males 11-15 slightly above 1 (mg/L air)
Females 16-20 slightly above 1 (mg/L air)

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: yes
After 14 days: 04-Dec-2008 (Group 1)
22-Jan-2009 (Group 2)
All surviving animals were transferred to the pathology unit and anaesthetised by an intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. All animals were necropsied and abnormalities recorded. The animals that died spontaneously during the exposure were transferred to the pathology unit and necropsied as soon as possible.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution. All collected organs/tissues are available for histopathological examination, if requested by the Sponsor under separate contractual agreement.
Tissue/Organs colleted: Head with nasopharyngeal tissues, Larynx, Lungs, instilled via trachea with formalin at approximately 30 cm H2O pressure, Trachea and All gross lesions

OBSERVATIONS
The day of exposure was named test day 1 and counting continued for the subsequent days of observation.
--> Viability /Mortality
Observations for viability were recorded once before exposure on the day of exposure, once per hour during exposure, once after exposure on test day 1 and twice daily during the observation period.
--> Clinical Signs
Each animal was examined once per hour during exposure, once after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal signs were detectable during exposure, as the animals were restrained in the exposure tubes.
--> Body Weights
The body weight of each animal was recorded on test days 1 (before exposure), 4, 8 and 15 (before necropsy).

Statistics:

No statistical analysis was performed as only two groups were allocated to the study.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

>= 1.3 - <= 6.1 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Observations for viability were recorded once before exposure on the day of exposure, once per hour during exposure, once after exposure on test day 1 and twice daily during the observation period.
Three males died during the exposure at 6.1 mg/L air and one female within two hours after the end of exposure period. Two females treated at 6.1 mg/L air were found dead on day 2 of the observation period. One female was found dead on day 2 after exposure at 1.3 mg/L air. All other animals survived the scheduled observation period

Clinical signs:

other: Each animal was examined once per hour during exposure, once after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal

Body weight:

The body weight of each animal was recorded on test days 1 (before exposure), 4, 8 and 15 (before necropsy).
Marked body weight loss was noted in all surviving animals treated at 6.1 mg/L air between test days 1 and 4. Thereafter normal body weight gain was recorded in these animals. Marginal body weight loss was seen in two males and one female treated at 1.3 mg/L air from test day 1 to 4. Thereafter these animals showed a normal body weight development. No effects on body weight were recorded in the remaining animals.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

LC50 = 1.3 mg/L air (aerosol). Substance classifies as Acute tox. inhalation. Cat. 4.

Executive summary:

Two groups of five male and five female albino rats [HanRcc:WIST(SPF)] each were exposed by nose-only, flow-past inhalation to the test item at a chemically determined mean concentration of 6.1 mg/L air or 1.3 mg/L air, respectively. All animals were observed for clinical signs and mortality during the inhalation exposure and the observation period of 14 days. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 4, 8 and 15 before necropsy. On day 15 all surviving animals were sacrificed and necropsied.

The ranges of aerosol concentration, temperature, relative humidity, oxygen content and airflow rate measured during the exposure were considered to be satisfactory for a study of this type. In addition, the test item was considered to be respirable to rats.

Three males and one female died during or shortly after the exposure at 6.1 mg/L air. Two females treated at 6.1 mg/L air and one female treated at 1.3 mg/L air were found dead on day 2 of the observation period. All other animals survived the scheduled observation period.

Mortality:

Aerosol Concentration	Males	Females	Both sexes
1,3mg/L air	0%	20%	10%
6,1 mg/L air	60%	60%	60%

Salivation, decreased spontaneous activity, rales and ruffled fur were observed in most of the animals of both groups. Tachypnea was seen in animals exposed at 6,1mg/ L air.

Marked body weight loss was observed in all surviving animals treated at 6.1 mg/L air between test days 1 and 4. Thereafter these animals showed a normal body weight development.

Marginal body weight loss was recorded in two males and one female treated at 1.3 mg/L air between test days 1 and 4. Thereafter these animals showed a normal body weight development.

There were no macroscopic findings that were considered to be related to treatment with the test item.

In conclusion, the LC50 of Servalesa L60 obtained in this study was estimated to be between 1.3 mg/L air and 6.1 mg/L air (chemically determined mean aerosol concentration). There was no indication of relevant sex-related differences in toxicity of the test item.