Diphosphoric acid, copper salt

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-04 to 2013-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

Principles of method if other than guideline:

Deviations from study plan:

Deviation No. 1 (02 May 2013)
Animals from Group 2 were acclimatised to the restraint tubes on the day of exposure.

Deviation No. 2 (02 May 2013)
Two male animals from Group 2 were slightly larger than 20% of the mean group weight of Group 1 males at the start of the test.

These deviations are considered not to affect the purpose or validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 10 July 2012, Date of signature 30 November 2012

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Diphosphoric acid, copper salt

EC Number:

233-279-4

EC Name:

Diphosphoric acid, copper salt

Cas Number:

10102-90-6

Molecular formula:

Cu.xH4O7P2

IUPAC Name:

diphosphoric acid, copper salt

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): IP37: Copper (II)-pyrophosphate
- CAS Number: 10102-90-6
- EC Number: 233-279-4
- Description: Pale blue powder
- Batch: 9000022795
- Analytical purity: >80% (based on Cu content > 34% )
- Impurities (identity and concentrations): <18% water, < 2% free orthophosphate, pyrophosphate
- Expiration date of the lot/batch: 29 September 2014
- Storage condition of test material: Room temperature, in the dark
- Other: No correction for purity was made.

Test animals

Species:

rat

Strain:

other: RCC Han : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 200g - 300g*
- Fasting period before study:
- Housing: animals were housed in groups of 3 by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes and provided with environmental enrichment items.
- Diet (e.g. ad libitum): ad libitum with the exception of the exposure period.
- Water (e.g. ad libitum): ad libitum with the exception of the exposure period.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

*2 Group 2 males were slightly lighter than 20% of the mean group weight of group 1 males, this was considered to be a slight deviations the 2 group 2 males were found to be out of the desired range by 16g and 13g respectively.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply. A particle separator was introduced before the aerosol entered the exposure chamber (Group 2 only) in order to help achieve the lower target atmosphere concentration (1mg/L) within the exposure chamber.
- Exposure chamber volume: The cylindrical exposure chamber had a volume of approximately 30 L (dimensions 28 cm diameter by 50 cm high)
- Method of holding animals in test chamber: Prior to the day of exposure (Group 1) and on the day of exposure (Group 2) each rat was acclimatised (for approximately 2 hours) to a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
Following an appropriate equilibrium period two groups, each of six rats (3 males and 3 females), were subjected to a single exposure to the test item for a period of 4 hours.
- Source and rate of air: Compressed air was supplied by means of an oil free compressor.
- Method of conditioning air: Air passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.
- System of generating particulates/aerosols: see attached figures
- Method of particle size determination: see below
- Treatment of exhaust air: no data
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Individual values are recorded in the tables included as Annexes.
The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure. See attached diagram of system (Figures 1 and 2)

TEST ATMOSPHERE
- Brief description of analytical method used: Homogenecity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test materials (Green JD et al, 1948)
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowbrough, East Sussex). The test atmosphere was generated to contain 19% oxygen. Individual values are given in the table Annexes attached.
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.
Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
The nominal chamber concentration was calculated by dividing the mass of test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
The nominal chamber concentration was calculated by dividing the mass of the test item used by the total volume of air passed through the chamber.
The nominal chamber concentrations were calculated by dividing the mass of the test item used by the total volume of air passed through the chamber during each exposure. The nominal concentrations were 170% and 335% of the actual mean achieved atmosphere concentration for Groups 1 and 2 respectively and shows that keeping the aerosol airborne was relatively straightforward for each exposure group.

- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: the particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds, UK). The devices consisted of six impactor stages (8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 µm cut points) with stainless steel collection substrates and a back up glass fiber filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and back up filter were weighed before and after sampling and the weight of the test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 µm cut points was calculated.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. The second concentration was selected after consideration of the results of the first exposure.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric method

Duration of exposure:

4 h

Concentrations:

Dose group 1: 5.16 mg/L
Dose group 2: 1.08 mg/L

No. of animals per sex per dose:

3 males and 3 females per dose group.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to 14 days. Any evidence of overt toxicity was recorded at each observation.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other:

Body Weight: Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 2, 3, 7 and 14.
Necropsy: At the end of the 14 day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.08 - < 5.16 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Group 1 (5.16 mg/L): 2/3 males died, 1/3 females died (total 3/6 deaths)
Group 2 (1.08 mg/L) No deaths

Clinical signs:

other: Clinical observations are given in tables 6-9 (attached). Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during a

Body weight:

Individual body weights, together with body weight changes are given in Table 10 and 11.

GROUP 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Reasonable body weight development was noted in all animals during the remainder of the recovery period.
GROUP 2 – All animals exhibited body weight losses on Day 1 post-exposure. Two male animals exhibited further body weight losses from Days 1 to 3 post-exposure. Reasonable body weight development was noted in all animals during the remainder of the recovery period.

Gross pathology:

Individual necropsy findings are given in Tables 12 and 13.

Two instances of dark patches on the lungs and one instance of pale patches on the lungs were noted in the surviving group 1 animals. With the exception of one instance of dark patches on the lungs no macroscopic abnormalities were detected at necropsy amongst Group 2 animals.

The following macroscopic abnormalities were detected in the animals that died during the course of the study (Group 1 only) at necropsy:

Lungs – abnormally dark
Liver – accentuated lobular pattern
Kidneys – dark
Stomach – gaseous distension
Small intestine – gaseous distension
Large intestine – gaseous distension
Due to the observations noted it is considered that the deaths noted during the study may have been mainly attributed to local toxicity.

Other findings:

DOSE GROUP 2 - 1.08 mg/L (clinical findings).
During exposure occasional instances of increased respiratory rate were noted. On removal from the chamber all animals exhibited increased respiratory rate and there were occasional instances of laboured respiration. One hour post-exposure, little or no change in the condition of animals in Group 2 were noted. One day after exposure all animals exhibited increased respiratory rate, laboured respiration and hunched posture, pilo-erection was also noted in male animals. Animals recovered to appear normal from Days 9 to 10 post-exposure.

Any other information on results incl. tables

Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during each exposure period and the actual concentration of the test item calculated. The mean values obtained were:

 

Group Number	Atmosphere Concentration
	Mean achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	5.16	0.20	8.77
2	1.08	0.08	3.62

 

The exposure chamber concentrations are given in Tables 1 and 2 and are presented graphically in Figures 3 and 4 (attached).

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T99) was 3 minutes*

*The test atmosphere was generated for 17 minutes and 21 minutes prior to animal insertion (for Groups 1 and 2 respectively) to ensure the test item concentration was being achieved.

 

Particle Size Distribution

Group Number	Mean achieved atmosphere concentration (mg/L)	Mean mass median aerodynamic diameter (µm)	Inhalable fraction (% < 4µm)	Geometric standard deviation
1	5.16	3.06	60.9	267
2	1.08	1.99	82.3	2.14

 

The particle size distribution data are presented in Tables 3 and 4 and graphs showing the distribution are presented in Figures 5 to 8 (attached).

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute inhalation LC50 was determined to be in the range from >1 mg/L to 5 mg/L for the test substance.

Executive summary:

Three out of six animals died at a mean achieved atmosphere concentration of 5.16 mg/L, whereas, no deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 1.08 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4hr LC50) of IP37: Copper (II)-pyrophosphate, in RccHanTM: IST strain rat, was in the range >1 mg/L to 5 mg/L (Category 4).