Dicalcium pyrophosphate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Mar - 07 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP - Guideline study Documented deviations were considered to have no impact on the quality / integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see 'Remarks on results including tables and figures'

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:

Dicalcium pyrophosphate

EC Number:

232-221-5

EC Name:

Dicalcium pyrophosphate

Cas Number:

7790-76-3

Molecular formula:

Ca2O7P2

IUPAC Name:

dicalcium pyrophosphate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Barcelona, Spain
- Age at study initiation: 8 weeks
- Weight at study initiation: 282-288 g (m) and 192 - 207 g (f)
- Housing: 4 animals per cage before treatment, 3 animals per cage after treatment
- Diet: Global Diet 2914 C (Harlan Teklad, UK), ad libitum, except when animals were restrained in the exposure tubes
- Water: Tap water, ad libitum, except when animals were restrained in the exposure tubes
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 - 23.2
- Humidity (%): 21 - 53
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.9 - <= 2.14 µm

Geometric standard deviation (GSD):

>= 2.47 - <= 6.74

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure chambers type EC-FPC-232 (anodised aluminium), equipped with glass exposure tubes were used.
- Exposure chamber volume: approximately 3 L
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
- Source and rate of air: Filtered air from a compressor. The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. The actual airflow rate was monitored hourly in each group during each exposure. The target range was 0.5 - 1.0 L/min through each inhalation tube.
- System of generating particulates/aerosols: A dust aerosol was generated from the sieved test item using a Rotating Brush Generator PALAS RBG 2000. The dust was diluted with filtered air from a compressor and conveyed via glass tubing from the generator to the exposure chamber. The flow rate through the exposure chamber was adjusted as necessary.
- Method of particle size determination: The particle size distribution was determined gravimetrically twice during exposure. The cumulative particle size distribution of the test aerosol was determined using a PIXE cascade impactor. The particle size distribution of the test item in the generated aerosol was measured by gravimetry analyzing the test item deposited on each stage of the cascade impactor.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 4 μm for the MMAD and 1.5 to 3 for the GSD.
- Temperature, humidity in air chamber: The temperature in the chamber was measured continuously during exposure using a thermohygrometer (Kimoth110, Kimo). The target range was 19 - 25°C. The results were reported approximately hourly from the start of the inhalation exposure.
The relative humidity in the chamber was measured continuously during exposure using a thermohygrometer (Kimoth110, Kimo). The target range was 30 - 70%. The results were reported approximately hourly from the start of the inhalation exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The test item usage was determined once per exposure by weighing the amount of the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.
Gravimetric determination of the aerosol concentration was performed at least once during each hour of exposure. Test aerosol samples were collected onto a Whatman filter (grade F319-04) using a filter sampling device. The sampling flow was similar to the air flow rate per exposure port. The duration of sampling was 5 minutes. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Mean Mass Median Aerodynamic Diameter (MMAD) of particle size distribution during exposure was calculated from two gravimetric measurements PSD #1 and PSD #2. Mean MMAD during exposure was 2.02 μm. This value is within the respirable range (1-4 μm) and appropriate for acute inhalation toxicity testing. Geometric Standard Deviation (GSD) on PSD #2 was above the upper limit of 3 but considered acceptable as more than 62% of particles were below the upper limit of 4 μm (see Table 1).

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: The starting dose (approximately 5 mg/L air, during 4 hours) was selected as no toxic effects were expected based on the available data. This concentration was determined during technical trials (see 'Any other information on materials and methods incl. tables').

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Nominal concentration: 5 mg/L
Analytical concentration: 5.09 mg/L

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were examined daily for mortality and morbidity. Clinical observations in response to treatment were performed on all animals hourly during exposure (only grossly abnormal signs), immediately and 1 h after exposure, and once daily thereafter until the end of the observation period. Any visible clinical signs and discomfort were recorded. All animals were weighed on the day of treatment, just before starting the inhalation period (Day 1 of study), on Day 2, 4, 5, 6, 8, 9 and immediately before sacrifice on Day 15 of study.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

No statistical analysis was performed.

Results and discussion

Preliminary study:

Technical trials were performed without animals and conducted before the animal phase of the corresponding dose group(s) to establish the conditions for aerosol generation, which included: Determination of target concentration and/or technical limit. Several tests were performed to establish the highest stable aerosol concentration achievable that could be maintained at least for 4 hours. Aerosol concentration was aimed to 5 mg/L air since the test item was expected to be non-toxic. A stable respirable aerosol at a maximum concentration of 2.5 mg/L air could be achieved using the TOPAS SAG 410 aerosol generator. Higher concentrations of dicalcium pyrophosphate could not be achieved using this generator. By contrast, a stable aerosol at approximately 5 mg/L could be obtained with the PALAS RBG 2000 generator. Aerosol concentration was determined by gravimetric analysis. Determination of particle size distribution: Mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were determined at the target concentration and calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). Dicalcium pyrophosphate aerosol at approximately 5 mg/L was found to be within the respirable range (1-4 μm). Therefore, starting dose was set at 5 mg/L.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.09 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals survived the scheduled observation period.

Clinical signs:

other: Dirty fur and chromorrhinorrhea were observed in all animals immediately after exposure. In addition, chromodacryorrhea was recorded immediately after exposure in 2 out of 3 males and in 2 out of 3 females. Dirty fur and chromorrhinorrhea were not longer

Body weight:

A slight decrease in body weight with respect to body weight at pre-treatment (approximately 2% less in males and 4% less in females) was observed in all animals from Day 1 of study to Day 2 of study. Body weight higher than body weight at pre-treatment was observed in all animals at Day 4 of study, with the exception of one female whose body weight was not higher than body weight at pretreatment until Day 6 of study. Thereafter, a normal body weight gain was observed in all animals.

Gross pathology:

No macroscopic findings were observed during necropsy.

Any other information on results incl. tables

Test atmosphere conditions

Temperature during exposure was considered to be satisfactory and within target range (19 - 25ºC). Relative humidity was below target range (30 - 70%). Data are presented in the following table:

Recording time (h:min from exposure start)	Temperature (ºC)	Relative Humidity (%)
0:46	21.3	13.1
1:11	21.8	13.4
2:06	21.8	12.9
3:09	21.7	13.8
Mean	21.7	13.3
SD	0.24	0.39
N	4	4

Mean oxygen and carbon dioxide concentrations were 20.9% and 0.04% respectively. These values are considered satisfactory for inhalation studies and within target range (at least 19% and below 1% respectively). Data are presented in the following table:

Recording time (h:min from exposure start)	Oxygen (%)	Carbon dioxide (%)
0:46	20.9	0.04
1:11	20.9	0.04
2:06	20.9	0.04
3:09	21.0	0.04
Mean	20.9	0.04
SD	0.05	0
N	4	4

 

Aerosol concentrations

The mean of the gravimetric concentrations during exposure was 5.09 mg/L air, as targeted. Data on aerosol concentrations are presented in the following table: 

Group A (5.09 mg/L air). Day 1 of study
Sampling starting time (h:min from exposure start)	Sampling volume (L)	Amount of test item on the filter (mg)	Gravimetric aerosol concentration (mg/L)
0:12	5.63	29.08	5.17
0:36	5.57	27.30	4.90
0:45	5.59	31.20	5.59
1:22	5.59	27.96	5.01
2:08	5.58	34.17	6.12
2:24	5.56	27.93	5.02
2:48	5.52	22.74	4.12
3:09	5.52	30.95	5.61
3:29	5.24	22.36	4.27
MEAN	5.53	28.19	5.09
SD	0.11	3.84	0.64
N	9	9	9

 

Summary of mortality and clinical signs.

Concentration (mg/L)	Animal number (sex)	Mortality	Clinical signs	Time point/duration
5.09	1 (male)	no	Chromorrhinorrhea	Immediately post-exposure
			Chromodacryorrhea	Immediately post-exposure – 1 h post-exposure
			Dirty fur	Immediately post-exposure
	2 (male)	no	Chromorrhinorrhea	Immediately post-exposure
			Chromodacryorrhea	Immediately post-exposure
			Dirty fur	Immediately post-exposure
	3 (male)	no	Piloerection	1 h post-exposure
			Chromorrhinorrhea	Immediately post-exposure
			Dirty fur	3 h exposure – Immediately post-exposure
	4 (female)	no	Piloerection	1 h post-exposure
			Chromorrhinorrhea	Immediately post-exposure – 1 h post-exposure
			Chromodacryorrhea	Immediately post-exposure
			Dirty fur	3 h exposure – 1 h post-exposure
	5 (female)	no	Chromorrhinorrhea	Immediately post-exposure – 1 h post-exposure
			Dirty fur	Immediately post-exposure – 1 h post-exposure
			Ptosis	Immediately post-exposure
	6 (female)	no	Piloerection	1 h post-exposure
			Chromorrhinorrhea	Immediately post-exposure
			Chromodacryorrhea	Immediately post-exposure – 1 h post-exposure
			Dirty fur	3 h exposure – 1 h post-exposure

 

Summary of body weight (gain)

Concentration (mg/L)	Animal number (sex)	Body weight (gain) (g)
		Day 1	Day 2	Day 4	Day 5	Day 6	Day 8	Day 9	Day 15
5.09	1 (male)	287.40	278.82 (-8.58)	299.40 (20.58)	308.25 (8.85)	316.68 (8.43)	327.80 (11.12)	334.27 (6.47)	376.44 (42.17)
	2 (male)	283.80	279.55 (-4.25)	292.70 (13.15)	301.08 (8.38)	305.11 (4.03)	310.50 (5.39)	322.82 (12.32)	354.47 31.65)
	3 (male)	282.96	277.19 (-5.77)	297.40 (20.21)	305.87 (8.47)	315.08 (9.21)	326.00 (10.92)	338.83 (12.83)	388.94 (50.11)
	4 (female)	192.29	185.96 (-6.33)	193.60 (7.64)	193.08 (-0.52)	194.77 (1.69)	197.10 (2.33)	199.29 (2.19)	207.45 (8.16)
	5 (female)	206.49	194.05 (-12.44)	196.00 (1.95)	206.00 (10.00)	213.04 (7.04)	200.00 (-13.04)	212.96 (12.96)	229.20 (16.24)
	6 (female)	200.19	194.32 (-5.87)	201.90 (7.58)	203.90 (2.00)	206.28 (2.38)	205.78 (-0.50)	211.77 (5.99)	226.91 (15.14)

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The LC50 of the test material in male and female rats was determined to be > 5.09 mg/L under the conditions of this study. There were no test material-related mortalities, clinical signs of systemic toxicity, adverse effects on body weight (gain) or abnormal necropsy findings.Therefore, the material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and is thus considered to be not acutely toxic by the inhalation route.

CLP: not classified