Pentacalcium hydroxide tris(orthophosphate)

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 - 27 May 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Due to agglomeration of the test substance the aerosol generator was blocked repeatedly and, consequently, it was only possible to expose the rats for a total of 2 h and 50 min. The animals were in the exposure tubes for a total of 6 h and 54 min until the exposure was ended.

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)

Version / remarks:

adopted Sep 2007

Deviations:

yes

Remarks:

Due to agglomeration of the test substance the aerosol generator was blocked repeatedly and it was only possible to expose the rats for a total of 2 h and 50 min. The animals were in the exposure tubes for a total of 6 h and 54 min.

GLP compliance:

yes (incl. QA statement)

Remarks:

Comunidad de Madrid, Consejera de Sanidad, Dirección General de Ordenación e Inspecctión, Spain

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pentacalcium hydroxide tris(orthophosphate)

EC Number:

235-330-6

EC Name:

Pentacalcium hydroxide tris(orthophosphate)

Cas Number:

12167-74-7

Molecular formula:

Ca5HO13P3

IUPAC Name:

Calcium hydroxycalcium(1+) phosphate (4:1:3)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: Sprague-Dawley (Crl:sd)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Barcelona, Spain
- Age at study initiation: 12 weeks
- Weight at study initiation: 442.3 - 463.5 g (males); 233.7 - 264.3 g (females)
- Fasting period before study: no
- Housing: animals were housed 4/cage/sex before study start and 3/cage/sex after study start. Enrichment material (nesting material, tubes and chewing blocks) was provided.
- Diet: Global Diet 2914C (Harlan Teklad, Oxon, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14.1 - 25.8
- Humidity (%): 23 - 68
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 May 2015 To: 27 May 2015

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.08 µm

Geometric standard deviation (GSD):

3.87 - 5.77

Remark on MMAD/GSD:

Geometric Standard Deviation (GSD) on the two PSD determinations (PSD #2 and PSD #3) were 3.87 and 5.77 respectively, which are above the target range (1.5 to 3). Nevertheless, these values were considered to be acceptable taking into account that more than 76% and 50% of particles were below upper limit of 4µm in PSD #2 and PSD #3 respectively. Hence, the particle size distributions obtained were considered to be respirable to rats and these incidents are considered to have no impact on the quality / integrity of the study

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: EC-FPC-232 anodised aluminium exposure chamber
- Exposure chamber volume: approximately 3 L in individual nose-only, flow-past chamber
- Method of holding animals in test chamber: separately in glass restraint tubes
- Source and rate of air: filtered air at 1 L/min
- System of generating particulates/aerosols: the test subtance was desiccated for 18 h to facilitate the generation of a respirable aerosol. The dust aerosol was generated from the desiccated test substance using a Rotating Brush Generator (PALAS RBG 2000) and the dust was diluted with filtered air from a compressor and lead in glass tubes from the generator to the exposure chamber
- Method of particle size determination: mass median aerodynamic diameter (MMAD) and 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.
- Temperature, humidity, pressure in air chamber: 21. 4 ± 0.55 °C, 16.9 ± 0.76%

TEST ATMOSPHERE
- Brief description of analytical method used: aerosol concentration was determined using gravimetric analysis at least once per hour during each hour of exposure.

VEHICLE
- Composition of vehicle (if applicable): air

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: determined gravimetrically 3 times during the exposure period (see Table 1 under 'any other information on materials and methods incl. tables').
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD: 3.08 µm; GSD: 3.87 and 5.77 (results from PSD#2 and #3 are considered to be acceptable as the majority of the particles were below the upper limit of 4 µm)

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: the starting dose of approximately 2 mg/L air was selected based on the available data and as this was the highest technically achievable dose during technical trials.

OTHER:
The test substance pentacalcium hydroxide tris(orthophosphate) is prone to agglomeration and aerosolisation could not be performed in its natural form. Therefore, the test substance was provided by the data owner as a commercial product whose specific manufacture process yields more spherical particles (without affecting the chemical identity), which were predicted to have a better aerosolisation than products from other manufacture processes.
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. These trial included determination of the target concentration and/or technical limit. Several tests were performed to establish the highest stable aerosol concentration achievable that could be maintained for at least 4 h. Aerosol concentration was initially aimed to 5 mg/L air since the test item was expected to be non-toxic. A stable aerosol concentration of approximately 2 mg/L air could be achieved with a PALAS RBG 2000 generator. Higher concentrations were not attempted since they would have required piston speed configurations that could damage the generator. Aerosol concentration was determined by gravimetric analysis.
Despite the attempts to aerosolize the test substance for the total duration of 4 h, blockages started to occur after the first hour of exposure and all attempts to complete the 4 hours exposure were unsuccessful. The study director decided to stop the aerosol generation after 6 hours and 54 minutes. The total cumulative exposure of animals to pentacalcium hydroxide tris(orthophosphate) (CAS No.: 12167-74-7, EC No.: 235-330-6) was 2 hours and 50 minutes.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

170 min

Remarks on duration:

Due to agglomeration of the test substance the aerosol generator was blocked repeatedly, therefore it was only possible to expose the rats for a total of 2 h and 50 min. The animals were in the exposure tubes for a total of 6 h and 54 min.

Concentrations:

2.35 mg/L (analytical concentration)

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: animals were observed for mortality and morbidity daily; clinical signs were recorded hourly during exposure (only grossly abnormal signs), immediately and 1 h after exposure, and once daily thereafter for the rest of the study period; the body weight was recorded on Day 1 prior to treatment, on Day 2, 4, 8, and on Day 15 prior to sacrifice.
- Necropsy of survivors performed: yes, with particular attention being paid to changes in the respiratory tract.

Statistics:

No statistical analysis was performed.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.35 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

170 min

Remarks on result:

other: Due to agglomeration of the test substance the aerosol generator was blocked repeatedly, therefore it was only possible to expose the rats for a total of 2 h and 50 min.

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: 2/3 males had wet fur after 2 h exposure, while 3/3 males and 3/3 females had wet fur immediately after exposure ended and 1 h post-dosing. 1/3 males had dirty fur 2 h after exposure ended. Chromorrhinorrhea was observed in 3/3 males and chromodacryorrea

Body weight:

A slight decrease in mean body weight (< 5%) was observed from Day 1 to Day 2 in 6/6 rats. On Day 4, 6/6 rats had gained weight, relative to Day 2, although the individual body weight in 2/3 males and 1/3 females was lower than the body weight recorded on Day 1. By Day 8, all rats had gained body weights that were higher than that recorded on Day 1.

Gross pathology:

In 1/3 males, 2 red foci were observed in the right lung. It is not clear if this effect is treatment-related. No other macroscopic findings were reported in any animal.

Any other information on results incl. tables

Table 2: Results

Target concentration [mg/L air]	Mortality	Clinical signs
	N	N
Males
2.35 mg/L	0/3	3/3
Females
2.35 mg/L	0/3	3/3

*N= Number of animals/ number of animals used

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No mortality occurred during the study period and the LC50 of pentacalcium hydroxide tris(orthophosphate) was considered to be > 2.35 mg/L in air (analytical). This was the highest technically achievable test concentration. Due to agglomeration of the test substance the aerosol generator was blocked repeatedly and, consequently, it was only possible to expose the rats for a total of 2 h and 50 min. The animals were held in the individual restraint tubes for a total of 6 h and 54 min until the exposure phase was ended, as opposed to the standard 4-h exposure period. Therefore, both for technical reasons and for animal welfare reasons it was justified to end the exposure phase. During the exposure period and the 14-day observation period the study was performed according to OECD guideline 436, with the exception of the total exposure period of the animals, for technical and animal welfare reasons.
Taking into account the lack of mortality at 2.35 mg/L, the highest technically achievable test concentration, and the lack of clearly toxicologically relevant effects on body weight or macroscopic findings, the study is considered to be In accordance with Regulation (EC) No. 1272/2008. The test substance is considered to be not classified as acutely toxic via the inhalation route.