Registration Dossier

Administrative data

Description of key information

Oral: LD50(rat, f) > 2000 mg/kg bw (OECD 420, GLP)
Inhalation: LC50(rat, m/f) > 5.09 mg/L (OECD 436, GLP)
Dermal: no study available

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Nov - 20 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 153 - 170 g
- Housing: The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet: 2014C Teklad Global Rodent diet (Harlan Teklad, Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: Arachis oil BP was considered to produce a formulation more suitable for dosing than distilled water

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: For the purpose of the study, the test item was freshly prepared, as required, as a suspension in vehicle.
The test item was formulated within two hours of being applied to the test system. It was assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
300 mg/kg bw: 1
2000 mg/kg bw: 5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were made ½, 1, 2, and 4 hours after dosing and subsequently once daily for fourteen days. Morbidity and mortality checks were made twice daily. Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.


Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
300 and 2000 mg/kg bw: There were no deaths.
Clinical signs:
300 and 2000 mg/kg bw: No signs of systemic toxicity were noted during the observation period.
Body weight:
300 and 2000 mg/kg bw: All animals showed expected gains in bodyweight over the observation period.
Gross pathology:
300 and 2000 mg/kg bw: No abnormalities were noted at necropsy.

Table 1. Summary of individual bodyweights and bodyweight changes

Dose level [mg/kg bw]

Animal number and sex

Bodyweight [g] at Day

Bodyweight gain [g] during Week

0

7

14

1

2

300

1-0 female

159

178

191

19

13

2000

2-0 female*

160

200

218

40

18

3-0 female

164

182

195

18

13

3-1 female

163

183

194

20

11

3-2 female

170

189

192

19

3

3-3 female

153

177

192

24

15

 * Due to a dose volume calculation error the animal received a dose of at least 2000 mg/kg. As no signs of systemic toxicity were observed, this error was considered not to have affected the outcome of the study.

Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar rat was estimated to be greater than 2000 mg/kg bw. Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and is thus considered to be not acutely toxic by the oral route.

CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw
Quality of whole database:
The available data comprise one study conducted following an OECD Guideline and under GLP conditions, with no or no relevant deviations or deficiencies which may affect the validity and reliability of the study results. Therefore, the available data are sufficient to fulfil the endpoint specific standard information requirements of Regulation (EC) No 1907/2006 (REACH), and are likewise sufficient for the purpose of classification and labelling in accordance with Regulation (EC) 1272/2008 (CLP).

Acute toxicity: via inhalation route

Link to relevant study records
Reference
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.
Qualifier:
according to
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. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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.
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.
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.

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)

 

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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating conc.
Value:
5 090 mg/m³
Quality of whole database:
The available data comprise one study conducted following an OECD Guideline and under GLP conditions, with no or no relevant deviations or deficiencies which may affect the validity and reliability of the study results. Therefore, the available data are sufficient to fulfil the endpoint specific standard information requirements of Regulation (EC) No 1907/2006 (REACH), and are likewise sufficient for the purpose of classification and labelling in accordance with Regulation (EC) 1272/2008 (CLP).

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute oral toxicity

The acute oral toxicity of dicalcium pyrophosphate was tested in female Wistar rats in a study following OECD Guideline 420 and under GLP conditions. In the absence of data regarding the toxicity of the test item, one single animal was tested first at a starting dose of 300 mg/kg bw given by oral gavage. In the absence of mortality and any other signs of toxicity, a second animal was tested at a nominal oral dose of 2000 mg/kg bw (the author reported that due to an error in the dose volume calculation, the actual dose received was at least 2000 mg/kg bw). In the absence of toxic effects, a group of 4 animals were orally treated with the test material at 2000 mg/kg bw. There were no deaths and no signs of systemic toxicity were noted during the 14-day observation period. All animals showed expected gains in body weight. No abnormalities were noted at necropsy. The oral LD50 of the test material in female rats was thus determined to be > 2000 mg/kg bw.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not acutely toxic by the oral route.

Acute inhalation toxicity

The acute inhalation toxicity of dicalcium pyrophosphate was evaluated in male and female Sprague Dawley rats by the acute toxic class (ATC) method according to OECD Guideline 436 and under GLP conditions. A group of three male and three female rats was exposed by nose-only, flow-past inhalation to the test material at a mean concentration of 5.09 mg/L air during 4 hours. This concentration was found to be the highest technically achievable concentration. The mean Mass Median Aerodynamic Diameter (MMAD) during exposure was 2.02 µm. More than 62% of the particles were < 4 µm, and nearly all particles (> 97%) were < 16 µm. The test material was therefore considered to be respirable to rats.

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 observed 1 h after exposure in any of the males, but was still present in all females, with the exception of 1 out of 3 females that only presented dirty fur. Similarly, chromodacryorrhea was not longer detected in any of the males 1 h after exposure, but was still present in 1 out of 3 females. Additional clinical signs recorded on Day 1 of study were palpebral ptosis (in 1 out of 3 females) and mild piloerection (in 1 out of 3 males and 2 out of 3 females). All these clinical signs were not longer present on Day 2 of study and no other clinical signs were observed from Day 2 of study to the end of the observation period.

A slight decrease in body weight was observed in all animals from Day 1 of study to Day 2 of study. This body weight loss was transient and body weight from all animals was higher at Day 4 of study than body weight at pre-treatment, with the exception of one female whose body weight was not higher than pre-exposure bodyweight until Day 6 of study.

The LC50 of the test material in male and female rats was determined to be > 5.09 mg/L.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not acutely toxic by the inhalation route.

Acute dermal toxicity

There are no studies available.


Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) for acute oral and inhalation toxicity.