Dicalcium pyrophosphate

Endpoint summary

Description of key information

Skin irritation: In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 the assessment of the endpoint ‘skin irritation or skin corrosion’  has been performed following the consecutive steps detailed in the Regulation. As such an in vitro skin corrosion study has been performed. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and is therefore submitted as supporting data.  The key study (Warren N, 2012) is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). 
Eye irritation: In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 an ex-vivo study has been performed prior to conducting an in vivo study. The in vivo study is sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and is therefore selected as the key study. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 06 November 2012 and 12 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspectection: 10 July 2012 Date of Signature: 07 September 2012

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 minute exposure & 42 hour post-exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 hours.

- Time after start of exposure:
15 minutes post-exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : H315, Category 2

Mean tissue viability is >50% : Non-Irritant (NI)

Irritation / corrosion parameter:

other: other: Viability of cells

Value:

112.2

Remarks on result:

other:

Remarks:

Basis: mean Viability of cells (%). Time point: Day 6. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: See relative mean viability below.. (migrated information)

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 112.2% after a 15-minute exposure.

Other effects:

No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 112.2% after a 15 minute exposure.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 8.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.697. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 9.4%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material ⊕	0.728	0.697	0.057	104.4	100*	8.2
	0.631			90.5
	0.732			105.0
Positive Control Material ⊕	0.032	0.038	0.015	4.6	5.4	2.2
	0.055			7.9
	0.026			3.7
Test Material	0.855	0.782	0.065	122.7	112.2	9.4
	0.729			104.6
	0.761			109.2

SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

⊕ = Control group shared with Harlan Laboratories Ltd, Project numbers 41205083, 41205113, 41205120, 41205125, 41205128 and 41205133

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be Non-Irritant (NI).
This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Dicalcium pyrophosphate is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 02 January 2013 and 10 January 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no minor deviations from standard test guidelines and no minor methodological deficiencies. This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 10 July 2012; Date of Signature on GLP certificate: 30 November 2012

Species:

rabbit

Strain:

other: New Zealand White (Hsdlf:NZW)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Leicestershire, UK.

- Age at study initiation: Twelve to twenty weeks old

- Weight at study initiation: 2.61 or 2.93 kg

- Housing: The animals were individually housed in suspended cages. 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 (e.g. ad libitum): ad libitum (2030C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd, Oxon, UK)

- Water (e.g. ad libitum): ad libitum.

- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 17 to 23°C

- Humidity (%): 30 to 70%

- Air changes (per hr): At least fifteen changes per hour

- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness


IN-LIFE DATES:
From: day 1 To:day 3

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit): 100 mg of the test material was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

VEHICLE

- Amount(s) applied (volume or weight with unit):Not applicable

- Concentration (if solution):Not applicable

- Lot/batch no. (if required):Not applicable

- Purity: Not applicable

Duration of treatment / exposure:

Up to 21 days (test item was not removed from the eyes).

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.

Number of animals or in vitro replicates:

2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, one additional animals was treated).

Details on study design:

REMOVAL OF TEST SUBSTANCE

- Washing (if done): Not applicable

- Time after start of exposure: Not applicable

INTERPRETATION OF RESULTS
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae = (A + B + C) * 2
Score for iris = D * 5
Score for cornea = (E * F) * 5

Using the numerical data obtained a modified version of the system described by Kay JH and Calandra J C (1962), J. Soc. Cosmet. Chem. 13, 281-289 (see below) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.

If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

The results were also interpreted according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

72824 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

72839 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

72824 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.25

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

72839 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.25

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

other: redness

Basis:

animal #1

Remarks:

72824 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.5

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

other: redness

Basis:

animal #2

Remarks:

72839 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.5

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

72824 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.5

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

72839 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.75

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.

No corneal effects were noted during the study.

Iridial inflammation was noted in both treated eyes one hour after treatment.

Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one hour after treatment. Minimal conjunctival irritation noted in both treated eyes at the 24-hour observation.

Both treated eyes appeared normal at the 48-hour observation.

Other effects:

Individual bodyweights and bodyweight changes are given in Table 3.
Both animals showed expected gain in bodyweight during the study.

Table 1. Individual Score and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	72824 Male				72839 Male
	IPR = 1				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
Degree of Opacity	0	0	0	0	0	0	0	0
Area of Cornea Involved	0	0	0	0	0	0	0	0
IRIS	1	0	0	0	1	0	0	0
CONJUNCTIVAE
Redness	1	1	0	0	1	1	0	0
Chemosis	1	1	0	0	2	1	0	0
Discharge	1	0	0	0	2	1	0	0

 

IPR =  Initial pain reaction

Table 2. Individual bodyweights and bodyweight changes

Rabbit number and sex	Individual bodyweight (kg)		Bodyweight change (kg)
72824 Male	Day 0	Day 3	0.07
	2.93	3.00
72839 Male	Day 0	Day 21	0.07
	2.61	2.68

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

This study is conducted according to the appropriate guidelines (OECD 405) and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement for REACH (Regulation (EC) No.1907/2006) as a key study for this endpoint. Study is sufficient for classification and labelling purposes, in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
This study has been selected as the key study in accordance with Regulation (EC) No. 1907/2006 (REACH) because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). No effects are noted.

Justification for selection of eye irritation endpoint:
This study has been selected as the key study in accordance with Regulation (EC) No. 1907/2006 (REACH) because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Justification for classification or non-classification

SKIN IRRITATION: No classification is proposed for skin irritation in accordance with Regulation (EC) No. 1272/2008 (EU CLP). This conclusion is based on reliable (Klimisch 1) and adequate data and as such no further testing is anticipated.

EYE IRRITATION: Study not scientifically justified according to Regulation (EC) No. 1907/2006 (REACH), Annex XI, Section 2: testing is technically not possible. According to the relevant OECD guideline (OECD 114) a study cannot be conducted on a substance that is a solid at room temperature, as dicalcium pyrophosphate is solid at room temperature no study is necessary.

There are no data to suggest that dicalcium pyrophosphate is irritating to the respiratory tract.