reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 26 May 2010 and 28 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate (multi-constituent)

IUPAC Name:

Reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate (multi-constituent)

Test material form:

solid

Details on test material:

Sponsor's identification: A reaction mass of monocalcium phosphate and dicalcium phosphate
Description : cream coloured granular solid
Purity : 82.1% (26.8% dicalcium phosphate and 55.3% monocalcium phosphate)
Batch number : Not supplied
Date received : 05 May 2010
Storage conditions: room temperature in the dark.

In vitro test system

Test system:

other: reconstituted human epidermal model

Source species:

other: reconstituted human epidermal model

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstituted human epidermal model

Source strain:

other: reconstituted human epidermal model

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Test method is validated for this system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN(TM)
- Tissue batch number(s):
Not given
- Delivery date:
26 May 2010 (date received)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
37°C
- Temperature of post-treatment incubation (if applicable):
37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
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
- Observable damage in the tissue due to washing:
None recorded

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
0.3 mg/mL
- Incubation time:
3 hours
- Spectrophotometer: Anthos 2001 miscoplate reader
- Wavelength - 540 nm



NUMBER OF REPLICATE TISSUES:
2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
N/A, no direct MTT interference


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One pre-incubation test and one test material

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to to skin if the mean tissue viability is:
- <35% after the 3-minute exposure period
- >=35% after the 3-minute exposure period and <35 % after the 60-minute exposure period
- >=35% after the 60-minute exposure period and <35 % after the 240-minute exposure period

- The test substance is considered to be non-corrosive to skin if the mean tissue viability is >=35% after the 240-minute exposure period

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution): N/A

VEHICLE
- N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% w/v

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): ca. 100%

Duration of treatment / exposure:

3, 60 or 240 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Test animals

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: topical

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
20 mg of the test material was applied to the epidermis surface.

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

VEHICLE
No vehicle used

Duration of treatment / exposure:

3, 60, 240 minute treatments

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
20 mg of the test materialwas applied to the epidermis surface.

- % 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 each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) 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. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.


SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240 minute treatments, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. 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 corrosivity potential was based upon relative viabilities for both exposure times according to the following:

3 minute exposure : <35 Corrosive (EU R35)

3 minute exposure : ≥35
and 60 minute exposure : <35 Corrosive (EU R34)

60 minute exposure : ≥35
and 240 minute exposure : <35 Corrosive (EU R34)

240 minute exposure : <35 Non-corrosive

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 240 minutes

Value:

109.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 60 minutes exposure

Value:

121.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean - after 3 minutes exposure

Value:

0.278

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

6.2Test Material, Positive Control Material and Negative Control Material

Mean OD540values and viabilities for the negative control, positive control and test material are given in Table 1.

 

The relative mean viability of the test material treated tissues was as follows:

240 minutes exposure:          109.6%

60 minutes exposure:121.9%

3 minutes exposure:              156.2%

 

The qualitative evaluation of tissue viability is given in Table 2.

Following the 3, 60 and 240-Minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 6.20/0 relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table 1. Mean OD₅₄₀Values and Viabilities for the Negative Control Material,

Positive Control Material and Test Material

Material	Exposure period	Mean OD540if duplicate tissues	Relative mean viability (%)
Negative control material	240 mins	0.178	100*
Positive control material	240 mins	0.011	6.2
Test material	240 mins	0.195	109.6
	60 mins	0.217	121.9
	3 mins	0.278	156.2

 

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

 

Table 2. Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

 

Material	Exposure period	Tissue 1	Tissue 2
Negative control material	240 mins	-	-
Positive control material	240 mins	++	++
Test material	240 mins	-	-
	60 mins	-	-
	3 mins	-	-

 

- = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be Non-Corrosive to the skin.

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). However, the study does support the conclusion that the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate has a low overall potential for skin irritation in vivo and the data can therefore be used to support the conclusions made in the key study.