Calcium hydrogen phosphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2016 to 22 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

yes

Remarks:

Tp° in the incubator > 0.2°C.The SD value of viability (%) was 37.4% instead of 18% max for negative control tissues but homogenous results were obtained in test item treated tissues and historical data. No impact on the conclusion of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Calcium hydrogen phosphonate

EC Number:

244-182-1

EC Name:

Calcium hydrogen phosphonate

Cas Number:

21056-98-4

Molecular formula:

Ca.H3O3P

IUPAC Name:

calcium phosphonate

Test material form:

solid: crystalline

Details on test material:

Purity : 95%
Batch number: PCAF151028 Rns
CAS number : 21056-98-4
Chemical name : Phosphonic acid, calcium salt (1:1), Calcium hydrogen phosphonate
Manufacturing date : 28/10/2015
Shelf life or Expiry date : 28/10/2018
Date of analysis : 27/11/2015

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis

Details on test system:

The 0.50 cm2 reconstructed epidermis (Episkin SA, RHE/S17 Batch No. 16-RHE-008)
Medium and Incubation T°C: 37°C

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

Amount applied per tissue : 16 mg

Duration of treatment / exposure:

Exposure period of 42 minutes, followed by rinsing. The rinsed tissues were checked for any coloration. There were incubated for a 41 hours and 05 minutes post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then the epidermis were put in contact with MTT solution. Observation of MTT-> formazan transformation by viable cells.

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

the exact % viability value was 118 considered 100

Other effects / acceptance of results:

In the preliminary tests, the test item was found not to have direct MTT reducing properties or significant colouring potential.
Main test: All acceptance criteria for the negative and positive controls were fulfilled except that the standard deviation value of viability percent of the tissues treated with the negative control was 37.4% instead of 18% (maximal value) as initially scheduled in the study plan. Considering the study results obtained in the tissues treated with the test item and the historical data, this deviation is considered as without impact on the conclusion of the study. Indeed, the results of the tissues treated with the test item are homogenous and all markedly above the cut-off of 50%. The study was therefore considered to be valid.
Following a 42 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 118% (considered 100%) with a standard deviation of 13%.
As the mean viability was > 50% after the MTT reduction, the IL-1 a concentrations in culture media samples retained were analyzed by ELISA
The mean IL-1a concentration for treated tissues was 7.08 pg/mL vs 169.54 pg/mL in the positive control. Due to this value being below 60 pg/mL, the results met the criteria for an in vitro classification as non-irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In vitro, the test item is considered to be non-irritant to the skin.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of calcium hydrogen phosphonate using the Episkin reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46).

 Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 42 minutes. At the end of the treatment period, each tissue was rinsed and incubated for 42 hours at 37.5°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In addition, the concentration of the inflammatory mediator IL-1awas evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 In the main test, all acceptance criteria for the negative and positive controls were fulfilled except that the standard deviation value of viability percent of the tissues treated with the negative control was 37.4% instead of 18% (maximal value) as initially scheduled in the study plan. Considering the study results obtained in the tissues treated with the test item and the historical data, this deviation is considered as without impact on the conclusion of the study. Indeed, the results of the tissues treated with the test item are homogenous and all markedly above the cut-off of 50%. The study was therefore considered to be valid.

Following a 42 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 118% (considered 100%) with a standard deviation of 13%.

As the mean viability was > 50% after the MTT reduction, the IL-1aconcentrations in culture media samples retained were analyzed by ELISA

The mean IL-1aconcentration for treated tissues was 7.08 pg/mL vs 169.54 pg/mL in the positive control. Due to this value being below 60 pg/mL, the results met the criteria for an in vitro classification as non-irritant to skin.

 Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be:

.            not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).