Calcium glycerophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September 2017 until 03 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 439 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2009)

Version / remarks:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Calcium glycerophosphate

EC Number:

248-328-5

EC Name:

Calcium glycerophosphate

Cas Number:

27214-00-2

Molecular formula:

C3H9O6P.Ca

IUPAC Name:

calcium glycerophosphate

Test material form:

solid: particulate/powder

Details on test material:

Batch: CGP0416003

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Calcium glycerophosphate
CAS No.: 27214-00-2
Batch: CGP0416003
Purity: 92.0% (w/w) (calculated) dose calculation was not adjusted to purity
Expiry Date: 29 June 2021
Appearance: White powder
Storage Conditions: At room temperature, protected from light and moisture*
Stability in Solvent: Stable in water (not quantified)
* only valid for storage conditions, not for test performance

In vitro test system

Test system:

human skin model

Remarks:

reconstituted human epidermis model

Source species:

human

Details on test system:

Cell Culture

Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLsÒ, 10 mmÆ).
EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 01 November 2017 (for additional test with the viable tissue) or 26 January 2016 (for main experiment), respectively. Each on day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
MTT Solution
On the day of testing the MTT concentrate (either from Sigma or from MatTek) was diluted with the MTT diluent (either with DMEM from Gibco or from MatTek; resulting: 1 mg/mL).

 Test for Direct MTT Reductionand Colour Interference
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose about 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2).
The colour of test item was very intensive (yellow) and the test item dyed the water during the incubation period. Therefore, an additional test with one viable tissue going through the entire test procedure, but treated with maintenance medium instead of MTT at the end of the performance, had to be performed.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose approximately about 25 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.
 Experimental Performance
 Pre-warming of EpiDerm™ Tissues
The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1.     air bubbles between agarose and insert were not > 30% of the total surface,
2.     liquid on top of the insert was removed with steriles cotton tips,
3.     if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) were pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22.25 (additional test) to 22.5 (main experiment) hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

Treatment
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, the vehicle control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 18 (additional test) or nearly 18 (main experiment) hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was about 42 hours.
MTT Assay
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2; the additional tissue was treated with maintenance medium), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for 69 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3´200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax®Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter.Mean values were calculated from 3 wells per tissue.

Control samples:

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

Duration of treatment / exposure:

60 minutes

Test system

Details on study design:

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

all

Value:

ca. 97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results after treatment with Calcium glycerophosphate and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	OD 570 nm Well 3	Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Viability [%]**
Blank		0.037	0.037	0.037	0.037
Negative Control	1	1.447	1.438	1.444	1.443	1.406	1.431	98.2	1.6	100.0
	2	1.468	1.481	1.469	1.473	1.436		100.3
	3	1.452	1.499	1.514	1.488	1.451		101.4
Positive Control	1	0.104	0.103	0.103	0.104	0.067	0.067	4.7	1.1	4.7
	2	0.105	0.104	0.104	0.104	0.067		4.7
	3	0.104	0.106	0.105	0.105	0.068		4.8
Test Item	1	1.370	1.382	1.383	1.378	1.341	1.392	93.7	3.5	97.3
	2	1.432	1.432	1.433	1.432	1.395		97.5
	3	1.480	1.479	1.468	1.476	1.439		100.5

 

*       Mean of three replicate wells after blank correction

**       relative absorbance per tissue [rounded values]

***     relative absorbance per treatment group [rounded values]

 

  The optical pre-experiment (colour interference pre-experiment) to investigate the test item’scolour change potential in water did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation withMTT-reagent did not show blue/purple colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 97.3% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin. 

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In this in vitro skin irritation study and under the experimental conditions reported, Calcium glycerophosphate is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitrostudy was performed to assess the irritation potential of Calcium glycerophosphate by means of the Human Skin Model Test.

The test item passed the MTT- and the Colour Interference pre-tests.

The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60minutes treatment. After further incubation for about 41.5 hours the tissues were treated withthe MTT solution for 3 hours following about 2.5 hours extraction of the colorant from thecells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance comparedwith the negative control to 4.7% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positiveand negative controls in the main test were below 10% (threshold of the "OECD Guidelinefor the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe mean relative absorbance value was reduced to 97.3% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.