Calcium glycerophosphate

Endpoint summary

Description of key information

Calcium glycerophosphate showed no irritatiom potential to skin (in vitro skin model) and eyes (BCOP) according to UN GHS and EU CLP regulation.

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:

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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

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.

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

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. 

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The test was performed on 26 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

physiological saline

Controls:

other: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline); negative control: Saline

Amount / concentration applied:

The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

SCORING SYSTEM:
Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3

Value:

ca. 2.71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0.00	0.072	0.069	1.08	1.04	No Category
	0		0.070		1.05
	0		0.065		0.98
Positive Control	125.00*		0.018*		125.27	130.61	Category 1
	131.00*		0.036*		131.54
	135.00*		0.002*		135.03
Calcium glycero-phosphate	3.00*		-0.002*		2.97	2.71	No Category
	3.00*		0.002*		3.03
	2.00*		0.009*		2.14

*corrected values

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

According to the current BCOP study and under the experimental conditions reported, Calcium glycerophosphate is not categorized (GHS).

Executive summary:

This in vitrostudy was performed to assess the corneal damage potential of Calcium glycerophosphate by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspensionin saline of the test item Calcium glycerophosphate, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae andopacity was measured again (t₂₄₀).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline) an increase of neither opacity nor permeability of thecorneae could be observed (mean IVIS = 1.04).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity of the corneae (mean IVIS =130.61) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Calcium glycerophosphate was tested assuspension. Relative to the negative control, the test item Calcium glycerophosphate did not cause a relevant increase of the corneal opacity or permeability. The calculated mean IVIS was 2.71 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

no classification

The test item showed no irritation in vitro on skin and eyes.