Calcium dibenzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-02 to 2017-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2015-07-28

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dibenzoate

EC Number:

218-235-4

EC Name:

Calcium dibenzoate

Cas Number:

2090-05-3

Molecular formula:

C7H6O2.1/2Ca

IUPAC Name:

calcium dibenzoate

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation at room temperature: solid powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, < 30 °C

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25834

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [mean ODtest item / positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

99.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.773).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.4 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.8 % - 3.9 %).
Please also refer to the field "An other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Result of the test item calcium dibenzoate

Name	Negative Control (NK)			Positive Control (PC)			Test material (TM)
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.755	1.853	1.950	0.112	0.104	0.087	1.729	1.858	1.799
	1.819	1.847	1.671	0.120	0.106	0.092	1.754	1.902	1.833
OD570(blank-corrected)	1.712	1.811	1.907	0.069	0.061	0.044	1.686	1.816	1.756
	1.776	1.804	1.628	0.078	0.063	0.049	1.711	1.859	1.790
mean OD570of the duplicates (blank-corrected)	1.744	1.808	1.767	0.073	0.062	0.047	1.699	1.837	1.773
total mean OD570of 3 replicate tissues (blank-corrected)	1.773*			0.061			1.770
SD OD570	0.032			0.013			0.069
relative tissue viability [%]	98.4	101.9	99.7	4.1	3.5	2.6	95.8	103.6	100.0
mean relative tissue viability [%]	100.0			3.4**			99.8
SD tissue viability [%]***	1.8			0.8			3.9
CV [% viabilities]	1.8			22.1			3.9

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is ≤  20 %.

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Table 2: Historical data

	Mean OD570±30nmNK	Mean Relative Viability [%] PC	SD Viability [%]
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Historical data were generatedfrom 2015 to 2016

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the criteria of Regulation (EC) No 1272/2008 and its subsequent amendments, Calcium dibenzoate does not have to be classified for skin irritation.