Carboxylic acids, di-, C5-9

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul to 20 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul to 20 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, National Institute of Pharmacy and Nutrition, Budapest, Hungary

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ (SM) (Manufacturer: SkinEthic, France)

Source strain:

other: not applicable

Justification for test system used:

The EPISKIN™ (SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN™ Small/ Human Epidermis (SM/13)
- Tissue batch number:
19-EKIN-029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
room temperature (23.6 - 24.2°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
after treatment (3 min, 1 and 4 h, respectively) the tissues were thoroughly rinsed with PBS, residual PBS was removed from the epidermal surface with a pipette without touching the epidermis

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
2 mL of 0.3 mg/mL MTT working solution
- Incubation time:
3 hours at 37°C, with 5% CO2, protected from light in a > 95% humidified atmosphere
- Spectrophotometer:
not reported
- Wavelength:
570 nm
- Filter:
not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer:
0.0 to 3.0 OD

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
EPISKIN™(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at the supplier, SkinEthic laboratories.
- Viability:
The quality of the EpiSkin™ tissue was assessed by undertaking a MTT cell viability test. The IC50 was 1.9 and 2.7 mg/mL for the two batches, respectively (acceptance criteria: 1.5 - 3.0 mg/mL).
- Barrier function:
habe ich nicht gefunden!!!!!!!!
- Morphology:
HES staining revealed multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multi-layered stratum corneum
- Contamination:
absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B anitigens was verified from the blood of donors; absence of bacteria, fungus and mycoplasam was verified on cells from donors

NUMBER OF REPLICATE TISSUES:
3 tissues each for the negative control, positive control and both treatment groups, respectively. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean viability of three individual tissues after 15 minutes exposure is less or equal to 50% of the negaitive controls.
- The test substance is considered to be non-corrosive to skin if the mean viability of three individual tissues after 15 minutes exposure is greater than 50% of the negaitive controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg (10 mg of the test item did not cover the epidermal surface)

VEHICLE
- Amount applied: before application of the test item, 10 µL distilled water was applied to the epidermal surface in order to improve further contact with the epidermal surface

NEGATIVE CONTROL
- Amount applied: 20 µL PBS

POSITIVE CONTROL
- Amount applied: 20 µL
- Concentration: 5% (w/v) SDS

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h at 37°C with 5% CO2 in an > 95% humidified atmosphere

Number of replicates:

Test item, negative and positve control: 3 replicates

Species:

other: human

Strain:

other: EPISKIN™ (SM) (Manufacturer: SkinEthic, France)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min (mean of three tissues)

Value:

11.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was in the recommended range (0.848) which satisfied the acceptability criterion of being 0.6-1.5. Standard deviation of the viability results for negative control samples was 4.1%.
The positive control treated tissues showed 3.7% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.6%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 6.6%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability %

Substance	Optical Density (OD)			Viability	Standard
		Measured	Blank corrected	(% RV)	deviation (SD)
Negative Control:	1	0.925	0.878	103.4	--
Phosphate buffered saline	2	0.857	0.810	95.4	--
	3	0.906	0.858	101.1	--
	mean	--	0.848	100.0	4.1
Positive Control:	1	0.079	0.031	3.7	--
5% (w/v) SDS solution	2	0.074	0.027	3.1	--
	3	0.085	0.037	4.4	--
	mean	--	0.032	3.7	0.6
Test Item:	1	0.189	0.142	16.7	--
Carboxylic Acids, di-, C4-11	2	0.161	0.114	13.4	--
	3	0.081	0.033	3.9	--
	mean	--	0.096	11.3	6.6

 

Notes:

1.          Mean blank value was0.048.

2.          Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3.          SDS: sodium dodecyl sulphate (5%(w/v))

 

Interpretation of results:

other: CLP/EU GHS criteria met, as the test item is already classified as Skin Corr. 1B according to Regulation (EC) No 1272/2008, no further classification for skin irritation potential is needed

Conclusions:

Skin Corr. 1B, based on results of a study according to OECD 431

Executive summary:

In this reliable and GLP-conform study according to OECD 439, exposure of disks of EPISKIN^TM

(SM) with the test item for 15 min, resulted in a mean cell viability of 11.3% compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin.

The experiment met the validity criteria, therefore the study was considered to be valid.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, National Institute of Pharmacy and Nutrition, Budapest, Hungary

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C5-9

EC Number:

701-329-9

Molecular formula:

C5di: C5H8O4 C6di: C6H10O4 C7di: C7H12O4 C8di: C8H14O4 C9di: C9H16O4

IUPAC Name:

Carboxylic acids, di-, C5-9

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ (SM) (Manufacturer: SkinEthic, France)

Source strain:

other: not applicable

Justification for test system used:

The EPISKIN™ (SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Small/ Human Epidermis (SM/13)
- Tissue batch numbers: 19-EKIN-029 used in Experiment I; Batch No.:19-EKIN-038 used in Experiment II

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.5 - 24.8°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after treatment (3 min, 1 and 4 h, respectively) the tissues were thoroughly rinsed with PBS, residual PBS was removed from the epidermal surface with a pipette without touching the epidermis

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT working solution
- Incubation time: 3 hours at 37°C, with 5% CO2, protected from light in a > 95% humidified atmosphere
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: 0.0 to 3.0 OD

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
EPISKIN™(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at the supplier, SkinEthic laboratories.
- Viability: The quality of the EpiSkin™ tissue was assessed by undertaking a MTT cell viability test. The IC50 was 1.9 and 2.7 mg/mL for the two batches, respectively (acceptance criteria: 1.5 - 3.0 mg/mL).
- Morphology: HES staining revealed multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multi-layered stratum corneum
- Contamination: absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B anitigens was verified from the blood of donors; absence of bacteria, fungus and mycoplasam was verified on cells from donors

NUMBER OF REPLICATE TISSUES: 2 tissues each for the negative control, positive control and both treatment groups, respectively. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in Experiment I (4 hour exposure).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431: The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 35%, or if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35%, or if the viability after 1 hour exposure is greater than or equal to 35 % and the viability after 4 hours exposure is less than 35%
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg

VEHICLE
- Amount applied: after application to the test system, 100 µL physiological saline was added to the test item to ensure good contact with the epidermis
- Lot/batch no.: 90352Y05-2 (B. Braun Pharamceuticals SA)

NEGATIVE CONTROL
- Amount applied: 50 µL 0.9% NaCl

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min, 1 h and 4 h

Number of replicates:

Test item: 2 replicates per time point (3 min, 1 and 4 h)
Positive control: 2 replicates at 1 and 4 hours
Negative control: 2 replicates per time point (3 min, 1 and 4 h)

Test animals

Species:

other: human

Strain:

other: EPISKIN™ (SM) (Manufacturer: SkinEthic, France)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

84.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 h

Value:

8.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD value of the two negative control tissues was in the recommended range (1.089 in Experiment I, and 0.970 and 0.824 in Experiment II for the 3 min and 1 h treatments, respectively) which satisfied the acceptability criterion of being 0.6- 1.5.
The positive control treated tissues showed 1.1% and 0.1% relative viability (Experiment I and II, respectively) when compared to the relevant negative control values, thus demonstrating the proper performance of the assay in each case.
The variability of the calculated viability values of the test item treated replicates were 13.1% (3 min treatment), 22.1% (1 h treatment), and 4.1% (4 h treatment).
The variability of the calculated viability values of the negative control treated replicates were 10.6% (3 min treatment), 0.4% (1 h treatment), and 0.2% (4 h treatment).
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples in Experiment I - (4 hours exposure)

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
Negative Control:	1	1.136	1.088	99.9
Physiological saline	2	1.138	1.090	100.1
(0.9% (w/v) NaCl)	mean	--	1.089	100.0
Positive Control:	1	0.053	0.006	0.5
Glacial acetic acid	2	0.067	0.019	1.7
	mean	--	0.012	1.1
Test Item:	1	0.146	0.099	9.0
Carboxylic Acids, di-, C4-11	2	0.142	0.095	8.7
	mean	--	0.097	8.9

Table 2: Optical Density (OD) and the calculated relative viability % of the samples in Experiment II (3 min and 3 hours exposure)

Substance	Optical Density (OD)			Viability
		Measured	Blank corrected	(% RV)
3 minutes exposure period
Negative Control:	1	1.069	1.022	105.3
Physiological saline	2	0.966	0.919	94.7
(0.9% (w/v) NaCl)	mean	--	0.970	100.0
Test Item:	1	0.817	0.770	79.3
Carboxylic Acids, di-, C4-11	2	0.925	0.877	90.4
	mean	--	0.824	84.9
1 hour exposure period
Negative Control:	1	0.873	0.825	100.2
Physiological saline	2	0.869	0.822	99.8
(0.9% (w/v) NaCl)	mean	--	0.824	100.0
Positive Control:	1	0.050	0.002	0.3
Glacial acetic acid	2	0.047	0.000	0.0
	mean	--	0.001	0.1
Test Item:	1	0.128	0.080	9.8
Carboxylic Acids, di-, C4-11	2	0.148	0.100	12.2
	mean	--	0.090	11.0

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria met, classified as Skin Corr. 1B according to Regulation (EC) No 1272/2008

Conclusions:

Skin Corr. 1B

Executive summary:

In this reliable and GLP-conform study according to OECD 431, exposure of disks of EPISKIN^TM (SM) with the test item for 1 and 4 hours, resulted in mean cell viabilities of 11.0 and 8.9%, repsectively, compared to the negative control. This is below the threshold of 35%, therefore the test item was considered as being corrosive to skin. Exposure of 3 minutes resulted in a mean cell viability of 84.9%. The experiment met the validity criteria, therefore the study was considered to be valid.