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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2017 - 13 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and acc ording to GLP principles.
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2017 - 17 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and acc ording to GLP principles.
Reason / purpose:
reference to other study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
Test system:
human skin model
Remarks:
EPISKIN Small Model (EPISKIN-SM, 0.38 cm2)
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN Small Model (EPISKIN-SM, 0.38 cm2)
- Tissue batch number(s):
17 EKIN 045
- Expiration date: 13 November 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
Volume of phosphate buffered saline and number of washing steps not specified
- Observable damage in the tissue due to washing:
None
- Modifications to validated SOP:
None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
0.3 mg/mL in PBS
- Incubation time:
3 hours at 37°C
- Spectrophotometer:
TECAN Infinite® M200 Pro Plate Reader
- Wavelength:
570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring= 23.3+/-0.3 [Acceptance criteria: >= 19.5]
- IC50 determination= 2.3 mg/mL [Acceptance criteria: 1.5-3.0 mg/mL]
- Morphology:
Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
No contamination

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- None, the test item did not interfere with the MTT endpoint.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test itema nd 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
21.55 to 30.76 mg of the solid test item directly applied on top of the skin tissue after moisture with 5 µl Milli-Q water.

VEHICLE
- No vehicle

NEGATIVE CONTROL
- Amount(s) applied:
25 µL

POSITIVE CONTROL
- Amount(s) applied:
25 µL
- Concentration: 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15-minute exposure
Value:
99
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
100%
Positive controls valid:
yes
Remarks:
7.0%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
No
- Direct-MTT reduction:
No
- Colour interference with MTT:
No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes [mean OD570 of the three tissues within the laboratory historical control data range: 0.422-1.547 and SD of the % viability =< 18]
- Acceptance criteria met for positive control:
Yes [mean relative viability =< 50% and SD of the % viability =< 18]
- Acceptance criteria met for variability between replicate measurements:
Yes [SD from individual % tissue viabilitiesof the three identically treated replicates =< 18]
- Range of historical values if different from the ones specified in the test guideline:
Negative control (OD570): 0.422 - 1.547
Positive control (OD570): 0.023 - 0.437
Historical data range obtained by collecting all data over the period of November 2014 to November 2017.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test substance, 1,3-Bis (4-hydroxy benzoyl) benzene is non-irritant in the in vitro skin irritation test.
Executive summary:

The assessment of the skin irritation potential of 1,3-Bis (4-hydroxy benzoyl) benzene was carried out, under GLP compliance, using an in vitro skin irritation test based on the guidelines described in: OECD No. 439 (adopted 28 July 2015) and EU Method B.46 (Amended by EC No. 640/2012 OJ No. L193, 20 July 2012).

The test consisted of topical application of 1,3-Bis (4-hydroxy benzoyl) benzene (21.55 to 30.76 mg of the solid test item with 5 µl Milli-Q water) on the skin tissue for 15 minutes. After exposure the skin tissue was thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The positive control had a mean cell viability of 7% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was <8%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after the 15 -minute treatment with 1,3-Bis (4-hydroxy benzoyl) benzene compared to the negative control tissues was 99%.

Since the mean relative tissue viability for the test item was above 50%, 1,3 -Bis (4 -hydroxy benzoyl) benzene is considered to be non-irritant.

Finally, 1,3-Bis (4-hydroxy benzoyl) benzene is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Identification: 1,3-Bis (4-hydroxy benzoyl) benzene
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
Specific details on test material used for the study:
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, U.S.A.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm Skin Model, EPI-200
- Tissue batch number(s):
27144 Kit I and Kit J

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 3 minutes or 1 hour at room temperature (26.4-27.6°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
Volume of phosphate buffered saline and number of washing steps not specified
- Observable damage in the tissue due to washing:
None
- Modifications to validated SOP:
None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time:
3 hours at 37°C in air containing 5% CO2
- Spectrophotometer:
TECAN Infinite® M200 Pro Plate Reader
- Wavelength:
570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
MTT QC assay, 4 hours, n=3: OD (540-570 nm)= 1.989+/-0.122 [Acceptance criteria: 1.0-3.0]
- Barrier function:
ET-50 assay, 100 µL 1% Triton X-100, 4 time-points, n=3, MTT assay: ET-50= 6.21 hrs [Acceptance criteria: 4.77-8.72 hrs]
- Morphology:
Presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination:
No contamination

NUMBER OF REPLICATE TISSUES:
4 (Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure) or 2 (2 tissues for negative control and 2 tissues for positive control)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- None, the test item did not interfere with the MTT endpoint.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
- A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
28.2 to 32.8 mg of the solid test item directly applied on top of the skin tissue after moisture with 25 µl Milli-Q water.

VEHICLE
- No vehicle

NEGATIVE CONTROL
- Amount(s) applied:
50 µL

POSITIVE CONTROL
- Amount(s) applied:
50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes or 1 hour
Number of replicates:
4 (two tissues for a 3-minute exposure to the test item and two for a 1-hour exposure) or 2 (two tissues for negative control and two tissues for positive control)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
114
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
100%
Positive controls valid:
yes
Remarks:
11%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
104
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
100%
Positive controls valid:
yes
Remarks:
7.6%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
No
- Direct-MTT reduction:
No
- Colour interference with MTT:
No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes [mean OD570 of the two tissues within the laboratory historical control data range: 1.324-2.615 (3-minute exposure) or 1.361-2.352 (1-hour exposure)]
- Acceptance criteria met for positive control:
Yes [mean relative viability following 1-hour exposure < 15%]
- Acceptance criteria met for variability between replicate measurements:
Yes [in the range 20 - 100% viability, CV =< 30%]
- Range of historical values if different from the ones specified in the test guideline:
Negative control (3-minute treatment, OD570): 1.324 - 2.615
Negative control (1-hour treatment, OD570): 1.361 - 2.352
Positive control (3-minute treatment, OD570): 0.0172 - 0.56
Positive control (1-hour treatment, OD570): 0.046 - 0.339
Historical data range obtained by collecting all data over the period of November 2013 to November 2016.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test substance, 1,3-Bis (4-hydroxy benzoyl) benzene, is not corrosive to the skin.
Executive summary:

The assessment of the corrosive potential to skin of 1,3-Bis (4-hydroxy benzoyl) benzene was carried out in compliance with GLP, using an in vitro skin corrosive test based on the guidelines described in: OECD No. 431 (adopted 29 July 2016) and EU Method B.40 BIS.

The test consisted of topical application of 1,3-Bis (4-hydroxy benzoyl) benzene (28.2 to 32.8 mg of the solid test item with 25 µl Milli-Q water) on the skin tissue for 3-minute and 1 -hour exposure. After exposure the skin tissue was thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

The test item did not interfere with the MTT endpoint. The positive control (Potassium hydroxide) had a mean relative tissue viability of 7.6 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (Milli-Q water) tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit =< 2.8) and the laboratory historical control data range (actual range 1.4955 – 1.8957). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was =< 13%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with 1,3 -Bis (4 -hydroxy benzoyl) benzene compared to the negative control tissues was 114% and 104% respectively.

Because the mean relative tissue viability for 1,3 -Bis (4 -hydroxy benzoyl) benzene was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, 1,3-Bis (4-hydroxy benzoyl) benzene is considered to be non-corrosive in the performed experiment.

The substance is not classified as corrosive to skin according to UN-GHS criteria.