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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 October 2020 - 16 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EC method B.40 bis: In vitro Skin Corrosion: Human Skin Model Test Council Regulation (EC) No. 2019/1390, adopted 31 July 2019.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylstyrene
EC Number:
202-889-2
EC Name:
3-methylstyrene
Cas Number:
100-80-1
Molecular formula:
C9H10
IUPAC Name:
1-methyl-3-vinylbenzene
Constituent 2
Chemical structure
Reference substance name:
4-methylstyrene
EC Number:
210-762-8
EC Name:
4-methylstyrene
Cas Number:
622-97-9
Molecular formula:
C9H10
IUPAC Name:
1-methyl-4-vinylbenzene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 June 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none, the test item was used as supplied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s):Lot no. 30897
- Production date: October 14,2020
- Date of initiation of testing: October 13,2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37℃

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 18 times in approximately 100 mL D-PBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan infinite 200Pro10
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD[540-570nm] (1.0-3.0):1.906±0.071
- Barrier function: ET-50[4.77-8.72 hrs]:6.52 hrs
- Contamination: Sterile

NUMBER OF REPLICATE TISSUES:
Two tissues were used per treatment for test item, positive and negative controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE/COLOUR INTERFERENCE
1. Functional check for MTT interference was performed as follows: Fifty μL of the test item were added to 1 mL of the MTT medium (1 mg MTT6/mL) and incubated at 37 °C, 5 % CO2, and 95 % relative humidity for 60 minutes. Untreated MTT solution was used as control. The test item displayed a slight purple discolouration. Due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed. The frozen tissues were stored in the freezer (-20 ± 5 °C). The test item was applied to two freeze-killed tissues9. In addition, two freeze-killed tissues were treated with the negative control. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDermTM tissues.

2. Functional check for colour interference was performed as follows: Fifty μL of the test item were mixed with 300 μL sterile deionised water and incubated at 37 °C, 5 % CO2 and 95 % relative humidity for 60 minutes. In addition, 50 μL of the test item were added to 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was observed in either case.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Fifty µL


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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Fifty µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 minutes and 60 minutes
Number of replicates:
2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 mins
Value:
85.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 85.4 % ± 3.7
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction and Colour interference with MTT: No discolouration of the test item with water or isopropanol was noted.

As the test item displayed a slight purple discolouration due to interaction of the test item with the MTT measurement (reduction of MTT), an additional test with freeze-killed tissues had to be performed. The OD correction factor for the 3-minute exposure was 0.0034, while the OD correction factor for the 1-hour exposure was determined as 0.0295.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control and positive control:
The mean optical density (OD) of the negative control of 2 tissues was 1.7940 (3-minute exposure) or 1.7560 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

The mean viability of cells treated with the positive reference item 8 N KOH was 6.7 % ± 0.1 % or 4.0 % ± 0.2 % (3-minute or 1-hour exposure, respectively) of the negative control and, hence well below the 15 % cut-off value at the 1-hour exposure.

- Acceptance criteria met for variability between replicate measurements:
The difference of viability between two tissue replicates (for tissues in the viability range between 20 – 100 % viability) was below the limit of acceptance of 30 %. Hence, all acceptance criteria were fulfilled

Historical data of negative and positive controls are presented in Appendix 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin corrosion test using the reconstructed human epidermal model EpiDerm, the test item Vinyltoluene was non-corrosive.
Executive summary:

In an in vitro skin corrosion in the human epidermal model EpiDerm (OECD 431/GLP), reconstructed human epidermis tissue was exposed to 50 µL of Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) for 3 and 60 minutes. Sterile deionised water was used for the negative control and 8N KOH was used for the positive control. After removal of the test substance, the tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at room temperature with shaking. The OD540 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.



The controls confirmed the validity of the study. The mean absolute OD570 of the 2 negative control tissues was ≥ 0.8 and ≤ 2.8 [1.7940 (3 min) and 1.7560 (60 mins)]. The mean relative tissue viability (% negative control) of the positive control was < 15% [6.7 % ± 0.1 % or 4.0 % ± 0.2 % (3-minute or 60 mins exposure, respectively)]. The colour of the test substance did not interfere with the endpoint. As the test item displayed a slight purple discolouration due to interaction of the test item with the MTT measurement (reduction of MTT), an additional test with freeze-killed tissues had to be performed. The OD correction factor for the 3-minute exposure was 0.0034, while the OD correction factor for the 1-hour exposure was determined as 0.0295.


 


The average viability of tissues treated by the test item, 1 Vinyltoluene were higher than the threshold values (50 % and 15% respectively): 85.4 % ± 3.7 after a 3-minute exposure period and 63.5 % ± 2.7 after a 1-hour exposure. The test item should be regarded as non-corrosive.

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