Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 06 June 2018 to 15 June 2018. Report Issued: 30 July 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bromoform
EC Number:
200-854-6
EC Name:
Bromoform
Cas Number:
75-25-2
Molecular formula:
CHBr3
IUPAC Name:
bromoform
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Test System: An EpiDerm™ Reconstructed Human Epidermis Model Kit was received from the supplier, MatTek, on 12 June 2018. The EpiDermTM Tissues (0.63cm2) lot number was 28624 and the assay Medium lot number was 060718MSA. Upon receipt of the Epiderm™ tissues, the sealed 24 well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2

PRE-INCUBATION
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT-loading.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- MTT incubation period: 3 hours.
- MTT extraction: After the 3 hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed. After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to three wells designated as blanks.
- Spectrophotometer: Labtech LT 4500 microplate reader and LT-com analysis software.
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Duplicate

PREDICTION MODEL / DECISION CRITERIA
- As given in the OECD 431 test guideline.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): As supplied

VEHICLE
No vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0N Potassium Hydroxide
Duration of treatment / exposure:
1 x 3 minute exposure period
1 x 60 minute exposure period
Duration of post-treatment incubation (if applicable):
None
Number of replicates:
Testing was conducted in duplicate for each treatment

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure period
Value:
81.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability
Positive controls validity:
valid
Remarks:
3.1% viability
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute Exposure Period
Value:
6.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability
Positive controls validity:
valid
Remarks:
3.1% viability
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the mean OD570 for the negative control treated tissues was 2.252 for the 3 Minute exposure period and 2.129 for the 60 Minute exposure period.
- Acceptance criteria met for positive control: Yes - The relative mean tissue viability for the positive control treated tissues was 0.065% relative to the negative control following the 60 Minute exposure period.
- Acceptance criteria met for variability between replicate measurements: The acceptance critiera was satisfied as in the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%.

Any other information on results incl. tables

Classification of corrosivity potemtial is based on relative viabilities for both exposure times according to the following table:

 

 Viability Measured after Exposure Time Points  Prediction to be considered according to the EU CLP Regulation (EC) No 1272/2008 and UN GHS
 STEP 1   
 < 50% after 3 minutes exposure  Corrosive

≥ 50% after 3minutes exposure AND

< 15% after 60 minutes exposure

Corrosive 

≥ 50% after 3 minutes exposure AND 

≥ 15% after 60 minutes exposure

Non-corrosive 

 STEP 2 for test items identified as corrosive in step 1

 

 

 < 25% after 3 minutes exposure

 H314, sub-categorry 1A

 ≥ 25% after 3 munites exposure

 H314, combination of sub-categories 1B and 1C

Applicant's summary and conclusion

Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Conclusions:
The test item was considered to be corrosive: UN GHS H314 Combination of sub categories 1B and 1C.
Executive summary:

Introduction

The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The test was designed to meet the following guidelines:


·        OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (29 July 2016)

·        Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Method

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

 Exposure Period     Percentage Viability      
 Negative Control  Positve Control  Test Item
 3 minutes  100* 3.1  81.4 
 60 minutes 100*  3.1  6.1 

*The mean viability of the negative control tissues is set at 100%

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be corrosive: UN GHS H314 Combination of sub‑categories 1B and 1C.