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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:
12 January - 04 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

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:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Council Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by In Vitro Life Science Laboratories, Bratislava, Slovak Republic
Details on animal used as source of test system:
N/A
Justification for test system used:
Guideline specific test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek, In Vitro Life Science Laboratories, Bratislava, Slovak Republic
- Tissue batch number(s): 23397
- Production date: 01 Mar 2017
- Shipping date: 01 Mar 2017
- Delivery date: 01 Mar 2017
- Date of initiation of testing: 01 Mar 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS - Volume and number of washing steps: the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Not reported
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Pass - details provided in CoA (OD540-570 was between 1.0 and 3.0).
- Barrier function: Pass - details provided in CoA (ET-50 was between 4.77 and 8.72 h).
- Morphology: Not reported
- Contamination: Pass - details provided in CoA (no contamination - tested for HIV-1-virus; Hep B; Hep C; bacteria; yeast and other fungi).
- Reproducibility: Coefficient of Variation between tissue replicates was ≤ 30%.

NUMBER OF REPLICATE TISSUES: 4 per test group (2 each for 3 min and 60 min exposure)

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if 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): 50 µL
- Concentration (if solution): Unchanged/ undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 M
Duration of treatment / exposure:
3 mins and 60 mins
Duration of post-treatment incubation (if applicable):
3 h MTT incubation followed by overnight isopropnaol extraction.
Number of replicates:
2 per treatment and exposure group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure (repeat)
Value:
89.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure (repeat test)
Value:
52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.357 for the 3-Minute exposureperiod and 1.338 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.6 and 8.0 % relative to the negative control following the 3 and 60-,minute exposure periods, respectively. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the positive and negative control were within the historical ranges achieved by the testing facility in previous tests (Feb 2008 - Feb 2017, n=46), thus confirming the acceptable functioning of the test system.

Any other information on results incl. tables

Table 1      Relative mean viabilities for each treatment group

 

Exposure Period

Percentage Viability (% SD / % CoV)*

Negative Control§

Positive Control

Test Item

3 minutes

100 (0.068 / 6.0)

6.2 (0.078 / 20.6)

89.1 (0.090 / 8.8)

60 minutes

100 (0.061 / 4.9)

-1.4 (0.034 / 23.2)

52.0 (0.022 / 3.4)

*mean of 2 replicates

§negative controls set to 100 %

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin under the conditions of the test.
Executive summary:

OECD 431 (2017) - The skin corrosivity potential of Reaction Mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

The test item was considered to be non-corrsive to the skin under the conditions of the test.

 

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 89.1 and 52.0 %, respectively. The quality criteria required for acceptance of the results was met.

 

Under the conditions of this study the test substance is not considered to be corrosive to the skin.