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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:
From 26-09-2016 to 08-11-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Xianyang Sanjing Technology Co., ltd./160612
- Expiration date of the lot/batch: June 18, 2018
- Purity: > 99.6 % w/w


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in a cool and dry place, away from light

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:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 minutes at room temperature and the remaining 35 minutes at 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues are then thoroughly rinsed with PBS
- Modifications to validated SOP: None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:Libra S22
- Wavelength:570 nm
- Filter: No external filter was used
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of substance/surface ratio 39.7 mg/cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution):5 % SDS (in H2O)
Duration of treatment / exposure:
60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at 37±1°C
Duration of post-treatment incubation (if applicable):
Approximately 42 hours (23 hours and 48 minutes of post-incubation; medium was replaced and tissues were incubated for another 18 hours, 21 minutes.)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
73.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
3.3
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: In the previously performed study Study No. 415/16/4AC: N,N′-m phenylenedimale-imide - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS-CETA 2016, Report No.: 16-612, direct reduction with the test substance was excluded.
- Colour interference with MTT: In the previously performed study Study No. 415/16/4AC: N,N′-m phenylenedimale-imide - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS-CETA 2016, Report No.: 16-612, colour interference was excluded.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 of the NC tissue was 2.378 ±0.115 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8. OD570 historical negative control range is 1.470 - 2.342.
- Acceptance criteria met for positive control: The mean viability of the PC tissues expressed as % of the negative control tissues is 3.3% which meets the acceptance criterion of ≤ 20 %. The 95±% confidence interval of historical positive control is 0.044-0.150.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance is < 18 %.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, the test substance N,N′-m-phenylenedimaleimide was not an irritant in the in vitro skin irritation test on human epidermal EpiDermTM tissues.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDermTM (16-650), PBS-moistened reconstructed human epidermis (RhE) tissue was exposed to 25 mg of N,N′-m-phenylenedimaleimide (99.6%) for 60±1 minutes ((25 minutes at room temperature and the remaining 35 minutes at 37±1°C). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and a 2 hour isopropyl alcohol extraction period followed. The  OD570 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 colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the test substance N,N′-m-phenylenedimaleimide was 73.9 % of negative control average value i.e. viability was > 50 %. The average viability of tissues treated by the positive control (5% SDS) was 3.3 % of negative control average value. According to these results, the test substance is not irritating.

This in vitro skin irritation study in the human epidermal model EpiDermTM is acceptable and satisfies the guideline requirement for an OECD 439 study.