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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:
07/02/2018 - 12/02/2018
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring / Batch 02/16
- Expiration date of the lot/batch: 01 January 2021
- Purity test date: 100%
- Physical state/Appearance: Yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as supplied.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: NA
Source strain:
other: Epiderm Reconstructed Human Epidermis Model Kit
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 06 February 2018
EpiSkinTM Tissues (0.38cm2) lot number : 18-EKIN-006
Maintenance Medium lot number : 18-MAIN3-005
Assay Medium lot number : 18-ESSC-005
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 per assay

Test animals

Species:
other: NA
Strain:
other: NA
Details on test animals and environmental conditions:
NA

Test system

Type of coverage:
other: NA
Preparation of test site:
other: NA
Vehicle:
other: NA
Controls:
other: NO
Amount / concentration applied:
NA
Duration of treatment / exposure:
NA
Observation period:
NA
Number of animals:
NA
Details on study design:
NA

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure
Value:
ca. 130.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 5.0% relative to the negative control treated tissues and the standard deviation value of the viability was 2.9%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.807 and the standard deviation value of the viability was 4.1%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 14.1%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a yellow color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD570 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1.

The relative mean viability of the test item treated tissues was 130.8% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Appendix 1 Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item OD570 of individual tissues Mean OD570 of triplicate tissues SD of OD570 Relative individual tissue viability (%) Relative mean viability (%) */- SD ofRelative Mean Viability (%)
Negative control 0,78 0,807 0,033 96,7 100 4,1
0,844 104,6
0,796 98,7
Positive control 0,021 0,04 0,023 2,6 5 2,9
0,066 8,2
0,033 4,1
Test item 1,154 1,055 0,113 143,1 130,8 14,1
0,932 115,5
1,08 133,9

Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period : prediction irritant if the relative mean tissue viability is < or = 50% (cat 1 or 2 under UN GHS) and prediction non irritant in the contrary case (Category 3 cannot be determined under US GHS).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply: UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

In a in vitro EPISKIN study (Lacey F.E, 2018), performed according to the OECD 439 Guideline and in compliance with GLP, Triplicate tissues were treated with the Butyl-NENA for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this.

At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density 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).

The relative mean viability of the test item treated tissues was 130.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

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

The test item was therefore classified as non-irritant. The following classification criteria apply: UN GHS Not classified for Irritation (category 3 can not be determined).