Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2013-09-26 to 2013-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The 12 wells plates with tissues were preincubated for 2h instead of 24h. Upon receiving instructions from the supplier, Episkin tissues were transferred to maintenance medium for 24h. This protocol deviation did not affected the study integrity.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
The 12 wells plates with tissues were preincubated for 2h instead of 24h. Upon receiving instructions from the supplier, Episkin tissues were transferred to maintenance medium for 24h. This protocol deviation did not affected the study integrity.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch: 110801
Purity: 99.8%
Test substance storage: At room temperature in the dark under nitrogen
Stable under storage conditions until 26 August 2018 (expiry date)
Reactivity: reactive to oxygen

Test animals

Species:
other: EPISKIN Small Model (human)
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment.
-Humid atmosphere of 80 - 100% (actual range 82 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark
-Temperature:37.0 ± 1.0°C (actual range 36.3 – 37.6°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Test system

Type of coverage:
open
Vehicle:
physiological saline
Amount / concentration applied:
10.0 to 13.3mg
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SYSTEM
Name: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-034)
Source: SkinEthic Laboratories, Lyon, France.
Rationale: One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.0 to 13.3 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
112
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables


Table 1 Mean absorption in thein vitroskin irritation test with the test substance

A

 B 

 

C

  Mean(OD570) +/-SD Negative Control 1.180

1.134 1.050  1.121 +/- 0.066

  The test substance 1.2021.2841.286  1.257 +/- 0.048

 


Table 2 Mean tissue viability in thein vitroskin irritation test with the test substance

 Mean tissue viability (Percentage od control)  Negative Control 100 The test substance 101 Positive Control 6

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other:
Conclusions:
The test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.