Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 December 2012 to 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to current test guidelines and GLP compliant

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Oxypyrion-Acetate
CAS Number: 1742-79-6
Batch number: 11085913
Purity: Not stated
Date of arrival: 20 November 2012
Storage conditions: Refrigerated (2 to 8°C)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
The test system was a three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation. Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevented the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.
EpiDermTM SIT (EPI-200) tissues were kept in their packaging at room temperature in a microbiological safety cabinet until the next step. The tissues were set up the day prior to treatment by placing each tissue onto 0.9 mL pre-warmed maintenance medium (supplied with the EpiDermTM SIT (EPI-200) tissues) in 6-well plates and incubating at 37°C.
The test was performed on a total of three tissues per test item, negative and positive control. Immediately prior to treatment initiation, the media under the tissues was replaced with 0.9 mL of pre-warmed assay medium (supplied with the EpiDermTM SIT (EPI-200) tissues).
Three tissues were treated by application of 25 mg of test item (with the tissue moistened with 25 µL sterile PBS) onto the surface of the tissues. The tissues were incubated at 37 ± 1°C, 5 ± 1% CO2 for a 35 ± 1 minute period. The plates were then removed from the incubator and placed into a sterile hood until the 60 ± 1 minute treatment period was complete for each tissue.
This procedure was repeated using a group of three tissues treated with 30 µL of the negative control and a further group of three tissues treated with 30 µL of the positive control.
Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42 hours.
At the end of the 42-hour incubation period, tissue viability was assessed by MTT assay.
Once all tissues were rinsed, they were transferred to wells containing 300 µL of the positive control.
FollowiL of 1 mg/mL MTT-medium and were incubated for 3 hours (37°C, 5% CO2). The protocol specified that at the end of the incubation period, the tissues would be removed from the MTT solution and any resultant colour formed in the tissues by the MTT assay would be extracted. However, in a deviation from protocol, the tissues were not removed from the MTT solution and 2 mL of isopropanol was added to the tissues whilst still in the MTT solution.
It was considered that this deviation may have impacted on the integrity or outcome of the study, therefore the test procedure was therefore repeated using fresh tissues, with the extraction procedure following the requirements of the protocol.
In the repeat test, at the end of the 42-hour incubation period all tissues were rinsed, transferred to wells containing 300 µL of the positive control.
FollowiL of 1 mg/mL MTT-medium and incubated for 3 hours (37°C, 5% CO2). The tissues were then removed from the MTT solution and placed in clean wells. Any resultant colour was extracted by flooding the tissue with 2 mL isopropanol and shaking for a further two hours at room temperature.
Upon completion of the extraction, each tissue was pierced using a hypodermic needle so that the extract could run through the tissue. Once drained, the tissue was discarded and the extract mixed by shaking for two hours. Two x 200 µL of the positive control.
FollowiL aliquots of each resultant extract were placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm using extraction solution as a blank. Tissue viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test was performed on a total of three tissues per test item, negative and positive control. Immediately prior to treatment initiation, the media under the tissues was replaced with 0.9 mL of pre-warmed assay medium (supplied with the EpiDermTM SIT (EPI-200) tissues).
Three tissues were treated by application of 25 mg of test item (with the tissue moistened with 25 µL sterile PBS) onto the surface of the tissues.
Duration of treatment / exposure:
The tissues were incubated at 37 ± 1°C, 5 ± 1% CO2 for a 35 ± 1 minute period. The plates were then removed from the incubator and placed into a sterile hood until the 60 ± 1 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues.
Duration of post-treatment incubation (if applicable):
The tissues were then placed on the appropriate medium and incubated for 42 hours.
Number of replicates:
three

Test animals

Species:
other:
Strain:
other:

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
5.8
Negative controls validity:
valid
Remarks:
100% viability
Positive controls validity:
valid
Remarks:
3.9% viability
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The group mean viability for the test item was 5.8%.
The group mean viability for the negative control was 100%.
The group mean viability for the positive control was 3.9%.

Any other information on results incl. tables

Test substance

OD570

Mean

Corrected mean

% relative survival

Mean

SD

CV

Negative

1.735

1.740

1.738

1.738

116.8

100.0

18.077

18.077

Negative

1.203

1.204

1.203

1.203

80.9

Negative

1.476

1.568

1.522

1.522

102.3

Test item

0.077

0.095

0.086

0.086

5.8

5.8

0.400

6.921

Test item

0.091

0.093

0.092

0.092

6.2

Test item

0.078

0.082

0.080

0.080

5.4

Positive

0.049

0.053

0.051

0.051

3.4

3.9

0.411

10.588

Positive

0.064

0.062

0.063

0.063

4.2

Positive

0.061

0.058

0.059

0.059

4.0

Blank

0.000

0.000

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The test item, Oxypyrion-Acetate, was considered to be irritant (GHS category 2) to the in vitro skin model EpiDerm SIT (EPI-200).