Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15.10.-18.10.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,1,1,2,3-pentachloropropane
- Substance type: organic
- Physical state: liquid
- Analytical purity: 99.940 % (w/w)
- Impurities (identity and concentrations): chlorinated hydrocarbons - 0.048 % (w/w)
- Lot/batch No.: 2-750
- Expiration date of the lot/batch: august 2014
- Storage condition of test material: dry place at room temperature

Test animals

Details on test animals and environmental conditions:
TISSUE:
The reconstructed human epidermal model EpiDerm (EPI-212 ver. 2.0, MatTek, Ashland, USA); Lot No. 18383, kit B

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
30 µL
Details on study design:
FUNCTIONAL CHECK IN TUBES - DIRECT MTT REDUCTION
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: 50 μL of the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

MTT TEST
- Test substance application:
The test substance (30 μL) is placed directly atop to the tissue. The material is spread on the tissue surface.
- Procedure:
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations (1 and 18±3hours), tissues are topically exposed to the test chemicals for 1 hour. Three tissues are used per test chemical (C1) and for the positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After 24±2 hours incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).
- OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank.

NEGATIVE CONTROL:
The absolute OD of the negative control (NC) tissues (treated with sterile phosphate buffered saline) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8 (see OECD Test Guideline 439, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, Adopted: July 26, 2013; Table 2: Acceptability ranges for negative control OD values of the test methods included in this TG, page 4).

POSITIVE CONTROL:
A 5 % sodium dodecyl sulphate (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
0.06
Remarks on result:
other:
Remarks:
Basis: other: viability of tissue. Time point: 60 min. Max. score: 1.885. Remarks: score: OD570 for test substance / max. score: OD570 for negative control. (migrated information)

In vivo

Irritant / corrosive response data:
see Table No. 1

Any other information on results incl. tables

Table No. 1: Measured value and viability

Treatment

Parameter

OD570

Mean

SD

Average viability

(% negative control)

1

2

3

negative control (NC)

(phosphate buffered saline)

OD570

1.908

1.809

1.938

1.885

0.055

100.0

viability (% NC)

101.2

96.0

102.8

100.0

2.9

1,1,1,2,3-pentachloropropane

OD570

0.066

0.059

0.056

0.060

0.004

3.2

viability (% NC)

3.5

3.1

3.0

3.2

0.2

positive control

(5% sodium dodecyl sulphate)

OD570

0.054

0.059

0.061

0.058

0.003

3.1

viability (% NC)

2.9

3.1

3.2

3.1

0.2

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Migrated information
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance was 3.2 % of negative control average value, i.e. viability was ≤ 50 %.
The effect of test substance was positive in EpiDerm model.
According to the classification criteria given in chapter 3.9. of this report, the test substance 1,1,1,2,3-Pentachloropropane is considered to be irritant to skin in accordance with UN GHS category 2.
Executive summary:

Test substance, 1,1,1,2,3-pentachloropropane was assayed for the in vitro skin irritation in human epidermal model EpiDerm. The test was performed according to the EU Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4, (1), (3)).

After pre-incubation of tissues, the test substance (30 μL) was placed directly atop the tissue. Length of exposition was 60 minutes. Nine tissues were used for the experiment, three per test substance, three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (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.

Under the above-described experimental design, average viability of tissues treated by the test substance was 3.2 % of negative control average value (i.e. viability was ≤ 50 %).

The effect of test substance was positive in EpiDerm model (the tissue was damaged).

According to the classification criteria given in chapter 3.9. of this report (see Method B.46. In vitro skin irritation: Reconstructed human epidermis model test. Commission Regulation (EC) No. 761/2009, 23rdJuly 2009; 2.2. Interpretation of results, L 220/29), the test substance 1,1,1,2,3-pentachloropropane is considered to be irritant to skin.