Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-14 to 2016-03-21
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviation.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
EC Number:
206-581-9
EC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Cas Number:
355-37-3
Molecular formula:
C6HF13
IUPAC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AGC Chemicals Europe Ltd; Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

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

OTHER SPECIFICS:

Purity/Composition: 100.00%
Molecular weight: 320

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Preparation and preincubation: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 21.5 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67- 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3- 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]

Application/Treatment of the test item
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five µL of the undiluted test item 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 ± 0.5 minutes at room temperature the tissues were washed with phosphate buffered saline to remove residual test item. At the end of the exposure, it was noted that no test item was left on the tissue, possibly due to its volatility. 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test Material
Value:
ca. 119
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Asahiklin™ AC-2000 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Asahiklin™ AC-2000 did not interact with the MTT endpoint.
- Colour interference with MTT: Asahiklin™ AC-2000 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Asahiklin™ AC-2000 did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline:

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the positive and negative control was less than 8%, indicating that the test system functioned properly. The standard deviation of the viability of the test item treated tissues was 27% which is above acceptance criteria. But, since all individual viabilities were clearly >50%, this does not influence the outcome of the test (study plan deviation 1).

Any other information on results incl. tables

Table 2. Mean absorption in the in vitro skin irritation test with Asahiklin™ AC-2000

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

SD

Negative control

1.066

1.052

1.024

1.047

± 0.022

Asahiklin™ AC-2000

1.568

1.157

1.026

1.250

± 0.283

Positive control

0.398

0.549

0.437

0.462

± 0.078

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table, the values are corrected for background absorption (0.0408). Isopropanol was used to

measure the background absorption.

 

Table 3. Mean tissue viability in the in vitro skin irritation test with Asahiklin™ AC-2000

 

Mean tissue viability

(percentage of control)

Negative control

100

Asahiklin™ AC-2000

119

Positive control

44

Applicant's summary and conclusion

Interpretation of results:
other: Not irritating
Remarks:
GHS Criteria
Conclusions:
Asahiklin™ AC-2000 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

In a key in vitro skin irritation test using a human skin model, the test material (Asahiklin™ AC-2000; purity 100%) was applied undiluted (25 µL) directly on top of the skin tissue (human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM))) for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.

 

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Asahiklin™ AC-2000 compared to the negative control tissues was 119%. Since the mean relative tissue viability for Asahiklin™ AC-2000 was above 50% after 15 ± 0.5 minutes treatment, Asahiklin™ AC-2000 is considered to be non-irritant.

 

The positive control had a mean cell viability of 44% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the positive and negative control was less than 8%, indicating that the test system functioned properly.

 

It was concluded that Asahiklin™ AC-2000 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.