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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines (OECD 431), and in compliance with the UK Good Laboratory Practice Regulations. All relevant validity criteria were met.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identity: Allylamine



In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
The positive and negative controls were in liquid form and were applied by dispensing a volume of 50 uL over each tissue using a pipette.
Liquids - The test substance was applied by dispensing a volume of 50 uL over each tissue using a positive displacement pipette.
Duration of treatment / exposure:
3 minutes and 1 hour.
Number of replicates:
2 per treatment

Test system

Type of coverage:
other: Human skin model
Controls:
not required
Amount / concentration applied:
The test substance was applied by dispensing a volume of 50 uL over each tissue using a positive displacement pipette
Duration of treatment / exposure:
The EpiDerm™ was exposed to the test material for 3 miutes and for 1 hour.
Observation period:
3 miutes and for 1 hour.
Number of animals:
Not applicable (in vitro test)
Details on study design:
At least one hour before dosing, the live tissues and the thawed freeze killed tissues were transferred to 6-well plates containing 0.9 mL pre-warmed assay medium per well, then incubated at 37±1°C in a humidified atmosphere of 5% CO2 in air.
During the pre-incubation period, two sets of 24-well plates were prepared. The first set were holding plates containing 300 µL assay medium (supplied in the Epi-200 kit) and the second set MTT assay plates containing 300 µL MTT medium (diluted MTT 1 mg/mL).
At least one hour after pre-incubation, the medium below the tissue inserts was replaced with 0.9 mL fresh assay medium. Plates awaiting sample application were returned to the incubator. Duplicate tissues were dosed for three minutes and duplicate tissues dosed for one hour with test substance, negative control or positive control.
Duplicate freeze killed tissues were dosed for three minutes and duplicate tissues dosed for one hour with the test substance. As freeze killed tissues may retain some reducing enzymes (which may reduce MTT), duplicate freeze killed tissues were left untreated for each of the two time points.
For practical purposes, dosing of the one hour tissues and the three minute tissues were performed as two separate batches. The one hour tissues were dosed first and returned to the incubator for the completion of the one hour dosing period. The three minute tissues were dosed during the one hour dosing period.
After the required dosing period, each tissue (including the killed tissue untreated control) was rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS), for a minimum of 20 rinses, and then blotted on absorbent paper. The inserts were then transferred to the holding plate.
When all tissues from one batch had been transferred to a holding plate, the inserts were transferred to the MTT assay plate which was then incubated for three hours at 37±1°C in a humidified atmosphere of 5% CO2 in air. After three hours, the tissues were rinsed with DPBS. After the three hour incubation, a white ring was noted at the bottom of the inserts around the edge of the tissues (live and killed) treated with the test substance.
After rinsing with DPBS the tissues were then transferred to 1 mL per well extraction solution (isopropanol) in 24-well plates. A further 1 mL was then added to each insert. The tissues were extracted overnight at room temperature, without shaking.
After the extraction period, triplicate 200 µL aliquots of extractant from each tissue were pipetted into the wells of flat-bottomed 96-well plates and the absorbance read at 540 nm.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute contact - Replicate 1
Value:
13.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute contact - Replicate 2
Value:
12.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour contact - Replicate 1
Value:
8.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour contact - Replicate 2
Value:
7.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: After the one hour incubation, the MTT solution control (orange/red) remained unchanged, The initial colour of the test substance, Allylamine/MTT mixture was red/purple and then purple/blue after the one hour incubation indicating the test substance had reduced the MTT. The initial red/purple colour of the test substance/MTT mixture was possibly due to the alkalinity of the test substance affecting the pH indicator (phenol red) present in the MTT solution. The pH of the test substance Allylamine (as a 10% v/v solution in distilled water) was 11.5, measured using pH indicator papers. As the test substance had reduced the MTT, freeze killed tissues (which have no metabolic activity but absorb and bind the test substance like viable tissues) were included in the assay together with the live tissues as a control.
- Colour interference with MTT: The test substance, Allylamine/water solution and water control were colourless after the 15 minute shaking period. Therefore, the test substance had not shown any potential for colouring water.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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: Yes

The mean optical density of each duplicate negative control value for the three minute and one hour contact were 1.828 and 2.022, respectively. Both values were above the minimum assay acceptance value of 0.8.

The mean relative tissue viability of the positive control, 8.0 N potassium hydroxide, for the one hour application was 4.4%. This value was below the maximum acceptable value of 15%.

The coefficient of variation (CV) was not applicable for the test substance, Allylamine, three minute and one hour applications and positive control one hour application, as the mean percentage viability was below the 20% - 100 % viability range. All other values did not exceed the CV value of 0.3.

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance, Allylamine, elicited a mean tissue viability of 12.8% for three minute contact and 8.4% for one hour contact and was predicted as corrosive in the EpiDerm™ skin corrosivity test.
Executive summary:

The objective of this test was to assess the skin corrosivity,in vitro, of the test substance, Allylamine.

The test substance was applied for three minutes and one hour to the EpiDerm™ three-dimensional human skin model. The model consisted of normal, human-derived epidermal keratinocytes, which had been cultured on 0.6 cm2inserts to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability of the multi layers was determined by mitochondrial dehydrogenase activity assessed by the reduction of MTT (3‑(4,5‑dimethylthiazol‑2‑yl)‑2, 5‑diphenyltetrazolium bromide)to a soluble, coloured, formazan product. The formazan produced wasquantified by spectrophotometric measurement. The prediction model uses the percentage viability values (compared to negative control viability) at three minute and one hour exposure times to identify corrosive and non-corrosive substances.

The test substance, Allylamine, elicited a mean tissue viability of 12.8% for three minute contact and 8.4% for one hour contact and was predicted as corrosive in the EpiDerm™ skin corrosivity test.