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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-Jan-2016 to 22-Feb-2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Details on animal used as source of test system:
n.a.; in vitro skin model
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:
Application/Treatment of the test item
In the first test the standard deviation of the test item treated replicates was above acceptance criteria therefore this test was rejected (not reported). However, since the controls and test item treated killed tissue met the acceptance criteria, the values of the non-treated and test item treated killed tissue were used and a repeat test was performed without the additional killed tissue. The test was performed on a total of 3 tissues per test item 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 item to the tissue and the solid test item (18.7 to 62.0 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. In addition three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. 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. 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
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour 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. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 18.7 to 62.0 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 10 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min
Value:
9.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
The test item was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that Isobutylparaben did interact with MTT.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Irrit. 2 (H315) is required according to Regulation (EC) No 1272/2008
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that isobutyl 4-hydroxybenzoate is irritant in the in vitro skin irritation test.
Based on the results of this test the test substance should be labelled, according to Regulation (EC) No 1272/2008 (CLP), as: Skin Irrit. 2 (H315).
Executive summary:

Isobutyl 4-hydroxybenzoate did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal procedure, three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by isobutyl 4-hydroxybenzoate was 8.4% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

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 isobutyl 4-hydroxybenzoate compared to the negative control tissues was 9.7%. Since the mean relative tissue viability was below 50% after 15±0.5 minutes treatment isobutyl 4-hydroxybenzoate is considered to be irritant.

 

The positive control had a mean cell viability of 9.7% 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 was less than 13%, indicating that the test system functioned properly.