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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:
22/01/2016-31/05/2016
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,5-dimethylbicyclo[3.2.1]octan-8-one oxime
EC Number:
278-079-8
EC Name:
1,5-dimethylbicyclo[3.2.1]octan-8-one oxime
Cas Number:
75147-23-8
Molecular formula:
C10H17NO
IUPAC Name:
N-[(8E)-1,5-dimethylbicyclo[3.2.1]octan-8-ylidene]hydroxylamine

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
PRE-TEST
1) Assessment of direct test Item reduction of MTT
- 25 +/- 2 mg of test item were added to 1 mL of MTT solution
- Incubation temperature: 37°C +/- 1.5 °C
- Incubation duration: 1h
- Assessment: blue/purple coloration of the MTT solution indicates chemical interference of the test chemical with MTT reduction.

2) Assessment of colored or staining materials
- 25 mg of test item were mixed with 300 µL of deionised water
- Incubation temperature water mixture: 37 +/- 1.5°C
- Incubation time water mixture: 1h
- Assessment: coloration of the test mixture indicates possible interation of the MTT measurement.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Kit Lot number(s): 23314

PRE-WARMING OF EPIDERM TISSUES
- Quality control EpiDermTM tissues:
1. air bubbles between agarose and insert are not > 30% of the total surface,
2. liquid on top of the insert is removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
- Pre-incubation in assay medium for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Transfer inserts from upper wells into the lower wells and continue pre-incubation for 23 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

MAIN EXPERIMENT

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1.5°C
- Temperature of post-treatment incubation (if applicable): 37 +/- 1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least 15 rinsings with DPBS.
- After the rinsigs, the inserts were submerged in DPBS at least 3 times and once again rinsed with DPBS from the inside and outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL in the MTT diluent
- Incubation time: 3h
- Extraction: with isopropanol solution for 67.3h at 2 - 8 °C
- Spectrophotometer: microplate reader Versamax Molecular Devices, Softmax Pro version 4.7.1
- Wavelength: 570 +/- 1 nm

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
- Negative control: Mean OD of 3 negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability.
- Test item or the positive control: individual relative tissue viability is calculated according to: relative viability (%) = {mean OD (test item/positive control) / mean OD negative control} * 100
The mean relative viability ± rel. standard deviation of 3 individual tissues was calculated and used for classification

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
- In vitro result: mean tissue viability =< 50% ; classification Irritant Category 2 (H315)
- In vitro result: mean tissue viability > 50% ; classification Non Irritant

VALIDATION
- Negative control: absolute OD negative control is indicator of tissue viability and should be ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: SD of 3 identical replicates should be < 18%.
- OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL: 25mg (+- 39 mg/cm2 according to guideline) in 25µL DPBS
Duration of treatment / exposure:
60 minutes: 35 minutes in incubator + 25 minutes on sterile bench at room temperature
Duration of post-treatment incubation (if applicable):
24 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: cell viability (%)
Run / experiment:
Mean of all runs
Value:
112.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean absorbance of the 3 tissues treated with the test item was 1.757. The mean relative absorbance value is calculated from this absorbance and corresponds to the cell viability. The cell viability was 112.4% for the test item with the negative control set to 100%. The threshold for classification for irritancy is ≤ 50%, consequently the test item was not irritant to skin.

Any other information on results incl. tables

Colour interference pre-experiment: No color changes observed; the test substance did not cause optical interference.

Pre-experiment assessing direct reduction of MTT by the test chemical: No color changes observed; the test substance did not cause direct reduction of MTT.

Detailed results:

Dose Group Exposure interval Tissue number Mean absorbance of 3 Wells Mean Absorbance of three wells blank corrected Mean Absorbance of 3 wells after blank correction* Rel. Absorbance (%) Tissue 1,2 + 3** Rel. Standard Deviation (%) Mean Rel. Absorbance (% of Negative Control)***
Blank     0.000          
Negative Control 60 min 1 1.532 1.495 1.563 95.6 4.1 100.0
2 1.644 1.591 100.6
3 1.717 1.631 103.8
Positive Control 60 min 1 0.118 0.081 0.080 5.2 4.4 5.1
2 0.118 0.107 4.8
3 0.104 0.132 5.3
Test item 60 min 1 1.773 1.735 1.757 111.0 4.9 112.4
2 1.904 1.875 118.4
3 1.706 1.731 107.8

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue (rounded value): 100*(absorbance tissue)/(mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100* (mean absorbance test item/positive control)/mean absorbance negative control)

Validity criteria: the validity criteria are met

- Absorbance negative control: within the acceptability criterion (mean OD ≥0.8 and ≤2.8)

- Positive control: decrease in the relative absorbance compared to the negative control

- Relative standard deviations: were below 5% (threshold < 18%)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test substance is not irritant to skin according to the OECD 439 in vitro test.
Executive summary:

In the current study the irritant effects of the test item were asessed in an in vitro test performed according to OECD 439 (in vitro Skin Irritation), EU B.46 (In vitro skin irritation: reconstructed human epidermis model test) and GLP. The test uses a Human Skin Model test and MTT as a read-out system. The Human Skin Model test can be used to identify chemicals that do not require classification for skin irritation or skin corrosion according to the UN GHS classification system. Consequently, a negative result in this test can also be used to conclude that no classification for Skin irritation Cat 2 or Skin corrosion Cat 1 is required under CLP. However, a positive result in this test does not allow to differentiate between Skin irritation and Skin corrosion.

In this in vitro test, the test chemical was topically applied to a 3 -dimensional reconstructed human epidermis (RhE) tissue. The tissue cell viability was measured as the degree of enzymatic conversion of the vital dye MTT by the viable cells to the corresponding blue MTT formazan salt, which is quantified after extraction from the tissue. The test item, positive and negative controls were tested in triplicate.

A pre-test is performed to ensure that the colour of the test chemical does not interfere with the MTT formazan measurement (optical interference), and that the test chemical does not cause (non-enzymatic) reduction of MTT into MTT formazan (chemical interference).

In the pre-test, buccoxime did not cause optical or chemical interference.

In the main study, the cell viability was found to be 112.4% with the negative control set to 100%. The threshold for classification for irritancy is ≤ 50%. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 5.1%. The validity criteria of the study were met.

In conclusion, the test item was found to be not irritant to the skin under the experimental conditions.