1,5-dimethylbicyclo[3.2.1]octan-8-one oxime

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.01-19.02.2016

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July, 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation's Reconstructed Human EpiOcular TM Model; 14 July 2014

Deviations:

no

Principles of method if other than guideline:

The EpiOcular Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. The test consists of a topical exposure of the neat test item to human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (3-4,5-dimethyl thiazole 2-yl)2,5-diphenyl-tetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. Reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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

Test animals / tissue source

Species:

human

Strain:

other: epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

CELL CULTURE
- Source: MatTek Corporation (Ashland, MA 01721, USA)
- Model: human-derived epidermal keratinocytes forming a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium.
- Surface: 0.6 cm2
- Storage conditions: 2 - 8 °C on medium-supplemented agarose gels

IN-LIFE DATES: From: 16.02.2016

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): 100 %

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

Ca. 18 hours

Number of animals or in vitro replicates:

duplicate

Details on study design:

PRE-TEST
1) Assessment of direct test Item reduction by MTT
- 50 mg of test item were added to 1 mL of a 1.0 mg/mL MTT solution in DMEM in a 6-well plate.
- Incubation temperature: 37°C +/- 1.5 °C
- Incubation duration: approx. 3 h
- 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
- 50 mg of test item were added to 1.0 mL of water and to 2 mL of isopropanol in 6-well plates.
- Incubation temperature water mixture: 37 +/- 1.5°C
- Incubation time water mixture: at least 1h
- Incubation temperature isopropanol mixture: room temperature
- Incubation time isopropanol mixture: 2-3 hours
- Assessment: coloration of the test mixtures indicates possible interation of the MTT measurement.

TEST KIT INFORMATION
- EpiOcular and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA 01721, USA)
- Test Kit Lot No: 21597


EXPERIMENTAL DESIGN
- Incubation: overnight at standard culture conditions for 30 minutes.
- Exposure conditions: standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH)
- Removal test item: extensively rinsing with Ca++Mg++-free DPBS
- Post- soak immersion: incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
- Post-treatment: blotting on absorbent material and incubation for 18 hours in assay medium at standard culture conditions.

CONTROLS
- Negative: deionised water
- Positive: methyl acetate

MTT
- Concentration solution: 1.0 mg/mL in DMEM
- Assessment of Direct Test Item Reduction by MTT: 3h incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air
- Assessment of Colored or Staining Materials: 1h incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air
- Assay: incubation for 180 ± 10 minutes at standard culture conditions and rinsed 3x with DPBS afterwards.

DATA EVALUATION
1) Mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) Mean ODBlk from each OD value of the same experiment (blank corrected values) was subtracted.
3) Mean value of 2 aliquots for each tissue (= corrected test item OD) was calculated.
4) Mean value of 2 relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

CALCULATIONS
1) % viability of the 2 relating tissues for each control and test item relative to the negative control (100% control) was calculated.
Viability [%]= 100 x (corrected test item OD / corrected mean negative control OD)
2) The difference of the viability between duplicate tissues Is calculated.
3) The mean test item viability (TI viability) was calculated and the test item is classified according to the prediction model.

VALIDITY CRITERIA
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

other: Cell viability (%)

Run / experiment:

Mean of all runs

Value:

82.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean absorbance of the 2 tissues exposed to the test item was 1.292. From this the relative absorbance value is calculated and this corresponds to the cell viability. For the test item the cell viability was calculated to be 82%, when the negative control was set to 100 %. The threshold for irritancy is ≤ 60%, consequently the test item was not irritant to eye.

Any other information on results incl. tables

Colour change pre-experiments: No change in color was observed.

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.

Results after treatment for 6 hours with test item and the controls:

Dose group	Mean Absorbance* Tissue 1 and 2	Mean Absorbance* of 2 tissues	Rel. Absorbance (%) Tissue 1 and 2**	Absolute value  of the difference of the  Rel. Absorbance (%) Tissue 1 and 2	Rel. Absorbance  (% of Negative Control)**
Negative Control	1.545	1.558	99.2	1.6	100.0
	1.610		100.8
Positive Control	0.635	0.612	40.8	2.9	39.3
	0.637		37.8
Test Item	1.197	1.292	76.9	12.2	82.9
	1.425		89.0

*Mean of two replicates wells after blank correction

** Relative absorbance (rounded values): 100 * (absorbance (test item/positive control)) / (absorbance negative control)

Validity criteria:

- The negative control OD is > 0.8 and < 2.5 ( 1.564 and 1.612)

- The mean relative viability of the of the positive control is below 50% of the negative control viability (39.3%).

- The difference of viability between the two relating tissues of a single item is < 20% in the same run (value between 1.6 % to 12.2%).

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item does not possess eye irritating potential under the test conditions set in an in vitro OECD 492 study.

Executive summary:

In the current study the eye irritation potential of the test item was assessed in an in vitro study according to OECD 492 guideline, MatTek Corporation Protocol EpiOcular Eye Irritation Test and under GLP. The EpiOcular EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. Consequently, a negative result in this test can also be used to conclude that no classification for Eye damage Cat 1 or Eye Irritation Cat 2 is required under CLP. However, a positive result in this test does not allow to differentiate between Eye damage Cat 1 and Eye Irritation Cat 2.

In this in vitro test, the test chemical was topically applied to a 3 -dimensional reconstructed human cornea-like eipithelium (RhCE ) tissue. The tissue viability was measured following exposure and a post-treatment incubation period. The RhCE tissue viability is 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 quantitatied after extraction from the tissue. The test item, positive and negative controls were tested in duplicate. The exposure duration was 6 hours.

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 test chemical does not cause chemical (non-enzymatic) reduction of MTT into MTT formazan (chemical interference).

In the pre-tests, buccoxime did not cause optical nor chemical interference.

In the main study, the cell viability was calculated to be 82%, when the negative control was set to 100 %. The threshold for irritancy is ≤ 60%. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 39.3%. The validity criteria of the study were met.

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