[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Endpoint summary

Description of key information

In vitro skin and eye irritation studies have been conducted on unchanged and undiluted test material and are, therefore, considered key in preference to an in vivo skin irritation study in which the test material contained 50% active component in white vaseline. The in vitro studies are also in accordance with the updated ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a, Version 5.0, December 2016.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

3rd revision

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT Kit
- Tissue batch number(s): 23314
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 16 February 2016
- Date of initiation of testing: Pre-incubation phase started 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 ºC
- Temperature of post-treatment incubation (if applicable): 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 300 μL, one washing step
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not applicable, microplate reader
- Wavelength: 570 ± 1 nm
- Filter: Not reported
- Filter bandwidth: 570 ± 1 nm
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control; 4.77 %
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: None reported
- Reproducibility: Relative standard deviation for mean viability; 14.79 % postive control. Relative standard deviation for mean absorption; 16.6 % postive control, 8.55 % negative control.

NUMBER OF REPLICATE TISSUES: 3 per control and test item treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues Not applicable
- Procedure used to prepare the killed tissues (if applicable): Not applicable
- N. of replicates : Not applicable
- Method of calculation used: Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 43.5 hours exposure is less than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): Undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5 % SLS in deioinsied water; concentration not reported

Duration of treatment / exposure:

43.5 hours

Duration of post-treatment incubation (if applicable):

67.5 hours

Number of replicates:

3 per test item, negative control or postive control

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 1

Value:

0.601

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 2

Value:

0.592

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Tissue 3

Value:

0.585

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Mean absorbance of three wells blank corrected

Run / experiment:

Mean

Value:

0.593

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 1

Value:

38.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 2

Value:

37.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Tissue 3

Value:

37.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

other: Relative absorbance (%) compared to blank

Run / experiment:

Mean

Value:

37.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Standard deviation: 1.4 %

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Did not show blue colour after 1 hour incubation
- Colour interference with MTT: No colour change

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, within the required range of OD ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: Yes, induced a decrease in absorbance of 5.1 % compared to the negative control

Results of treatment with test item and the controls

Dose Group	Exposure Interval	Tissue No.	Absorbance 570 nm (Well 1)	Absorbance 570 nm (Well 2)	Absorbance 570 nm (Well 3)	Mean Absorbance of 3 Wells*	Mean Absorbance of three wells blank corrected	Mean Absorbance of three tissues after blank correction	Rel. Absorbance (%) 1, 2 + 3**	Relative Standard Deviation (%)	Mean. Rel. Absorbance (% of negative control)***
Blank			0.037	0.038	0.038	0.038	0.000
Negative Control	60 mins	1	1.540	1.548	1.508	1.532	1.495	1.563	95.6	4.1	100.0
		2	1.644	1.591	1.593	1.609	1.572		100.6
		3	1.717	1.631	1.630	1.659	1.622		103.8
Positive Control	60 mins	1	0.117	0.120	0.118	0.118	0.081	0.080	5.2	4.4	5.1
		2	0.118	0.107	0.116	0.113	0.076		4.8
		3	0.104	0.132	0.124	0.120	0.083		5.3
Test item	60 mins	1	0.646	0.628	0.643	0.639	0.601	0.593	38.5	1.4	37.9
		2	0.617	0.645	0.627	0.629	0.592		37.9
		3	0.613	0.625	0.630	0.623	0.585		37.4

 * Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 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})

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

and EU CLP criteria

Conclusions:

The test item is a category 2 irritant to skin according to UN GHS regulations based on a human skin model test conducted following OECD 439, EU B.46 and UN GHS guidelines and performed according to GLP.

Executive summary:

The test item was assessed for skin irritation in a human skin model test conducted following OECD 439, EU B.46 and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicate that the test item is a Category 2 irritant to skin according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2016 to 18 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Test item sourced from test sponsor, batch number: 10300010
- Expiration date of the lot/batch: January 2017
- Purity test date: Sum of Main Isomer: 96.30 Area %, Sum Isomers: 99.73 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have indicated this method as a sufficiently promising predictor for eye irritancy potential. 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.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 μL
- Concentration (if solution): Undiluted test item

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

3 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model (June 2015) followed.
- RhCE tissue construct used, including batch number: EpiOcular™ kits and MTT-100 kits purchased. EpiOcular™ lot no. 21597
- Doses of test chemical and control substances used: 50 μL of undiluted test item, methyl acetate or deionised water.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 30 minutes exposure at 37 ± 1.5 ºC; post-exposure immersion for 12 minutes at room temperature; post-exposure incubation for 120 minutes at 37 ± 1.5 ºC
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Not applicable
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 per test item, postive control or negative control.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Measured at 570 nm with a microplate reader without a reference filter. Absorbance values calculated with computer software.
- Description of the method used to quantify MTT formazan:
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: If the test item-treated tissue viability is > 60 % relative to the negative control treated tissue viability, the test item is labelled non-irritant. If viability is ≤ 60 % of treated negative control, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
- Complete supporting information for the specific RhCE tissue construct used: Method validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- Reference to historical data of the RhCE tissue construct: Historic data from July 2015 to February 2016 show the postive control range of viabiltiies of 6.90 % - 40. 4 %. The range of absorbance for historic postive control data is 0.107 - 0.849 and for negative control data 1.27 - 2.05.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Historical report data reported.
- Positive and negative control means and acceptance ranges based on historical data: Positve control - mean viability: 32.0 %, range: 6.90 - 40.4 %, mean absorption: 0.538, range 0.107 - 0.849. Negative control - mean absorption: 1.65, range 1.27 - 2.05
- Acceptable variability between tissue replicates for positive and negative controls: < 20 %
- Acceptable variability between tissue replicates for the test chemical: < 20 %

Irritation parameter:

other: Mean absorbance

Run / experiment:

2 Tissues

Value:

2.025

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Relative absorbance (% of negative control)

Run / experiment:

Mean of 2 tissues

Value:

100.2

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: None reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Validity criteria met

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control OD is > 1.0 and < 2.5 (1.99 and 2.053). The difference of viability between the two relating tissues of the postive control was 3.1 % (must be < 20%)
- Acceptance criteria met for positive control: Mean relative viability of the positive control is below 50% of the negative control viability (40.4%). The difference of viability between the two relating tissues of the postive control was 3.2 % (must be < 20%)
- Range of historical values if different from the ones specified in the test guideline: Not applicable

Irritant / corrosive response data:

Irritating effects were not observed following 30 minutes incubation with the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, under the experimental conditions reported, the test item does not possess an eye irritating potential.

Executive summary:

The test item was assessed for eye irritation in a human cornea model test conducted following OECD 492, and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item was assessed for skin irritation in a human skin model test conducted following OECD 439, EU B.46 and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicate that the test item is a Category 2 irritant to skin according to the UN GHS regulations.

The test item was assessed for eye irritation in a human cornea model test conducted following OECD 492, and UN GHS guidelines in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.