1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to OECD 438 and under GLP conditions for indicating severe eye damage. The test is, however, not fully validated for severe eye irritation but the results are sufficiently adequate to conclude on the eye irritation effect of the substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 438 ("Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants")

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

EC Number:

251-649-3

EC Name:

1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one

Cas Number:

33704-61-9

Molecular formula:

C14H22O

IUPAC Name:

1,1,2,3,3-pentamethyl-2,3,4,5,6,7-hexahydro-1H-inden-4-one

Test animals / tissue source

Species:

other: Chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

TEST ANIMALS (eye-donors)
- Source: Poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, The Netherlands
- Age at study initiation: 7 weeks old
- Weight at study initiation: approx. 1.5-2.5 kg

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 uL
- Concentration: Undiluted (100%)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

240 minutes (examination at 0, 30, 75, 120, 180 and 240 minutes after treatment)

Number of animals or in vitro replicates:

Number of eyes used:
- Test group: 3
- Positive control: 3
- Negative control: 1

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: Rinsing with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: Corneal thickness and corneal opacity were scored at different time points, while fluorescein retention was scored only at 30 minutes after exposure. These endpoint were related to 4 classes of eye irritation (not; slightly; moderately; severely irritating) and finally reconciled to the classification criteria as applicable in EU-CLP, in accordance with the decision criteria in OECD438. Additionally, morphological and microscopic effects were examined.

TOOL USED TO ASSESS SCORE: Slit lamp microscope.

HISTOPATHOLOGY: After treatment, all eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde and corneas were embedded in parraffin wax, sectioned and stained. Histopathological examination was not performed, but the microscopic slides were filed in the archive.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Irritancy score category III

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Irritancy score category III

Results 3

Irritation parameter:

corneal swelling 

Run / experiment:

Mean %

Value:

20

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Irritancy score category III

Results 4

Irritation parameter:

in vitro irritation score

Remarks:

Irritation index = maximum mean corneal swelling + maximum mean opacity (x20) + mean fluorescein score (x20)

Run / experiment:

Mean

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

max score 200

Other effects / acceptance of results:

The test substance caused moderate swelling (20%) of the cornea, moderate or moderate to severe corneal opacity (2.2) and moderate or moderate to severe fluorescein retention (2.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Wrinkling of the epithelium followed by erosion/loosening of the top-layer of the epithelium were observed in the test substance group. Severe loosening of the epithelium was observed in the positive control group.

Any other information on results incl. tables

Please find in the table below the summary results.

 

Test material	Maximum score for:			Irritation categories	Irritation index	Classification (EU-CLP)
	Swelling %	Opacity	Fluorescein retention
Cashmeran	20	2.2	2.3	III;III;III	110	2
Saline (negative control)	0	0.0	0.0	Not applicable, one eye tested
BAC 5% (w/v) (positive control)	27	3.0	3.0	III;IV;IV	147	1

 

EC/GHS-CLASSIFICATION (CLP) OF EYE IRRITANTS EU classifcation sceme ICE
Regulatory Classification	Combinations of the three categories
NC/NI = not classified/not irritating	3 x I
	2 x I, 1 x II
	2 x II, 1 x I
EU/CLP Category 2 [4]: Irritating to eyes	3 x II[3]
	2 x II, 1 x III
	1 x I, 1 x II, 1 x III[1,3]
	3 x III
	2 x III, 1 x II
	2 x III, 1 x I
	2 x I, 1 x IV[1]
	2 x II, 1 x IV[1]
	2 x III, 1 x IV[2]
	1 x II, 1 x III, 1 x IV[1]
EU/CLP Category 1 [4]: Irreversible effects on the eye/serious damage to eyes	3 x IV
	2 x IV, 1 x III
	2 x IV, 1 x I[1]
	2 x IV, 1 x I[1]
	immediate corneal opacity score 3 in at least 2 corneas corneal opacity score 4 in at least 2 corneas severe loosening of epithelium in at least one cornea

[1] Combinations of categories less likely to occur.
[2] The combination of 2 x III, and 1 x IV can be considered as a borderline case between irritating and severely irritating.
[3] These combinations can be considered as a borderline case between Category 2 and not irritating.
[4] Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.

Applicant's summary and conclusion

Interpretation of results:

other: Serious eye irritation in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance is an eye irritant in the isolated chicken eye test (OECD guideline 438)

Executive summary:

The eye irritating potential of Cashmeran was determined in an isolated chicken eye test performed according to OECD guideline 438 and under GLP conditions.

The test substance caused moderate swelling (20%) of the cornea, moderate or moderate to severe corneal opacity (2.2) and moderate or moderate to severe fluorescein retention (2.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Furthermore, the positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants. The irritation index for Cashmeran as defined in the OECD guideline was 110 out of a maximum of 240.

Based on the irritation index for Cashmeran the substance does not need to be classified as severely irritating but according to the EU-CLP classification schemes of the ICE, the substance should be classified as irritating and Category 2: “Irritating to eyes”.