Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-09-07 to 2004-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Kißlegg, Germany
- Strain/sex: Albino rabbit, Crl:KBL(NZW)BR, female (f)
- Age at study initiation: approx. 17 weeks
- Weight at study initiation: 3.45 to 3.91kg (mean: 3.60 kg)
- Housing: separately in special rabbit cages (Becker, type K99/30 KU, floor area approx. 5400 cm2, a shelter with an integrated sitting board of 1820 cm2; overall height: 60cm) with plastic grids placed on mobile racks.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: more than 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-30°C
- Humidity: 29-71%
- Photoperiod: 12 hrs dark / 12 hrs light
The room temperature and relative atmospheric humidity in the animal room was transiently outside the target range of 16 to 22°C and >40%, respectively. However, this short deviation did not influence the integrity of the study.

IN-LIFE DATES: From: 2004-09-15 To: 2004-09-24

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0,1 mL, neat substance

Observation period (in vivo):

1 hour after treatment, after 24, 48, and 72 hours, then daily up to day 8 of the experimental part.

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

-- Identification and adaptation
Healthy rabbits were allocated to the study group. The animals were kept in the experimental room for more than 7 days to allow for acclimatization. Each animal was uniquely identified by a tattoo in the ear displaying the animal number. Individual cage cards were affixed to each cage displaying the study number, test material, day of treatment, and animal number.

-- Assignment
3 female rabbits were used for this study.

-- Housing and diet
All rabbits were housed in an air-conditioned room of about 50 m^2. Lighting was controlled by a timer to provide a 12-hour light and a 12-hour dark regime.
They were kept separately in special rabbit cages (manufacturer: Becker; type K99/30 KU, floor area about 5400 cm^2, a shelter with an integrated sitting board of about 1820 cm^2; overall height: 60 cm) with plastic grids placed on mobile racks. The collection pans underneath the cages were cleaned at least three times a week. The cages were cleaned before the start of the study.
Temperature and relative humidity were measured using a thermohygrograph. The room temperature varied from 17 to 30 °C and the relative humidity from 29 to 71 %.

The rabbits received a commercial diet for rabbits, Provimi Kliba 3418.0 ad libitum and ssniff K snack, as well as fresh community tap water from Makrolon drinking bottles ad libitum.

The diet is checked periodically according to the specifications of the manufacturer by an independent laboratory, approved by the German government. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics.
The drinking water is periodically analyzed according to the German regulations for human drinking water.

–- Administration and dose level
To ensure that only rabbits with normal eyes were included in the study, approximately 24 hours before treatment ophthalmological examinations were performed after instillation of a 0.15 % fluorescein solution (Dr. Thilo & Co.) using an ophthalmoscope according to Eisenhut (Basel). Animals with eye defects, injury or irritation were excluded.
0.1 mL test material was instilled into the conjunctival sac of the left eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test material was placed. The right eye remained untreated and served as control. After instillation, the eyelids were closed for about 30 seconds.

-- Observation for clinical symptoms
The rabbits were examined for eye irritation and for changes in behavior and general condition 1 hour after treatment, after 24, 48, and 72 hours, then daily up to day 8 of the experimental part.
Eye changes were evaluated according to the DRAIZE- , OECD- and EEC recommendations.

-- Evaluation of eye reactions

- Cornea Scores
A) Opacity-degree of density (area most dense taken for reading)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling
of normal lustre), details of iris clearly visible 1
Easily discernible translucent area, details of iris slightly obscured 2
Nacrous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

B) Area of cornea involved not evaluated (Rating according to the DRAIZE method)

- Iris
Normal 0
Markedly deepened rugae, congestion, swelling, moderate
circumcorneal hyperemia, or injection, any of these or combination
of any thereof, iris still reacting to light (sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2

- Conjunctivae
A) Redness (refers to palpebral and bulbar conjunctivae, cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2
Diffuse beefy red 3

B) Chemosis: lids and/or nictating membranes
No swelling 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4

C) Discharge (Rating according to the DRAIZE method )
No discharge 0
Any amount different from normal (does not include small
amounts observed in inner canthus of normal animals) 1
Discharge with moistening of the lids and hairs just adjacent to lids 2
Discharge with moistening of the lids and hairs,
and of a considerable area around the eye 3

Mean score of all animals = Mean grading for irritations of cornea, iris, and conjunctivae per time point
Mean score per animal = Mean grading for each animal of irritations of cornea, iris, and conjunctivae (1, 24, 48, and 72 hours after application)
Maximum value = Maximum grading of a sign of irritation within a period

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 1 day

Remarks on result:

probability of weak irritation

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 3 days

Remarks on result:

probability of weak irritation

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No signs of irritation could be observed at the cornea and iris. The conjunctivar showed redness (score 1) from the first reading (2 animals) up to experimental day 3 (1 animal), maximally. Thereafter no signs of irritation were observed. Furthermore, discharge (score 1) was seen one hour after treatment. These effects were regarded as transient signs of irritation, based on the mechanical properties of the test item (highly viscous liquid).The untreated eyes were unchanged.

Other effects:

No clinical signs or mortality were observed. Body weight development was inconspicious.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Slight redness of the conjunctivae and discharge were regarded as transient signs of irritation, based on the mechanical properties of the test item (highly viscous liquid). The test material should not be classified as eye irritant according to EU Directive 67/548/EC amended and repealed by EU Regulation No. 1272/2008 and CLP.

Executive summary:

Purpose

The purpose of this primary eye irritation assay was to provide information on possible health hazards in case of acute eye contact with a test material and serve as a a rational basis for risk assessment to the eye irritating potential of the test item in man.

Study Design

To test for eye irritation a test according to the OECD-Guidelines for Testing of Chemicals No. 405, the annex to Directive 92/69 EEC, and the recommendations of DRAIZE (1959) was performed.

Results

No signs of irritation were observed at the cornea or iris. One hour and 24 hours after treatment, the conjunctivae showed redness (score 1) in two animals. Furthermore, discharge (score I) was seen one hour after treatment. Thereafter no signs of irritation were observed. The untreated eyes were unchanged.

Conclusion

The test material must not be classified as eye irritant according to EU Directive 67/548/EC amended and repealed by EU Regulation No. 1272/2008 and CLP.