bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-05-23 to 2006-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-510-2

EC Name:

-

Cas Number:

872182-46-2

Molecular formula:

C28H46O7

IUPAC Name:

1,3-bis(2-ethylhexyl) 2-[(4-hydroxy-3,5-dimethoxyphenyl)methyl]propanedioate

Test material form:

liquid: viscous

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Strain: Albino rabbit, Crl:KBL(NZW)BR, female (f)
- Source: Charles River Wiga GmbH, Kißlegg
- Age at study initiation: about 23 weeks
- Weight at study initiation: 3.94 kg (mean)
- Housing: Becker, type K99/30 KU, floor area approx. 5400 cm2
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 22°C
- Humidity: 39 - 60%
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: From: 2006-06-01 To: 2006-06-14

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0,1 ml

Duration of treatment / exposure:

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 period (in vivo):

The rabbits were examined for eye irritation and for changes in behavior and general condition 1 hour after treatment, after 24, 48, or 72 hours, then daily up to day 15 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 them to acclimatize.
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. The mean initial body weight at the start of the experimental part was 3.94 kg.

-- Housing and diet
All rabbits were housed in an air-conditioned room of about 50 m2. A timer to provide a 12-hour light and a 12-hour dark regime controlled lighting.
They were kept separately in special rabbit cages (manufacturer: Becker; type K99/30 KU, floor area about 5400 cm2, a shelter with an integrated sitting board of about 1820 cm2; overall height: 60 cm) with plastic grids placed on mobile racks. The collection pans underneath the cages were cleaned at Ieast three times per week. The cages were also cleaned before the start of the experimental part.
Temperature and relative humidity were measured using a thermohygrograph. The room temperature varied from 19 to 22 °C and the relative humidity varied from 39 to 60 %.
The relative atmospheric humidity in the animal room was transiently outside the target range of > 40 %. This minor and short deviation did not influence the integrity of the study.
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.
According to the specifications given by manufacturer, the diet had been checked by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics.

The drinking water was periodically analyzed according to the German regulations for human drinking water.

-- Preparation
The test material, a liquid, was administered undiluted.


–- Administration and dose level
To ensure that only rabbits with normal eyes were included in the study, approximately 24 hours before treatment ophthalmologic examinations were performed after instillation of a 0.15 % fluorescein solution (Dr. Thilo & Co.) using an ophthalmoscope. 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.
The study consisted of an initial test with one rabbit and a confirmatory test with two further animals.


-- 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, or 72 hours, then daily up to day 15 of the experimental part.

Eye changes were evaluated according to 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
A) 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

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

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: 48 h

Results 8

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Results 9

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No signs of irritation could be observed at the cornea or iris. The conjunctivae showed redness (score 1) at the first (1 hour) and second reading (24 hours). Furthermore discharge (score 1) at the first reading was seen. Thereafter no signs of irritation were observed. The untreated eyes were unchanged.

Other effects:

No signs of clinical toxicity were detected.
All animals survived the observation period.
Body weight development of the treated rabbits was inconspicuous.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Except for some slight reddening and discharge of the conjunctivae within the first 24 hours, no signs of irritation were observed. According to the EEC-Directive 2001/59 the test material should not be regarded as an irritant to the eyes.

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

The test item was tested for its eye irritating properties. Prior to testing, the HET-CAM test was performed and no irritating potential could be detected. The test for primary eye irritation was started as an initial test with one animal and followed by the confirmatory test with two further animals.
For the test of primary eye irritation, 0.1 mL of the test material was applied into the conjunctival sac of rabbits. The first examination of the eyes followed 1 hour after instillation. The other examinations were performed daily for further 7 days.

Results

No signs of irritation could be observed at the cornea or iris. The conjunctivae showed redness (score 1) at the first (1 hour) and second reading (24 hours). Furthermore discharge (score 1) at the first reading was seen. Thereafter no signs of irritation were observed. The untreated eyes were unchanged.

Evaluation of all animals (mean score):

Day (after treatment)	1 (1h)	2 (24 h)	3 (48h)	4 (72 h)
Cornea  A	0	0	0	0
Iris  A	0	0	0	0
Conjunctivae  A	1	1	0	0
Conjunctivae  B	0	0	0	0

Evaluation of each animal:

Animal No	Mean score (24, 48, 72 h)			Maximum value (24, 48, 72 h)
	13	14	15	13	14	15
Cornea  A	0	0	0	0	0	0
Iris  A	0	0	0	0	0	0
Conjunctivae  A	0.33	0.33	0.33	1	1	1
Conjunctivae  B	0	0	0	0	0	0

Conclusion

Except for some slight reddening and discharge of the conjunctivae within the first 24 hours, no signs of irritation were observed. According to the EEC-Directive 2001/59 the test material should not be regarded as an irritant to the eyes.