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

Endpoint summary

Description of key information

Based on the results of tests in rabbits, the test material is not considered to be irritating to the skin or to the eyes. The studies were performed according to international accepted Guidelines and under GLP regulation and, therefore, of very high quality (Klimisch 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-04-26 to 2006-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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 19 to 20 weeks
- Weight at study initiation: 4,14 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: 42 - 71%
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: From: 2006-05-03 To: 2006-05-25

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0,5 ml

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48, 72 hours after removal of patches up to 15 days after start of treatment

Number of animals:

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 study was 4.14 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.

Animals 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 least three times per 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 19 to 22 °C and the relative humidity varied from 42 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.

According to the specifications given by the 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.



Grading scale for evaluation (according to OECD Guideline):

Erythema and eschar formation
Scores
--------------------------------------------------
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar
formation preventing grading of erythema 4
--------------------------------------------------
Maximum possible: 4
--------------------------------------------------

Edema formation
Scores
--------------------------------------------------
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well
defined by definite raising) 2
Moderate edema (raised approx. 1 mm) 3
Severe edema (raised more than 1 mm
and extending beyond area of exposure) 4
--------------------------------------------------
Maximum possible: 4
--------------------------------------------------

Total possible irritation score (maximum): 8

Mean score = Mean grading for erythema or edema of all the rabbits per time point
Mean cumulative score = Mean grading of all readings for erythema and edema per time point
Mean score per animal = Mean grading for each animal of erythema or edema (24, 48, and 72 hours after removal of the patches)

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Other effects:

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

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, undiluted, induced a weak erythema of the skin up to day 7. According to the EEC-Directive 2001/59 the test material should not be classified as a skin irritant.

Executive summary:

Purpose

The purpose of this assay was to identify the skin irritation/corrosion potential of the test substance when applied to the intact skin of previously shaven rabbits for a 4 hours period under semiocclusive conditions. This study should provide a rational basis for risk assessment to the irritating potential of the test item in man.

Study Design
To test for primary skin irritation, 0.5 mL of the test material, was spread onto 6 cm²patches and applied to the intact skin of previously shaven rabbits for a 4 hours period under semiocclusive conditions. The study was started as an initial test with one animal and followed by the confirmatory test with two further animals. The first examination of the treated skin sites followed 1 hour after removal of the patches. Thereafter, examinations were performed daily for further 14 days.

Results

Under the conditions of the present study, erythema (score 1) from day 1 up to day 7, and in one animal edema (score 1) from day 3 up to day 5 of the experimental part, were seen. Furthermore all three animals showed scales from day 6 up to day 11 of the experimental part.

Evaluation of all animals (mean score):

Day (after treatment)	1 (1h)	2 (24 h)	3 (48h)	4 (72 h)
Erythema	1	1	1	1
Edema	0	0	0.33	0.33

Evaluation of each animal:

Animal No	Mean score (24, 48, 72 h)			Maximum value (24, 48, 72 h)
	16	17	18	16	17	18
Erythema	1	1	1	1	1	1
Edema	0.67	0	0	1	0	0

Conclusion

The test item, undiluted, induced a weak irritation of the skin up to day 7.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

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)

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

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

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

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

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

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

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on in vivo studies in rabbits, no skin or eye irritating potential has been observed for the test material. As the test system used is very sensitive with regard to skin and eye irritation, the results of the local tolerance studies performed with this substance are relevant for human risk assessment.

Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin irritation and eye damaging potential under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.