Dihexyl fumarate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM D6355

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

4% dilution in petrolatum

Species:

other: Human

Strain:

not specified

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

other: 4% in petrolatum

Controls:

not specified

Duration of treatment / exposure:

48h

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Tested at 4% in petrolatum. it produced no irritation after a 48-hr closed-patch test on human subjects

Interpretation of results:

study cannot be used for classification

Conclusions:

Tested at 4% in petrolatum. it produced no irritation after a 48-hr closed-patch test on human subjects

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

no

Species:

rabbit

Strain:

not specified

Type of coverage:

occlusive

Preparation of test site:

abraded

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Duration of treatment / exposure:

24h

Observation period:

48h

Irritation parameter:

other: erythema

Basis:

mean

Time point:

48 h

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Dihexyl fumarate applied full strength to intact or abraded rabbit skin for 24 hr under occlusion produced erythema lasting 24 hr

Remarks:

Dihexyl fumarate applied full strength to intact or abraded rabbit skin for 24 hr under occlusion produced erythema lasting 24 hr

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

produced erythema lasting 24 hr

Interpretation of results:

study cannot be used for classification

Conclusions:

produced erythema lasting 24 hr

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

The study was conducted between 12 April 2018 and 12 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

yes

Remarks:

This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Dihexyl fumarate (EC 242-833-4)
Batch: 800317920
Purity: 98.6%
Physical state/Appearance: Clear colorless liquid
Expiry Date: 25 January 2019
Storage Conditions: Room temperature in the dark

Species:

other: Eyes from adult cattle

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the appropriate corneas

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was required for this study.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Corneal Epithelium Condition

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

 In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	0.4
Negative Control	2.4
Positive Control	59.8

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	1	3	4	3		0.000
	3	1	2	3	2		0.000
	6	1	3	3	2		0.008
					2.3*		0.003¨		2.4
Positive Control	7	2	30	33	31	28.7	1.990	1.987
	10	1	33	35	34	31.7	2.145	2.142
	11	2	32	37	35	32.7	1.625	1.622
						31.0·		1.917·	59.8
Test Item	12	1	3	2	1	0.0	0.072	0.069
	14	2	3	2	0	0.0	0.004	0.001
	15	3	3	2	-1	0.0	0.002	0.000
						0.0·		0.024·	0.4

OD= Optical density           * = Mean of the post-incubation -pre‑treatment values           ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	2	Clear	Clear
	3	Clear	Clear
	6	Clear	Clear
Positive Control	7	Cloudy	Cloudy
	10	Cloudy	Cloudy
	11	Cloudy	Cloudy
Test Item	12	Clear	Clear
	14	Clear	Clear
	15	Clear	Clear

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for classification: EU CLP

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS	EU CLP (Regulation (EC) No 1272/2008)
≤ 3	No Category	Not classified for irritation
>3; ≤ 55	No prediction can be made	No prediction can be made
> 55	Category 1	Category 1 H318: Causes serious eye damage

 

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	0.4
Negative Control	2.4
Positive Control	59.8

 

Conclusion

No category. Not requiring classification to UN GHS or EU CLP.

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

In the 1976 Levenstein study, Dihexyl fumarate was applied neat to abraded rabbit skin for 24 hr under occlusion which produced erythema lasting 24 hr.

Furthermore, in the 1976 Epstein study, the compound was assessed in a human patch test. It produced no irritation after a 48-hr closed-patch test on human subjects at 4% in petrolatum.

As such it was determined that the substance should not be classified for skin irritation/corrosion.

In the Bovine Corneal Opacity and Permeability study,  the response to the test item was lower than the negative control, and therefore it was determined that the test item did not require classification in accordance with both UN GHS and EU CLP.