Diisopropyl ether

Endpoint summary

Description of key information

Results of in vitro and in vivo skin irritation studies on diisopropyl ether performed according to test guidelines have demonstrated that the compound is not a skin irritant. An in vitro eye irritation study on diisopropyl ether which was performed according to the ICCVAM Test Method Evaluation Report: Appendix G – ICCVAM recommended Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) Method Protocol, demonstrated that the compound possesses severe irritation potential.

In a GLP in vitro Bovine Corneal Opacity and Permeability (BCOP) test, diethyl ether was reported to not cause serious eye damage (Remmele, 2010). In an in vivo eye irritation study on diisopropyl ether, which was conducted in accordance with OECD Guideline for the Testing of Chemicals No. 405 and in compliance with GLP, diisopropyl ether did not induce significant or irreversible damage to the rabbit eye (Mallaun, 2010).

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with detailed documentation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage de Gerome (Quartier Labaste - F40260 Linxe)
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 2.26 to 3.07 kg
- Housing: Individual cages
- Diet (e.g. ad libitum): ad libitum, SDS-C15
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

Skin reactions were appreciated 1, 24, 48, and 72 hours after removal of the patch

Number of animals:

Initially, a single animal was treated. After consideration of the cutaneous responses produced in the first treated animal, two additional animals were treated during 4 hours.

Details on study design:

TEST SITE
- Area of exposure: 6 square centimetre patch
- Type of wrap if used: semi-occlusive dressing secured with surgical adhesive tape


REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removal of patch treated area was rinsed with distilled water.
- Time after start of exposure: 4 hours


SCORING SYSTEM: Local dermal irritation was assessed using the prescribed OECD numerical system

Erythema and eschar formation:

No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar formation
(injuries in depth) preventing erythema reading 4



Oedema formation:

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending
beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24 hours

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 48 hours

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 72 hours

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24 hours

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 3 to 6 days

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 48 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 3 to 6 days

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 72 hours

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 3 to 6 days

Irritant / corrosive response data:

A very slight to well defined erythema, associated with a very slight to slight oedema, was noted on the treated area of all animals, 1 hour after patch removal. The oedematous reactions were totally reversible between day 3 and day 6 and the erythematous reactions were totally reversible on day 6.

Other effects:

Regarding the cutaneous aspect, a slight dryness was noted on day 3 or day 6 and was totally reversible on day 10 in two animals and remained on day 14 in the other one.

Interpretation of results:

other: Not classified

Remarks:

Criteria used for interpretation of results: other: CLP (EC 1272/2008)

Conclusions:

The results obtained in this in-vivo study conclude that the test item diisopropyl ether does not have to be classified for skin irritation and is therefore, considered not to be a skin irritant.

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, according to accepted guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of GLP not signed.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Hillcrest, Dodgeford Lane, Belton, Loughborough, Leics, LE12 9TE / UK.
- Age at study initiation: 11 weeks.
- Weight at study initiation: 2298 - 2415 g
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet (e.g. ad libitum): Pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 25/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum.
- Acclimation period: 23-Sep-2010 to 27-Sep-2010 (one animal); 23-Sep-2010 to 28-Sep-2010 (two animals).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C.
- Humidity (%): 30-70%.
- Air changes (per hr): 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL/animal.

Duration of treatment / exposure:

Single exposure.

Observation period (in vivo):

72 hours with observations at 1, 24, 48, and 72 hours.

Number of animals or in vitro replicates:

3 animals.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not reported.

SCORING SYSTEM: The eye reactions were assessed according to the numerical scoring system listed in the Commission Regulation (EC) No 440/2008, B.5, at approximately 1, 24, 48 and 72 hours after administration. Scleral reddening and ocular discharge were also assessed.

Commission Regulation (EC) No 440/ 2008, 30 May 2008 - Grading of Ocular Lesions.

1. Cornea
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 luster), details of iris clearly visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Nacreous area, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4

2. Area of cornea involved
Zero: 0
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4

3. 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

4. Conjunctivae
-Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye)
Blood vessels normal: 0
Some blood vessels definitely hyperemic (injected): 1
Diffuse, crimson color, individual vessels not easily discernible: 2
Diffuse beefy red: 3

-Chemosis: lids and/or nictitating membranes
No swelling: 0
Any swelling above normal (including nictitating 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

Additionally, ocular discharge, reddening of the sclerae and staining of conjunctivae, sclerae and cornea by the test item were assessed according to the following scheme:

1. Ocular Discharge
No discharge: 0
Slight: Any amount different to normal (does not include small amount observed in inner canthus of normal animal): 1
Moderate: Discharge with moistening of the lids and hair just adjacent to the lids: 2
Marked: Discharge with moistening of the lids and hairs, and a considerable area around the eye (running): 3

2. Sclerae
-Redness
Normal: Blood vessels normal: 0
Slight reddening: Some blood vessels definitely hyperemic (injected): 1
Moderate reddening: Diffuse, crimson color, individual vessels not easily discernible: 2
Marked reddening: Diffuse beefy red: 3

3. Staining of Conjunctivae, Sclerae and Cornea by the Test Item
Not observed: 0
Slight staining: 1
Marked staining: 2

The test item was classified according to the referred classification criteria: Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and
1999/45/EC, and amending Regulation (EC) No 1907/2006.

TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).

Irritation parameter:

cornea opacity score

Remarks:

Cornea opacity and area of corneal opacity

Basis:

mean

Time point:

other: 1, 24, 48, and 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 1, 24, 48, and 72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #1

Time point:

other: 1 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #2

Time point:

other: 1 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #3

Time point:

other: 1 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #1

Time point:

other: 24 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #2

Time point:

other: 24 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

animal #3

Time point:

other: 24 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

-Redness

Basis:

mean

Time point:

other: 48 and 72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 1, 24, 48, and 72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

other: -Sclera

Basis:

animal #1

Time point:

other: 1 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

other: -Sclera

Basis:

animal #2

Time point:

other: 1 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

other: -Sclera

Basis:

animal #3

Time point:

other: 1 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

other: -Sclera

Basis:

mean

Time point:

other: 24, 48, and 72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

other: -Ocular discharge

Basis:

mean

Time point:

other: 1, 24, 48, and 72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible

Irritant / corrosive response data:

1. Irritation: The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, respectively. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.33 and 0.00 for reddening and 0.00 for chemosis for all three animals.
Slight reddening of the conjunctivae was noted in all three males 1 hour after instillation and persisted in one animal (No. 91) up to the 24-hour reading. Slight reddening of the sclerae was recorded in two animals (Nos. 90 and 91) at the 1-hour reading. No abnormal findings were observed in the treated eyes of any animals 48 hours after instillation.

2. Coloration: No staining produced by the test item was observed in the treated eyes.

3. Corrosion: No corrosion of the cornea was observed at any of the reading times.

Refer to Table 1.

Other effects:

1. Viability / Mortality: No intercurrent deaths occurred during the course of the study.
2. Clinical Signs: No clinical signs were recorded throughout the entire observation period.
3. Body Weights: The body weight of the animals was within the range commonly recorded for this strain and age.
4. Macroscopic Findings: No necropsy was performed at the end of the study.

Table 1: Eye Irritation Scores – Mean Values after 24, 48, and 72 hours
Animal number	Sex	Corneal Opacity	Iris Light Reflex	Conjunctivae
				Redness	Chemosis
90	Male	0.00	0.00	0.00	0.00
91	Male	0.00	0.00	0.33	0.00
92	Male	0.00	0.00	0.00	0.00

Interpretation of results:

other: Not classified

Remarks:

Criteria used for interpretation of results: other: CLP (EC 1272/2008)

Conclusions:

Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), diisopropyl ether does not have to be classified with respect to eye irritation in rabbits, following the results of this in-vivo study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in vitro skin irritation study on diisopropyl ether was identified, which was performed according to a draft OECD Guideline for the Testing of Chemicals (in vitro skin irritation: reconstructed human epidermis test method) and in compliance with GLP (Paulus, 2010). In this study, 30 µL diisopropyl ether was applied undiluted to the human skin model EpiDerm for 60 minutes followed by a cell viability test {3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide (MTT) assay}. The test was performed in triplicate. Dulbecco’s Phosphate Buffered Saline (DPBS) buffer and 5% sodium dodecylsulphate (SDS) solution were used as negative and positive controls, respectively. The positive control showed clear irritating effects, which was within the range of historical data of the test facility. The absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5 and variation among replicates was acceptable for the negative control, positive control and test item (<18%). The relative absorbance values for the test item were increased to 102.3%, which was above the threshold for irritation (50%). Therefore, diisopropyl ether is considered as a non-irritant in the in vitro Human Skin Model Test. 

Based on the results of the in vitro skin irritation study, an in vivo skin irritation study was conducted in rabbits (Colas, 2010). The study was performed according to OECD Guideline for the Testing of Chemicals No. 404 and in compliance with GLP. In this study, 3 New Zealand White rabbits were exposed to 0.5 mL of undiluted diisopropyl ether under semi-occlusive conditions. Animals were exposed to the test compound for 4 hours and observations were recorded at 1, 24, 48, and 72 hours and up to 14 days after removal of the patch and residual test substance. In all animals, a very slight to well defined erythema and a very slight to slight oedema were noted 1 hour after patch removal. The oedematous reactions were fully reversed between Day 3 and 6 and the erythematous reactions were fully reversed on Day 6. In 3 animals, a slight dryness was noted on Day 3 or Day 6, which was fully reversed on Day 10 in two animals and remained on Day 14 in the other animal. Based on the results of this study, diisopropyl ether was considered not to be a skin irritant, and therefore, is not classified according to CLP criteria. The in vivo study was determined to be the key study, which is supported by the results of the in vitro test.

Eye Irritation:

An in vitro eye irritation study on diisopropyl ether was identified, which was performed according to the ICCVAM Test Method Evaluation Report: Appendix G – ICCVAM recommended Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) Method Protocol and in compliance with GLP (Paulus, 2010). In this study, 300 µL of diisopropyl ether was placed onto the surface of the of hen’s eggs, which had been incubated at 38 °C and 59% humidity for 9 days. The was observed for hemorrhage, vascular lysis, and coagulation for 300 seconds at room temperature. Physiological sodium chloride solution was used as the negative control and 1% SDS solution and 0.1M sodium hydroxide solution were used as positive controls. The positive controls induced severe irritation and the negative control induced no irritation on the blood vessels. The mean irritation score for diisopropyl ether was 19.15, which corresponds to severely irritating. Under the conditions of the study, diisopropyl ether was considered to possess severe irritation potential. 

Another in vitro eye irritation study was identified, which was performed according to OECD Guideline for the Testing of Chemicals No. 437 and in compliance with GLP (Remmele, 2010). In this study, bovine corneas were washed with Eagle’s minimum essential medium (EMEM) then exposed to 750 µL of diisopropyl ether for 10 minutes. Corneas were washed 3 times with EMEM (containing phenol red) and once with EMEM (without phenol red) and then incubated for another 2 hours. Observations of opacity and permeability were taken. Highly de-ionized water and 1% (w/v) sodium hydroxide solution were used as the negative and positive controls, respectively. An in vitro irritancy score (IVIS) was assessed. Both the negative and positive control results were within the historical control data range. A mean IVIS of 29.7 was reported for diisopropyl ether. Based on the observed results and applying the evaluation criteria, diisopropyl ether does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP test) under the test conditions. The test method does not allow a detailed decision upon the degree of eye irritation. The result does not exclude an irritation potential of the test substance.

For final decision upon the classification with regard to eye irritation, results from an in vivo study were needed. Subsequently, an in vivo eye irritation study on diisopropyl ether was conducted according to OECD Guideline for the Testing of Chemicals No. 405 and in compliance with GLP (Mallaun, 2010). The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.33 and 0.00 for reddening and 0.00 for chemosis for all three animals. Mild, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, as well as ocular discharge were reported. These effects were reversible and were no longer evident 48 hours after treatment for all animals. No abnormal findings were observed in the cornea or for the iris light reflex of any animals at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed. According to CLP classification (EC 1272/2008), diisopropyl ether is not classified with respect to eye irritation in rabbits.

Justification for selection of skin irritation / corrosion endpoint:

in-vivo study

Justification for selection of eye irritation endpoint:

in-vivo study

Justification for classification or non-classification

Skin irritation: The results of the in vivo test in rabbits according to OECD TG 404 indicate that the substance di-isopropyl ether was not a skin irritant. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.2.

 

Eye irritation: According to the Guidance on the Application of the CLP Criteria (ECHA Reference ECHA-09-G-02-EN), a positive result in the HET-CAM or the BCOP is classified as serious eye damage Category 1, while negative results in the HET-CAM and the BCOP must be followed by further testing. In a follow-up in vivo study, diisopropyl ether did not induce significant or irreversible damage to the rabbit eye. As a result, diisopropyl ether does not have to be classified with respect to eye irritation according to Regulation (EC) No 1272/2008, Annex I section 3.2.