Butyl ethyl ether

Endpoint summary

Description of key information

The skin irritant potential of butyl ethyl ether (98.4 % purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SM^TM). Based on the results the target substance can be considered as non-irritant to the skin.

The eye irritation potential of butyl ethyl ether (98.4% purity) was examined in an in vivo study according to OECD 405. Only a slight irritant effect (grade 1) which was fully reversible within 6 days could be observed. Based on these results butyl ethyl ether can be considered as non-irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-10 to 2016-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS "Category 2". Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 16-EKIN-046
- Expiration date: November 21, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable):
a) 37 ± 1 °C (5.0% CO2 for 42 ± 1 h) in 2 mL pre-warmed fresh maintenance medium
b) 37 ± 1 °C (5.0% CO2 for 3 h ± 5 min) in 2 mL pre-warmed MTT medium

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
* washed with DPBS; excess DPBS was removed by blotting bottom with blotting paper;
* after post-incubation: excess medium was removed by blotting bottom on absorbent paper

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2.
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered irritant to the skin if the viability after 15 minutes exposure and 42 h of post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure and 42 h is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²) of the test item was dispensed directly atop the EPISKIN-SM tissue using a positive displacement pipette. The test item was spread to match size of the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277) in aqua dest.

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

73.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For detailed results see box "Any other information on results".

Results of the  Pre-Experiments

The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixtures of 10 µL of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_living equalled 0%.

Table 1: Results of the test item butyl ethyl ether

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.760 0.824	0.715 0.735	0.741 0.795	0.235 0.182	0.176 0.180	0.185 0.153	0.573 0.573	0.532 0.523	0.607 0.618
OD570 (Blank corrected	0.717 0.781	0.672 0.693	0.698 0.752	0.192 0.139	0.133 0.137	0.142 0.110	0.531 0.530	0.489 0.480	0.565 0.575
Mean OD570 of the Duplicates (Blank corrected)	0.749	0.683	0.725	0.166	0.135	0.126	0.530	0.485	0.570
Total Mean OD570 of 3 Replicate Tissues (Blank corrected)	0.719*			0.142			0.528
SD OD570	0.034			0.021			0.043
Relative Tissue Viabilities [%]	104.2	94.9	100.9	23.0	18.8	17.6	73.7	67.4	79.3
Mean Relative Tissue Viabilities [%]	100.0			19.8**			73.5
SD Tissue Viabilities [%]***	4.7			2.9			5.9
CV [% Viability]	4.7			14.5			8.1

*               Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

**             Mean relative tissue viability of the three positive control tissues is ≤ 40%.

***           Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570Blank	0.043	< 0.1	pass
Mean Absolute OD570NK	0.762	0.6 ≤ NK ≤ 1.5	pass
Mean Relative Viability PC [%]	19.8	≤ 40%	pass
Max. SD of % Viability	5.9	≤ 18%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritant effects in an in vitro skin irritation study conducted according to OECD 439.

Executive summary:

In an in vitro skin irritation study according to OECD Guideline 439 (Reconstructed Human Epidermis Test), the potential of buthl ethyl ether (98.4% purity) to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM™ (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study the test item was applied topically to the EPISKIN-SM™ tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The test item showed no irritant effects and is therefore considered to be non-irritating to the skin.

 

 

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:

weight of evidence

Study period:

2017-01-17 to 2017-04-10

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 02 October 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: animal 1: approx. 44 weeks old; animal 2: approx. 41 weeks old
- Weight at study initiation: > 2 kg
- Housing: ABS-plastic or Noryl rabbit cages, floor 4200 cm², in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum - Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (e.g. ad libitum): ad libitum - Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test item was applied to the test site

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects in animal no. 2, the observation period was extended up to 6 days after dosing.

Number of animals or in vitro replicates:

2 rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eye was not rinsed after application.

SCORING SYSTEM: Grading system for ocular lesions according to OECD 405

TOOL USED TO ASSESS SCORE: At the end of the observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

TEST PROCEDURE:
Initial test: The in vivo test was performed initially using one animal.
Confirmatory test: The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, one additional animal was treated in the same manner. According to OECD 405, section 23, treatment of a third animal was omitted as results from the second animal were sufficient to allow for hazard classification determination.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 6 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

animal #2

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

Irritant / corrosive response data:

After the application into the eyes of two male NZW rabbits the test item produced slight irritant but not corrosive effects in both animals, which were fully reversible within 72 hours in animal no. 1 and within 6 days in animal no. 2. Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal. Conjunctival redness, chemosis and hypersecretion were observed in both animals.

Other effects:

Neither mortality nor significant clinical signs of toxicity but local effects were observed. Moreover, the body weight development of all animals was within the expected range.

Table 1: Eye Irritation Scores - Animal No. 1
Observation	Animal No. 1, Eye Irritation Scores Post-Application After								Average Score (24, 48 and 72 hours)
	1 hour		24 hours		48 hours		72 hours
	Test item	Control	Test item	Control	Test item	Control	Test item	Control
Redness	0	0	1	0	1	0	0	0	0.67
Chemosis	1	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0
Cornea	0	0	0	0	0	0	0	0	0

Table 2: Eye Irritation Scores - Animal No. 2
Observation	Animal No. 2, Eye Irritation Scores Post-Application After								Average Score (24, 48 and 72 hours)
	1 hour		24 hours		48 hours		72 hours
	Test item	Control	Test item	Control	Test item	Control	Test item	Control
Redness	1	0	1	0	1	0	1	0	1
Chemosis	1	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0
Cornea	0	0	0	0	0	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo acute eye irritation/corrosion study in rabbits, butyl ethyl ether was tested negative regarding irritation to the eye.

Executive summary:

In a primary eye irritation study 0.1 mL of butyl ethyl ether (98.4 % purity) was applied into the conjunctival sac of one eye of two male New Zealand White Rabbits, Crl: KBL (NZW). The treated eye was not rinsed after the application. The untreated eye served as control. Control and treated animals were observed for a period of 72 hours.

Under the conditions of the present study, a single ocular application of the test item to rabbits at a dose of 0.1 mL produced slight irritant but not corrosive effects in both animals, which were fully reversible within 72 hours in animal no. 1 and within 6 days in animal no. 2. Neither mortalities nor significant clinical signs of toxicity were observed. Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.

On the basis of the test results and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008, the substance should be not classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Butyl ethyl ether (98.4 % purity) was tested negative for acute dermal irritating properties in a study conducted according to OECD 439. In a screening study for identifying ocular corrosives and severe irritants (BCOP, OECD 437) no prediction could be made regarding the classification of butyl ethyl ether and further testing was required. Thus, an acute eye irritation/corrosion study in vivo (OECD 405) was conducted in rabbits. Only slight irritating effects (grade 1) were observed in both animals. These effects were fully reversible within 72 hours to 6 days. Based on the results, butyl etyhyl ether is considered to be not irritating to the skin and/or eye.

Justification for classification or non-classification

Butyl ethyl ether (98.4 % purity) was tested negative for acute dermal irritating and eye irritating/corrosive properties in studies conducted according to OECD 439 and OECD 405. Therefore, butyl ethyl ether does not warrant classification for skin and/or eye irritation.