Reaction mass of trans-5-hexyldihydro-4-methylfuran-2(3H)-one and cis-5-hexyldihydro-4-methylfuran-2(3H)-one

Endpoint summary

Description of key information

Skin irritation: The test substance was considered to be irritant to skin in the in vitro Human Skin Model Test.
Skin corrosion: The test substance was considered to be non-corrosive to skin in the in vitro Human Skin Model Test.
Eye irritation: The test substance is considered to be not irritating to rabbits' eyes. 

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:

2015-03-20 and 2015-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-conform study according to guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Species:

other: EPISKIN Standard Model

Strain:

other: EPISKIN Standard Model

Type of coverage:

other: 30 µL (47 µL/cm2) of the undiluted test item was dispensed directly on top the skin surface.

Preparation of test site:

other: The epidemis in the upper wells were preincubated for 60 minutes (37 ± 1.5°C, 5 ± 1% CO2, 95 ± 5% RH). The inserts were transferred from upper wells into the lower wells and preincubation was continued for 4.25 hours (37 ± 1.5°C, 5 ± 1% CO2, 95 ± 5% RH).

Vehicle:

unchanged (no vehicle)

Controls:

other: 5% Sodium Lauryl Sulphate in deionised water (positive control) and Dulbecco's Phosphate Buffered Saline (negative control) were used.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (47 µL/cm2)

Duration of treatment / exposure:

The complete exposure period of the skin equivalents was 60 minutes. According to the protocol of the test kit supplier, the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2 within 60 minutes. Outside the incubator the 6-well plates were placed in a sterile bench at room temperature for 25 minutes in sum.

Observation period:

not applicable

Number of animals:

Three replicates per test, negative control and positive control respectively were used.

Details on study design:

TEST SITE
- Area of exposure: surface of epidermis 0.6 cm2

REMOVAL OF TEST SUBSTANCE
- Washing: Tissues were gently rinsed with DPBS at least 15 times and afterwards submerged in DPBS at least three times. Subsequently, the tissue was once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the tissues and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
- Time after start of exposure: 60 min after exposure

SCORING SYSTEM: Cell viability by using the MTT test

Irritation / corrosion parameter:

other: other: cell viability (test item)

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min treatment + 42 h post exposure. Remarks: 25.5% relative absorbance (to negative control). (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (positive control)

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min treatment + 42 h post exposure. Remarks: 6.8% relative absorbance (to negative control). (migrated information)

Irritant / corrosive response data:

Cell viability: negative control
No. 1: 110.3%
No. 2: 98.8%
No. 3: 90.8%
Mean: 100%
Mean absorbance: 1.532

The absorbance value obtained for the positive control was 0.104 and this result corresponds to 6.8% viability when compared to the results obtained from the negative controls.
Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 25.5% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Other effects:

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance is irritant to skin.

Executive summary:

The irritation potential of the test substance was investigated by using the in vitro Human Skin Model Test (OECD 439, EU B.46). Each three tissues of the human skin model EpiDerm were treated with 30 µL of the test item, the negative (Dulbecco's Phosphate Buffered Saline) or the positive control (5% sodium lauryl sulfate) for 60 minutes. Cell viability was determined using the MTT assay. The absorbance value obtained for the positive control was 0.104 and this result corresponds to 6.8% viability when compared to the results obtained from the negative controls. After treatment with the test item Aprifloren the mean relative absorbance value decreased to 25.5% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. Thus, the test substance was considered to be irritant to skin under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2015-05-27 and 2015-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-conform and according to OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

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: Cheonan Yonam College, Laboratory Animal Center
- Age at study initiation: 11 weeks old
- Weight at study initiation: 2.10–2.17 kg
- Housing: individually ((during the quarantine/acclimation and observation periods)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Initial test (June 1 - 8); Confirmatory test (June 1 - 10)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Measurement value: 20.4–21.4
- Humidity (%): Measurement value: 47.5–59.8
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated left eye

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL

Duration of treatment / exposure:

The eyelid was gently held together for approximately one second.

Observation period (in vivo):

1, 24, 48 and 72 hours after application

Number of animals or in vitro replicates:

1 (initial test), 2 (confirmatory test); males

Details on study design:

SCORING SYSTEM: according to OECD scoring system

TOOL USED TO ASSESS SCORE: slit lamp (scores of cornea, iris and conjunctivae); fluorescein paper strips (cornea injury at 24 hours after test substance application)

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24 h/48 h/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24 h/ 48 h/ 72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24 h/ 48 h/ 72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24 h/48 h/ 72 h

Score:

0

Max. score:

4

Other effects:

- No abnormal signs or symptoms were observed in any animal throughout the course of the study.
- No clinical signs of pain and distress were evident in three animals immediately after test substance application and at 1, 24, 48 and 72 hours after application.
- All animals exhibited normal body weight gains. The mean body weight gain throughout the observation period was 0.11 kg.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, the test substance, the test substance, did not show any evidence of eye irritation or corrosion in New Zealand White rabbits under the conditions of this study.

Executive summary:

This study was conducted to evaluate the potential eye irritation/corrosion after a single application of the test substance in three 11-week-old male New Zealand White rabbits.In the initial test, one-tenth (0.1) mL of the test substance (100% test substance) was instilled to the conjunctivae sac of the right eye of one animal after gently pulling over the lower eyelid away from the eyeball. There was a negative response after application in the initial test. Therefore, a confirmatory test was conducted using two animals. Eye irritation was scored according to the method of Draize at 1, 24, 48 and 72 hours after test substance application. Mean 24, 48, 72 hour scores of the three animals were: cornea: 0; iris: 0; conjunctival redness: 0 and chemosis: 0. Based on these results, the test substance is considered to be non-irritating in rabbits' eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

Key study (skin irritation)

The irritation potential of the test substance was investigated by using the in vitro Human Skin Model Test (OECD 439, EU B.46). Each three tissues of the human skin model EpiDerm were treated with 30 µL of the test item, the negative (Dulbecco's Phosphate Buffered Saline) or the positive control (5% sodium lauryl sulfate) for 60 minutes. Cell viability was determined using the MTT assay. The absorbance value obtained for the positive control was 0.104 and this result corresponds to 6.8% viability when compared to the results obtained from the negative controls. After treatment with the test item Aprifloren the mean relative absorbance value decreased to 25.5% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. Thus, the test substance was considered to be irritant to skin under the test conditions chosen.

Key study (skin corrosion)

The corrosive potential of the test item was examined by using the Human Skin Model Test with EpiDerm tissues models according to OECD TG 431 and EU method B.40. In the pre-tests, the test item passed the MTT- and the Colour Interference. Independent duplicate tissues of EpiDerm were exposed to the test item, the negative control or positive control for 3 minutes and 1 hour, respectively. After washing, a 3 -hours incubation period with MTT followed. After exposure to the test item the relative absorbance value increased slightly to 116.1% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 116.5%. Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

Supporting study

A 10% solution of the test material in white mineral oil was examined according to OECD 404 and EU method B.4 to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The 10% test material solution produced a mean erythema and edema score of 0, respectively, at the 24, 48, 72 -h observations. No corrosive effects were noted.

Supporting study

A 20% solution of the test material in white mineral oil was examined according to OECD 404 and EU method B.4 to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The 20% test material solution produced very slight to well-defined erythema and slight edema to rabbits' skin which were fully reversible. 3-minute and 1-hour semi-occluded applications of the test material to the intact skin of one rabbit produced no corrosive effects. A 20% solution of the test substance in white mineral oil was not considered to be irritant or corrosive to rabbits' skin.

Eye irritation

Key study

This study was conducted to evaluate the potential eye irritation/corrosion after a single application of the test substance in three 11-week-old male New Zealand White rabbits.In the initial test, one-tenth (0.1) mL of the test substance (100% test substance) was instilled to the conjunctivae sac of the right eye of one animal after gently pulling over the lower eyelid away from the eyeball. There was a negative response after application in the initial test. Therefore, a confirmatory test was conducted using two animals. Eye irritation was scored according to the method of Draize at 1, 24, 48 and 72 hours after test substance application. Mean 24, 48, 72 hour scores of the three animals were: cornea: 0; iris: 0; conjunctival redness: 0 and chemosis: 0. Based on these results, the test substance is considered to be non-irritating in rabbits' eyes.

Supporting study

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (too). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging ( CLP/EPA/GHS (Cat 1)). In conclusion, the test item is not categorized (GHS).

Justification for selection of skin irritation / corrosion endpoint:
Only one reliable GLP-conform skin irritation study which is according to OECD guideline and EU method.

Justification for selection of eye irritation endpoint:
GLP and guideline conform study.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

On the basis of the available data the substance is considered to be classified for skin irritation cat. 2, labelled with H315 but is not considered to be corrosive to skin or irritating to eye under Regulation (EC) No 1272/2008 (CLP).