5-hexyldihydro-5-methylfuran-2(3H)-one

Endpoint summary

Description of key information

An weight of evidence approach determined irritating properties of the pure test substance to skin in vitro. 

The test substance was found to be slightly irritating for the eye but without classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-11-09 to 2015-12-07

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase onversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number: Lot number: 23305
- Shipping date: 2015-12-01
- Date of initiation of testing: 2015-12-01 (pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: at least 15 times, volume not provided
- Observable damage in the tissue due to washing: no data provided
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 ± 1 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5 % SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

post-treatment: 41.5 h
MTT: 3 h treatment and 69 h extraction

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

10.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.9 %

Remarks on result:

positive indication of irritation

Other effects:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Dose Group	Exposure Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Standard Deviation [%]	Mean rel. Absorbance [% of Negative Control]**
Negative control	1 h	1.705	1.552	1.747	1.668	6.1	100.0
Positive control	1 h	0.075	0.083	0.085	0.081	6.4	4.9
Test item	1 h	0.196	0.177	0.15	0.174	13.1	10.4

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

 

The mean relative absorbance value of the test item, corresponding to the cell viability, was reduced to 10.4 % (threshold for irritancy:≤50 %), consequently the test item was irritant to skin.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test substance was determined to be irritant in the in vitro human skin model test with EpiDermTM.

Executive summary:

The in vitro Human Skin Model Test with EpiDerm™ study according to OECD 439 was performed to assess the irritation potential of the test substance. Independent triplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (PBS) or the positive control (5 % SDS in water) for 1 hour. 30 μL of the test item were dispensed directly onto triplicate EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test substance the mean tissue viability decreased to 10.4 % after 1 h exposure. This value is below the threshold for irritation, which is defined to be < 50 %. Therefore, the test substance was considered to be skin irritating. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test was irritating (Category 2) to skin according to EU CLP and UN GHS.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-03-17 to 2016-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional subcategory 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Kit
- Tissue batch number: 23327
- Delivery date: 2016-04-05
- Date of initiation of testing: 2016-04-05

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, for exposure of 3 ± 0.5 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times, no defined volume
- Modifications to validated SOP: There was the following alteration from the MatTek test protocol: Concerning exposure conditions (chapter 3.6.2): Only the 6-well plates for the 60 ± 5 minutes exposure period was placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) during treatment, the wells for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer and the test item did not dye water.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (Cat. 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (Cat. 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: Controls for MTT assay

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL (79.4 μL/cm2)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3 min and 1 h

Duration of post-treatment incubation (if applicable):

3 h MTT incubation, 22 h extraction

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

109.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0 %

Positive controls validity:

valid

Remarks:

4.6 %

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure

Value:

133.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0 %

Positive controls validity:

valid

Remarks:

3.6 %

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: the mean viability of the tissue replicates treated with the positive control for 1 hour, was <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates was ≤ 30%

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to non-corrosive in the in vitro human skin model test with EpiDermTM.

Executive summary:

The in vitro Human Skin Model Test with EpiDerm™ study according to OECD 431 was performed to assess the corrosion potential of the test substance. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) for 3 min or 1 hour. 50 μL of the test item were dispensed directly onto EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test item the relative absorbance value did not decrease (109.3 %) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 133.4 %. Both values did not exceed 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. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test substance was non corrosive to skin according to EU CLP and UN GHS.

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:

1984-09-25 to 1984-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.14-2.42 kg
- Housing: individually, in suspended metal cages
- Diet: ad libitum, Rabbit Diet, A.W. Tindall Limited, Holbeach, Lincolnshire
- Water: ad libitum, tap water
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 2.0
- Humidity (%): 65-68
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of each animal

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: 100 %

Duration of treatment / exposure:

Single treatment.
Test substance was not removed from the eye (only self-cleaning of eye).

Observation period (in vivo):

max. 21 days
Observations done after 1, 24, 48 and 72 h and as necessary at day 7, 14 and 21.

Number of animals or in vitro replicates:

3 (sex not specified)

Details on study design:

SCORING SYSTEM:
- Assessment of damage/irritation: Draize J.H., 1959, Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics"
- classification of ocular irritation potential: modified version of the system described by Kay J.H. and Calandra J.C., J.Soc. Cosmet, Chem. 1962 13 281 to 289

TOOL USED TO ASSESS SCORE: standard opthalmoscope

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.11

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #1

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:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis 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:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

chemosis score

Basis:

animal #2

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:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

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 #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Other effects:

1 h after application dulling of the cornea was observed in all animas. After 24 h the effect was not observed anymore.

Mean scores

Animal	Mean score redness (24/48/2 h)	Mean score chemosis (24/48/72 h)
1	0.00	0
2	0.33	0
3	0.00	0
Mean	0.11	0

Individual scores

Animal		1					2					3
Time after treatment [h]		1	24	48	72	7 days	1	24	48	72	7 days	1	24	48	72	7 days
Cornea
	E=Degree of opacity	D	0	0	0	-	D	0	0	0	-	D	0	0	0	-
	F=Area of opacity	2	0	0	0	-	1	0	0	0	-	2	0	0	0	-
	Score (E*F)*5	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
Iris
	D	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
	Score (D*5)	0	0	0	0	-	0	0	0	0	-	0	0	0	0	-
Conjuntivae
	A=Redness	1	0	0	0	-	2	1	0	0	-	1	0	0	0	-
	B=Chemosis	1	0	0	0	-	1	0	0	0	-	1	0	0	0	-
	C=Discharge	2	0	0	0	-	2	0	0	0	-	1	0	0	0	-
	Score (A+B+C)*2	8	0	0	0	-	10	2	0	0	-	6	0	0	0	-
Total Scores		8	0	0	0	-	10	2	0	0	-	6	0	0	0	-

D=Dulling of the cornea

Interpretation of results:

GHS criteria not met

Conclusions:

A study in rabbits was conducted to evaulate the irritation potential of the test substance in the eye. The results showed a minimal irritation effect of the test substance on rabbit eyes.

Executive summary:

The potential of the test substance to cause eye irritation was tested using rabbits. The animals were observed for 72 hours with data recorded and up to 21 days if necessary. The mean score for cornea opacity and iris were 0 for 24/48/72 h. For all animals a reversible dulling of the cornea was observed 1 h after treatment. For conjuntivae redness it was 0.11 and for chemosis 0 at 24/48/72 h. For all animals the individual mean scores for cornea opacity and iris were 0 for 24/48/72 h. Animal 1 had a score for conjuntivae redness of 0 and 0 for chemosis at 24/48/72 h. Animal 2 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 3 had a score for conjuntivae redness of 0 and 0 for chemosis at 24/48/72 h. All effects observed were reversible within 24 to 48 h. Due to these results the test substance was found to be slightly irritating for the eye but without classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

In a weight of evidence approach the pure test substance was tested in two in vitro studies.

An in vitro skin irritation test (OECD 439) was conducted to evaluate the skin irritation potential of the test substance. Undiluted test substance (30 µL) was applied to the human skin modell (EpiDerm) and incubated for 1 h. The viability of cells was evaluated with MTT test and measurement of optical density. As the mean viability was determined to be less than 50 %, to be precise 10.4 %, the test substance is considered skin irritating.

The in vitro Human Skin Model Test with EpiDerm™ study according to OECD 431 was performed to assess the corrosion potential of the test substance. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) for 3 min or 1 hour. After exposure of the tissues to the test item the relative absorbance value did not decrease (109.3 %) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 133.4 %. Both values did not exceed 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.

Furthermore data from in vivo studies with diluted test substance are available.

A skin irritation test was performed (supporting study). A range finding test with 2 animals was conducted. The exposure time was 24 h and concentrations 100, 75, 50, 25 and 5 or 10 % were applied. The range finding test revealed a very slight erythema reaction to concentrations of 100, 75 and 50 % test substance. The main study was conducted using 3 rabbits with the highest non irritating concentration from the range finding test of 25 % and exposure times of 4, 12 and 24 hours. The animals were observed for 72 hours. The mean erythema and edema score for 24/48/72h were 0 for all exposure times. Due to these results the 25 % test substance was found to be not skin irritating and without classification.

A preliminary siting test for the guinea pig maximization test was conducted (supporting study). Therefore the test substance was applied to clipped skin of 4 guinea pigs. The test substance was applied undiluted (100 %) and with a concentration of 50 %. The skin was covered with an occlusive patch and exposed to the test substance for 24 h. The effects were evaluated 1, 24 and 48 h after the removal of the test substance. None of the guinea pigs showed any skin reactions. Therefore the test substance was not irritating to the skin of guinea pigs in with a concentration of 50 and 100%.

A maximization test in 25 human volunteers was conducted to test the sensitising potential of the test substance. The pre test (supporting study, see IUCLID section 7.10.5) for this study showed that the 2 % test substance had no skin irritation properties. The occlusive application of the test substance on the volar forearm or back without additional treatment for 48 h caused no skin irritation.

Conclusion

The in vivo tests (including the human test) were conducted with diluted test substance or under no standard application and observational conditions. In the range finding test a indication of irritating properties of the test substance in high concentrations was already provided. The in vitro tests using the human skin model determined a clearly irritation but non-corrosive function of the test substance. In conclusion, it can be stated that the 100 % test substance was irritating (Category 2) to skin according to EU CLP and UN GHS.

 

Eye irritation/corrosion:

The potential of the test substance to cause eye irritation was tested using rabbits. The animals were observed for 72 hours with data recorded and up to 21 days if necessary. The mean score for cornea opacity and iris were 0 for 24/48/72 h. For all animals a reversible dulling of the cornea was observed 1 h after treatment. For conjuntivae redness it was 0.11 and for chemosis 0 at 24/48/72 h. For all animals the individual mean scores for cornea opacity and iris were 0 for 24/48/72 h. Animal 1 had a score for conjuntivae redness of 0 and 0 for chemosis at 24/48/72 h. Animal 2 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 3 had a score for conjuntivae redness of 0 and 0 for chemosis at 24/48/72 h. All effects observed were reversible within 24 to 48 h. Due to these results the test substance was found to be slightly irritating for the eye but without classification.

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation/corrosion, the test item is classified into category 2 (H315: Causes skin irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.

Based on available data on eye irritation/corrosion, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.