α-acetyl-γ-butyrolactone

Endpoint summary

Description of key information

Skin irritation/corrosion:

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method

EPISKIN-SM. The study was conducted in accordance with OECD TG 431. Following exposure with the test substance after 4 hours, the mean cell viability was 91.6% compared to the negative control value. Therefore, the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method EpiDerm™. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 39 % (rounded). The test item was thus shown to be irritating to reconstructed human skin in vitro.

Eye Irritation:

The irritation effects of the test item were evaluated according to the OECD No. 438. After the zero reference measurements, the eyes were held in a horizontal position and the treatments (test item, positive, nagative controls) were applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with isotonic saline solution. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant.

According to OECD TG 405 a single instillation of 0.1 ml of the undiluted test item into the conjunctival sac of three rabbits caused slight conjunctival erythema accompanied by edema and corneal opacity which disappeared completely in two animals after one week, while the eye of the third animal had cleared after two weeks. For these reactions a primary irritation score of 3.25 was calculated, corresponding to the classification "moderately irritating". This evaluation and is consistent with the in vitro result.

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

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)

Version / remarks:

adopted in 2015

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:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

60 min (35 min at 37 °C and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for treatment and control groups

Irritation / corrosion parameter:

% tissue viability

Value:

38.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

other: positive

Conclusions:

In this in vitro skin irritation test in EpiDerm™ tissue in accordance with OECD 439 and in compliance with GLP, the mean cell viability compared to the negative control was 38.5% after 60 min exposure with the test substance. All validity criteria were within acceptable limits. In conclusion, the test substance was irritating to human epidermis tissue. However, since the RhE test method cannot resolve between irritation and corrosion, further information on skin corrosion would be required to decide on its final classification or hazard potential.

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method EpiDerm™.

The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 39 % (rounded). The test item was thus shown to be irritating

to reconstructed human skin in vitro.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (Manufacturer: SkinEthic, France
- Tissue batch number(s):Batch No.:14-EKIN-016, Expiry Date: 12 May 2014)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μl
- Concentration (if solution): undiluted
NEGATIVE CONTROL
- Amount(s) applied: 50 μl
- Concentration (if solution): 0.9% NaCl in water
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.4-23.9°C) covered with the plate lids.

Number of replicates:

Two replicates per test item per time point were used. Two negative controls and two positive controls were also run in the assay. Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in the study.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

expousre period: 4 hours

Value:

91.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item had no colouring potential or intrinsic colour. Therefore, the Non Specific Colour % (NSC %) determination was not necessary.
However, colour change (purple) was observed after three hours of incubation of the test item in MTT solution, thus the test material interacted with the MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability.

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method

EPISKIN-SM. The study was conducted in accordance with OECD TG 431.

Following exposure with the test substance after 4 hours, the mean cell viability was 91.6% compared to the negative control value. Therefore, the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Species:

other: chicken

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

Details on study design:

NEGATIVE CONTROL USED : physiological saline (0.9% NaCl solution)

POSITIVE CONTROL USED : benzalkonium chloride solution (5%, w/v)

APPLICATION DOSE AND EXPOSURE TIME : 30 µl, 10 seconds

OBSERVATION PERIOD : at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed with 20 ml isotonic saline

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

2.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean at up to 75 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean up to 240 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

None

Positive Control

Observation Value ICE Class*	Value ICE Class*	ICE Class*
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	-7%	II
Mean maximum corneal opacity	3.50	IV
Mean fluorescein retention	2.83	IV
Other Observations	Loosening of epithelium was observed in two eyes (at 75 and 120 minutes after the post-treatment rinse)
Overall ICE Class*	2xI 1xIII

Negative Control

Observation Value ICE Class*	Value ICE Class*	ICE Class*
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00
Other Observations	None
Overall ICE Class*	3xIII

Test item

Observation Value ICE Class*	Value ICE Class*	ICE Class*
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.50	III
Mean fluorescein retention	2.33
Other Observations	None
Overall ICE Class*	2xI 1xIII

Interpretation of results:

other: positive

Conclusions:

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant.

Executive summary:

The irritation effects of the test item were evaluated according to the OECD No. 438. After the zero reference measurements, the eyes were held in a horizontal position and the treatments (test item, positive, nagative controls) were applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with isotonic saline solution. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Based on this in vitro eye irritation test in isolated chicken eyes, the test item is not classified as severe irritant and not classified as non-irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29.01.-12.02.1991

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:

(1987)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml/animal

Duration of treatment / exposure:

At least 14 days following instillation (no washout after 24 hours)

Observation period (in vivo):

1, 24, 48 and 72 hours, 7 and 14 days after administration.

Number of animals or in vitro replicates:

3 (1 male, 2 females)

Details on study design:

To calculate the irritation effect all scores of each animal at each of the following reading times (24, 48, 72 hours) were used in calculating the respective mean values.
The primary irritation score was calculated by totalling the individual cumulative scores at 1, 24, 48 and 72 hours and then divided by the number of figures.

Irritation parameter:

other: Primary irritation score

Basis:

mean

Time point:

other: 1/24/48/72 h

Score:

3.25

Max. score:

13

Reversibility:

fully reversible

Irritant / corrosive response data:

Generally slight diffuse corneal opacity was observed in the animals nos. 67
(male) and 68 (female) from 1 to 48 hours after treatment. Generally slight
diffuse corneal opacity was observed in the animal no. 69 (female) at 1 to 72
hours. At day 7 slightly spotted diffuse corneal opacity was observed in the
same animal only.

Other effects:

No acute clinical symptoms were observed in the animals during the test and observation period, and no mortality occurred.
The body weight gain of all rabbits was similar.

Interpretation of results:

other: positive

Executive summary:

According to OECD TG 405 a single instillation of 0.1 ml of the undiluted test item into the conjunctival sac of three rabbits caused slight conjunctival erythema accompanied by edema and corneal opacity which disappeared completely in two animals after one week, while the eye of the third animal had cleared after two weeks. For these reactions a primary irritation score of 3.25 was calculated, corresponding to the classification "moderately irritating".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results for skin irritation/corrosion a classification in Category 2 (H 315: Causes skin irritation) according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.

Based on the study results for eye irritation a classification in Category 2 ( H319: Causes serious eye irritation) according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.