3-methylbutyl butyrate

Endpoint summary

Description of key information

In the key skin irritation study, the test item was found not to be irritating to the skin in the in vitro human epidermis model assay (OECD 439).

             

In the key eye irritation study, the test item was found not to be irritating to the eyes of rabbit (OECD 405).

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:

2015-01-22 to 2015-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

without any impact on the outcome of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

without any impact on the outcome of the study

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm TM tissues (Epi-200-SIT Kit)

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: The test substance was added into the insert atop the corresponding EpiDerm™ triplicate tissues
- Amount/test concentration: 30 µL
- Duration of treatment: 60 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1 °C in the remaining period at room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C

CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5 % SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: Tissues washed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times

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

- Viability:
Positive control: mean viability: 5.6 %; relative standard deviation: 2.4 %; range of viabilities: 2.9 - 11.3 %.
-Absorption:
Positive control: mean absorption: 0.098; relative standard deviation: 0.038 %; range of absorbance: 0.030 - 0.194
Negative control: mean absorption: 1.796; relative standard deviation: 0.281%, range of absorbance: 1.397 - 2.651

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Yes. Evaluation of colour interference: 30 µL of the test item mixed with 300 µL deionised water was incubated for 60 minutes at 37 ± 1.5 °C. Since the colour of the test item mixture did not change during the incubation period, additional testing with viable tissues was not required.
Evaluation of direct reduction of MTT: 30 µL of the test item added to MTT (1 mg/mL). Incubated in the dark at room temperature for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50 %; irritant (I), H315 (category 2)
mean tissue viability > 50 %; non-irritant (NI)
Amount/concentration applied :undiluted
Duration of treatment / exposure: 60 minutes

Acceptability of the Assay
Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is > 0.8 and = 2.8.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is at or below 20 %.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be < 18 %.
OD values should not be below historically established boundaries.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

115.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative absorbance

Other effects / acceptance of results:

- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test
- After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD = 0.8 and = 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system; the mean relative absorbance value of the positive control was 3.8 % compared to the negative control.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions reported, the test substance is not irritating to skin.

Executive summary:

The skin irritant potential of the test item was determined by means of the Human Skin Model Test (OECD 439). The relative absorbance value of the test item was 115.2% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of <= 50%. There, the test item is not considered to possess an irritant potential.

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:

12 - 16 April 1999

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:

Feb. 1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29.12.1992

GLP compliance:

yes

Species:

rabbit

Strain:

other: albino Mol:Russian (SPF)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Mollegaard Breeding and Research Centre A/S, Ejby, DK-4623 Lille Skensved
- Weight at study initiation: 2.2 - 2.7 kg
- Housing: individually in PPO cages (floor area: 2576 cm2) with perforated floor.
- Diet (e.g. ad libitum): pelleted complete rabbit diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 10 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness

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
- Concentration (if solution): undiluted

Duration of treatment / exposure:

24 h

Observation period (in vivo):

1, 24, 48, 72 h

Number of animals or in vitro replicates:

4

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsing with 20 mL 0.9 % sodium chloride solution
- Time after start of exposure: 24 h

SCORING SYSTEM: according to OECD 405

TOOL USED TO ASSESS SCORE: visual examination with UV-light after fluorescein was instilled

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hr

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions reported, the test substance shall not be classified as irritating to the eye.

Executive summary:

The eye irritant effect of the test substance was investigated according to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", 29.12.1992.

 

Four female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing.

Very slight to well-defined signs of irritation were observed among the rabbits.

The following mean values, based on the results from the 24, 48 and 72 tour readings, were calculated:

 

Substance	Cornea opacity	Iris lesion	Redness of conjunctiva	Oedema of conjunctiva
Isoamylbyturate	0.0	0.0	0.3	0.2

 

According to the Regulation (EC) No 1272/2008 the test substance shall not be classified as irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation, key study

The skin irritant potential of the test item was determined by means of the Human Skin Model Test (OECD 439). The relative absorbance value of the test item was 115.2% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of <= 50%. There, the test item is not considered to possess an irritant potential.

 

In vivo eye irritation, key study

The eye irritant effect of the test substance was investigated according to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", 29.12.1992.

 

Four female albino rabbits were exposed to 0.1 ml of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing.

Very slight to well-defined signs of irritation were observed among the rabbits.

The following mean values, based on the results from the 24, 48 and 72 tour readings, were calculated:

 

Substance	Cornea opacity	Iris lesion	Redness of conjunctiva	Oedema of conjunctiva
Isoamylbyturate	0.0	0.0	0.3	0.2

 

According to the Regulation (EC) No 1272/2008 the test substance shall not be classified as irritating to the eye.

Justification for classification or non-classification

Based on the available data, the substance does not require classification for skin or eye irritation according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.