3-methylbutyl isovalerate

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:

2015-06-12 to 2015-10-15

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epi-200 kits purchased from MatTek Corporation (Bratislava, Slovakia), containing EpiDerm™ tissues on agarose.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues (Epi-200 SIT kits, MatTek)
- Tissue batch number(s): Lot No.: 21678
- Delivery date: 30 June, 2015
- Date of initiation of testing: 30 June, 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37±1°C, remaining period of treatment at room temperature on a sterile bench
- Temperature of post-treatment incubation (if applicable): 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 (if done): 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: 3h
- Spectrophotometer: Versamax®Molecular Devices, Softmax Pro (version 4.7.1)
- Wavelength: 570 nm
- Filter: ±1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- 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 37 ± 1.5 °C for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item is determined and classification is recommended if the mean relative tissue viability of three individual tissues is reduced to less than or equal to 50% of the negative control.
Mean tissue viability =50% = irritant (I)
Mean tissue viability >50% = non-irritant (NI)

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 positive control

Amount/concentration applied:

30 µL (47 µL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue.

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

114.8

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 6.5% 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. Therefore, the test substance is not classified according to CLP.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test substance by means of the Human Skin Model Test.

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. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 µl of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue.

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.

After treatment with the test substance the mean relative absorbance value was 114.8% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of = 50%. Therefore, the test substance is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance is not irritating to 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:

key study

Study period:

1999-09-06 to 1999-09-14

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

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: 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
- Source: Charles River, Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kißlegg
- Weight at study initiation: 1.8 to 1.9 kg
- Housing: individually in PPO cages
- Diet (e.g. ad libitum): pelleted complete rabbit diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+-3°c
- Humidity (%): 55%+-15%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 ml per application

Duration of treatment / exposure:

24 h

Observation period (in vivo):

1 h, 24 h, 48 h, 72 h and 7 d

Number of animals or in vitro replicates:

4 females

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 after exposure

SCORING SYSTEM:
- According to OECD guideline.

TOOL USED TO ASSESS SCORE: fluorescein / UV-lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.6

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.1

Max. score:

0.33

Reversibility:

fully reversible

Scores for ocular lesions

Rabbit No.		Individual mean score from readings after 24, 48 and 72 hours
1623	Cornea opacity, degree	0.00
	Iris	0.00
	Conjunctiva Redness	0.67
	Chemosis	0.00
1624	Cornea opacity, degree	0.00
	Iris	0.00
	Conjunctiva Redness	1.00
	Chemosis	0.33
1625	Cornea opacity, degree	0.00
	Iris	0.00
	Conjunctiva Redness	0.67
	Chemosis	0.00
1626	Cornea opacity, degree	0.00
	Iris	0.00
	Conjunctiva	0.00
	Chemosis	0.00

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is non irritating to the eye according to GHS criteria.

Executive summary:

The test item was tested for eye irritation in an in-vivo test in rabbits (according to OECD 405 and GLP). Four rabbits had the test item instilled in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours as well as day 7 after dosing. Slight signs of irritation were observed on the treated eyes. By 7 days, 4 of 4 treated eyes had returned to normal. It is concluded that the substance is not irritating to the eyes of rabbits under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an older acute dermal toxicity study the skin irritation potential of the test item was assessed in 10 rabbits, showing slight redness (9 of 10 animals), moderate redness (1 of 10 animals), slight edema (5 of 10 animals) and moderate edema (5 of 10 animals). This old study was considered as disregarded study as it is insufficiently documented for the endpoint “skin irritation”, and therefore less reliable.

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.