3-methylbutyl butyrate

Endpoint summary

Description of key information

As a weight of evidence approach, three in vitro assays were carried out, namely DPRA, LuSens and h-CLAT for isoamyl isovalerate as a read-across substance. The DPRA study was inconclusive due to the insolubility of the test compound in the peptides. The h-CLAT assay was positive regarding the sensitization as the substance activated dendritic cells. All data obtained in the LuSens assay compiled with acceptance evaluation criteria, and showed a negative result regarding sensitizing. Based on the overall inconclusive result of the three in vitro assays an in vivo test (guinea pig maximization test) using the read across substance isoamyl isovalerate was conducted. Based on the in vivo data the substance is considered not to be sensitizing to skin. Using this read across approach, the test item is considered not to be sensitizing to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04.Feb-23.May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

It is suspected that false positive LLNA results could be caused by some kind of detergent effect. Seeing that "emulsion formation" was the cause of the inconclusive output of the in vitro test battery, no LLNA test was conducted.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Microbiological status of animals, when known: SPF
- Age at study initiation: 5 weeks
- Weight at study initiation: 337–395 g
- Housing: (during the study) stainless wire mesh cages, 210W×350D×180H (mm), one animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 – 23.8 °C (permissible range: 18.0 - 24.0 °C)
- Humidity (%): 33.9 – 52.1 % (permissible range: 30.0 - 70.0 %)
- Air changes (per hr): 10 – 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route:

intradermal and epicutaneous

Vehicle:

olive oil

Concentration / amount:

25% / 0.1 mL (intradermal), 100 % / 0.2 mL (topical application)

Day(s)/duration:

Day 0 Intradermal; Day 7 topical application for 48 h

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% / 0.1 mL

Day(s)/duration:

24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 (5 for control)

Details on study design:

RANGE FINDING TESTS: Based on the results of the preliminary test the concentrations for the main study was determined

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (first induction: intradermal exposure; second induction: epicutaneous exposure)
- Exposure period: 20 days
- Test groups: 1
- Control group: 1
- Site: Six sites, three each on the left and right sides of the midline of the shoulder region for injection and two sites, one on the left and one on the right sidesfor application (region of the first induction sites)
- Frequency of applications: once
- Duration: 48 h
- Concentrations: 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21
- Exposure period: 3 days
- Test groups: 1
- Control group: 1
- Site: two sites, one on the left (for test substance) and one on the right (for vehicle) sides (region of the induction sites)
- Concentrations: 100%
- Evaluation (hr after challenge): 24 and 48 hours

Positive control substance(s):

yes

Remarks:

CDNB (1-Chloro-2,4- dinitrobenzene)

Positive control results:

The laboratory routinely uses a positive control so a positive control was not used in this assay.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

not measured/tested

Remarks:

The laboratory routinely uses a positive control so a positive control was not used in this assay.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

Remarks:

The laboratory routinely uses a positive control so a positive control was not used in this assay.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not produce skin sensitization under the conditions of this study.

Executive summary:

A Guinea pig maximization test was performed (OECD 406). In the test substance group, the 25% test substance was injected intradermally for the first induction. The second induction was concluded with the 100% test substance occluded for 48 hours. The challenge was conducted with the 100% test substance and olive oil occluded for 24 hours. As a result, no skin reactions such as redness or swelling were observed in any animal at 24 and 48 hours after challenge patch removal. Based on the results, isoamyl isovalerate item did not produce skin sensitization under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitising properties of the target substance isoamyl butyrate were predicted from the source substance isoamyl isovalerate.

 

DPRA, WoE (read-across)

The reactivity of the test item isoamyl isovalerate towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test item was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The test substance was soluble in acetonitrile at a concentration of 100 mM. The samples of the test substance with both peptides were emulsions at the time of preparation and after 24 hours. Thus all samples were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be -1.09%. The mean K-peptide depletion, caused by the test substance was determined to be -1.20%. Calculation of mean peptide depletion is not applicable due to insolubility of the test substance in the cysteine and lysine peptide samples. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model, it was concluded that the test item shows a minimal chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test item the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

 

ARE-Nrf2 Luciferase Test Method, WoE (read-across)

The keratinocyte activating potential of the test item isoamyl isovalerate was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75 % cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed: At concentrations used in the main experiment the test substance was an emulsion in 4 % DMSO in culture medium 3 (4 x stock preparations) and a solution in 1 % DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. After 48 hours of exposure to the test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments; calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

 

h-CLAT, WoE (read-across)

The potential of the test item isoamyl isovalerate to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test after 24 hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry.A total of 3 valid experiments were performed. At concentrations used in the main experiment the test substance was an emulsion (concentrations of 529 µg/mL and above) or soluble in culture medium (2 x stock preparations). Final concentrations were solutions at all concentrations at the time of application and after 24 hours. Calculation of EC150 (the concentration resulting in a RFI of 150) for CD86 was not applicable. The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated to be 482 µg/mL (experiment 2) and 757 µg/mL (experiment 3), respectively. In summary, after 24 hours of exposure to the test substance, CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test item induces dendritic cell activation.

 

Guinea pig maximization test, WoE (read-across)

A Guinea pig maximization test was performed (OECD 406) with the test substance isoamyl isovalerate. In the test substance group, the 25% test substance was injected intradermally for the first induction. The second induction was concluded with the 100% test substance occluded for 48 hours. The challenge was conducted with the 100% test substance and olive oil occluded for 24 hours. As a result, no skin reactions such as redness or swelling were observed in any animal at 24 and 48 hours after challenge patch removal. Based on the results, isoamyl isovalerate item did not produce skin sensitization under the conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available in vitro and in vivo results, the substance is not classified as sensitising according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.