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Description of key information

For this enpoint, read-across information on the source substance ethyl hexanoate is available.

In a combination of three in vitro methods, protein reactivity (DPRA), activation of keratinocytes (LuSens) and activation of dendritic cells (h-CLAT) were measured to assess the potential for skin sensitization using the in vitro Skin Sensitization Turnkey Testing Strategy to address key events of the adverse outcome pathway (AOP).

Based on the results and applying the evaluation criteria ethyl hexanoate is not peptide reactive, does not activate keratinocytes and activates dendritic cells, and is according to the evaluation criteria predicted not to be a skin sensitizer.

As a consequence, the target substance butyl butyrate was also considered to be not a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16.03.2015 - 23.03.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across from ethyl hexanoate to butyl butyrate is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. A full read-across justification including comparison of toxicological profiles will be included in section 13 of the IUCLID dossier.
Reason / purpose:
read-across source
Key result
Parameter:
other: Peptide depletion
Run / experiment:
C-containing peptide (mM)
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Peptide depletion
Run / experiment:
K-containing peptide (mM)
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean peptide depletion
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The mean peptide depletion of the positive control was 34.77%.
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance ethyl hexanoate shows no chemical reactivity in the DPRA test under the current conditions. Read-across was done from ethyl hexanoate to butyl butyrate based on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate. Therefore, the test substance butyl butyrate is to be considered as a nonsensitiser.
Executive summary:

Read-across was done from ethyl hexanoate.

In the current study the skin sensitisation effect of the test item was assessed in an in chemico assay according to OECD 442C.

This method quantifies the remaining concentrations of cysteine- or lysine-containing peptides after 24 hours incubation with the test item at 25 +/-2.5 °C. The percentage of depletion of cysteine- and lysine-peptides is calculated and used in a prediction model to assign the test chemical to one of four reactivity classes and in this way discriminate between sensitisers and non-sensitisers.

The test item was dissolved at a 100 mM concentration in acetonitrile (ACN) and 3 samples of the test item were incubated with a typical concentration of 0.667 mM of cysteine in a pH 7.5 phosphate buffer or 0.667 mM of lysine in a pH 10.2 ammonium acetate buffer. Incubation was done for 24 hours in the dark at 25 +/- 2.5°C. The remaining non-depleted concentrations were determined by HPLC with gradient elution and UV-detector at 220 nm.

The mean C-peptide depletion, caused by the test substance was determined to be -0.52%.

The mean K-peptide depletin, caused by the test substance was determined to be -0.04%.

Negative depletions are considered to be "zero" for the calculation of the mean peptide depletion, resulting in 0.0% for the test item. Based on the results and the prediction model, it is concluded that ethyl hexanoate shows no chemical reactivity in the DPRA test under the current conditions.

Based on this result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate, butyl butyrate is considered to be not sensitising.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23.04.2015 - 28.08.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across from ethyl hexanoate to butyl butyrate is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. A full read-across justification including comparison of toxicological profiles will be included in section 13 of the IUCLID dossier.
Reason / purpose:
read-across source
Key result
Parameter:
other: RFI CD54 mean (%) and RFI CD54 mean (%)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance ethyl hexanoate induces dendritic cell activation (CD54 expression increased =200% in at least one assay) in the h-CLAT test under the current conditions. Read-across was done from ethyl hexanoate to butyl butyrate based on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate. Therefore, the test substance butyl butyrate is to be considered as a sensitiser.
Executive summary:

Read-across was done from ethyl hexanoate.

In the current study the skin sensitisation potential of the test item was assessed according to a new guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT) accessed on 01 September 2014.

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the human Cell Line Test (h-CLAT). The test substance was incubated with the human pro-monocytic cell line THP-1 for 24 hours at 37°C and the membrane markers expression was measured by flow cytometry through staining with FITC labeled anti-human-CD86/anti human CD54 antibody and propidium iodide.

In order to determine the concentration suitable for the main test a pre-test was performed.

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main experiment the test item was tested at concentrations of 566 μg/mL onwards (1st experiment: 2027, 1689, 1408, 1173, 977, 815, 679 and 566 µg/mL; 2nd experiment: 1173, 977, 815, 679, 566, 471, 393 and 327 µg/mL). No precipitates were noticed in any concentration after 24 hours.

Relative fluorescence intensity and concurrent relative viability were determined in 2 main experiments. Calculation of an EC150 (the concentration resulting in a RFI of 150) for CD86 and an EC200 (the concentration resulting in a RFI of 200) for CD54 was not applicable.

In summary, after 24 hours of exposure to the test substance ethyl hexanoate, 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 test substance ethyl hexanoate

induces dendritic cell activation.

Based on this result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate, butyl butyrate is considered to be a sensitiser.

.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23.03.2015 - 27.04.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across from ethyl hexanoate to butyl butyrate is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. A full read-across justification including comparison of toxicological profiles will be included in section 13 of the IUCLID
dossier.
Reason / purpose:
read-across source
Parameter:
other: fold increase and cell viability
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance ethyl hexanoate did not show any keratinocyte activating potential in the LuSens test under the current conditions. Read-across was done from ethyl hexanoate to butyl butyrate based on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate. Therefore, the test substance butyl butyrate is to be considered as a nonsensitiser.
Executive summary:

Read-across was done from ethyl hexanoate.

In the current study on ethyl hexanoate the skin sensitising potential of the test item was assessed according to OECD 442D without significant deviations and GLP.

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for 48 hours at 37°C and the antioxidant response element (ARE) dependent luciferase activity was measured.

In order to determine the concentrations suitable for the main experiment a pre-test was performed in which cells were exposed to 9 concentrations and the 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 the luciferase activity was measured and in parallel a MTT assay was performed to assess the cytotoxicity of the test substance. The experiment was done in triplicate. The test substance was dissolved in 4% DMSO and in the end 1% DMSO was present in culture at all concentrations. No precipitates were noticed in any preparations.

The acceptance criteria were met.

Exposure to the test substance did not induce a statistical significant 1.5 fold increase in luciferase activity in LuSens cells while 70% viability was reached. Concluding, ethyl hexanoate did not show any keratinocyte activating potential and thus is to be considered as a non-sensitiser.

Based on this result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate, butyl butyrate is considered to be not sensitising.

.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For this endpoint there are 3 studies available on the read-across substance ethyl hexanoate.

To evaluate the skin sensitisation effects of ethyl hexanoate a combination of 3 in vitro methods addressing the key events of the adverse outcome pathway (AOP) for skin sensitization were performed. The tests are the Direct Peptide Reactivity Assay (DPRA), the ARE Reporter Assay (LuSens) and the Dendritic Cell Line Activation Assay (h-CLAT).

DPRA test

In the DPRA test (OECD 442C) the reactivity of ethyl hexanoate towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated. Incubation of the synthetic peptides with ethyl hexanoate was done for 24 hours and the remaining concentrations of cysteine- or lysine-containing peptides were determined.

The mean C-peptide depletion, caused by the test substance was determined to be -0.52%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.04%.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it is concluded that ethyl hexanoate shows no chemical reactivity in the DPRA test under the current conditions.

LuSens test

In the LuSens assay (OECD 442D) the keratinocyte activating potential of ethyl hexanoate was evaluated. Ethyl hexanoate was incubated with a luciferase reporter cell line (LuSens cells) for 48 hours and the antioxidant response element (ARE) dependent luciferase activity was measured, in parallel to a MTT assay to assess the cytotoxicity. No precipitates were noticed in any preparations. The acceptance criteria were met. After exposure to ethyl hexanoate activity in LuSens cells was not induced significantly in at least two consecutive concentrations affording at least 70% viability in at least two independent experiments. It has to be concluded that test substance ethyl hexanoate does not have a keratinocyte activating potential.

h-CLAT test

In the third in vitro study (OECD 442E) the potential of ethyl hexanoate to induce the expression of the cell membrane markers CD86 and CD54 in the human Cell Line Test (h-CLAT) was evaluated. In the human pro-monocytic cell line THP-1 the change in the expression of the cell membrane markers is measured by flow cytometry after 24 hours of test substance exposure through staining with FITC labeled anti-human-CD86/anti human CD54 antibody and propidium iodide.

 

No precipitates were noticed at any concentration.Calculation of an EC150 (the concentration resulting in a RFI of 150) for CD86 and an EC200 (the concentration resulting in a RFI of 200) for CD54 was not applicable.

In summary, after 24 hours of exposure to test substance ethyl hexanoate 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 test substance ethyl hexanoate induces dendritic cell activation.

The test battery evaluation uses the results of the three individual assays reflecting three key events along the adverse outcome pathway leading to skin sensitization. In the test battery a weight of evidence approach is made in such a way that any 2 out of 3 tests determine the overall result (2 positive test results drive the prediction of a sensitizer, while 2 negative test results drive the prediction of a non-sensitizer). In the case of ethyl hexanoate 2 of the tests are negative and 1 is positive. Applying the evaluation criteria ethyl hexanoate is predicted not to be a skin sensitizer.

 

Conclusion

Based on this result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate, butyl butyrate is not considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The criteria to evaluate a substance for the skin sensitisation endpoint using the in vitro Skin Sensitization Turnkey Testing Strategy to address key events of the adverse outcome pathway (AOP) according to the CLP Regulation (EC) No 1272/2008 were fulfilled. In the case of ethyl hexanoate 2 of the tests are negative and 1 is positive. Applying the evaluation criteria ethyl hexanoate is predicted not to be a skin sensitizer.

Based on this result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and butyl butyrate, butyl butyrate is not considered to be a skin sensitizer.