Registration Dossier

Administrative data

Description of key information

For this enpoint, read-across information on the source substance 3-methylbutyl isovalerate is available.

As the combined results of 3 in vitro / in chemico tests resulted in an inconclusive outcome, the substance was tested in vivo in a GPMT test. In this test, 3-methylbutyl isovalerate was found to not be a skin sensitizer.

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

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation, other
Remarks:
an in vivo skin sensitization (non-LLNA) study was performed based on ambigious results in the in vitro test battery
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read-across from 3-methylbutyl isovalerate to ethyl isobutyrate 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
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

CLINICAL OBSERVATIONS: No abnormal signs or symptoms were observed in any animal of any group throughout the course of the study.

BODY WEIGHTS: All animals exhibited normal body weight gain.

POSITIVE CONTROL: The sensitization rate of CDNB was 100% and the sensitization grade was classified to be ‘V (Extreme)’. Consequently, the study was considered to be valid.

Interpretation of results:
GHS criteria not met
Conclusions:
Read-across was done from 3-methylbutyl isovalerate. Based on this result, and on the structural, chemical and toxicological similarities between 3-methylbutyl isovalerate and ethyl isobutyrate, ethyl isobutyrate did not provoke an allergic response, and is therefore not sensitising.
Executive summary:

Read-across was done from 3-methylbutyl isovalerate. In a study on 3-methylbutyl isovaleratew the skin sensitizing potential was assesssed by means of a maximization test using guinea pigs according to OECD 406 and GLP.

 

In a preliminary test topical application did not reveal any evidence of adverse skin reactions, therefore, the induction sites in the main test for each group were pretreated with 10% sodium dodecyl sulfate prior to the second induction.

There were 2 groups, one for 3-methylbutyl isovalerate, this group consisted of 10 animals, while the second group was the control group made of 5 animals. In the test group, a concentration of 25% of 3-methylbutyl isovalerate was injected intradermally for the first induction. The second induction was conducted with 100% of 3-methylbutyl isovalerate. The sites were occluded for 48 hours, whereafter the challenge was conducted with 100% of the test substance. Hereafter, the sites are occluded for 24 hours. In the control group, the first and second inductions were done with olive oil and the challenges with the 100% of the test substance and olive oil.

Both in the control and the test group no skin reactions such as redness or swelling were observed in any animal at 24 and 48 hours after the challenge patch was removed. During the observation period, no abnormalities in clinical signs or body weight gain were observed in any animal of any group. Based on the result of this study, 3-methylbutyl isovalerate did not produce skin sensitization under the conditions of this study. Therefore, 3-methylbutyl isovalerate does not have to be classified as sensitising.

Based on this result, and on the structural, chemical and toxicological similarities between 3-methylbutyl isovalerate and ethyl isobutyrate, ethyl isobutyrate is considered to be not sensitising.

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

For this endpoint there are 4 studies available on the read-across substance 3-methylbutyl isovalerate.

In a first step to evaluate the skin sensitisation effects of 3-methylbutyl isovalerate 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 3-methylbutyl isovalerate towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated. Incubation of the synthetic peptides with 3-methylbutyl isovalerate was done for 24 hours and the remaining concentrations of cysteine- or lysine-containing peptides were determined.

The mean C-peptide depletion was -1.09% and the mean K-peptide depletion was -1.20%.

Based on the results it was concluded that 3-methylbutyl isovalerate shows a minimal chemical reactivity in the DPRA under the test conditions. However, due to the limited solubility of 3-methylbutyl isovalerate the samples with both peptides were emulsions and the result could therefore be underpredictive. Following the OECD TG 442C the study should be considered as “inconclusive".

LuSens test

In the LuSens assay (OECD 442D) the keratinocyte activating potential of 3-methylbutyl isovalerate was evaluated. 3-methylbutyl isovalerate 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. Exposure to 3-methylbutyl isovalerate did not induce a statistical significant increase in the luciferase activity in LuSens cells while 70% viability was reached. In other words, 3-methylbutyl isovalerate is to be considered as a non-sensitiser.

h-CLAT test

In the third in vitro study (OECD 442E) the potential of 3-methylbutyl isovalerate 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. The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated to be 482 µg/mL in experiment 2 and 757 µg/mL in experiment 3. 3-methylbutyl isovalerate induced CD54 expression in THP-1 cells with at least 50% viability in at least two independent experiments, thus it induces dendritic cell activation.

Conclusion in vitro tests

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 test substance to be a non-sensitizer).

In the case of 3-methylbutyl isovalerate 1 of the test is inconclusive, 1 is negative and 1 is positive. The test battery indicates contradictory results and thus an evaluation is not possible. For this reason a follow-up in vivo test was necessary.

In vivo study

In the in vivo study the skin sensitizing potential of the test item was assessed in a maximization test using guinea pigs according to OECD 406 and GLP.

In a preliminary test topical application did not reveal any evidence of adverse skin reactions, therefore, the induction sites in the main test for each group was pretreated with 10% sodium dodecyl sulfate prior to the second induction. There were 2 groups, one for the test substance and one for the negative control. In the test group, a concentration of 25% of the test substance was injected intradermally for the first induction. The second induction was conducted with 100% of the test substance. The sites were occluded for 48 hours, whereafter the challenge was conducted with 100% of the test substance. Hereafter, the sites are occluded for 24 hours. In the control group, the first and second inductions was done with olive oil and the challenges with the 100% of the test substance and olive oil.

Both in the control and the test group no skin reactions such as redness or swelling were observed in any animal at 24 and 48 hours after the challenge patch was removed. During the observation period, no abnormalities in clinical signs or body weight gain were observed in any animal of any group. Based on the result of this study, the test substance did not produce skin sensitization under the conditions of this study. Thus, the test article does not have to be classified as sensitising in the in vivo study.

Based on this result, and on the structural, chemical and toxicological similarities between 3-methylbutyl isovalerate and ethyl isobutyrate, ethyl isobutyrate is not considered to provoke skin sensitization in a maximization test with guinea pigs.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The criteria to classify a substance for the skin sensitisation endpoint using the results of a maximization study according to the CLP Regulation (EC) No 1272/2008 are set out in Tables 3.4.3 and 3.4.4 for the sub-categories. Since no animals showed an allergic response in the GPMT test with 3-methylbutyl isovalerate, this substance does not have to be classified as sensitising.

Based on this result, and on the structural, chemical and toxicological similarities between the 3-methylbutyl isovalerate and ethyl isobutyrate, ethyl isobutyrate is not classified as sensitising.