3-methyl-2-butenyl acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Additional information:

A Local Lymph Node Assay (LLNA) was performed in mice according to OECD TG 429 and EU Method B.42 under GLP conditions (BASF 2013; 58V0362/10X295). Prenyl acetate was applied at concentrations of 25, 50% (w/w), and 100% (undiluted) with acetone:olive oil (4+1 v/v) as vehicle.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group.

A statistically significant increase in DPM values and also in lymph node weight and lymph node cell count was observed in animals treated with 100% (undiluted) prenyl acetate in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in the high dose group (index of 2.01). An EC3 value could not be calculated, since all obtained S.I.´s are below the threshold value of 3; namely 0.98, 1.10 and 2.92 at concentrations of 25, 50% and 100% prenyl acetate. However, if the Stimulation Index of the high dose group is adjusted upwards, the Stimulation Index would be 3, resulting in an EC3 value of about 100%. As no signs of local skin irritation were noted, irritation can be excluded as cause for the distinct and statistically significant increase in lymphocyte proliferation present in the high dose group.

Thus, although the observed reactions regarding DPM values were borderline and only noted at the high concentration, a weak skin sensitizing potential of the test substance  cannot be fully excluded under the cesting conditions chosen in this study.

 

Due to the borderline results obtained with the LLNA, additional confirmatory in vitro assays have been performed. A combination of several methods addressing major steps of the skin sensitization process (protein reactivity, activation of keratinocytes and activation of dendritic cells) have been used. The individual assays are combined into a strategy to qualitatively assess the skin sensitizing potential of prenyl acetate in vitro.

 

A Direct Peptide Reactivity Assay (DPRA) has been performed under GLP (BASF SE, 64V0362/10A475, 2012). The reactivity of the test substance prenyl acetate towards proteins with nucleophilic side chains such as cysteine or lysine residues was evaluated.

For this purpose prenyl acetate was incubated with synthetic peptides in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide) for approx. 24 hours at room temperature. Additionally, vehicle controls and co-elution controls were analyzed. The remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 258 nm.

The mean C-peptide depletion, caused by prenyl acetate was determined to be 3.0%. The mean K-peptide depletion, caused by the test substance was determined to be 0.5%. Thus, the mean peptide depletion was calculated to be 1.8%.

Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that prenyl acetate shows a minimal chemical reactivity in the DPRA under the test conditions chosen. According to the classification tree model described by Gerberick et al. for substances with known molecular weight the minimal reactive test substance Prenyl acetate (mean peptide depletion < 6.38 %) is a non-sensitizer.

 

The myeloid U937 skin sensitization test (MUSST) is a dendritic cell activation test to predict skin sensitizing potential and was performed under GLP (BASF SE, 65V0362/10A476, 2012). The human pro-monocytic cell line U937 was used as model for dendritic cells. After 48 hours of test substance exposure using concentrations between 125 and 2000μg/mL, the cell membrane marker CD86 was quantitatively detected using flow cytometry.

A relevant induction of CD 86 expression in U937 cells was found after treatment with prenyl acetate. From this it has to be concluded that prenyl acetate induces dendritic cell activation under the chosen testing conditions.

 

The ARE Reporter Assay (LuSens), i.e. a keratinocyte activation assay, was performed with prenyl acetate under GLP (BASF SE, 66V0362/10A477, 2013).

LuSens cells, a transgenic keratinocyte cell line derived from HaCaT cells, were incubated with the prenyl acetate using six concentrations between 804 to 2000μg/mL, in culture medium containing 1% DMSO (6271-15600 µM; MW=128.2 g/mol). After 48 hours, luciferase activity was measured as read out for the respective reporter gene activation via the Keap1/Nrf2/ARE signaling pathway. In parallel a MTT assay was performed to assess cytotoxicity. Prenyl acetate significantly induced luciferase activity in LuSens cells at all concentrations tested. However, no concentration response relationship has been observed. Overall, it has been concluded that prenyl acetate has a keratinocyte activating potential under the chosen testing conditions.

 

The individual test methods, the evaluation criteria and the experimental results are summarized in the following table:

Test method	Endpoint	Evaluation criteria	Experimental result	Test result
DirectPeptideReactivity Assay (DPRA)	Peptide depletion	positive if ≥6.38% mean peptide depletion; negative if <6.38% mean peptide depletion	1.8%mean peptide depletion	negative
KeratinocyteActivation Assay-LuSens	Luciferase activity	positive if ≥1.5-fold luciferase activity when viability is >70%of the vehicle control; negative if <1.5 -fold luciferase activity	Activation of keratinocytes (≥1.5-fold increasedluciferaseactivity)	positive
Dendritic Cell LineActivationAssayMyeloidU937SkinSensitization Test(MUSST)	CD86expression	positive if ≥1.2 -fold of CD86 when viability is >70% of the control; negative if <1.2 -fold of CD86	Induction of CD 86 expression (≥1.2-fold of the control)	positive

 

Taking the individual and combined sensitivities and specificities of the three in vitro assays into account the decision matrix below is used to predict the skin sensitizer potential of the test substance:

DPRA	LuSens	MUSST	Test strategy prediction
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

 

Based on the results and applying the evaluation criteria, prenyl acetate is predicted to be a skin sensitizer under the described test conditions in vitro.

 

The database has been extended by an additional in vitro skin sensitization test using a human keratinocytes cell line (HaCaT), i.e. a KeratinoSens assay (Givaudan 2013). The KeratinoSens assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response. Prenyl acetate has been tested at concentrations between 1 - 1000 µM in DMSO. Prenyl acetate was not cytotoxic in the tested concentration range, did not induce luciferase gene expression above the threshold of 1.5 at any concentration and was thus rated negative. Therefore, the outcome of this study contradicts the findings observed in the LuSens (BASF SE, 66V0362/10A477, 2013), possibly due to the difference in concentrations tested.

 

In a human maximization test, 20% prenyl acetate in petrolatum was applied to 25 healthy male and female subjects (Kligman 1977). Applications were made under occlusion to the same site on the forearms or backs of all subjects for five alternate-day 48-hour periods. Patch test sites were pretreated for 24 hours with 2.5% aqueous sodium lauryl sulfate (SLS) under occlusion. Following a 10-day rest period, a challenge patch was applied to a fresh site on the backs for 48 hours under occlusion. The challenge sites were pretreated for 1 hour with 5% - 10% aqueous SLS under occlusion.No skin sensitization reactions were observed.

Furthermore, in two human repeated insult patch tests with 9 and 35 volunteers, no skin sensitization reactions have been observed after 9 dermal semiocclusive induction applications with 3% prenyl acetate in denatured Alcohol 39C (estimated to be approx. 2100 µg/cm2), followed by a single challenge application under same conditions (IFF 1971, IFF 1972).

 

Equivocal findings in an animal study, contradicting results in in vitro tests and no evidence of a skin sensitizing potential in human tests at lower but more realistic use concentrations are available, therefore, an independent expert in the field of skin sensitization has been asked for an overall assessment of the skin sensitizing properties of prenyl acetate. The following has been concluded:

The results of the present LLNA were found to be not entirely clear, and it is in fact unreasonable to proceed to a firm conclusion that prenyl acetate is a skin sensitiser on the basis of this information. At best, the data is equivocal – a strict interpretation is that the standard LLNA is negative (SI = 2.9, which is <3), but that would be to ignore the cell count data. Either a second study should be completed, or other data should be examined carefully in a weight of evidence argument.The overall in vitro test results were found to lead to the conclusion that prenyl acetate is not to be considered a skin sensitiser. However, based on the the test design used for the KeratinoSens assay, a repeat study including this highest concentration could provide further reassurance. The negative human data were considered an important factor in the conclusion that prenyl acetate is not a skin sensitizer, although human use concentrations have been low.

It has been concluded, that if certain aspects of the information (i.e. the LuSens, MUSST and local lymph node cell count) existed in isolation, then there would be a case for a precautionary classification. However, the existence of a formally negative LLNA, a convincingly negative human maximisation test, two wholly negative results from the two in vitro alternatives which are on the threshold of formal validation combined with several decades of human exposure and zero evidence of skin allergy make an overwhelming argument that prenyl acetate should not be regarded as a skin sensitiser. For further details, see attachment 1.

 

Overall, inconclusive results on skin sensitization of prenyl acetate exist. According to the opinion of an independent expert, the classification of prenyl acetate as skin sensitizer is not warrented. In order to substantiate the classification decision for this endpoint, a further adequate test has been performed, i.e. a Buehler test acc. to OECD TG 406 and GLP (BASF SE, 32H0362/10X379; 2014). The aim of this study was to evaluate the possible allergenic activity of prenyl acetate after repeated topical administration in guinea pigs, representing a relevant and accepted test system to address the given use conditions as fragrance material in a variety of consumer products like cleaning agents, fine fragrances and cosmetics.

Twenty female Dunkin-Hartley guinea-pigs were treated for induction by 3 topical applications of undiluted prenyl acetate under occlusive dressing. As vehicle control, ten animals have been treated with liquid paraffin accordingly. Following a 13-day rest period, animals were treated with a single topical application of undiluted prenyl acetate and liquid paraffin for 6 hours under occlusive dressing for challenge.

In the treatment group, no macroscopic cutaneous reactions attributable to allergy were recorded during induction or challenge phase, i.e. 24 and 48 hours after removal of the patch.

No cutaneous intolerance reaction was recorded in animals from the negative control group at the application sites treated with undiluted prenyl acetate. No cutaneous intolerance reaction was recorded at the applications sites of all animals treated with liquid paraffin.

In conclusion, prenyl acetate is not found to be a skin sensitizer under the chosen experimental conditions.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No data available.

Justification for classification or non-classification

Conflicting results in vitro and inconclusive data in vivo (LLNA) have to be weighed against a negative Buehler test, representing a valid test system to address use conditions as fragrance material in consumer products. Furthermore, convincingly negative human maximisation test data combined with several decades of human exposure and zero evidence of skin allergy exist. Therefore, in a weight of evidence, the present data on dermal sensitization do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.