Registration Dossier

Administrative data

Description of key information

Direct Peptide Reactivity Assay (OECD 442C): not peptide reactive (BASF 2019; 67V0080/13V062)

LuSens assay (OECD 442D): does not activate keratinocytes (BASF 2019; 67V0080/13V062)

Human repeated insult patch test: no skin irritation/sensitization (Epstein 1984)

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Additional information:

The skin sensitizing potential of tetrahydro-2-isobutyl-4-methyl-2H-pyran was assessed in a combination of three in vitro methods (“2 out of 3” ITS), addressing key events of the adverse outcome pathway (AOP) for skin sensitization as defined by the OECD (BASF 2019; 67V0080/13V062). This in vitro skin sensitization turnkey testing strategy covers protein binding in chemico via the Direct Peptide Reactivity Assay (OECD 442C), activation of keratinocytes via the LuSens assay (OECD 442D) and activation of dendritic cells via the h-CLAT (OECD 442E). Of note, the molecular initiating event of protein binding also occurs in the cell-based assays (LuSens, h-CLAT) and is not solely detected via the DPRA. However, in the case for tetrahydro-2-isobutyl-4-methyl-2H-pyran, the results derived with DPRA and LuSens were sufficient for a final assessment and testing in the h-CLAT was waived.

 

In the DPRA, the reactivity of tetrahydro-2-isobutyl-4-methyl-2H-pyran towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated. Tetrahydro-2-isobutyl-4-methyl-2H-pyran was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by HPLC with gradient elution and UV-detection at 220 nm. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide).

The samples of tetrahydro-2-isobutyl-4-methyl-2H-pyran were solutions with the C-peptide and emulsions with the K-peptide at the time of preparation and after 24 hours. No co-elution of test substance and peptides was present.

The mean depletion caused by the test substance was determined to be 5.17% (C-peptide) and -0.45% (K-peptide). Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable.

Based on the observed results, it was concluded that tetrahydro-2-isobutyl-4-methyl-2H-pyran shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

 

In the LuSens assay, tetrahydro-2-isobutyl-4-methyl-2H-pyran 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. Luciferase activity was measured after 48-hour exposure and a MTT assay was performed to assess cytotoxicity of the test substance in parallel. A total of 2 valid experiments were performed. No evident cytotoxicity was observed when tested up to the limit concentration of 2000 µM in a cytotoxicity prestest.

The mean fold induction of luciferase activity was <= 1.27 at all concentrations tested. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. Thus, 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 after 48 hours of exposure to tetrahydro-2-isobutyl-4-methyl-2H-pyran. It was concluded, that tetrahydro-2-isobutyl-4-methyl-2H-pyran does not have a keratinocyte activating potential.

 

Testing tetrahydro-2-isobutyl-4-methyl-2H-pyran in two different non-animal methods addressing different key events of the skin sensitization AOP resulted in two negative results in the DPRA and LuSens. Based on these results, tetrahydro-2-isobutyl-4-methyl-2H-pyran is not peptide reactive and does not activate keratinocytes. Thus, tetrahydro-2-isobutyl-4-methyl-2H-pyran is predicted not to be a skin sensitizer.

 

This has been confirmed in a human study on skin sensitization, i.e. a human repeated insult patch test (Epstein 1984). In this HRIPT, 10% tetrahydro-2-isobutyl-4-methyl-2H-pyran in DEP was applied to 23 human volunteers under occlusion for 5 alternate day 48-hour periods. Patch sites were pretreated for 24 hours with 7.5% aqueous SLS under occlusion for the inital patch. Following a 10-14 day rest period challenge patches were applied under occlusion to fresh sites for 48 hours. Challenge applications were preceded by 30-minute application of 7.5% aqueous SLS under occlusion whereas the test material was applied without- SLS treatment on another side of the back. SLS pretreatment at the challenge patches produced irritation in nearly half of the subjects-, and there were some tape reactions, but no significant irritation or evidence of sensitization was observed with the test material. Therefore, tetrahydro-2-isobutyl-4-methyl-2H-pyran at a 10% concentration in DEP produced no evidence of skin irritation or skin sensitization under the chosen testing conditions. 

 

Skin sensitization studies using substances with high structural similarities but varying in the numbers of double bonds as putative reactive structural feature (dehydro-rose oxide containing 2 double bonds or rose oxide containing 1 double bond) have been included as supportive evidence. Furthermore, the assessment of a skin sensitizing potential of tetrahydro-2-isobutyl-4-methyl-2H-pyran has been assessed in an in silico tool, i.e. the OECD QSAR toolbox. Based on the outcome of the OECD QSAR toolbox, the skin sensitization potential of tetrahydro-2-isobutyl-4-methyl-2H-pyran is predicted negative from category members using read-across to 5 values from 5 nearest neighbours in analogy to the profile of the target chemical.

 

In a Buehler test acc. to OECD guideline 406 and GLP, 2-[2-methylprop-1-en-1-yl]-4-methylenetetrahydropyran has been applied to Dunkin-Hartley guinea pigs. For induction, the undiluted test substance has been applied dermally (occlusive) for 6 hours on day 0, 7 and 14. After a resting period of 14 days, the undiluted test substance was applied dermally (occlusive) for 6 hours for challenge and skin findings were assessed 24 and 48 hours after patch removal. The challenge with the undiluted test substance did not cause any skin reactions neither in animals of the control group nor in test group animals 24 and 48 hours after removal of the patch. Therefore, 2 -[2-methylprop-1-en-1-yl]-4-methylenetetrahydropyran does not have a sensitizing effect on the skin under the test conditions chosen.

 

In a Buehler test equivalent to OECD guideline 406 conducted with male and female Hartley guinea pigs, a 0.3 ml aliquot of 50% rose oxide in diethyl phthalate (DEP) was placed under occlusion to the clipped left shoulder for 6 hours for induction. This procedure was repeated at the same site, once a week for the next 2 weeks, until a total of 3 exposures were applied. After a 2-week rest period, 0.5, 1.5, and 5% test substance in DEP were applied to previously untreated sites for 6 hours under occlusion. No skin sensitization was observed for rose oxide under the chosen testing conditions (RIFM 1993).

 

In the in vitro skin sensitization turnkey testing strategy tetrahydro-2-isobutyl-4-methyl-2H-pyran was determined not to be peptide reactive and does not activate keratinocytes. Thus, tetrahydro-2-isobutyl-4-methyl-2H-pyran is predicted not to be a skin sensitizer. In line, a human repeated insult patch test using 10% tetrahydro-2-isobutyl-4-methyl-2H-pyran in DEP showed no evidence of skin irritation or skin sensitization. Taking together these study results in a weight of evidence, tetrahydro-2-isobutyl-4-methyl-2H-pyran is not considered to be a skin sensitizer.

As supporting evidence, the skin sensitization potential of tetrahydro-2-isobutyl-4-methyl-2H-pyran is predicted negative from 5 category members using the OECD QSAR toolbox. Furthermore, no skin sensitization potential has been observed for structurally similar substances with putative reactive structural features containing 1 or 2 double bonds as a worst case, i.e. rose oxide and dehydrorose oxide. 

Respiratory sensitisation

Endpoint conclusion
Additional information:

No data available

Justification for classification or non-classification

The present data on dermal sensitization do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted.