Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl propionate

Endpoint summary

Description of key information

Isobornyl propionate is not considered a skin sensitiser based on information of the analogue Isobornyl acetate. This substance is tested in an in vitro battery using DPRA, KeratinoSens and hClat (OECD TG 442 C/D and E), where it is negative in the first two assays and positive in the latter. An LLNA up to 25% was negative.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information from an analogue is used

Justification for type of information:

The skin sensitisation information is based on read across from Isobornyl acetate. The read across rationale is presented in the Skin sensitisation Endpoint summary. The read across rationale is also presented there.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

other: Mean Peptide depletion of Cysteine and Lysine (%)

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Negative

Remarks:

Results obtained from DPRA

Key result

Run / experiment:

other: Mean Percent Peptide Depletion (%)

Parameter:

other: Cysteine reactivity

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from DPRA

Key result

Run / experiment:

other: Mean Percent Peptide Depletion (%)

Parameter:

other: Lysine reactivity

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from DPRA

Key result

Parameter:

other: EC 1.5 (µM)

Remarks:

The concentration for gene induction above the threshold (1.5 fold) as compared to the DMSO solvent controls

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from Keratinosens assay

Key result

Run / experiment:

mean

Parameter:

other: IC50 (µM)

Remarks:

MTT

Value:

170.14

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from Keratinosens assay

Key result

Parameter:

other: Imax

Remarks:

Maximal Induction

Value:

0.95

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from Keratinosens assay

Parameter:

other: CImax (µM)

Remarks:

Concentration for maximal gene induction

Value:

3.91

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from Keratinosens assay

Key result

Run / experiment:

other: Experiment 3 and 4 (average)

Parameter:

other: EC200% for CD54 (μg/mL)

Value:

74.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive

Remarks:

Result obtained from h-CLAT with read-across substance Isobornyl acetate (CAS 125-12-2)

Run / experiment:

other: Experiment 2

Parameter:

other: EC150% for CD86 (μg/mL)

Value:

80

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Result obtained from h-CLAT

Interpretation of results:

other: Not a skin sensitiser

Remarks:

according to EU CLP (EC no. 1272/2008 and its amendments).

Conclusions:

Isobornyl propionate is not a skin sensitiser based on negative results of Isobornyl acetate in the DPRA and the KeratinoSens, despite positive results in the h-Clat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For Isobornyl propionate a human maximization test is available which was negative up to 10% but this information is insufficient for fullfilling the requirements. Therefore information from Isobornyl acetate is presented which can be used for read across. First the in vitro information data are presented, followed by the LLNA. The LLNA is considered supporting because the concentration used was up to 25%, without a clear indication for not using 100%, The read across rationale is presented thereafter.

 

In vitro test: DPRA assay (OECD422C) with Isobornyl acetate

The Direct Peptide Reactivity Assay (similar to OECD TG 442C) was used in a weight-of-evidence approach to assess the skin sensitizing potential of the test substance. Synthetic peptides containing cysteine or lysine were reacted with 100 mM test article for 24± 2 hours in a ratio of 1:10 (cysteine) or 1:50 (Lysine). Acetonitrile was used as vehicle and Cinnamic aldehyde was used as positive control. After the incubation period the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection. The positive control resulted in 75.4 and 59.8% depletion of the respective peptides. The test substance gave a mean peptide depletion of 0.4% and 0.0% for Cysteine and Lysine, respectively. The mean peptide depletion of Cysteine and Lysine was 0.2%. Based on these results, the test is negative.

 

In vitro battery: Keratinosens assay (OECD422D) with Isobornyl acetate

A keratinosens assays was performed similar to OECD TG 442D. This assay was used in a weight-of-evidence approach to assess the skin sensitizing potential of the test substance. The test article, dissolved in DMSO, was tested in three definitive keratinosens assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test articles were tested at 12 concentrations ranging from 0.977 to 2000 µM. The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 µM. The experimental design of this study consisted of three definitive assays to determine the maximal induction(Imax),the concentration for maximal gene induction (CImax), the EC1.5 value(concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test articles. For each definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with the test articles for 48 hours, and assessed for luciferase induction (3plates) and cytotoxicity (1 plate). For the test item an EC 1.5 value > 2000 µM, a mean IC50 of 170.14 µM, a maximal induction of 0.95 and a Concentration for maximal induction of 3.91 µM was found. The positive control, Cinnamic aldehyde resulted in an EC 1.5 value of 14.55 µM and the mean IC50 was > 64 µM. Based on these results, the test is negative.

 

In vitro battery: h-CLAT assay (OECD422E) with Isobornyl acetate

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in a GLP-compliant Human Cell Line Activation Test (h-CLAT) according to OECD TG 442E. The test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry. In order to determine the concentrations suitable for the main experiment two pre-tests (non-GLP) were performed. Cells were exposed to 9 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 to be 88 μg/mL (result of the 2nd pre-test). In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human- CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed (the first one was invalid). At concentrations used in the valid main experiments the test substance (500 x stock solutions) was soluble in DMSO and soluble in 0.2% DMSO (final concentrations). After 24 hours precipitates were not noticed in any concentration. In experiment 2 the cell viability was too low when the EC150% and EC200% were exceeded and is negative. In experiment 3 the EC150% was exceeded in CD86 and EC200% in CD54 both at >=88 ug/ml and thus considered positive. In experiment 4 the EC200% was exceeded in CD54 at >=88 ug/ml and therefore considered positive. In 2/3 tests a positive result was found and therefore the substance induces dendritic cell activation in the h-CLAT under the test conditions chosen.

Isobornyl propionate LLNA

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node according to OECD TG 429 and GLP principles (Syngenta, 2007). Test concentrations were set by the sponsor based on acute oral toxicity data. Per dose group 4 female CBA/Ca mice were included. The application of the test substance at concentrations of 1, 2.5, 5, 10 and 25% (w/v) in a vehicle of 1:3 EtOH:DEP for 3 consecutive days did not result in an increase in the proliferation index of more than 3 fold (1.7 fold for the 25%) compared to the vehicle control. The positive control hexylcinnamaldehyde proved the validity of the protocol. No effects on body weight, skin irritation nor systemic toxicity (clinical signs) were reported. Based on the results, the substance was not considered to be a skin sensitizer.

Overall conclusion: The test substance does not have a skin sensitization potential based on the key information of three in vitro skin sensitization tests. The ITS proposed by Bauch et al. 2012, presents that when 2/3 in vitro results are negative the substance can be considered a non-skin sensitiser. In addition, the LLNA is negative up to 25%. Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

Skin sensitisation of Isobornyl propionate (Cas no. 2756-56-1) using read across from Isobornyl acetate (125-12-2).

Introduction and hypothesis for the analogue approach

Isobornyl propionate has an exo-1,7,7-Trimethylbicyclo[2.2.1]heptane-2-ol (Isobornyl alcohol) backbone to which a propionic ester group is attached. For this substance insufficient skin sensitisation information is not available. In accordance with Article 13 of REACH, lacking information can be generated by other means i.e. applying alternative methods such as QSARs, grouping and read-across. The analogue approach is selected since for the structurally related analogues Isobornyl acetate information is available which can be used for read across.

Hypothesis: The skin sensitisation of Isobornyl propionate is the same as for Isobornyl acetate

Available information: Isobornyl propionate was tested in a Human maximization test and no sensitisation was observed at a concentration of 10% (RIFM information). For Isobornyl acetate a battery of in vitro tests are available: DPRA, KeratinoSens and h-Clat (OECD TG 442C/D and E, respectively), which were negative, negative and positive, respectively. As two out of 3 in vitro tests are negative, it is not considered a skin sensitiser. Also for Isobornyl acetate a Local lymph node assay is available (according to OECD 429, Rel. 2) which is negative for concentrations up to 25%.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including relevant physico-chemical properties.

Purity / Impurities

Isobornyl propionate is a mono-constituent >=88%, with impurities similar to the parent substance and < 10%.

Analogue approach justification

According to REACH Annex XI 1.5 read-across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read-across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection:For Isobornyl propionate the one methyl shorter Isobornyl acetate was selected as an analogue being the closest analogue for which skin sensitisation information is available. E.g. for Isobornyl butyrate no such information was found on the ECHA site or in the RIFM database.

Structural similarities and differences:Isobornyl propionate and Isobornyl acetate have the same backbone and functional ester group. The only difference between the two is the propionate and acetate group, respectively, which will result in a higher molecular weight of the first one.

Skin absorption:For Isobornyl –propionate and acetate the liquid appearance, molecular weight and other physico-chemical properties i.e. log Kow indicate that some skin absorption can occur and that there is only a minimal difference between the two.

Reactivity: Isobornyl-propionate and acetate are similarly reactive because these are almost the same substances with one methyl difference in the alkyl-ester chain, which has a slight impact on the log Kow but not on reactivity. The OECD Toolbox 2.2 shows a similar non-reactive profile (data not shown).

Uncertainty of the prediction: There are no uncertainties other than those already addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix.

Conclusions for hazard and risk assessment

For Isobornyl propionate limited skin sensitisation information is available insufficient to fulfil the endpoint. Information from two analogues can be used for read across. When using read across the result should be applicable for classification and labelling and risk assessment as well as presented with reliable and adequate documentation. This documentation is presented in the current document. For Isobornyl acetate results of in vitro skin sensitisation is available (OECD TG 442 C/D and E) and an LLNA (OECD TG 429 up to 25%) which both present absence of skin sensitisation.

The ITS proposed by Bauch et al. 2012, presents that when 2/3 in vitro results are negative the substance can be considered a non-skin sensitiser. In addition, the LLNA is negative up to 25%.

Using this information for Isobornyl propionate this substance is not a sensitizer either.

Final conclusion on hazard and risk assessment: Isobornyl propionate is not a skin sensitizer.

Data matrix to support the read across to Isobornyl propionate from Isobornyl acetate

Common names	Isobornyl propionate	Isobornyl acetate
	Target	Source
Chemical structures
CAS no.	2756-56-1	125-12-2
EINECS	220-410-5	204-727-6
REACH registration	2018	Registered
Empirical formula	C13H22O2	C12H20O2
Molecular weight	210.32	196.29
Phys-chem properties
Physical state	Liquid	Liquid
Log Kow (measured)	5.0 (IFF measured)	4.3 (Simonich, EpiSuite)
Human health
Skin sensitisation	Not sensitising (Read across)	Not sensitising based on Negative results in DPRA an KeratinoSens (OECD TG 442C and D), despite positive in h-CLAT (OECD TG 442E)
	Not sensitising (Read across)	Not sensitising (OECD TG 429 up to 25%)

 

Reference

Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Respiratory sensitisation can be assessed using human data such as indicated in R7.3.5.2 of the ECHA guidance (2017) that indicate respiratory reactions e. g. from consumer experience or occupational exposure. In case no such data are available the respiratory sensitisation can be assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2017), which says that if the substance is not a skin sensitiser, it is unlikely to be a respiratory sensitiser.

Justification for classification or non-classification

The substance dose not need to be classified for skin or respiratory sensitisation according to EU CLP (EC No.1272/2008 and its amendments).