Bisisopropyl peroxydicarbonate

Endpoint summary

Description of key information

Skin sensitisation

The Adverse Outcome Pathway (AOP) provides the mechanistic basis for the integration of skin sensitisation-related information (OECD, Series on Testing & Assessment No. 168 and 256). The different steps of the AOP were evaluated using in silico, in vitro and in vivo studies.

 

Step 1) Dermal Bioavailability:

A limited dermal bioavailability of 3% was estimated for diisopropyl peroxydicarbonate using the IH SkinPerm model.

 

Steps 2 – 4; Key Event 1) Protein-Binding Reactions, Reactivity and Metabolism:

Isopropanol and peroxydicarbonic acid were identified as possible skin metabolites of diisopropyl peroxydicarbonate with the OECD QSAR Toolbox.

Diisopropyl peroxydicarbonate and both metabolites does not show any binding protein potential or alert for skin sensitization according to the specific profilers of the OECD QSAR Toolbox.

A DPRA assay was performed on diisopropyl peroxydicarbonate but was considered to be incompatible and the final outcome was inconclusive (Michel, 2018). Using the Pred-Skin QSAR model, diisopropyl peroxydicarbonate was predicted to be positive in the DPRA.

Isopropanol was negative in the DPRA (Natsch et al., 2013).

 

Step 5; Key Event 2) Biochemical Pathways Related to Skin Sensitisation:

No data available.

 

Step 6; Key Event 2) Events in Keratinocyte:

Diisopropyl peroxydicarbonate was negative in the KeratinoSens assay (Richez, 2017) and therefore was considered to have no potential to activate the Nrf2 transcription factor. Using the Pred-Skin QSAR model, diisopropyl peroxydicarbonate was also predicted to be negative in the KeratinoSens assay.

According to the OECD TG 442D (2015), isopropanol is a recommended negative reference substance for the KeratinoSens assay (Natsch et al., 2013).

 

Step 6; Key Event 3) Events in Dendritic cell:

Diisopropyl peroxydicarbonate was tested following the h-CLAT test method (Gerbeix, 2018). However, the study was considered inconclusive, and the model not adapted to assess the skin sensitizing potential of this particular chemical. Using the Pred-Skin QSAR model, diisopropyl peroxydicarbonate was predicted to be negative in the h-CLAT assay.

According to the OECD QSAR Toolbox, no alert was found for Protein Binding Potency h-CLAT for diisopropyl peroxydicarbonate and both metabolites.

Isopropanol was negative in the U937-CD86 test (Natsch et al., 2013) and according to the OECD TG 442E (2017), it is a recommended negative substance for demonstrating technical proficiency with the IL-8 Luc assay.

 

Step 7; Key Event 4) Events in Lymphocytes:

Using the Pred-Skin QSAR model, diisopropyl peroxydicarbonate was predicted to be non-sensitizer in the LLNA.

According to the OECD TG 429 (2010), isopropanol is a recommended negative reference substance for the LLNA assay (Basketter et al., 1998; Gerberick et al., 2004).

 

Steps 9-11; In Vivo Skin Sensitisation:

The skin sensitization potential of a closely related product, di-n-propyl peroxydicarbonate (CAS reg. no. 16066-38-9), was tested according to the Buehler method (Doubs, 1996). Di-n-propyl peroxydicarbonat was not considered to be a contact sensitizer in guinea pigs.

The allergic contact dermatitis potential of diisopropyl peroxydicarbonate was estimated in guinea pig and human with the three QSAR models (CASE Ultra, Leadscope, SciQSAR) of the Danish QSAR data base. All results were negative.

 

In summary, the experimental and/or estimated parameters relevant for the skin sensitization were:

	Protein binding	KeratinoSens assay	Dendritic cell activation	LLNA	In vivo skin sensitization
Diisopropyl peroxydicarbonate	Negative1 Positive6	Negative2, 6	Negative1, 6	Negative6	Negative3*, 4
Isopropanol	Negative1, 2	Negative2	Negative1, 2	Negative2	Negative5*
Peroxydicarbonic acid	Negative1	No data	Negative1	No data	No data

1 OECD QSAR toolbox; 2 experimental result; 3 analogue substance; 4 Danish QSAR database; 5 ECHA dissemination database; 6 Pred-Skin QSAR; *Buehler assay

Overall, diisopropyl peroxydicarbonate is not expected to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA assay

The reactivity of diisopropyl peroxydicarbonate was evaluated on synthetic cysteine and lysine peptides (Michel, 2018). This test is part of a tiered strategy for skin sensitization assessment. The design of this study was based on the OECD guideline No. 442C and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

The reactivity of the test item was evaluatedin chemicoby monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The test item was dissolved at 100 mM inacetonitrile without sonication. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide:

.            for the cysteine peptide, the mean depletion value was 100%,

.            for the lysine peptide, the mean depletion value was 80.94%.

The mean of the percent cysteine and percent lysine depletions was equal to 90.47%. Accordingly, the test item was considered to have high peptide reactivity. However, the test item is known to be a strong oxidizing agent which can lead to the formation of the cysteine dimer and over estimated the peptide depletion. Moreover, based on information provided by the Sponsor, degradation of the test item in contact with the vehicle can lead to isopropyl alcohol, acetone and acetal dehyde which are known to denature and precipitate the proteins. Therefore, based on the above information, the positive outcome is to be considered inconclusive.

Under the experimental conditions of this study, diisopropyl peroxidicarbonate was considered to have high peptide reactivity. However, due to the nature and instability of the test item, the DPRA assay is considered incompatible with this test item and the final outcome is inconclusive.

KeratinoSens assay

The potential ofdiisopropyl peroxydicarbonatetoactivate the Nrf2 transcription factor was evaluated in the KeratinoSens cell line (Richez, 2017). This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.The design of this study was based on the OECD Guideline No. 442D and the study was performed in compliance with CiToxLAB France Standard Operating Procedures and with the OECD Principles of Good Laboratory Practice.

Thisin vitrotest uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence.In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

All acceptance criteria were fulfilled for the positive and negative controls in each run; both runs were therefore considered as validated.

Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

.  no precipitate/emulsion was observed in any test item treated wells at the end of the 48-hour treatment period in either run,

.  a high decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations = 1000 µM during the first run and = 2000 µM in the second run,

.  the corresponding IC₃₀and IC₅₀were calculated to be 758.36 and 838.88 µM, respectively, during the first run and 1490.01 and 1636.52 µM, respectively, during the second run,

.  in both runs, no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations,

.  the I_maxvalues were 1.25 and 1.09, during the first and second run respectively.

No EC_1.5was calculated in either run.

The geometric means IC₃₀andIC₅₀of the two validated runs were calculated to be 1063 and 1171.7 µM, respectively.

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a lack of skin sensitisation potential.

Under the experimental conditions of this study, diisopropyl peroxydicarbonate was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Diisopropyl peroxydicarbonate is not expected to be a skin sensitizer, no classification is warranted according to CLP/GHS criteria.