Di-tert-pentyl peroxide

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2018 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Investigation the potential of di-tert amyl peroxide (DTA) and di-tert butyl peroxide (DTB) to elicit cytotoxicity in primary rat hepatocytes.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Cytotoxicity in cultured rat hepatocytes

Specific details on test material used for the study:

Test Item Name: di-tert amyl peroxide (DTA)
Lot/Batch: 12679-00 JH201803X00255
Purity: 98.1%
Appearance: Colourless liquid
Re-test Date/Expiry: 11/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Test Item Name: di-tert butyl peroxide (DTB)
Lot/Batch: 12678-00 JH201803H00107
Purity: 99.5%
Appearance: Colourless liquid
Re-test Date/Expiry: 04/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Positive control:

Test Item Name: Menadione sodium bisulfite (Men)
Lot/Batch: SLBR3541V
Purity: 99%
Appearance: Off-white powder
Re-test Date/Expiry: June 2020
Supplier: Sigma
Storage Details: At approximately -20 oC.

Test Item Name: Cumene hydroperoxide (CH)
Lot/Batch: BCBS6305V
Purity: 89.7%
Appearance: Colourless liquid
Re-test Date/Expiry: Not stated
Supplier: Sigma
Storage Details: At approximately 4 oC.

Details on results:

DTA and DTB did not induce a significant cytotoxicity in rat hepatocyte up to 100 µM after 24 or 48 hours of culture. In the same conditions, the positive control substances menadione and cumene hydroperoxide induced a significant cytotoxicity.

Table 1. Hepatotoxicity of DTA and DTB

Data are expressed as percentage of control (mean ± SD, n = 6 replicates). The number of significant figures reflects the error in the estimate.

Concentration	DTA		DTB
	24 hours	48 hours	24 hours	48 hours
0.000	100 ± 22	100 ± 14	100 ± 18	100.0 ± 8.2
0.015	078.9 ± 5.0	107.2 ± 5.3	075.2 ± 5.7	088.4 ± 6.5
0.045	080.0 ± 8.3	112 ± 16	076.5 ± 7.6	087.3 ± 8.4
0.136	078.9 ± 6.8	112 ± 13	078.9 ± 9.6	094.5 ± 9.6
0.410	090.1 ± 9.4	112 ± 13	078 ± 12	091 ± 11
1.200	088 ± 11	106 ± 16	081.0 ± 8.9	093 ± 12
3.700	079.0 ± 10.0	116 ± 13	078 ± 10	088.9 ± 8.6
11.000	084.5 ± 9.0	111 ± 13	079.5 ± 7.3	088 ± 12
33.000	084.9 ± 10.0	118 ± 14	072.7 ± 7.1	083.0 ± 8.2
100.000	071.0 ± 4.0	102.0 ± 5.8	064.5 ± 4.6	070.3 ± 4.7

 

Table 2. Hepatotoxicity of Men and CH

Data are expressed as percentage of control (mean ± SD, n = 6 replicates). The number of significant figures reflects the error in the estimate.

Concentration	Menadione		Cumene hydroperoxide
	24 hours	48 hours	24 hours	48 hours
0.000	100.0 ± 9.9	100.0 ± 7.6	100.0 ± 8.1	100 ± 16
0.015	129 ± 13	128 ± 13	106.9 ± 9.7	161 ± 45
0.045	130 ± 10	126 ± 12	112.0 ± 8.9	157 ± 58
0.136	135 ± 12	137 ± 17	107.6 ± 9.4	183 ± 51
0.410	137 ± 14	130 ± 19	109 ± 12	210 ± 36
1.200	142 ± 18	134 ± 20	101.9 ± 8.4	171 ± 40
3.700	133 ± 12	127 ± 23	108 ± 12	139 ± 26
11.000	135 ± 21	127 ± 24	095 ± 11	140 ± 28
33.000	002.07 ± 0.40	001.22 ± 0.18	076 ± 15	109 ± 26
100.000	001.032 ± 0.064	000.438 ± 0.047	002.72 ± 0.71	003.3 ± 1.8

 

Executive summary:

DTA and DTB did not display any significant cytotoxicity towards rat hepatocytes at concentrations of up to 100 µM. By way of contrast, almost complete cytotoxicity was observed when rat hepatocytes were treated with cumene hydroperoxide at the same concentration of 100 µM. Therefore, both peroxides are less toxic towards rat hepatocytes than cumene hydroperoxide.

Reference 2

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2018 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Investigation the potential of di-tert amyl peroxide (DTA) and di-tert butyl peroxide (DTB) to elicit lipid peroxidation in primary rat hepatocytes. Lipid peroxidation was evaluated using the Click-iT Lipid Peroxidation Imaging Kit.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Reactive oxygen species (ROS) production in rat hepatocytes

Specific details on test material used for the study:

Test Item Name: di-tert amyl peroxide (DTA)
Lot/Batch: 12679-00 JH201803X00255
Purity: 98.1%
Appearance: Colourless liquid
Re-test Date/Expiry: 11/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Test Item Name: di-tert butyl peroxide (DTB)
Lot/Batch: 12678-00 JH201803H00107
Purity: 99.5%
Appearance: Colourless liquid
Re-test Date/Expiry: 04/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Positive control:

Test Item Name: Cumene hydroperoxide (CH)
Lot/Batch: BCBS6305V
Purity: 89.7%
Appearance: Colourless liquid
Re-test Date/Expiry: Not stated
Supplier: Sigma
Storage Details: At approximately 4 oC.

Details on results:

DTA showed weak, non-significant, evidence for dose-dependent effects on peroxidation (approximately 1.5 fold-control). DTB showed weak, statistically-significant, evidence for dose-dependent effects on peroxidation (up to 2 fold-control).

CH is a classic inducer of lipid peroxidation (Weiss & Estabrook, 1986). The mechanism involves CYP450-mediated homolytic cleavage of CH to produce the cumyloxyl radical that can abstract hydrogens from lipids to initiate peroxidation(Weiss & Estabrook, 1986). Therefore, it was surprising that statistical support for increased lipid peroxidation with increased concentrations of CH was only observed in two out of three experiments. Moreover, in both of the experiments that supported dose-dependent increases in lipid peroxidation, the effect sizes were small (increases of approximately 1.7 – 2 fold-control at the highest dose tested (100 µM)). One possible explanation is suggested by the observation that previous researchers have had to use millimolar concentrations of CH to drive appreciable shifts in lipid peroxidation (see e.g.(Gebhardt, 1997)). A second and possibly related factor relates to the need for CYP450-mediated metabolism of CH to initiate lipid peroxidation; this suggests that induction of CYP450 may be needed to drive peroxidation to an appreciable extent in hepatocytes. Experimental support for this has been published (CADENAS et al, 1981).

The data suggest that lipid peroxidation is weakly elevated in response to both DTA and DTB. However, formal trend analysis only provides statistical support for this in the case of DTB. Overall, in light of the weak effects, and failure to detect robust peroxidation in response to the Reference Item CH, it is not possible to draw any firm conclusions on the basis of these data.

CADENAS E, WEFERS H & SIES H (1981) Low-Level Chemiluminescence of Isolated Hepatocytes.Eur. J. Biochem.119:531–536 Available at: https://doi.org/10.1111/j.1432-1033.1981.tb05640.x

Gebhardt R (1997) Antioxidative and Protective Properties of Extracts from Leaves of the Artichoke (Cynara scolymus L.) against Hydroperoxide-Induced Oxidative Stress in Cultured Rat Hepatocytes.Toxicol. Appl. Pharmacol.144:279–286 Available at: http://www.sciencedirect.com/science/article/pii/S0041008X97981308

Weiss RH & Estabrook RW (1986) The mechanism of cumene hydroperoxide-dependent lipid peroxidation: The function of cytochrome P-450.Arch. Biochem. Biophys.251:348–360 Available at: http://www.sciencedirect.com/science/article/pii/0003986186900822

Executive summary:

Lipid peroxidation induced by DTA and DTB in primary rat hepatocytes was evaluated using the Click-iT Lipid Peroxidation Imaging Kit. Cumene hydroperoxide (CH) was used as a positive control. Cumene hydroperoxide, did not reproducibly increase peroxidation across three independent experiments. On one occasion, there was no statistically-significant, dose-dependent, increase in peroxidation. On the other two occasions, there were statistically-significant, dose-dependent effects on lipid peroxidation. However, the increases in peroxidation was mild (1.7 – 2.2 fold-control). Both Test Items were assayed for peroxidation on a single occasion. DTA showed weak, non-significant, evidence for dose-dependent effects on peroxidation (approximately 1.5 fold-control). DTB showed weak, statistically-significant, evidence for dose-dependent effects on peroxidation (up to 2 fold-control).

Reference 3

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2018 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Investigation the potential of di-tert amyl peroxide (DTA) and di-tert butyl peroxide (DTB) to elicit production of reactive oxygen species (ROS) in primary rat hepatocytes. ROS were quantified using the ROS-sensitive dye CellROX Green.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: Reactive oxygen species (ROS) production in rat hepatocytes

Specific details on test material used for the study:

Test Item Name: di-tert amyl peroxide (DTA)
Lot/Batch: 12679-00 JH201803X00255
Purity: 98.1%
Appearance: Colourless liquid
Re-test Date/Expiry: 11/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Test Item Name: di-tert butyl peroxide (DTB)
Lot/Batch: 12678-00 JH201803H00107
Purity: 99.5%
Appearance: Colourless liquid
Re-test Date/Expiry: 04/09/2018
Supplier: Arkema
Storage Details: Stored at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Positive control:

Test Item Name: Menadione sodium bisulfite (Men)
Lot/Batch: SLBR3541V
Purity: 99%
Appearance: Off-white powder
Re-test Date/Expiry: June 2020
Supplier: Sigma
Storage Details: At approximately -20 oC.

Test Item Name: Cumene hydroperoxide (CH)
Lot/Batch: BCBS6305V
Purity: 89.7%
Appearance: Colourless liquid
Re-test Date/Expiry: Not stated
Supplier: Sigma
Storage Details: At approximately 4 oC.

Details on results:

DTA caused a mild but statistically significant and dose-dependent increase in ROS (maximum of 1.6 fold-control). DTB also caused a statistically significant, dose-dependent, elevation in ROS production (maximum of 2.6 fold-control).

CellROX Green is primarily a sensor of hydroxyl free radicals and superoxide; it does not detect hydrogen peroxide (personal communication with manufacturer). Men is a classic redox cycler and generates superoxide in mitochondria(Monkset al, 1992). The ROS assay developed during the course of this work successfully revealed that in all three independent experiments ROS production was increased at 2 hours in rat hepatocytes subjected to increasing concentrations of Men. The increases were more modest than were anticipated at the outset, ranging from 2- to 4 fold-control in individual experiments. One possible explanation for the modest increases in ROS seen with CellROX Green after Men treatment in this Test System is that hepatocytes express large amounts of manganese:superoxide dismutase (MnSOD) in their mitochondria, which may suppress superoxide accumulation, while increasing hydrogen peroxide production.

The ROS experiments provide some support for the hypothesis that DTA and DTB both mildly elevate ROS production in rat hepatocytes, at least at 2 hours. Visual inspection, and formal trend analysis, of the data indicate small dose-dependent increases in ROS production by both Items (estimated at up to 1.5 fold-control). The nature of the putative ROS generated by the organic peroxides remains unclear, as does the underlying mechanism of production.

Executive summary:

The Reference Item, Menadione, robustly elevated ROS levels in rat hepatocytes by 2 – 4 fold-control in all three independent experiments carried out. Both Test Items were assayed for effects on ROS production on a single occasion. DTA caused a mild but statistically significant and dose-dependent increase in ROS (maximum of 1.6 fold-control). DTB also caused a statistically significant, dose-dependent, elevation in ROS production (maximum of 2.6 fold-control).

Description of key information

The potential for di-tert-amyl peroxide (DTA) and di-tert-butyl peroxide (DTB) to elicit production of reactive oxygen species (ROS), or lipid peroxidation was evaluated in primary rat hepatocytes (McMahon, 2019). ROS were quantified using the ROS-sensitive dye CellROX Green. Menadione (Men) was used as a positive control for ROS experiments. Lipid peroxidation was evaluated using the Click-iT Lipid Peroxidation Imaging Kit. Cumene hydroperoxide (CH) was used as a positive control for lipid peroxidation. Preliminary cytotoxicity experiments were performed.

Preliminary cytotoxicity experiments at concentrations of up to 100 µM were performed. Both Reference Items displayed considerable cytotoxicity towards rat hepatocytes within this range. However, DTA and DTB were non-cytotoxic. Therefore, it was decided to increase the DTA and DTB concentrations to up to 400 µM for the subsequent ROS and peroxidation assays. The Reference Item, Men, robustly elevated ROS levels in rat hepatocytes by 2 – 4 fold-control in all three independent experiments carried out. Both Test Items were assayed for effects on ROS production on a single occasion. DTA caused a mild but statistically significant and dose-dependent increase in ROS (maximum of 1.6 fold-control). DTB also caused a statistically significant, dose-dependent, elevation in ROS production (maximum of 2.6 fold-control).

The Reference Item, cumene hydroperoxide, did not reproducibly increase peroxidation across three independent experiments. On one occasion, there was no statistically-significant, dose-dependent, increase in peroxidation. On the other two occasions, there were statistically-significant, dose-dependent effects on lipid peroxidation. However, the increases in peroxidation was mild (1.7 – 2.2 fold-control). Both Test Items were assayed for peroxidation on a single occasion. DTA showed weak, non-significant, evidence for dose-dependent effects on peroxidation (approximately 1.5 fold-control). DTB showed weak, statistically-significant, evidence for dose-dependent effects on peroxidation (up to 2 fold-control).

Both organic peroxides, DTA and DTB, are both less cytotoxic to rat hepatocytes than is the organic hydroperoxide CH. DTA and DTB are capable of elevating ROS production in rat hepatocytes at 2 hours. Lipid peroxidation was not reproducibly or substantially elevated by the classic inducer CH at 2 hours in the Test System. Therefore, it was not possible to reach a conclusion on the ability of DTA or DTB to affect lipid peroxidation.