Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-03-03 to 2008-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although there is no guideline, the method should be considered as valid (currently undergoing validation within the EU), the methods are clearly described (when including the reference article) and the results may be used for the chemical safety assessment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Trout S9 in vitro metabolism assay according to CE cowan Ellsberry, SD Dyer, S Erhardt, MJ Bernhard, AL Roe, ME Dowty, AV Weisbrod. 2008. Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70:1804-1817

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
none

Radiolabelling:

no

Details on sampling:

Sampling intervals for both active and heat-deactivated S9 were 0, 15, 30, 60, 90 and 120 minutes. Each reaction was terminated by the addition of an equal volume (0.5 mL) of ice-cold hexane, the sample was vortexed to mix and placed on an ice-bath for 30 minutes to halt the biological processes and extract the analytes. Precipitated proteins were removed from the solution by centrifugation.The hexane layer was recovered and used for further analysis as described in the analytical section.

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

The test compound (CAS# 78-63-7) was added to a concentration of nominal 5 μM as a 1 mM stock prepared in ethanol using a 50 mL aliquot.

Test organisms (species):

other: Trout, but specie was not specified.

Details on test organisms:

The fish were supplied by the Oden State Fish Hatchery located in Alanson, MI and ranged in weight from 63 to 335 g each. They were held for eleven days under standard aquaculture conditions at water temperature of 11.7°C. The fish were fed Silver Cup pellets ad libitum, provided by the fish hatchery. The fish were anesthetized with MS222 (tricaine methanesulfonate) until immobile prior to sacrificing. The liver was dissected from the carcass, weighed and immediately flash-frozen in liquid nitrogen. The livers were stored at approximately -80 °C until processed. The trout livers were, minced, and mixed with buffer in a 4:1 ratio of buffer to liver (v:w). The buffer (pH 7.4) was composed of 50 mM potassium phosphate, 0.15 M potassium chloride, and 0.2 M sucrose. The tissue was homogenized on ice using Teflon tipped pestle with glass mortar. The homogenate was placed into chilled centrifuge tubes and centrifuged at 12,000 g for 30 min at 4°C. The supernatant (S9 fraction) was carefully removed and transferred to a chilled bottle or beaker and frozen in 2 mL aliquots on dry ice and transferred to an -80 °C freezer for storage. The total protein concentration (25 mg/mL) was determined using the Pierce BCA™ method (Pierce, Rockford, IL).

Route of exposure:

other: in vitro exposure

Test type:

other: In vitro metabolism assay

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

120 min

Hardness:

Not applicable

Test temperature:

15°C in a temperature controled shaking plateform incubator

pH:

No data

Dissolved oxygen:

Not applicable

TOC:

Not applicable

Salinity:

Not applicable

Details on test conditions:

The frozen S9 fraction was defrosted by running under a cool stream of water and diluted to a solution of 2 mg/mL protein with reaction buffer held on ice. The reaction buffer consisted of 0.1 M Tris buffer containing 150 mM KCl and 10 mM MgCl2 (pH 7.4). An NADPH regeneration system was added to the solution to ensure adequate energy for the reactions. It was composed of iso-citrate, NADP+, and iso-citrate dehydrogenase. Isocitrate
was added to the reaction buffer to a concentration of 7 mM. Iso-citrate dehydrogenase (Type IV, Porcine Heart, Sigma-Aldrich) was added to a concentration of 0.5 units/mL. The reaction was initiated by the addition of 50 μL of 50 mM NADPH prepared in 1% sodium bicarbonate solution. Reactions were initiated approximately 30 seconds apart to allow for sample collection and vortexing at each time interval. Triplicate reaction solutions containing approximately 2 mg/mL protein of active trout liver S9 in reaction buffer were prepared. The reaction solutions were incubated at 15oC in a temperature controled shaking platform incubator over a 2 hour period.
Heat deactivated control reaction solutions were prepared in triplicate with trout liver S9 fraction inactivated by placing a 50 mL centrifuge tube of the diluted S9 preparation in boiling water for approximately 5 minutes. Data generated by the incubation of the heat deactivated S9 with the test substance were used to evalaute for any non-biological dissipation processes such as binding or hydrolysis.
Each reaction mixture was prepared as a total of 5 mL and was sub-divided into 0.5 mL aliquots in 4 mL vials for incubation.

Nominal and measured concentrations:

Nominal concentration : 5 µM

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Kinetics were assumed to be first order and the data were fitted using an exponential curve. A half life was determined for the loss of parent using the equation below. These data were extrapolated to whole organism rates of metabolism and finally bioconcentration factor (or BCF) as described by Cowan–Ellsberry et al., (2008). The BCF was predicted based on the mass-balance model described by Arnot and Gobas (2003, 2004). The steady state model described by:
BCF= k1/(k2+kE+kg+kMET)
where k1 is the gill uptake rate constant (day-1*kg-1)), k2 is the gill elimination rate constant (day-1), kE is the elimination rate constant through fecal egestion (day-1), kG is the growth dilution rate constant (day-1) and kMET is the metabolic transformation rate constant (day-1). The details for extrapolation of all rate constants other than kMET are described in Arnot and Gobas (2003, 2004).
The data required to parameterize this BCF model, in addition to kMET, are the log Kow of the chemical (6.55 estimated for CAS# 78-63-7), environmental temperature, and the fish physical and physiological parameters of body wet weight and fraction of lipid to the whole fish body.

Type:

BCF

Value:

839

Basis:

other: modelled value with in vitro extrapolation

Calculation basis:

kinetic

Remarks on result:

other: kMET= 0.116 day-1 assuming only hepatic flow influence the distribution of CAS# 78-63-7.

Type:

BCF

Value:

521

Basis:

other: modelled value with in vitro extrapolation

Calculation basis:

kinetic

Remarks on result:

other: kMET= 0.189 day-1 assuming portal and arterial flow influence the distribution of CAS# 78-63-7.

Remarks:

Conc.in environment / dose:5 µM

Details on kinetic parameters:

If only the hepatic blood flow was used to extrapolate the rate of metabolism to the whole body rate constant kMET, the resulting kMET was calculated as 0.116 day-1. If both the portal and arterial flow were assumed to influence the distribution of CAS# 78-63-7 throughout the organism, then the kMET was calculated as 0.189 day-1.

Metabolites:

All samples were analyzed for the presence of a proposed primary degradation product, tbutanol. However, quantifiable amounts were non-detectable in any sample. It is not surprising that t-butanol was not detectable in the biologically active samples. Liver tissue is known to have high levels of alcohol dehydrogenase activity, which have also been detected in fish tissues such as liver, gut and gill (Nagai et al., 1997). The alcohol would be rapidly converted to its concomitant aldehyde and then its organic acid equivalent prior to elimination from the body.

Results with reference substance (positive control):

Not applicable

Details on results:

CAS# 78-63-7 dissipated in biologically active S9 reaction mixture only. In the biologically active S9 fraction, CAS# 78-63-7 dissipated more rapidly with a half life of 210 min and rate constant of 0.0033 min-1. Because the dissipation of CAS# 78-63-7 was observed in only the biological active S9 fractions, the rate constant observed in the biological active solution was not corrected for changes observed in the heat deactivated system. The resulting rate of in vitro metabolism was calculated as 0.3410 (μmoles CAS# 78-63-7/gm protein/ hour) using the half- life information. The rate of reaction was then used as input for the Gobas model as an estimation of metabolism.

Peroxides such as CAS#78-63-7 are known to under go thermal decomposition and homolysis. The estimated rate of decomposition of the test compound at 15°C was 87,443 days or more than 200 years (Degussa, 2008). At the temperature used for the conduct of this study, the impact of thermal decomposition should be minimal based on these estimates and we can be assured that the observed loss is a result of metabolism or other abiotic degradation processes.

Using the Gobas model, and assuming no metabolism, the bioconcentration factor (BCF) for CAS# 78-63-7 is calculated as 31,819.51or a log BCF of 4.50. The addition of metabolism to the equation brings the calculated BCF down significantly.

Reported statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the data were conducted using Microsoft Excel spreadsheets and data in full precision mode (15 digits of accuracy).

kMET Calculation	Trout w/ arterial hepatic blood flow	Trout with arterial and portal hepatic blood flow
CLm Intrinsic clearance in the in vitro test (ml/hr*cells)	71.49	71.49
CLi -- Intrinsic Clearance in liver (ml/Kg*h)	49.13	49.13
CLi -- Intrinsic Clearance in liver (L/Kg*d)	1.18	1.18
CLh -- Hepatic Clearance incorporating blood flow (L/Kg*d))	0.65	1.05
kMET(1/day)	0.12	0.19
BCF Calculation
K1 - uptake rate (L/Kg*day)	1.00E+02	1.00E+02
K2- rate of elimination via resp (1/day)	0	0
Ke - rate of chemical egestion via fecal material (kg/kg/day)	0	0
Kg is rate of growth (1/day)	0	0
BCF = k1/(k2+ke+kg+kMET)	839.12	521.49
BCF with kMET= 0	31819.51	31819.51

Validity criteria fulfilled:

not applicable

Conclusions:

The BCF value of the substance was determined with and without metabolism being taken into account. The calculation is based on the Arnot Gobas BCF mass balance model run with and without metabolism. When metabolic rate (Kmet) is set to 0, the model calculates a BCF ot 46097. When Kmet is experimentally determined using two methods: arterial hepatic and arterial hepatic and portal, blood flow extrapolation further to a trout hepatocyte in vitro study, BCFs are calculated as 766 and 443 L/Kg respectively.
The in vitro rate of metabolism was determined for the organic peroxide CAS# 78-63-7. CAS# 78-63-7 dissipated rapidly under biologically active conditions. A half- life for metabolism was 210 min or 3.5 hours. These data were used to extrapolate in vivo rate constants of metabolism or kMET of 0.116 day-1 or 0.189 day-1 using hepatic portal blood flow or hepatic portal and arterial blood flow, respectively. The use of these kMET values
in conjunction with the Gobas model dropped the calculated BCF from 31,819.51 kg day- 1 (assuming no metabolism) to 521 or 839 kg day-1.

Executive summary:

The rate of fish metabolism for the organic peroxide CAS# 78-63-7 was determined in vitro using a trout liver S9 fraction. Each reaction mixture contained 2 mg/mL S9 protein and the concentration of CAS# 78-63-7 was approximately 5 μM. The experiment was conducted in triplicate, at a temperature of 15° C along with controls containing heat deactivated S9 protein and approximately 5 μM CAS# 78-63-7. Sub-samples were taken and extracted over the course of 2 hours to determine the rate of degradation due to metabolism and abiotic processes. The reaction was halted by the addition of an equal volume of cold hexane to each sub-sample, the solutions thoroughly vortexed and centrifuged to separate phases. The upper hexane layer was removed and analyzed by GC/MS to determine the concentration of the peroxide and t-butanol, a known homolysis degradation product. First order kinetics for dissipation of the parent peroxide was assumed and the percent parent remaining was plotted against time and fitted to an exponential curve. Kinetics were determined for the biologically active S9 fraction only. No loss of parent peroxide was seen in the heat deactivated samples. The biologically active rate constant was used to calculate the in vitro half-life of metabolism for CAS# 78-63-7 of 210 minutes. The half- life was used to calculate a rate of metabolism of 0.341 (μmoles /gm protein/ hour). The in vitro rate of metabolism was extrapolated to a whole body rate constant of metabolism (kMET) using the technique described by Cowan-Ellsberry et al. (2008). The kMET was then used as input in the Gobas bioaccumulation model to provide an estimate of metabolism in the calculation for bioconcentration factor (BCF). The original BCF for CAS# 78-63-7 calculated using the Gobas model assuming no metabolism was 31,819.51 kg/day. With the addition of metabolism, BCF ranged from 521 to 839 kg/day. Samples were analyzed for t-butanol, a proposed metabolite, but residues were non-detectable in all samples at an LLQ of 8.901 ng/mL. It is not surprising that t-butanol was not detectable in the biologically active samples. Liver tissue is known to have high levels of alcohol dehydrogenase activity, which have also been detected in fish tissues such as liver, gut and gill (Nagai et al., 1997). The alcohol is rapidly converted to its concomitant aldehyde and then its organic acid equivalent prior to elimination from the body.

Description of key information

Two studies on bioaccumulation were available on TRIGONOX 101.

An in vivo study evaluated the BCF on Cyprinus carpio (fish): but these results were not used for the chemical safety assessment due to the incompleteness of the information. However, an in vitro method (fish liver S9 metabolism assay) was evaluated as valid with restrictions, and used in this assessment.

Key value for chemical safety assessment

BCF (aquatic species):

839 L/kg ww

Additional information

Two studies on bioaccumulation were available on TRIGONOX 101.

The first study (Erhardt, 2008) was evaluated as the key study. This study assessed the bioaccumulation of TRIGONOX 101 using the in vitro trout liver S9 metabolism assay (no guideline followed). The BCF value was determined with and without metabolism being taken into account. The calculation is based on the Arnot Gobas BCF mass balance model run with and without metabolism. When metabolic rate (Kmet) is set to 0, the model calculates a BCF ot 46097. When Kmet is experimentally determined using two methods: arterial hepatic and arterial hepatic and portal, blood flow extrapolation further to a trout hepatocyte in vitro study, BCFs are calculated as 766 and 443 L/Kg respectively. The in vitro rate of metabolism was determined for this organic peroxide. The substance dissipated rapidly under biologically active conditions. A half- life for metabolism was 210 min or 3.5 hours. These data were used to extrapolate in vivo rate constants of metabolism or Kmet of 0.116 day^-1or 0.189 day^-1using hepatic portal blood flow or hepatic portal and arterial blood flow, respectively. The use of these kMET values in conjunction with the Gobas model dropped the calculated BCF from 31,819.51(assuming no metabolism) to 521 or 839.

The second study (author unknown, 2004) was evaluated as the weight of evidence. This study assessed the bioconcentration of TRIGONOX 101 on Cyprinus carpio, following "Methods concerning the testing of new chemical substances" (Kanpo n°. 5, Yakuhattsu n°.615, 49th Unit, n°.392, 1974, partially revised in 1998), which is similar to OECD Guideline 305. Results demonstrated that a BCF steady-state of 3690 and 2250 L/Kg (for primary and secondary concentration areas respectively).

As the experimental BCF study was incomplete missing numerous details allowing it to be validated, the BCF and as further experimental data are available from an in vitro study which is deamed to be valid, it is concluded that the BCF is between 521 and 839.