Registration Dossier

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
other: OECD319b
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
The aim of the OECD 319B is to determine the in vitro intrinsic clearance (CL in vitro int) of a test chemical using the substrate depletion approach. This can be applied to increase modelled predictions of bioaccumulation in fish. In short the clearance rate can be inputted into toxicokinetic models for fish bioaccumulation or extrapolated to a whole body biotransformation rate. See test guideline. The aim of this assay is to ultimately reduce animal testing in groups of similar substances, by more extensive use of such alternative tests. In addition the assay can be implemented in a simpler fashion to determine if a product is transformed and can be useful to aid degradation product identification. In this memo the
implementation of the assay for Trout and Rat liver is summarized and one test chemical was tested using the assay in order to establish if biotransformation occurred. No intrinsic clearance rate was calculated at this stage.
GLP compliance:
no
Radiolabelling:
no
Conclusions:
No metabolic activity was observed for the test chemical.
Executive summary:

The test chemical was used during the implementation of the OECD 319B assay. Performance of the assay was assessed using testosterone as a standard to demonstrate activity using both trout and rat liver S9 fractions. Multiple tests were conducted to establish an optimal concentration for use in subsequent tests as a positive control. After which, both rat and trout liver fractions gave an acceptable levels of testosterone metabolism and showed a sufficient level of reproducibility. Should definitive tests

take place an additional standard for phase II enzyme activity and day to day test variation still requires validation.

The test substance di(tert-butylperoxyisopropyl)benzene was tested with a rat liver S9 fraction at 50 and 200 μ/moles. No metabolic activity was observed for the test chemical. The meta isomer only was quantified. The reference material at 50 μ/moles performed normally demonstrating sufficient phase 1 activity in the cell line used.

Description of key information

Short description of key information on bioaccumulation potential result:

Limited substance specific data on metabolism and kinetics are available.

The test substance di(tert-butylperoxyisopropyl)benzene was tested with a rat liver S9 fraction at 50 and 200 μ/moles. No metabolic activity was observed for the test chemical. The meta isomer only was quantified. The reference material at 50 μ/moles performed normally demonstrating sufficient phase 1 activity in the cell line used.

Based on physical-chemical characteristics, no or only limited absorption by the dermal and inhalation routes is expected.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

No data on toxicokinetics, metabolism and distribution are available for the source and target substance. This is not a testing requirement according to EU REACH. The assessment of the toxicokinetics is based on the available toxicological data and its physicochemical properties as suggested by the REACH Guidance Chapter R.7c.

 

The molecular weight is 338.48 g/mol for the meta and para isomers. The substances are poorly soluble in water (0.04 and 0.02 mg/L, respectively) and have a very low vapor pressure (0.00012 Pa at 20 °C). The logKow of both meta and para isomers is >6.5 a logBCF of 3.96 was calculated. Based on its physico–chemical properties, the substances are not likely to penetrate skin to a large extent as both are solids, nearly insoluble in water and with a Log Kow not in favor of dermal uptake. The low absorption rate is further supported by the acute dermal toxicity study results for the source substance. Moreover, both substances are not a skin irritant; therefore, no increase of skin absorption is expected by damaged skin. Based on the low solubility in water and its high lipophilic properties, the substances could be absorbed in a large extent following oral exposure. The high absorption rate is further supported by the repeated oral toxicity study results where adverse effects were observed. Based on the very low vapor pressure and on the high granulometry of the powder fraction, inhalation exposure is unlikely. However, if exposed, it is likely the substance will also be absorbed.

Therefore, for both substance in their risk assessments, according to the REACH Guidance, default values of 100, 10 and 100% will be used for oral, dermal and inhalation absorptions, respectively.

 

Two studies are available that investigated the breakdown of the source substance, contain both the meta- and the para-isomer.The first study (Erhardt et al, 2009) concluded that rapid metabolism was seen with a metabolic half- life of 67 minutes or 1.13 hours. However, as noted by ECHA in 2017 in a decision for 25155-25-3,“Some critical remarks can be made to the in vitro metabolism study itself (--,2009). In the study, the concentration of tert-butanol, a potential metabolite ofthe registered substance, is followed as well as the parent substance.The disappearance of the parent compound from the biologically active 59 incubation issignificant, but at the same time highly variable. The coefficient of variation for the threereplicates are on average about 50%, but for the 5th time point of the biologically activetreatment even exceeds 100%. Thus, the rate of metabolism has wide confidenceintervals, but this is not addressed in the registration dossier. Further, on average, theamount disappeared from the heat-treated deactivated test is similar to the biologicallyactive 59 incubation. However, the lowest amounts of parent compound are present atthe intermediate time points (3,4,5) for the deactivated incubation, while they are present at the last time points (4,5,6) for the biologically active S9 incubation. Theamount of the metabolite in the heat-treated deactivated incubation is about ten timeshigher than in the biologically active 59 incubation, which can be explained by the factthat the $9 incubation metabolizes tert-butanol as well. However, in both treatmentsthere is no significant trend of the concentration of tert-butanol in time at all. This is incontradiction with the assumption that tert-butanol is formed as a result of metabolicactivity during the test. Finally, there is no complete mass balance in this kind of testand without the quantification of all metabolites, the dissipation of the parent compoundcan be caused by other processes as well. From other tests, such as the reproductiontest with Daphnia magna, it has become clear that it is extremely difficult to maintainaqueous concentrations of the registered substance in a static exposure system.”

 

Therefore, an additional study (Kean, 2019) was performed with the source substance to try and reproduce the finding from 2009. This study is a non-GLP adaptation of the OECD 319B assay. Performance of the assay was assessed using testosterone as a standard to demonstrate activity using both trout and rat liver S9 fractions. Multiple tests were conducted to establish an optimal concentration for use in subsequent tests as a positive control. After which, both rat and trout liver fractions gave an acceptable levels of testosterone metabolism and showed a sufficient level of reproducibility. The source substance was tested with a rat liver S9 fraction at 50 and 200 μ/moles, the meta isomer only was quantified. No metabolic activity was observed in this assay. The results from 2009 were therefore not confirmed.

 

Based on these findings it can be assumed that the toxicity observed is caused by the parent substance and/or its breakdown products. The data presented in appendix II shows that in (eco)toxicological studies the source and target substance have similar toxicological effects and that therefore similartoxicokinetics, metabolism and distribution profile for the parents andbreakdown products are also expected.