Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

Key value for chemical safety assessment

Absorption rate - dermal (%):
7

Additional information

Although no key study on toxicokinetics according to current guidelines is available, various quantitative data on the toxicokinetic behavior of Lysmeral from different experimental animals (rat, mouse, rabbit, guinea pig, dog and rhesus monkey) and humans exist. Based on its physico-chemical properties, i.e. water solubility (33 mg/l at 20°C), partition coefficient log Pow (4.2 at 24°C), molecular weight (204 g/mol) and vapour pressure (0.25 Pa at 20°C), Lysmeral is considered to have a high bioavailability via the oral route and a limited bioavailability via the inhalation route. After acute and repeated oral and dermal administration of Lysmeral to experimental animals and humans there is clear evidence of systemic absorption. However, in humans only limited percutaneous absorption of Lysmeral is observed especially when compared to the rat. 

After semiocclusive dermal application of 14C-lysmeral (14.7 µCi or 11.37 mg test substance in 70% ethanol on 10 cm2 back skin) on 3 human volunteers for 6 hours, a mean of 1.4% (range 0.8 - 2.4%) of the applied dose was excreted in urine within 24 hours, whereas radioactivity was below the detection limit in urine samples of later time points and in all faeces and blood plasma samples (Huntingdon Research Centre, 1994). Taking into account, that the chosen vehicle promotes dermal penetration of the applied test substance, these data indicate very limited percutaneous absorption of lysmeral in humans. 

In an in vitro study acc. to OECD 428 and GLP, penetration of 14C-Lysmeral through and into human skin was assessed by single topical application of target doses of 5 and 95 μg/cm² in different preparations, representative of in-market cosmetic formulations for 24 hours to split thickness human skin membranes mounted on modified Franz-type diffusion cells (BASF 2016; 10B0089/10B020). The formulations consisted of a hydro-alcoholic preparation with 1.9 weight % of Lysmeral in 70 % ethanol and 0.1 weight % Lysmeral in “silicone in water”, “water in oil” and “oil in water” based type of formulations. Diffusion cells were operated in a flow-through mode with tap water as the receptor (incl. 0.01 % sodium azide for the 72 hours experiments). In order to investigate the behavior of Lysmeral that remained on the skin surface after the 24 hours skin wash, additional prolonged experiments were performed to characterize the total radioactive residues in the skin preparation 72 hours after application. This has been done to determine whether a movement of 14C-Lysmeral from the skin reservoir to the receptor fluid occurs, allowing to exclude Lysmeral in the epidermis as systemically available and to differentiate between extractable (potentially absorbable) and non-extractable (bound, non-absorbable) residues of Lysmeral. Receptor fluid was collected in fractions by continuous collection over defined intervals over 24 or 72 hours. After the exposure or sampling time, skin membranes were washed with washing fluid (Sodium-laurylethersulfate, diluted 1:140 w/w in tap water) followed by tap water. 14C-Lysmeral was recovered from all compartments of each diffusion cell. The stratum corneum was removed by tape stripping (20 tape strips were taken). The remaining skin of the 24h experiments was separated into dermis and epidermis and analyzed separately whereas the remaining skin of the 72h experiments was not separated but extracted in a tissue lyzer with methanol immediately after the stripping procedure. Both extractable and non-extractable fractions were measured by liquid scintillation counting (LSC). For the other samples, weighed aliquots of each native receptor fluid or of the solvents used for extraction of the tape strips, epidermis and dermis and equipment extracts were analyzed by LCS as well.

A substantial amount of 14C-Lysmeral was recovered in the charcoal filter when applied in the alcoholic or “silicone in water” formulation (>50% of the applied dose). The skin washes after 24 hours contained a predominant fraction of 14C-Lysmeral when applied in the “oil in water” (48-56%) and “water in oil” formulation (50-54%), whereas lower fractions were found for the other vehicles (12-14%). Mean absorbed doses were 3.5 - 5.3% of the applied dose in the 24 hours absorption experiments and 5.0 - 7.8% of the applied dose in the 72 hours absorption experiments. Especially when applied in a hydroalcoholic and “oil in water” vehicle, no evident differences in the absorbed doses of 14C-lysmeral were found 48 hours after removal. Only minor amounts of 14C-Lysmeral were associated with the skin after the exposure period. In the 24 hours experiments, these amounts were differentiated into dermis and epidermis compartments and accounted to 0.64 - 0.78% and 0.69 - 1.50% of the applied dose, respectively. In the 72 hours experiments, the residues of 14C-Lysmeral in the skin preparations were differentiated into an extractable and non-extractable portion. The non-extractable portion of Lysmeral in living skin is assumed to be bound to the skin matrix and therefore represent a non-absorbable fraction. Based on the findings from these experiments 20 - 40% of the fraction found in living skin was not extractable and therefore potentially not absorbable. For the “oil in water” formulation, the residues of 14C-Lysmeral in the stratum corneum and the remaining skin appeared to be higher directly after test substance removal when compared to the respective 72 hours experiment, however, the absorbed dose did not significantly differ between experiments (4.77 versus 4.97%). Furthermore, the overall comparability of fractions found in the tape strips, living skin and absorbed doses observed for other vehicles indicate, that the post application phase does not significantly alter the distribution of 14C-Lysmeral in these compartments.

 

Overall, the percentage of systemically available lysmeral has been calculated based on the absorbed dose and the test substance content in remaining skin (dermis+epidermis), substracted by the non-extractable/ non-systemically available fraction determined in the living skin. Accordingly, the percentage of dermally absorbed lysmeral was calculated to be between 5 and 7% with the highest values obtained for the hydroalcoholic vehicle.

 

% non-extractable skin fraction

Ethanol in Water

 = (0.32) * 100 / (0.32 + 1.31) = 20%

Silicone in Water

 = (0.25) * 100 / (0.25 + 0.71) = 26%

Water in Oil

 = (0.24) * 100 / (0.24 + 0.50) = 32%

Oil in Water

 = (0.18) * 100 / (0.18 + 0.28) = 39%

% systemically available lysmeral

Ethanol in Water

 = 5.31+((1.5+0.71)*(100-20)%) = 7.08%

Silicone in Water

 = 3.5+((0.96+0.64)*(100-26)%) = 4.68%

Water in Oil

 = 4.83+((0.74+0.73)*(100-32)%) = 5.83%

Oil in Water

 = 4.77+((0.69+0.78)*(100-39)%) = 5.67%

 

 

In rats, occlusive dermal application of 14C-Lysmeral in 70% ethanol at a single dose of 6.8 mg/kg bw (0.2 mg/cm2) for a maximum of 6 hours (2 animals/time point i.e. 0.5, 1, 3, 6, 12, 24, 48, 72, 120 h sacrifice) showed a good bioavailability and rapid urinary excretion (Huntingdon Research Centre, 1995). Up to 120 hours after application of 14C-Lysmeral, a mean cumulative total of 14.6% of the dose was excreted in urine, 0.8% was recovered in cage washings and 2% was excreted via faeces, whereas levels in expired air traps were not detectable. Two animals per time point each were examined for tissue distribution and remaining dose at the application site. 120 hours after application of the test substance, the remaining radioactivity in all tissues investigated – excluding the skin at the application site – amounted to 1.2% of the applied radioactivity. The maximum urinary excretion rate (1.95μg equivalents/hour) was observed 6 - 12 hours after dermal application of 14C-Lysmeral. Dermal radioactivity concentrations were more persistent and declined with a half-life of approx. 152 hours compared to other tissues ranging from 10 - 93 hours (see Attachment 1).

The highest concentration of the absorbed 14C radiolabel was recovered in the liver (Cmax = 15.6 µg/g tissue representing 0.826 % of given dose/g tissue). Overall, a relation of Tmax and the blood perfusion rate of a respective tissue is indicated based on the findings for highly perfused tissues (i .e. lungs, heart) and poorly perfused tissues (i.e. skin, fat).

Although pharmacokinetic variables were not reported for testes in this study, a maximum concentration in this tissue was determined to be 0.008% of the topically applied dose per gram tissue 1 hour post application. The mean total proportion of dose in excreta and tissues was about 19%, which represents the apparent level of absorption of radioactivity into the systemic circulation. Overall, a distribution predominately to the liver has been observed after dermal administration and can be assumed for the oral route as well.

 

After oral administration of radiolabelled 14C-Lysmeral in a single dose of 25 and 100 mg/kg to 4 male lbm:RORO (SPF) rats per dose via gavage (Huntingdon Research Centre, 1995), a rapid absorption of the radioactive compound for both doses applied and proportionate plasma maximum concentration (Cmax) has been observed (see Attachment 2). In contrast, the AUC was found to increase disproportionate to the dose applied which is interpreted to be indicative for a saturation of the renal clearance.

When compared to the dermal toxicokinetic study in rats described above, dermal administration of lysmeral revealed 7 fold lower Cmax plasma values compared to oral administration taking into account the doses applied (0.5 μg/g after dermal administration of 6.75 mg/kg bw lysmeral vs. 14.3 μg/mL after oral administration of 25 mg/kg bw lysmeral). This comparison demonstrates, that systemic bioavailability after dermal administration is considerably lower in rats compared to that after oral administration. Since rat skin is more permeable to dermally applied substances than human skin, and taking into account the results of a dermal penetration study in human volunteers, very limited percutaneous absorption and systemic bioavailability of lysmeral is expected in humans.

Further, blood plasma kinetics of Lysmeral and the metabolite lysmerylic acid in rodents was studied in male Wistar rats (BASF SE 2006A) and male C57BL/6NCrl mice (BASF SE 2006B) after oral application of a single dose of 50 mg/kg bw of each Lysmeral and lysmerylic acid by gavage. Blood was taken retroorbitally, 3 days before gavage, directly after the first oral application (i.e. 20 minutes for mice and 10 minutes for rats), as well as 2, 4, 8, and 24 hours after application and blood plasma was analysed for Lysmeral and lysmerylic acid by HPLC/MS.

After application of Lysmeral, no unchanged parent compound was detectable in any plasma sample of both rodent species. In the male rat, lysmerylic acid was detected in all plasma samples and highest plasma concentration was observed 4 hours after application of Lysmeral or directly after application of lysmerylic acid (see Attachment 3). In the male mouse, highest plasma concentration of lysmerylic acid was observed directly after application of both Lysmeral and lysmerylic acid. No species difference in the toxicokinetic parameters were found after application lysmerylic acid, whereas some difference in Tmax and Cmax was evident between rat and mouse after oral application of Lysmeral.

 

Excretion of the expected urinary metabolites of Lysmeral, i.e. tert.-butyl benzoic acid (TBBA) and tert.-butyl benzoyl hippuric acid (TBHA) has been compared in the rat, mouse, guinea pig, dog and rhesus monkey (Roche 1985A). Urine was collected for 24 hours after the last bolus oral administration of Lysmeral for 5 consecutive days in the 5 species mentioned above. The doses ranged from 45 to 400 mg/kg bw/d differing between species (rat: 50-400 mg/kg bw/d; mouse, guinea pig, and rhesus monkey: 100 mg/kg bw/d; dog: 45 mg/kg bw/d). Urinalysis of tert-butylbenzoic acid (TBBA) and tert-butylhippuric acid (TBHA) was performed by GC/MS. In the control group of all species, no TBBA and TBHA were found. Considering the relation between TBBA and TBHA, the main urinary metabolite in orally treated rats, dogs and rhesus monkeys was found to be TBBA, whereas in the guinea pig and mouse TBHA resulting from glycine conjugation predominates (see Attachment 4). Surprisingly, urinary TBHA amounts in the rat were very low compared to other rodent species in this study, thus glycine conjugation or urinary TBHA excretion might not occur in the same rate as it does in other rodents. The urinary TBBA amounts in one of the two rhesus monkeys was found to be comparable to rat amounts, whereas the other monkey showed 2-3 fold lower TBBA amounts than the rat. 

 

Similarly, species specific differences in the urinary excretion of TBBA have been observed after oral application of p-tert-butylbenzaldehyde (TBB) or p-tert-butyltoluene (TBT).  After 5-day oral administration of 12.5 and 50 mg/kg bw/day p-tert-butylbenzaldehyde (TBB) or 25 and 100 mg/kg bw/day p-tert-butyltoluene (TBT) to rats, p-tert-butylbenzoic acid (TBBA) was identified as metabolite in the urine 24 hours after the last administration, but not the secondary metabolite p-tert-butylhippuric acid (TBHA) was found. Amounts in urine yielded 17.2 mg TBBA /kg bw after administration of TBT (100 mg/kg bw/d) and 12.7 mg TBBA/ kg bw after administration of TBB (50 mg/kg bw/ day), being approximately 2-3 fold higher compared to TBBA urine amounts observed after administration of comparable lysmeral doses to rats. No further metabolites have been investigated and glucuronic acid conjugates could not be identified by the analytical method used (Givaudan 1982B).

On the occasion of different 5 day oral toxicity studies in mice, guinea pigs and dogs, 100 mg/kg bw/d TBB or TBT was administered for 5 days, urine was collected for 24 h after the last administration and analyzed for TBBA and TBHA by GC analysis. After application of TBB, TBBA was determined as metabolite in urine samples of treated dogs, guinea pigs and as very low amounts in the urine of mice. However, higher TBHA amounts were found in urine samples of treated mice and guinea pigs compared to TBBA, whereas TBHA amounts tended to be lower in the urine of dogs than TBBA amounts (Givaudan 1985). A similar pattern was observed after application of TBT. TBBA was determined as metabolite in urine samples of treated dogs and at very low amounts in guinea pigs but not in mice. Higher TBHA amounts were found in urine samples of treated mice and guinea pigs compared to TBBA, whereas TBHA amounts were lower in the urine of dogs than respective TBBA amounts (Givaudan 1985).

The excretion kinetics of lysmeral in humans was investigated in an explorative study in human volunteers to develop a human biomonitoring (HBM) method including identification of suitable biomarkers of exposure in human urine (Scherer 2016). As a pilot study, the preliminary analytical method was applied to a urine sample collected prior to and all fractions voided up to 48 h after using a lysmeral-containing sunscreen (5g containing 6.5 mg/g lysmeral) by a volunteer (male nonsmoker, 65 years old). Further, 5 healthy subjects (3 females, 2 males) were orally dosed once with 5.26 mg lysmeral, dissolved in ethanol and applied as a chocolate coated eatable waffle cup containing and approximately 20 mL coffee, milk or water, depending on the choice of the volunteers. Urine was collected immediately before and.for 48 h after administration (all urine voids completely collected in separate fractions with the time of voiding being free). Lysmeral associated metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid and 4-tert-butylbenzoic acid (TBBA) were determined by UPLC-MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Due to high variations concerning precision and accuracy during method validation the metabolite TBHA was not included as a reliable parameter for HBM and not followed up in the oral excretion kinetic study.

In the dermal pilot study with one volunteer, peak amounts of lysmerol and lysmerylic acid were excreted into the urine about 3–6 h after, whereas TBBA and TBHA appeared about 12 h after dermal application. TBBA represented 0.67% of the applied dermal dose, followed by TBHA (0.04 %), lysmerol (0.02 %), and lysmerylic acid (0.012 %). In total, the lysmeral-related analytes represented 0.75% of the dermally applied dose.

Oral uptake resulted in peak amounts of the 4 metabolites between 3 and 6 h after application with lysmerol and lysmerylic acid appearing slightly earlier in the urine than the secondary metabolites hydroxyl-lysmerylic acid and TBBA. A rapid urinary excretion was observed, since more than 90% of all measured lysmeral metabolites were excreted after 12 h, and the excretion was found to be complete by 48 h after the oral intake. The sum of the 4 metabolites assessed in urine reflected about 16.5% of the applied dose. TBBA represented about 14.3% of the administered dose, followed by lysmerol, yielding 1.82% of the dose. The urinary fraction of hydroxy-lysmerylic acid and lysmerylic acid was 0.20% and 0.16% of the applied dose, respectively. Lysmeral itself was detectable after enzymatic deconjugation , but in very low amounts, i.e. <0.003% of the dose applied. Average times for peak excretion (tmax) were 2.2 h and 4.64 h for lysmerol and TBBA and 3.1 h for both lysmerylic acid and hydroxyl-lysmerylic acid. The elimination half-lives (t½) were found to be lower for the primary metabolites lysmerol and lysmerylic acid (1.19 h and 1.25 h, respectively) than for the secondary metabolites hydroxyl-lysmerylic acid and TBBA (1.39 h and 1.40 h, respectively), showing that the primary metabolites are excreted more rapidly.

Based on the results obtained by this exploratory excretion kinetic study, urinary conversion factors (CF) were deduced to allow the back-calculation of absolute lysmeral uptake doses from creatinine standardized urinary metabolite levels of spot urines samples of 40 adult volunteers from the general population. Back-calculation based on these CF resulted in median daily exposure doses of 224 µg/d lysmeral (range: 67-2218 µg/d) using all metabolites or 140 µg/d (range: 12-2249 µg/d) using all lysmeral specific metabolites (excluding TBBA as metabolite of potential lysmeral independent exposure sources). 

 

Besides the selective assessment of specific metabolites in animals an men mentioned above, a detailed in vivo study to cover the full metabolic range of Lysmeral is not available. To close this gap, a comparative in vitro metabolism study has been performed in order to study relevant Lysmeral specific metabolic pathways in different species(BASF SE 2010; BASF 09B0089/10B001). For this purpose liver microsomes and hepatocytes of male Han-Wistar rats, male CD1-mice, male white New Zealand rabbits and male humans were incubated with14C-lysmeral at nominal substrate concentrations of 10, 50 and 100 µM. Metabolic profiles were detected and quantified by Radio-HPLC after appropriate work up procedures of received incubates. Structure elucidation of formed metabolites was performed from14C-lysmeral incubates (100 µM) of liver microsomes and hepatocytes of rats and humans by LC/MS-analyses.

In liver microsomes, an oxidation of14C-lysmeral to its corresponding carboxylic acid (M7-lysmerylic acid) or a reduction to its corresponding alcohol (M9 - lysmerol), further oxidized at the tert-butyl group to form a hydroxy-metabolite (M3), was observed (see Attachment 5). In hepatocytes, oxidation to lysmerylic acid was confirmed and its further dehydrogenation (most probably by hydroxylation and dehydration) to (E)-3-(4-tert-Butyl-phenyl)-2-methyl-acrylic acid (M16) was observed. Putative decarboxylation of lysmerylic acid, followed by oxidation to the propanoic acid derivative and beta-oxidation led to the identified metabolite p-tert-butyl-benzoic acid (TBBA -M15). This metabolite was conjugated with glycine to form p-tert-butyl-hippuric acid (TBHA -M12) in rodents. In addition to these metabolites, glucuronic acid conjugates of metabolites M3, M7, M9, and M16 were detected.

The qualitative evaluation of the metabolic profiles of different species largely confirmed in vivo findings. The two lower test concentrations chosen (10, 50 µM) reflect plasma levels observed after oral administration of no adverse testicular effect levels of Lysmeral whereas 100 µM covers plasma levels obtained after doses exerting testicular toxicity. Cmax for Lysmeral metabolites in plasma were 14 µg/ml or approx. 70 µM (assuming the molecular weight for Lysmeral) after oral application of 25 mg/kg bw (Huntington Research Center, 1995). Oral application of 50 mg/kg bw Lysmeral yielded a Cmax of 9 µg/ml or approx. 40 µM lysmerylic acid, i.e. the main metabolite (BASF SE 2006A). The Radio-HPLC chromatograms were used to assign ROI values (region of interest = integrated peak area under Radio-HPLC) of each characterized metabolite in order to receive relative amounts of each metabolite in the respective metabolic profile. These ROI values were used for comparison of the incubation concentrations and species tested. As summarized in Attachment 6, Lysmeral was metabolized nearly completely in the hepatocytes of all species whereas lysmerylic acid (M7) was quantitatively the main metabolite. The metabolite M16 ((E)-3-(4-tert-Butyl-phenyl)-2-methyl-acrylic acid) was more pronounced in hepatocytes of rats than in hepatocytes of mice or humans (not detected in hepatocytes of rabbits). In line with findings in vivo, species differences in metabolic profiles were seen for M12, representing TBHA, which was more pronounced in mice (4.9 – 27.1 % ROI) than in rats (3.5 – 3.6 % ROI). TBHA was not detectable in incubates of hepatocytes of rabbits and humans.

In rat hepatocytes, an increase of TBBA (M15) levels was found, and the incubation with lower lysmeral concentrations resulted in higher TBBA levels. When compared to other rodent or non-rodent animal species, rats showed the highest concentration of TBBA. Whereas this metabolite contributed to 8.3 – 29.3 % ROI in hepatocyte cultures of rats, it was ≤ 0.5 % ROI in mice, ≤ 2.0 % ROI in rabbits. The concentrations observed in humans were found to be approx. 4 fold lower than in rat hepatocytes for corresponding tested lysmeral concentrations, ranging from 1.9 – 7.5 % ROI. Furthermore, the concentrations of TBBA observed in the human system were similar to those found in the rabbit system at the 50 µM and 100 µM doses (the doses most relevant to plasma levels obtained after doses exerting testicular toxicity). These quantitative differences in TBBA formation between human and rat hepatocytes were apparently less pronounced or absent in the 24 hour urine samples of rats and rhesus monkeys. However, the TBBA detected in the 24 hour urine represent a cumulative amount of the excreted metabolite, to which the concentrations in the supernatant of the hepatocyte cultures cannot be compared to. These concentrations in hepatocyte supernatants are seen as directly proportional to given plasma concentrations in vivo. Since no comparative in vitro data for rhesus monkey hepatocytes are available and only two individual animals have been assessed in the primate study, the inclusion of these data for the overall assessment is questionable. The plasma concentrations (i.e. Cmax) represent a more relevant parameter in respect of the thresholded testicular toxicity observed for lysmeral, the data from the comparative in vitro metabolism study in hepatocytes are considered to better demonstrate species differences in lysmeral metabolism.

In vitro data in hepatocytes indicate an inhibititory capacity of TBBA on hepatic lipogenesis and gluconeogenesis (McCune et al. 1982; see Chapter Toxicity to reproduction). Addition of glycine, which represents a relevant substrate to form the respective hippurate (TBHA), did not affect TBBA inhibition of lipogenesis in the rat cells. These in vitro findings underline the lack of efficient TBHA formation capacity observed in rats in vivo. Furthermore, coenzyme A (CoA), acetyl-CoA and citrate levels were decreased in these cells. A formation and accumulation of p-tert.-benzoyl-CoA conjugates was suggested by the authors, although this could not be confirmed by the analytical methods used in the study at that time.

In a very recent study, a sensitive method in detecting specific CoA conjugates has been applied in plated rat and human primary hepatocytes by using high resolution mass spectrometry linked to liquid chromatography (LC-HRMS), (Givaudan 2017; Laue et al. 2017; see Chapter Toxicity to reproduction). Plateable primary male Sprague-Dawley rat hepatocytes were seeded and incubated at a density of 450,000 cells/mL on 48-well plates coated with collagen. Primary human hepatocytes were cultivated in a comparable manner. The rat and human hepatocytes were incubated with Lysmeral, Lysmeral-like materials and their relevant metabolites for 0.5-22 h. 

As postulated initially by Cune et al., the recent study demonstrated, that TBBA and Lysmeral is rapidly and dose dependently transformed by rat hepatocytes to TBBA-CoA and an accumulation to stable levels occurs within 0.5-4 hours. This stabilization over time indicates, that TBBA - once conjugated to CoA - is not rapidly and/or quantitatively transferred to secondary acceptors such as glycine. Physiological CoA conjugate levels such as oleoyl-CoA, palmitoyl-CoA or arachidonoyl-CoA were clearly below (<0.1 µM) the TBBA-CoA levels (1-2 µM), which indicates hepatotoxicity due to a competitive inhibition of other CoA dependent cellular processes. In contrast to TBBA-CoA levels, the direct CoA conjugate of lysmerylic acid is only transiently formed at low levels within 0.5-4 hours and not detectable after 22 hours incubation with Lysmeral. In cultures incubated with benzoic acid, only negligible amounts of benzoyl-CoA were found after 0.5 hours incubation but benzoyl-glycine (hippuric acid) was formed. In line with the in vivo metabolism data in rats,lysmeral or TBBA treated hepatocytes formed no TBBA-glycine conjugates.

Plated human hepatocytes were incubated with lysmeral and TBBA under identical conditions as rat hepatocytes, except for a slight adjustment ofseeding density to account for small difference in cell size and slightly differences in media to provide optimal culture conditions. The amount and the kinetics of TBBA-CoA formation was fundamentally different between human and rat hepatocytes. Lysmeral incubation for 0.5 hours resulted in approx. 5 fold lower TBBA-CoA levels compared to rat hepatocytes and a strong decrease was found over time. No differences in the kinetics of the octanoyl-CoA (i.e. an endogenously formed and the most prominent CoA conjugate) was observed in untreated rat and human hepatocytes, excluding the possibility of a general loss of CoA conjugation capabilities by cell culturing over time in human cells. Findings for the human hepatocyte lots tested were highly comparable which supports absence of effects due to a donor or sex differences. In human hepatocytes, amounts and kinetics of Lysmerylic acid-CoA were comparable to TBBA-CoA and resemble Lysmerylic acid-CoA formation in rat hepatocytes, whereas a sustained accumulation of TBBA-CoA was a unique finding only seen in rat hepatocytes. Furthermore, similar results were obtained after incubation of rat and human hepatocytes with TBBA. Lower levels ofTBBA-CoA were detected in human compared to rat hepatocytes at 0.5 hours of incubation and a rapid and almost complete decrease of TBBA-CoA was observed within 22 hours of incubation.

 

Overall, species specific differences in the formation of metabolites have been clearly identified both in vitro and in vivo between responder (e.g. rat) and non responder species (e.g. mouse, rabbit) with respect to reproductive toxicity. The species specific organ toxicity after repeated oral application of Lysmeral can be attributed to the toxic metabolite TBBA (see chapter "Toxicity to reproduction"). In vitro studies show significantly lower production of TBBA in humans than in rats, with human TBBA production similar to that observed in rabbits at toxicologically relevant doses. Furthermore, the intracellular formation of stable levels of TBBA coenzyme A complexes is a rat specific effect and does not appear in human cells.

The present data on Lysmeral do not indicate a bioaccumulation potential.