2-methyl-2H-isothiazol-3-one

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2003 - 29 October 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Objective of study:

other: mass balance, pharmacokinetics, tissue distribution and metabolism

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled in the 4,5 position of the ring.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Adult male and female Sprague-Dawley rats from Charles River Laboratories, Inc. (Raleigh, NC, Kingston, NY, and Portage, MI) were used for this study. Animals were ca. 7-10 weeks old and weighed ~214-251 g at dosing. Upon receipt, each animal was given an ear tag with a unique identification number and marked on the tail with indelible ink.

Acclimation
All animals were retained for 5 days and acclimated to the study cages for ~24 hr before dosing. Prior to dosing, the animals were examined every day for any signs of abnormalities indicative of health problems.

Housing and Maintenance
Upon arrival, rats were housed <5/cage in polycarbonate cages. During testing, rats were housed in stainless steel metabolism cages. Environmental controls in the animal room were set to maintain a temperature of 19-25 C, a relative humidity of 50 +/- 20%, and intermittent light and dark cycles of ~12 hr. Environmental condition records were maintained in the study file.

Food and Water
During acclimation, fresh water was available ad libitum through a bottle dispenser attached to each cage. Samples of the water were analyzed by International Hydronics Corporation, Rocky Hill, NJ, for selected organophosphates and chlorinated hydrocarbons.

Food was available to the animals ad libitum throughout the acclimation and testing periods, except during the fasting period overnight prior to dosing and until 4 hr post-dose. While rats were housed in polycarbonate cages, they received Certified Purina Rodent chow@ #5002 in pellet form. During the testing periods, rats received Certified Purina Rodent Chow #5002 in powder form. Feed was analyzed by the manufacturer for nutritional contents and any contamination that may have adversely affected the health of the animals. There were no known contaminants in the food or water that would have adversely affected the results of this study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Animals were orally administered 5 or 50 mg/kg RH-573.

Duration and frequency of treatment / exposure:

Animals were orally gavaged once during the study.

Remarks:

Doses / Concentrations:
Three groups of 3-4 rats/sex were dosed with 5 mg/kg of RH-573 and two groups of 3-4 rats/sex were dosed with 50 mg/kg of RH-573.

14C-RH-573 Dose Solution Concentration
The mean concentration of I4C-RH-573 in the low dose solution ranged from 0.64-0.70 mg/g; the mean concentration of 14c-RH-573 in the hlgh dose solution was 5 mg/g. The formulation was homogeneous. The mean concentration at pre- and post-dose was used in dose calculation. The mean radiochemical purity of the test substance in the dose solution was 93.12%. The data indicate that I4C-RH-573 was stable over the dosing time frame.

No. of animals per sex per dose / concentration:

A total of 11 rat/sex were dosed with 5 mg/kg and 7 rats/sex were dosed with 50 mg/kg RH-573.

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

no data

Details on study design:

Dose Administration
Each treated rat received a single dose by oral gavage at the nominal dose based upon individual body weights taken prior to dosing. The actual amount of dose solution administered to each animal was determined by weighing the loaded dose syringe before dose administration and the emptied syringe after dose administration.

Sample Collection
Urine, Cage Rinse, and Feces
Urine was collected at pre-dose, 0-24, 24-48, 48-72, and 72-96 hr post-dose. The samples were collected into tared sample cups and freeze-trapped using dry ice to avoid atmospheric oxidation, evaporation, and bacterial degradation. Cages were rinsed with NANOPURE water at 24, 48, and 72 hr post-dose; at the end of the study, cages were thoroughly washed with IPA/water (1:1). All cage rinse/wash samples were collected in tared sample containers. The feces samples were collected at the same timepoints as the urine at room temperature from the cage screen, weighed, and stored frozen upon collection. Urine, cage rinse, and feces were collected at the time of sacrifice from animals in Group E.

Blood and Plasma
Blood (~0.4 mL) was collected at 1, 3, 6, 24, 48, and 72 hr post-dose from the tail veins of Groups B and D rats. At 96 hr post-dose, animals from all dose groups were euthanized by an overdose of CO2. Blood (~5 mL) was collected by cardiac puncture. Sodium heparin was used as anticoagulant. Duplicate aliquots of blood were combusted for total radioactivity; remaining blood was centrifuged at 4C and 2500 rpm for 10 min to obtain plasma. Duplicate aliquots of plasma were assayed directly by LSC for radioactivity concentration.

Tissues
At the time of sacrifice, the following tissues were collected from Groups A, C, and E: liver, fat, kidneys, bone marrow (femur bone), heart, lungs, brain, testes (M), ovaries (F), muscle (hind leg), spleen, adrenals, thyroids, and remaining carcass. The tissues were stored at ~-20C until analysis.

Sample Preparation for Radioactivity Analysis
Blood and Plasma
Blood was mixed by hand inversion of the collection tubes. Duplicate subsamples were prepared and weighed (~0.07-0.1 g) for combustion followed by LSC counting.

Duplicate subsamples of plasma were prepared, weighed (~0.07-0.1 g when sample size allowed), and counted directly by LSC.

Urine, Cage Rinse, and Feces
Urine and cage rinse were mixed thoroughly, duplicate aliquots (~0.1 mL for urine, ~1 mL for cage rinse) were weighed into scintillation vials and mixed with scintillation cocktail and assayed directly by LSC. Feces were homogenized with 3-5x (w/v) NANOPURE water, and triplicate aliquots of homogenate equivalent to ~100 mg fresh feces weight were combusted in a Harvey Biological Sample Oxidizer followed by LSC counting. When samples were expected to contain very high radioactivity, smaller sample sizes were analyzed.

Tissues
Tissues were homogenized with 2x (w/v) NANOPURE water. Duplicate aliquots equivalent to approximately 100 mg fresh tissue weight (when sample size allows) were combusted in a Harvey Biological Sample Oxidizer followed by LSC counting. Bone marrow, adrenals, ovaries, and thyroids were combusted directly; fat was minced and combusted directly.

Levels of radioactivity in plasma, urine, and feces extract samples were determined by counting aliquots directly in a liquid scintillation counter (LSC). Total radioactive residue (TRR) levels in rat tissues, feces, or post-extraction solid (PES) samples were determined by combusting aliquots of homogenized samples in a Harvey OX-500 or OX-300 Biological Sample Oxidizer. The evolved [14C]CO2 was counted in 15 mL of Harvey Scintillation Carbon-14 Cocktail. Samples analyzed to produce excretion data were either counted in duplicate (direct counting) or triplicate (combustion analysis). Samples analyzed for product identification and metabolite profiling were usually limited to single or duplicate analysis, depending on sample availability. Samples were routinely assayed with a Beckman LS 3801, LS 5000TD, or LS 6000TA liquid scintillation counter for 10 min or until the 2-sigma error was less than or equal to 2%, whichever came first. For HPLC fractions collected, the counting time will be 2 min. Quench correction was performed using an external standard method. Oxidizer efficiencies were validated by combusting a known amount of [14C]-mannitol.

For total radioactive residue (TRR) levels in rat tissues, plasma, urine, cage rinse, and feces, the scintillation spectrometer was set to zero background. Control and treated samples for each matrix were counted, and counts per minute (cpm) were automatically converted to disintegrations per minute (dpm). The dpm from the control sample was subtracted from the treated sample; the net dpm per aliquot was used for subsequent calculations.

HPLC eluates were collected in Deepwell LumaPlate 96~plates ( 0.25 minlwell) and dried by a SpeedVac system. The dried radioactive eluates were counted for 5 min using a Packard TopCount NXT Microplate counter.

Sample Preparation For Metabolite Profiling
In general, matrix samples from each group were pooled by one time interval and gender.

Urine
Group A and Group C 0-24 hr urine samples were pooled proportionally by each group and gender for each individual rat. Duplicate aliquots were analyzed by LSC. Prior to the HPLC analysis, the urine samples were filtered with Waters Alliance Filtration Manifold filter, GHP 0.45 um. Duplicate aliquots of each filtrate were analyzed by LSC.

Feces
Homogenized feces 0-24 and 24-48 hr samples from individual rats in Group A and Group C were proportionally (~30% each) pooled within the group and gender. Total radioactive residue (TRR) levels in pooled feces were determined by combusting triplicate aliquots of homogenized samples. A subsample from each pooled sample was used for extraction.

Prior to HPLC analysis, the pooled feces samples were extracted and concentrated according to the following method:
In a 50-mL polypropylene centrifuge tube, feces pooled were mixed with ~5x v/w CH30H, and placed on a Wrist Action shaker for 10 min at high speed. The samples were centrifuged at 4C for 10 min at 3000 rpm, and the supernatant was transferred to another tube. The resulting precipitate was then vortex-mixed with a mixture of H20 and CH30H (1:9, 5x v/w), followed by shaking for 10 min. Upon shaking, the samples were sonicated for 15 min, followed by centrifugation at 4C for 10 min at 3000 rpm, and the supernatants were combined with the first supernatant. The volume of the supernatants was adjusted to 25 mL using methanol, and aliquots were taken for LSC. Triplicate aliquots (ca. 50 mg) of dned PES were combusted with a Harvey Biological Sample Oxidizer and radioassayed by LSC for the determination of radioactivity.

The same procedure as described above was followed with a pre-dose feces sample with fortified 14C-RH-573 to evaluate the extractability and extraction stability of he parent compound.


Determination of Metabolite Profiles
The metabolite radioprofiles of 14C-RH-573 in urine were obtained on a waters 2695 HPLC System coupled with a Packed Radiomatic Series 500TR Flow Scintillation Analyzer on-line.

The metabolite radioprofiles of 14C-RH-573 in feces were determined by a waters 2695 HPLC System. Fractions of chromatography effluent were collected in Deepwell LumaPlate -96 by time (0.25 min/fraction). The plates were dried by a Savant SpeedVac prior to the radioactivity assay by TopCount NXT Counter. Radioactive peaks were integrated to determine the percent distribution of individual radioactive peaks or regions in each sample.

The reference standard, RH-573, was also analyzed under the same HPLC conditions as described above, and its retention time was determined.

Isolation and Purification of Metabolites for LC/MS Analysis
Due to a large amount of co-eluting matrices, the direct LCMS analyses of either urine sample or feces extract could not provide any definitive molecular ions for most of the metabolites. One urine sample GA-M-U24, and one feces extract, GC-F-F24, were subjected to preparative HPLC and the eluates were collected every 15 seconds into 2-mL 96-well fraction collection plates. Multiple collections were made for each sample in order to obtain a sufficient quantity of metabolites for subsequent HPLC or LCMS and/or LCMS/MS analyses. The following fractions were pooled for the secondary analyses.

Isolation and Purification of Metabolites for LCIMS and/or NMR Analysis
Isolation and Purification of M9
One urine sample, Group C male rat urine, was subjected to the initial preparative HPLC. The eluates from the retention time region of 15.25-16.25 min were subjected to an additional preparative HPLC. The major peak at the retention time region of 42 min was subjected to High Resolution TOF LC/MS to obtain accurate mass of the metabolite.

Isolation and Purification of M12
One urine sample, Group C male rat urine, was subjected to the initial preparative HPLC. The eluates from the retention time region of 24-24.5 min were subjected to an additional preparative HPLC. The major peak at the retention time region of 44 min was dried, followed by reconstitution in water using sonication. The reconstituted sample was subjected to solid phase cartridge cleanup using a C18 cartridge (Phenomenex strada C18-T, 55 pum, 500 mg) to remove salts used in tine mobiie phase. The retained metaboiite was eluted with methanol and the eluate was completely dried under nitrogen stream, followed by freeze-drying. Dried M12 isolate was reconstituted with d5-pyridine (99 atom %D) in a 3-mm NMR capillary tube and subjected to NMR Analyses (Bruker AVANCE 500).

Derivation of M12 Isolate
The remaining MI2 isolate was mixed with diazomethane in diethyl ether and the mixture was left at ambient temperature for 2 hr. Upon derivation, diethyl ether was removed by nitrogen stream. The dry sample was reconstituted with methanol, which was subjected to LC/MS analysis.

Deuterium Exchange of MI2 Isolate
After NMR analyses, a subsample of the M12 isolate in d5-pyridine was dried to remove pyridine, followed by reconstitution in CH3CN and DzO. M12 isolate was analyzed by LC/MS before and after deuterium exchange.

Identification of 14C-RH-573 and Its Metabolites by LC/MS
The isolates from the urine and feces extracts were analyzed by LC/MS. A urine sample was also directly analyzed by LC/MS.

Identification of Metabolite M12 by NMR
The purified M12 isolate was subjected to NMR analyses. Rohm & Haas Co conducted the NMR analyses.

Phamacokinetic Analysis
Pharmacokinetic parameters were estimated by non-compartmental techniques using validated WinNonlin software (Pharsight, version 3.2). The maximum concentration (Cmax) was obtained using the plasma concentrations of individual rats. The area under the plasma concentration-time curve over time was computed using the linear trapezoidal method. The first-order terminal elimination rate constant was determined by linear regression of the terminal phase of the log phase of the log plasma concentration curve. The apparent terminal half-life (t1/2) was calculated as t1/2 = 0.693Kel. The area under the curve from time zero to time infinity (AUC0-inf) was calculated as AUC0-inf= AUC0-t + Ct/ Kel. The plasma concentration versus time curve follows a biphasic elimination. The initial (faster phase) rate of elimination was estimated by using data points from 1 hr to 24 hr. The initial elimination half-life was determined from this parameter.

Details on dosing and sampling:

Dose Administration
Each treated rat received a single dose by oral gavage at the nominal dose based upon individual body weights taken prior to dosing. The actual amount of dose solution administered to each animal was determined by weighing the loaded dose syringe before dose administration and the emptied syringe after dose administration.

Sample Collection
Urine, Cage Rinse, and Feces
Urine was collected at pre-dose, 0-24, 24-48, 48-72, and 72-96 hr post-dose. The samples were collected into tared sample cups and freeze-trapped using dry ice to avoid atmospheric oxidation, evaporation, and bacterial degradation. Cages were rinsed with NANOPURE water at 24, 48, and 72 hr post-dose; at the end of the study, cages were thoroughly washed with IPA/water (1:1). All cage rinse/wash samples were collected in tared sample containers. The feces samples were collected at the same timepoints as the urine at room temperature from the cage screen, weighed, and stored frozen upon collection. Urine, cage rinse, and feces were collected at the time of sacrifice from animals in Group E.

Blood and Plasma
Blood (~0.4 mL) was collected at 1, 3, 6, 24, 48, and 72 hr post-dose from the tail veins of Groups B and D rats. At 96 hr post-dose, animals from all dose groups were euthanized by an overdose of CO2. Blood (~5 mL) was collected by cardiac puncture.

Statistics:

Not applicable.

Preliminary studies:

Not applicable.

Details on absorption:

Following oral administration of l4C-RH-573, a majority of radioactivity was recovered in urine and cage rinse, with a lesser amount recovered in feces. Most of the radioactivity (80 to 87% of dose) was recovered within 24 hr post-dose. In low dose groups, 65.21%, 4.52%, and 20.65% of dosed radioactivities were recovered in urine, cage rinse, and feces, respectively, from male rats; 56.04%, 8.37%, and 29.11% were recovered in urine, cage rinse, and feces, respectively, from female rats. In high dose groups, 49.59%, 6.45%, and 33.51% were recovered in urine, cage rinse, and feces, respectively, from male rats whereas 47.13%, 6.12%, and 37.39% were recovered in urine, cage rinse, and feces, respectively, from female rats.

Details on distribution in tissues:

Radioactivity concentration in selected tissues from low and high dose mass balance groups at 96 hr post-dose and from Group E at 1 hr post-dose were determined by combustion. Total recoveries of radioactivity in blood, fat, and muscle were calculated based on total mass in the animal body. The tissue radioactivity amounted to 2.32% to 4.14% of the dose in low dose groups and 1.75% to 2.48% of the dose in high dose groups. After 96 hr the majority of the dose remaining in tissues was found in the blood (1.51 to 2.63% of dose in blood of low dose and 1.21 to 1.71% of dose in blood at high dose). Blood, thyroid, and lungs contained the highest radioactivity in low dose groups; thyroid, lungs, spleen, kidneys, and heart contained the highest radioactivity in high dose groups (when concentrations were expressed as ug equivalents per g of tissue).

At 1 hr post-dose, following oral low dose, more radioactivity was found in the kidneys followed by thyroid, liver, and blood (when concentrations were expressed as ug equivalents per g of tissue).

Details on excretion:

Following oral administration of l4C-RH-573, a majority of radioactivity was recovered in urine and cage rinse, with a lesser amount recovered in feces. Most of the radioactivity (80 to 87% of dose) was recovered within 24 hr post-dose. In low dose groups, 65.21%, 4.52%, and 20.65% of dosed radioactivities were recovered in urine, cage rinse, and feces, respectively, from male rats; 56.04%, 8.37%, and 29.11% were recovered in urine, cage rinse, and feces, respectively, from female rats. In high dose groups, 49.59%, 6.45%, and 33.51% were recovered in urine, cage rinse, and feces, respectively, from male rats whereas 47.13%, 6.12%, and 37.39% were recovered in urine, cage rinse, and feces, respectively, from female rats.

Metabolites identified:

yes

Details on metabolites:

Percent Distribution of Metabolites
Metabolites were designated according to their retention times. The retention times obtained during the HPLC radioprofiling were generally similar to those obtained during the LC/MS and LC/MS/MS analyses, which used the same HPLC conditions as for the radioprofiling. The slight shift in retention times observed between runs was consistent with all the metabolites, based on the results from the analysis of the reference standards. The metabolite numbers assigned were consistent between matrices. [Note that percent dose values of each metabolite expressed in two decimal places did not represent the number of significant figures. Two decimal places were used to obtain cumulative values closer to the starting percent dose values.]

Urine
Four urine samples were directly analyzed by RP-HPLC for radioprofiling. Metabolites MI, M3, M9, and M12 each accounted for >5% of the dose in at least one of the urine samples, and all others accounted for 6 % of the dose. M1, M3, M4, M5, M7, M8, M9, and M12 in urine were further analyzed by LC/MS and/or LC/MS/MS for their characterization and identification.

Feces
The pooled feces samples were analyzed for TRR. The recovery of radioactivity from the extracted feces samples was quantitative, ranging from ~95% to ~110%. The PES ranged from 0.29% to 9.84% of the dose, and was not further analyzed. The extracts from the pooled feces samples were concentrated prior to analysis by RP-HPLC.

The results from a fortified feces extraction demonstrated that I4C-RH-573 could be quantitatively recovered in the extract: >93% of the spiked 14C-RH-573 was recovered in the extract. The HPLC metabolite profile of the extract showed a major radioactive peak corresponding to RH-573, demonstrating that the parent compound remained stable throughout the extraction procedure. Parent compound was not detected in fecal samples analyzed. The metabolites were assigned according to their retention times. The major radioactive peak in the feces extracts was not retained in the HPLC conditions used. The unretained radioactive peak, designated as M2, was isolated from Group C female 24-hr feces extract and the isolated fraction was analyzed by NP-HPLC. The fraction was composed of four radioactive components, each designated as M2-A, M2-B, M2-C, and M2-D.

Identification of 14C-RH-573 and Its Metabolites
Among 23 metabolites detected from rat urine and feces samples during the initial HPLC radioprofiling, metabolites MI, M2, M3, M9, and M12 each accounted for >5% of the dose in at least one of the urine or feces samples. They were further analyzed by LC/MS and LC/MS/MS for their structures. The urine and isolated metabolite samples from each matrix were analyzed by radio-HPLC coupled with mass spectrometry. Molecular ions corresponding to individual radioactive peaks were obtained. For most of the metabolites, the proposed structures were derived from MS/MS analysis. Whenever applicable, the resulting LC/MS and MS/MS data were compared to those from reference standards. The mass spectral data of isolated metabolites MI, M2, M3, and M5 still did not provide any definitive molecular ion using RP-HPLC. NP-HPLC was used in the LC/ESI-MS analyses for MI, M2, M3, and M5 analyses. Following the discussion of RH-573, the metabolites were discussed by order of elution. Pre-dose urine samples (control urine) were also analyzed by LC/UV/RAM/(+)ESI/MS, and the data was used to distinguish metabolites from the matrix.

Due to the poor UV absorption ability of the reference standards, the retention times of the reference standards was established by LC/MS.

14C-RH-573.
The protonated molecular ion ([M+H]+) was observed at m/z 116 (HPLC Rt ca. 16.5 min). Characteristic fragment ions were observed at m/z 101, 85, 71, and 58. Unchanged RH-573 was not found in urine and fecal extracts.

Metabolite M1
M1 was detected at Rt ~3.6 min in the urine sample by radioprofiling. An initial direct LC/MS analysis of a urine sample using the same HPLC condition could not provide any definitive molecular ion corresponding to M1 due to a large amount of the co-eluting matrices. Therefore, the M1 isolate was analyzed by LC/MS using NP-HPLC. M1 eluted at Rt 39.5 min and yielded mass ion peaks at m/z 118 and m/z 135 corresponding to [M+H]+ and an adduct ion [M+NH~]+ by NP-HPLC. It indicated that the MW of the compound was 117. The molecular weight and HPLC retention time of M1 were consistent with those of the reference standard N-methyl malonamic acid. LC-ESI(+)/MS/MS analysis of MI. also yielded similar product ions to those of N-methyl malonamic acid. Thus, M1 was identified as N-methyl malonamic acid. Although not distinctive, M1 was then directly detected from the urine sample using LC/MS. Structure of M1 is: CH3NHCOCH2COOH

Metabolites M2-A. M2-B. and M2-C
M2 was detected as a major component by radioprofiling at Rt ~4.25 min in feces extract. Due to a large amount of co-eluting matrices, the direct LC/IMS analyses of the feces extract sample could not provide any definitive molecular ion of M2. M2 was isolated using a preparative HPLC from a feces extract. Under an acidic condition using NP-HPLC, the M2 isolate showed one minor and three major radioactive components at Rt ~4.7, ~37, and ~52.2 min. The LC/MS analysis of the M2 isolate under LC/MS Condition4 (neutral mobile phase) yielded four detectable radioactive peaks at ~4.2, 34.6, 35.2, and 50.3 min. The four peaks were designated as M2-A, M2-B, M2-C, and M2-D. Although their retention order did not change, M2-A, M2-B, M2-C, and M2-D eluted at slightly earlier Rt's under the neutral LC condition, indicating that these components might have had an acidic functional group on their structures.

M2-A did not yield any distinctive molecular ions under either LC/MS condition. M2-B and M2-C yielded the molecular ion [M+H]+ at m/z 176 and 204 in positive ion mode, indicating the MW of each compound as 175 and 203 daltons, respectively.

Based on the LC/MS/MS data, the MS fragmentation schemes of M2-B and M2-C are proposed. The parent compound can undergo reductive ring cleavage followed by glutathione conjugation, which in turn can be further metabolized to form a cystein conjugate. S-substituted cysteins may undergo transamination yielding the corresponding thiopyruvic acids, which are further metabolized to thiolactic and thioacetic (thioglycolic) acids. Although the conjugation position and stereo confirmation could not be assigned from limited MS/MS data, the position is most likely to be on C5 position rather than C4 position according to the published findings that glutathione conjugation favors on the B-position of an a, B-unsaturated carbonyl group.

The LC-ESI(+)/MS and LC-ESI(-)/MS analyses of M2-D yielded characteristic 14C -isotope peaks at m/z 321 and 319, corresponding to [(M+~)+H]+an d [(M+2)-H]-, respectively. None of the other metabolites showed a characteristic 14C-isotope peak, which indicated that 14C-incorporation into the molecule might have been increased through the polymerization of the parent or one of the metabolites. The LC-ESI(+)/MS/MS spectrum yielded a characteristic fragment ion at m/z 288, which is considered to result from the loss of CH3NH2, indicating the presence of CH3NH-moiety in the molecule. The characteristic fragment ion at m/z 273 was considered to result from the loss of HCOOH, indicating that the molecule contained the free carboxylic acid moiety. The ion at m/z 237 could have resulted from the loss of 2x H20 from the ion at m/z 273. Although the LC-ESI(-)/MS/MS did not yield distinctive fragment ions under the mass spectral conditions used, the fragment ion at m/z 300 (resulting from the loss of 17 amu) indicated that it contains a primary amine. However, the mass spectral data are not sufficient enough to propose any reasonable structure of M2-D.
HOOCCH2SCHCHCONHCH3
M2-B

HOOCCOCH2SCHCHCONHCH3
M2-C
Metabolite M3
M3 was detected by radioprofiling at Rt ~5.2 min. Due to a large amount of co-eluting matrices, the direct LC/MS analyses of the urine sample or the M3 isolate from urine using LC/MS could not provide any definitive molecular ion of M3. M3 eluted at Rt ~3.5 rnin under the same NP-HPLC condition as used for M1, indicating that it might be a less polar and less acidic compound than MI. A much less polar mobile phase, methylene chloride, was used in the HPLC condition in order to get M3 retained in the column. A post column addition of 0.4% formic acid aqueous solution was made to the column effluent before the MS instrument to improve the ionization. LC/RAM/(+)ESI-MS analysis of M3 exhibited characteristic mass ion peaks corresponding to (M+H)+/(M+NH4)+ ions at m/z 104/121 at Rt ~25 min, suggesting a MW of 104. Although the molecular weight of M3 was the same as that of malonamic acid, the retention time of M3 did not match with that of malonamic acid. While M3 eluted at Rt ~3.6 min, malonamic acid eluted at Rt ~43 min.

Based on the LC/MS/MS data, the structure for M3 was proposed as follows: CH3NHCOCH2CH2OH

Although not distinctive, M3 was then detectable from the urine isolate.
Metabolites M4, M5, and M6
M4, M5, and M6 were detected by radioprofiling at Rt ~7, ~8, and ~9 min in urine, respectively. A direct LC/MS analysis of the compounds in urine showed distinctive peaks corresponding to the mass ion peak at m/z 311 for [M+H]+ The molecular ion was 196 amu higher than that of the parent compound, indicating that these compounds were conjugated metabolites. MS/MS analyses of the three compounds in the positive ion mode also yielded very similar fragment ions at m/z 247, 216, 205, 174, and 132. Thus, these three components were considered to be conjugated isomers. Based on the LC/MS and MS/MS data, M4, M5, and M6 were proposed as mercapturic acid conjugates of RH-573 after the reductive ring cleavage, followed by S-methylation and oxidation to form sulfoxide. Proposed structure for M4, M5 and M6 is: CH3CONHCH(COOH)CH2SCH(SOCH3)CH2CONHCH3

Metabolites M7-A and M7-B
M7 was detected by radioprofiling at Rt ~12 min in urine. A direct LC/MS analysis of the compounds in urine showed two distinctive peaks corresponding to the mass ion peak at m/z 148 and 245 for [M+H]+. The two compounds co-eluted at a similar retention time. The molecular ion of M7-A was 32 amu higher than that of the parent compound, indicating that an oxygen atom may have been added. MS/MS analyses of the compound in the positive ion mode yielded a distinctive fragment ion at m/z 84 in addition to other fragment ions at 130, 117, and 101. Based on the LC/MS and MS/MS data, M7-A was proposed to be formed from reductive S-N bond cleavage of RH-573, followed by S-methylation and oxidation to form sulfoxide. Based on the data, the structure of M7-A was proposed as: CH3SOCHCHCONHCH3

The molecular ion of M7-B was 129 amu higher than that of the parent compound, indicating that it's a conjugated metabolite. MS/MS analyses of the
compound in the positive ion mode yielded distinctive fragment ions at m/z 203, 172, 158, and 116. Based on the LC/MS and MS/MS data, M7-B was proposed to be formed from an oxidation product (sulfone) of the parent compound. The proposed structure is: CH3CONHCH(COOH)CH2SCCCONHCH3

M8 -A and M8 -B
M8 was detected by radioprofiling at Rt ~13 min in urine. A direct LC/MS analysis of the compounds in urine showed two distinctive peaks corresponding to the mass ion peak at m/z 148 and 245 for [M+H]+. The two compounds co-eluted at a similar retention time. The molecular ion of M8-A was 32 amu higher than that of the parent compound, indicating that an oxygen atom may have been added. MS/MS analyses of the compound in the positive ion mode yielded a distinctive fragment ion at m/z 117 in addition to other small fragment ions. The mass spectrum was different from that of M7-A, indicating that they are different compounds with the same molecular weights. Based on the LC/MS and MS/MS data, M8-A was proposed to be formed from oxidation of RH-573.

The molecular ion of M8-B was 129 amu higher than that of the parent compound, indicating that it's a conjugated metabolite. MS/MS analyses of the compound in the positive ion mode yielded similar fragment ions as shown with M7-B indicating that they might be isomers. Based on the LC/MS and MS/MS data, M7-B was proposed to be formed from an oxidation product (sulfone) of the parent compound. The proposed structure is: CH3CONHCH(COOH)CH2SCCCONHCH3

Metabolites M9-A and M9-B
M9 was detected by radioprofiling at Rt ~15.7 min in urine, which showed one peak. Due to a large amount of co-eluting matrices, the direct LC/MS analyses of the urine sample or M9 isolate could not provide any definitive molecular ion of the metabolite. An NP-HPLC was used in LC/MS analyses. The M9 isolate showed two radioactive peaks at Rt 37.4 and 38.4 min, both peaks corresponding to the mass ion peak at m/z 247 for [M+H]+ and an adduct ion at m/z 264 for [M+NH4]+. The molecular ion was 131 amu higher than that of the parent compound, indicating that these two compounds were conjugated metabolites. MS/MS analyses of the two compounds in the positive ion mode also yielded very similar fragment ions at m/z 230, 216, 187, 174, and 132. Thus, these two components in the M9 isolate were considered to be two conjugated isomers, designated as M9-A and M9-B. Based on the LC/MS and MS/MS data, M9-A and M9-B were proposed as a pair of mercapturic acid conjugates of RH-573 after the loss of the sulfur atom from the isothiazolinone ring. APCID-LCI(+)IESI/MSNS of a product ion at m/z 174 from the two compounds yielded the same fragment ions, supporting the theory that the compounds were isomers. The conjugation position and stereo confirmation could not be assigned from the limited data. Based on the reported findings that glutathione conjugation favors on the B-position of an a, B-unsaturated carbonyl group, the isomer was considered to be a stereo isomer rather than a positional isomer. The proposed structure is: CH3CONHCH(COOH)CH2SCHCHCONHCH3

The exact mass of M9B was determined to be 247.0753. The accurate mass of the formula of C9HI4N2O4S w as calculated as 247.075253, which yielded mass accuracy of <0.2 ppm with the experimental value of M9B. In addition, it also indicated the presence of a sulfur atom showing a distinctive peak at m/z 249 corresponding to [M+H+2]+at the ratio of ~4% of the molecular ion, demonstrating that it resulted from 34S isotope peak.

Metabolite M12
M12 was detected by radioprofiling at Rt ~25 min in urine. LC/RAM/ESI(+)MS analysis of the MI2 isolate produced an intense molecular ion [M+H]+ at m/z 295 and an adduct ion [M+Na]+at m/z 317, suggesting a MW of 294. The molecular ion was 180 amu higher than that of the parent compound, and 48 amu higher than that of M9, indicating an additional sulfinyl moiety or S-methyl moiety to M9. LC/ESI(+)-MS/MS of M12 yielded major fragment ions at m/z 247, 216 (247-CH3NH2), 205 (247-CH2CO), 174 (216- CH2CO), and 132 (from the mercapturic acid moiety).

LC/RAM/ESI(+)MS analysis of methylated product of M12 yielded an intense quasi-molecular ion [M+H]+ at m/z 309 and an adduct ion [M+Na]+ at m/z 322, indicating the presence of a carboxylic acid moiety.

The mass spectral data of M12 was obtained before deuterium exchange. Based on the results, MS ion fragmentation indicated that there were three exchangeable protons in M12, one in carboxylic acid, and the others in two amide moieties. The ESI(+)MS/MS spectrum of deuterated M12 was obtained from the ion at m/z 310 corresponding to [d3-M+Na]+ because its intensity was much higher than that of the ion at m/z 299.

Purified M12 was subjected to NMR analysis. 1H-1H COSY (clearly showed four pairs of protons that were J-coupled. It indicated that the protons were on adjacent carbons. The amide proton at 8.82 showed a correlation with the methyl signal at 2.87 (J=4.5 Hz), which indicated an N-S bond cleavage on the isothiazolinone ring. The methine proton at 5.02 had a correlation with the methylene protons at 3.03, which indicated that the CH=CH double bond of RH-573 was saturated and connected to a conjugated moiety. The methane proton at 5.33 displayed a correlation with the amide proton at 8.92 and methylene protons at 3.67, which confirmed the moiety of proposed mercapturic acid conjugates. Assignments of the proton and carbon chemical shifts were made based on the 1H-1H COSY, HMQC, and HMBC analyses. Based on the NMR and MS data, the structure for M12 was confirmed as follows: CH3CONHCH(COOH)CH2SCH(CCH3)CH2CONHCH3

The body weights of the rats on the day of dosing ranged from ~214-277 g. The rats received a mean dose of 4.9-5.1 mgikg in the low dose groups, and 49.2-50.0 mg/kg in the high dose groups; the target dose was 5 and 50 mg/kg in the respective groups. No untoward behavioral changes, ill health, or reaction to treatment were observed for the duration of the study.

Mass Balance

Following oral administration of ^l4C-RH-573, a majority of radioactivity was recovered in urine and cage rinse, with a lesser amount recovered in feces. Most of the radioactivity (80 to 87% of dose) was recovered within 24 hr post-dose. In low dose groups, 65.21%, 4.52%, and 20.65% of dosed radioactivities were recovered in urine, cage rinse, and feces, respectively, from male rats; 56.04%, 8.37%, and 29.11% were recovered in urine, cage rinse, and feces, respectively, from female rats. In high dose groups, 49.59%, 6.45%, and 33.51% were recovered in urine, cage rinse, and feces, respectively, from male rats whereas 47.13%, 6.12%, and 37.39% were recovered in urine, cage rinse, and feces, respectively, from female rats.

Total mean recovery from urine, cage rinse, feces, and tissues ranged from 91.71 % to 96.00%.

Plasma and Blood Concentrations and Pharmacokinetics

T_max was 1 hr in the low dose group for both male and female animals; it was 1.7 hr in the high dose group for males and 3 hr for females. Initial rates of elimination of ¹⁴C-label from plasma were rapid and ranged from 3 to 6 hr in low and high dose groups.

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
RH-573 was extensively metabolized in the rat. Based on the metabolites in feces and urine characterized by LC/MS and LC/MS/MS as well as NMR, the major metabolic pathways could be proposed as follows: 1) Phase I metabolites derived from a) oxidative cleavage of the isothiazolinone moiety with the loss of the sulfur atom and the C4, C5 double bond reduction of the moiety (MI and M3); b) oxidation of the intact parent compound (M8A), and c) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A); 2) Phase I1 metabolites derived from a) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A), followed by glutathione conjugation, with further degradation of the glutathione moiety to yield a cysteine conjugate, whch then formed a mercapturic acid moiety (M4, M5, and M6); b) reductive cleavage of the S-N bond, followed by methylation and glutathione conjugation, with further metabolism of the glutathione moiety to yield a cysteine conjugate, which then formed a mercapturic acid moiety (M12); c) reductive cleavage of the S-N bond, followed by the formation of mercapturic acid conjugate (M9); d) oxidation of the intact parent compound, followed by loss of sulfur dioxide, then formation of mercapturic acid conjugate (M7B and M8B); and e) reductive cleavage of the S-N bond, followed by the formation of cysteine conjugate, then transamination of the cysteine moiety (M2C), followed by decarboxylation and further oxidation (M2B).

Executive summary:

The mass balance, pharmacokinetics, tissue distribution and metabolism of [4,5-¹⁴C]-RH-573 were investigated in rat.

¹⁴C-RH-573 was administered to male and female rats at low (5 mg/kg) and high (50 mg/kg) dose levels. Urine, cage rinse, and feces were collected from rats at 24-hr intervals for a total of 96 hr. In separate groups, blood and plasma were collected at 1, 3, 6, 24, 48, 72, and 96 hr post-dose. Selected tissues were collected from a low dose group at 1 hr (Tmax) post-dose; the same tissues were also collected from the low and high dose mass balance groups. All samples were assayed for radioactivity concentrations. The percentages of the doses recovered in the excreta and tissues from the mass balance groups were calculated. PK parameters were also determined from the blood and plasma data.

The excretion of ¹⁴C-RH-573 (and metabolites) was rapid in the rat. A majority of the radioactivity was excreted from the rats in 24 hr (80-87%); most of the radioactivity was recovered in urine and cage rinse (53-70%), and a lesser amount was found in feces (21-37%). Tissues contained 1.9-3.6% of dosed radioactivity. Total mean recovery of radioactivity ranged from 92-96%.

Tmax was 1 hr in low dose groups (both gender); in high dose groups it was 1.7 hr in males and 3 hr in females. Blood concentration was higher than plasma. The half-life of elimination (T_l/2 initial) from plasma was 3-6 hr and not dose-dependent. No gender difference was observed.

Pooled urine and feces were analyzed according to their collection interval, gender, and dose group for the parent compound and corresponding metabolites using HPLC with radioactivity monitoring. The abundance of each product, expressed as the percentage of the administered dose, was determined for each HPLC radioactive region. In general, metabolites were identified by comparing their retention times with those of reference standards, as well as by LC/MS/MS analyses.

Twenty-three radioactive components (designated as M1 - M23) were observed in urine and feces samples from the HPLC radioprofiling. Among these, M1 (N-Methyl malonamic acid) and M12 (3-mercapturic acid conjugate of 3-thiomethyl-N-methylpropionamide) were detected as the major components in the urine. M3 (N-methyl-3- hydroxyl-propionamide) was also detected in urine at levels in the range of ~4% to 5% of the dose. M2 contained at least three components and was the major component detected in the feces. Other metabolites such as M9-A/M9-B were also proposed as mercapturate conjugates. Parent compound was not detected in either urine or feces samples.

Sixty-nine (69%) to seventy-seven (77%) percent cumulative dose from urine and feces in all 4 dose groups was profiled by HPLC radiochromatography and identified/characterized. Feces post-extraction solids (PES) accounted for 0.43% to 9.84% of the dose. The major radioactive peaks that accounted for >1% of the dose were either identified or characterized.

In summary, RH-573 was extensively metabolized in the rat. Based on the metabolites in feces and urine characterized by LC/MS and LC/MS/MS as well as NMR, the major metabolic pathways could be proposed as follows: 1) Phase I metabolites derived from a) oxidative cleavage of the isothiazolinone moiety with the loss of the sulfur atom and the C₄, C₅ double bond reduction of the moiety (MI and M3); b) oxidation of the intact parent compound (M8A), and c) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A); 2) Phase I1 metabolites derived from a) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A), followed by glutathione conjugation, with further degradation of the glutathione moiety to yield a cysteine conjugate, whch then formed a mercapturic acid moiety (M4, M5, and M6); b) reductive cleavage of the S-N bond, followed by methylation and glutathione conjugation, with further metabolism of the glutathione moiety to yield a cysteine conjugate, which then formed a mercapturic acid moiety (M12); c) reductive cleavage of the S-N bond, followed by the formation of mercapturic acid conjugate (M9); d) oxidation of the intact parent compound, followed by loss of sulfur dioxide, then formation of mercapturic acid conjugate (M7B and M8B); and e) reductive cleavage of the S-N bond, followed by the formation of cysteine conjugate, then transamination of the cysteine moiety (M2C), followed by decarboxylation and further oxidation (M2B).

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January - 20 April 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: OECD Guideline #28

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Species:

human

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Extraneous tissue was removed from human whole skin samples obtained from surgery or post mortem. The skin samples were immersed in water at 60C for 40-45 seconds and the epidermis teased away from the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminum foil until required for use.

Type of coverage:

occlusive

Vehicle:

other: See test material for the various formulations examined.

Duration of exposure:

24 hour

Doses:

Aqueous solutions of MI (313 ug, 104.3 ug and 52.2 ug MI/ml) and three undiluted formulations were examined

No. of animals per group:

six skin membranes obtained from at least 3 subjects were used for each dose group.

Control animals:

no

Details on in vitro test system (if applicable):

Assembly of Diffusion Cells
The type of glass diffusion cell used in this study has an exposed membrane area of 2.54 cm(2). Discs of approximately 3.3 cm diameter of prepared skin membrane were mounted, dermal side down, in diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32 +/- 1C.

Measurement of Membrane Integrity
Membrane integrity was determined by measurement of the electrical resistance across the skin membrane. membranes with a measured resistance of <10kOhmes (Davies et al., 2004). were regarded as having a lower integrity than normal and not used for exposure to the test materials.

Measurement of Test Substance Absorption
Cells were selected such that each application was represented by six intact membranes from at least three different subjects.

The receptor chambers of the cells, containing small magnetic stirrer bars, were filled with a recorded volume of water as the receptor fluid. MI is soluble in water and this choice of receptor fluid ensured that the test substance could freely partition into the receptor fluid from the skin membrane and never reached a concentration that limited its diffusion.

A pre-treatment sample (0.5 ml) was taken from each receptor chamber for analysis by LSC. An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed. The receptor chambers were stirred continuously throughout the exposure period.

Prior to dosing the aqueous solutions, the cells were placed in a water bath maintained at normal skin temperature of 32 +/-1 C to allow the receptor fluid temperature to equilibriate. For the formulations, which were applied by weight, this was not possible and the cells were not placed in the waterbath until after dosing.

The three aqueous solutions of MI (313 ug, 104.3 ug and 52.2 ug MI/ml) and the undiluted formulations were applied evenly to the skin membranes at rates of 20 ul/cm(2) and 20 mg/cm(2), respectively, and occluded for the duration of the exposure period (24 hr). These applications were designed to simulate as close as possible the potential human dermal exposure to the formulations during normal use. These occluded conditions however, may over-predict true MI absorption. Initial experiments, dosed as above but without occlusion, showed that MI was too volatile to obtain meaningful data.

At recorded intervals (1, 2, 3, 6, 8, 12 and 24 hr), 0.5 ml samples of the receptor fluid were taken for analysis. The volume of fluid in the receptor chamber was maintained by the addition of 0.5 ml fresh receptor fluid to the chamber immediately after the removal of each sample.

Measurement of Mass Balance
After the final receptor fluid sample had been taken, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid which was also discarded. The donor chamber was carefully removed, washed with water and the sample analyzed for MI by LSC.

The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol, and with further sponges pre-wetted with water. Decontamination was shown to be complete following assessment of residual radioactivity levels on the skin surface with a Geiger counter. The sponges were digested in Soluene 350 and made up to a recorded volume. A sample was taken for analysis.

To assess penetration through the stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape (eg Scotch 3M Magic Tape, 1.25 cm wide), to a maximum of 5 strips. In some cases, it was not possible to take the full 5 tape strips, as the epidermis began to tear during the process described below. in such cases, tape stripping was discontinued at first tearing and the last tape strip taken was digested with the remaining epidermis, so as not to underestimate residues in the remaining epidermis compartment.

The surface of the skin was allowed to dry naturally. A strip of adhesive tape was pressed onto the skin surface and then carefully peeled off to remove the stratum corneum. the adhesive strips were soaked in Soluene 350 to extract any test material. The strips were extracted individually and sequentially numbered prior to analysis by LSC.

The remaining epidermis was carefully removed from the receptor chamber and digested in Soluene 350 and the whole digest analysed.

Reference:
Davies, D.J., Ward, R.J. and Heylings, J.R. (2004). Multi-species Assessment of Electrical Resistance as a Skin Integrity Marker for In vitro Percutaneous Absorption Studies. Toxicology In Vitro 18:351-358.

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

Aqueous solutions
The fastest rate of MI absorption from all aqueous applications was between 8-12 hr after dosing. Little significant absorption occurred before 6 hr. After 12 hr absorption rapidly slowed, most probably due to depletion of the applied MI reservoir, giving 3 phase of sigmoidal profiles.

313 ug/ml solution
During the first 8 hr of exposure the mean absorption rate for MI was 0.017 ug/cm(2)/hr, with 2.53% (0.158 ug/cm(2)) of the dose being absorbed within that time. Over the following 4 hours, the mean rate had increased to 0.577 ug/cm(2)/hr, with 39.4% (2.47 ug/cm(2)) being absorbed by 12 hr. By the end of the exposure period at 24 hr, 54.7% (3.42 ug/cm(2)) of the dose had been absorbed.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 10.8%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 65.5%.

By the end of the exposure period, only 7.03% of the dose was washed from the surface of the epidermis and 1.55% was recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 86.9%.

104.3 ug/ml solution
Between 0-8 hr after dosing, the mean MI absorption rate was 0.007 ug/cm(2)/h, with 3.32% (0.069 ug/cm(2)) of the dose being absorbed by 8 hr. During the 8-12 hr period, the rate was 0.132 ug/cm(2)/hr, with 28.6% (0.598 ug/cm(2)) having been absorbed by 12 hr after application. By the end of the exposure period at 24hr, 38.0% (0.793 ug/cm(2)) of the dose had been absorbed.

The MI remaining in the epidermis after tape stripping was 22.1%, giving a total potentially systemically available proportion of the dose was 60.1% after 24 hr exposure.

By the end of the exposure period, only 15.0% of the dose was washed from the surface of the epidermis and 4.27% was recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 89.9%.

52.2 ug/ml solution
During the 0-8 hr period after dosing, the mean MI absorption rate was 0.005 ug/cm(2)/hr, with 4.68% (0.049 ug/cm(2)) of the dose being absorbed by 8 hr. Over the 8-12 hr period, the rate was 0.045 ug/cm(2)/hr, with 21.8% (0.228 ug/cm(2)) having been absorbed by 12 hr after application. By the end of the exposure period at 24 hr, 29.8% (0.312 ug/cm(2)) of the dose had been absorbed.

The MI remaining in the epidermis after tape stripping after 24 hr exposure was 35.5%, thus the total potentially systemically available proportion of the dose was 65.3%.

By the end of the exposure period, 14.1% of the dose was washed from the surface of the epidermis, with 4.48% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 94.7%.

Formulations
The absorption profiles from the formulations was noticeably different from the aqueous solution, in that the profiles over the 24 hr exposure period were only 2 phase, with a period of slowly increasing absorption of between 6-12 hr, depending on the formulation, followed by a steady state period of absorption up to the end of the exposure period.

100 ug/ml shampoo
During the first 6 hr of exposure, the mean rate of MI absorption was 0.007 ug/cm(2))/hr, with 2.32% (0.046 ug/cm(2)) of the dose being absorbed by 6 hr. A maximum, steady state rate of 0.030 ug/cm(2)/hr was maintained until the end of the exposure period at 24 hr, by which time 29.5% of the dose had been absorbed.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 20.2%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 49.7%.

By the end of the exposure period, 29.7% of the dose was washed from the surface of the epidermis, with 4.06% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 91.4%.

100 ug/ml body lotion
Between 0-12 hr after dosing, the mean MI absorption rate was 0.004 ug/cm(2)/h, with 2.25% (0.045 ug/cm(2)) of the dose being absorbed by 12 hr. During the 12-24 hr steady state period, the rate was 0.011 ug/cm(2)/hr, with 8.98% (0.180 ug/cm(2)) having been absorbed by 24 hr.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 16.9%, thus the total potentially systemically available proportion of the dose was 25.9%.

By the end of the exposure period, 69.4% of the dose was washed from the surface of the epidermis, with 3.86% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 103%.

100 ug/ml facial cream
During the 0-8 hr period after dosing, the mean MI absorption rate was 0.005 ug/cm(2)/hr, with 1.99% (0.040 ug/cm(2)) of the dose being absorbed by 8 hr. Over the 8-24 hr period to the end of the exposure period, the rate was 0.022 ug/cm(2)/hr, with a total 19.6% (0.391 ug/cm(2)) having been absorbed.

The MI remaining in the epidermis after tape stripping after 24 hr exposure was 16.5%, thus the total potentially systemically available proportion of the dose was 36.1%.

By the end of the exposure period, 49.1% of the dose was washed form the surface of the epidermis, with 2.11% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 96.0%.

Total recovery:

In the aqueous solutions, total recovery ranged from 86.9 - 94.7%.

Conversion factor human vs. animal skin:

Study was conducted on human skin

Table 1 Summary of MI Absorption from Aqueous Solutions through Human Epidermis

	Mean Absorption Rates
Details of Application of Test Materials	Time Period (hr)	Absorption rate (ug/cm^2/hr + SEM)
313 ug/ml solution	0 -8	0.017 + 0.006
	8 -12	0.577 + 0.103
	0 - 24	0.160 + 0.014
104.3 ug/ml solution	0 -8	0.007 + 0.004
	8 - 12	0.132 + 0.005
	0 - 24	0.037 + 0.005
52.2 ug./ml solution	0 - 8	0.005 + 0.002
	8 - 12	0.045 + 0.010
	0 - 34	0.015 + 0.002

Table 2 Summary of MI Absorption from Formulations through Human Epidermis

	Mean Absorption Rates
Details of Application of Test Materials	Time Period (hr)	Absorption rate (ug/cm^2/hr + SEM)
100 ug/ml Shampoo	0 -6	0.007 + 0.002
	6 - 24	0.030 + 0.005
	0 - 24	0.026 + 0.004
100 ug/ml Body Lotion	0 - 12	0.004 + 0.001
	12 - 24	0.011 + 0.002
	0 - 24	0.007 + 0.001
100 ug/ml Facial Cream	0 - 8	0.005 + 0.001
	8 - 24	0.022 + 0.004
	0 - 24	0.017 + 0.003

Conclusions:

Given the reactive nature of MI, [14C]-label present in the epidermis or having passed through the epidermis may represent parent compound or ring-opened degradation products/metabolites. It was not possible to determine if the [14C]-material, present in the tissue at the end of the study, was permanently bound or available for further absorption. Expressing the results as [14C]-MI equivalents the folowing conclusions can be drawn:

MI in aqueous solutions was readily absorbed across the epidermis following a 24 hr occluded exposure-29.8, 38.0 and 54.7% of applied dose at MI concentrations of 52.2, 104 and 313 ug/ml, respectively. When including [14C]-label retained in the epidermis, 65.3, 60.1 and 75.5% of the applied dose was 'potentially' systemically available at MI concentrations of 52.2, 104 and 313 ug/ml, respectively.

When MI (100 ug/ml) was formulated in a shampoo, body lotion and facial cream, 29.5, 9.0 and 19.6% of the applied dose was absorbed across the epidermis (24 hr, occluded exposure), respectively. When including [14C]-label retained in the epidermis, 49.7, 25.9 and 36.1% of the applied dose was 'potentially' systemically available from a shampoo, body lotion and facial cream, respectively, at 100 ug/ml MI concentration.

Overall, the rates of absorption of MI (100 ug/ml concentration) across human epidermis were slower when formulated in personal care formulations (0.007 to 0.026 ug/cm(2)/hr during 24 hr exposure) compared to the rate of absorption of MI (104 ug/ml concentration) in aqueous vehicle (0.037 ug/cm(2)/hr during 24 hr exposure). In addition, rates of absorption MI, in either aqueous solution or in formulations, were minimal within the first 6 hours of exposure.

Executive summary:

Study Design

The absorption of 2 -methyl-4 -isothiazolin-3 -one (MI: RH-573) from aqueous solutions (nominally 300, 100 and 50 ug MI/ml) and from three 100 ug MI/ml formulations, (shampoo, body lotion and facial cream) has been measured in vitro through human epidermis. The aqueous solutions were applied to the epidermal membranes at a rate of 20 ul/cm(2), while the formulations were applied at 20 mg/cm². The absorption of MI from these doses was measured under occlusion over an exposure period of 24 hr. The absorption process was followed using [¹⁴C]-labelled MI, which was incorporated into the doses. At the end of the exposure period, the distribution in the test system was measured. The results in this study are reported as [¹⁴C]-MI equivalents, subsequently referred to as MI.

Results

Aqueous Solutions

The fastest rates of MI absorption from all the aqueous applications was between 8 -12 after dosing. Little significant absorption occurred before 6 hr. After 12 hr, absorption rapidly slowed, most probably due to depletion of the applied MI reservoir.

313 ug/ml solution

The fastest rate of MI absorption occurred between 8 -12 hr at 0.577 ug/cm(2)/hr, with 39.4% (2.47 ug/cm2)) being absorbed by 12 hr. By the end of the exposure period at 24 hr, 54.7% (3.42 ug/cm²) of the dose had been absorbed. The residual MI in the epidermis after tape stripping at the end of the exposure period was 10.8%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 65.5%.

The total recovery of dosed [¹⁴C]-MI equivalents was 86.9%.

104.3 ug/ml solution

During the 8-12 hr period, the rate was 0.132 ug/cm²/hr, with 28.6% (0.598 ug/cm²) having been absorbed by 12 hr after application. By the end of the exposure period at 24hr, 38.0% (0.793 ug/cm²) of the dose had been absorbed. The MI remaining in the epidermis after tape stripping was 22.1%, giving a total potentially systemically available proportion of the dose was 60.1% after 24 hr exposure.

The total recovery of dosed [¹⁴C]-MI equivalents was 89.9%.

52.2 ug/ml solution

Over the 8-12 hr period, the rate was 0.045 ug/cm²/hr, with 21.8% (0.228 ug/cm²) having been absorbed by 12 hr after application. By the end of the exposure period at 24 hr, 29.8% (0.312 ug/cm²) of the dose had been absorbed. The MI remaining in the epidermis after tape stripping after 24 hr exposure was 35.5%, thus the total potentially systemically available proportion of the dose was 65.3%.

The total recovery of dosed [¹⁴C]-MI equivalents was 94.7%.

Formulations

The absorption profiles from the formulations was noticeably different from the aqueous solution, in that the profiles over the 24 hr exposure period were only 2 phase, with a period of slowly increasing absorption of between 6-12 hr, depending on the formulation, followed by a steady state period of absorption up to the end of the exposure period.

100 ug/ml shampoo

A maximum, steady state rate of 0.030 ug/cm²/hr was maintained until the end of the exposure period at 24 hr, by which time 29.5% of the dose had been absorbed. The residual MI in the epidermis after tape stripping at the end of the exposure period was 20.2%, giving a total potentially systemically available proportion of the dose was 49.7%.

The total recovery of dosed [¹⁴C]-MI equivalents was 91.4%.

100 ug/ml body lotion

During the 12-24 hr steady state period, the rate was 0.011 ug/cm²/hr, with 8.98% (0.180 ug/cm²) having been absorbed by 24 hr. The residual MI in the epidermis after tape stripping at the end of the exposure period was 16.9%, thus the total potentially systemically available proportion of the dose was 25.9%.

The total recovery of dosed [¹⁴C]-MI equivalents was 103%.

100 ug/ml facial cream

Over the 8-24 hr period to the end of the exposure period, the rate was 0.022 ug/cm²/hr, with a total 19.6% (0.391 ug/cm²) having been absorbed. The MI remaining in the epidermis after tape stripping after 24 hr exposure was 16.5%, giving a total systemically available proportion of the dose was 36.1%.

The total recovery of dosed [¹⁴C]-MI equivalents was 96.0%.

Conclusions

Given the reactive nature of MI, [¹⁴C]-label present in the epidermis or having passed through the epidermis may represent parent compound or ring-opened degradation products/metabolites. It was not possible to determine if the [¹⁴C]-material, present in the tissue at the end of the study, was permanently bound or available for further absorption. Expressing the results as [¹⁴C]-MI equivalents the folowing conclusions can be drawn:

MI in aqueous solutions was readily absorbed across the epidermis following a 24 hr occluded exposure-29.8, 38.0 and 54.7% of applied dose at MI concentrations of 52.2, 104 and 313 ug/ml, respectively. When including [¹⁴C]-label retained in the epidermis, 65.3, 60.1 and 75.5% of the applied dose was 'potentially' systemically available at MI concentrations of 52.2, 104 and 313 ug/ml, respectively.

When MI (100 ug/ml) was formulated in a shampoo, body lotion and facial cream, 29.5, 9.0 and 19.6% of the applied dose was absorbed across the epidermis (24 hr, occluded exposure), respectively. When including [¹⁴C]-label retained in the epidermis, 49.7, 25.9 and 36.1% of the applied dose was 'potentially' systemically available from a shampoo, body lotion and facial cream, respectively, at 100 ug/ml MI concentration.

Overall, the rates of absorption of MI (100 ug/ml concentration) across human epidermis were slower when formulated in personal care formulations (0.007 to 0.026 ug/cm²/hr during 24 hr exposure) compared to the rate of absorption of MI (104 ug/ml concentration) in aqueous vehicle (0.037 ug/cm²/hr during 24 hr exposure). In addition, rates of absorption MI, in either aqueous solution or in formulations, were minimal within the first 6 hours of exposure.

Description of key information

The following 2 studies were considered key for this endpoint -

Toxicokinetic study in rats -

RH-573 was extensively metabolized in the rat. Based on the metabolites in feces and urine characterized by LC/MS and LC/MS/MS as well as NMR, the major metabolic pathways could be proposed as follows: 1) Phase I metabolites derived from a) oxidative cleavage of the isothiazolinone moiety with the loss of the sulfur atom and the C4, C5 double bond reduction of the moiety (MI and M3); b) oxidation of the intact parent compound (M8A), and c) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A); 2) Phase I1 metabolites derived from a) reductive cleavage of the S-N bond, followed by methylation and oxidation (M7A), followed by glutathione conjugation, with further degradation of the glutathione moiety to yield a cysteine conjugate, whch then formed a mercapturic acid moiety (M4, M5, and M6); b) reductive cleavage of the S-N bond, followed by methylation and glutathione conjugation, with further metabolism of the glutathione moiety to yield a cysteine conjugate, which then formed a mercapturic acid moiety (M12); c) reductive cleavage of the S-N bond, followed by the formation of mercapturic acid conjugate (M9); d) oxidation of the intact parent compound, followed by loss of sulfur dioxide, then formation of mercapturic acid conjugate (M7B and M8B); and e) reductive cleavage of the S-N bond, followed by the formation of cysteine conjugate, then transamination of the cysteine moiety (M2C), followed by decarboxylation and further

oxidation (M2B).

Dermal absorption study in humans -

Given the reactive nature of 2-methylisothiazol-3(2H)-one (MI), [14C] -label present in the epidermis or having passed through the epidermis may represent parent compound or ring-opened degradation products/metabolites. It was not possible to determine if the [14C]-material, present in the tissue at the end of the study, was permanently bound or available for further absorption. Expressing the results as [14C]-MI equivalents the folowing conclusions can be drawn:

MI in aqueous solutions was readily absorbed across the epidermis following a 24 hr occluded exposure-29.8, 38.0 and 54.7% of applied dose at MI concentrations of 52.2, 104 and 313 ug/

ml, respectively. When including [14C]-label retained in the epidermis, 65.3, 60.1 and 75.5% of the applied dose was 'potentially' systemically available at MI concentrations of 52.2, 104 and 313 ug/ml, respectively.

When MI (100 ug/ml) was formulated in a shampoo, body lotion and facial cream, 29.5, 9.0 and 19.6% of the applied dose was absorbed across the epidermis (24 hr, occluded exposure), respectively. When including [14C]-label retained in the epidermis, 49.7, 25.9 and 36.1% of the applied dose was 'potentially' systemically available from a shampoo, body lotion and facial cream, respectively, at 100 ug/ml MI concentration.

Overall, the rates of absorption of MI (100 ug/ml concentration) across human epidermis were slower when formulated in personal care formulations (0.007 to 0.026 ug/cm2/hr during 24 hr exposure)

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential