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Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard protocol toxicokinetic study conducted according to GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Remarks:
C14 AMP
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
- Species and Sex: Rats (male and female)- Age at Study Start: ~ 8-10 weeks (~200 g body weight)- Strain and Justification: Fischer 344 rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, the reliability of the commercial supplier, and because the rat is the preferred species for pharmacokinetic/metabolism studies.- Supplier and Location: Jugular vein cannulated rats were obtained from Taconic Inc. (Germantown, New York). Non-cannulated animals were obtained from Charles River Laboratories Inc. (Raleigh, North Carolina). - Physical and Acclimation: Upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International), each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes. Non-cannulated animals were housed two per cage in stainless steel cages and acclimated to the laboratoryenvironment for at least one week prior to use, including at least two days in metabolism cages. The stainless steel cages had wire-mesh floors and were suspended above catch pans. Cages contained a hanging feeder and a pressure activated lixit valve type watering system. Jugular vein cannulated rats (surgery performed by the supplier) were acclimated in metabolism cages for approximately four days prior to use. Rodent jackets (Alice King Chatham, Los Angeles, California) were placed on the animals dosed dermally to protect the application site from grooming. The dermally dosed animals were acclimated with rodent jackets following a scheduled regimen (2 hours on day-1 and 4 hours on day-2 followed by 24 hours/day from day-3 until sacrifice).- Housing: Following administration of the test material, the animals were housed one per cage in glass Roth-type metabolism cages, which are designed for the separation and collection of urine, feces, CO2, and organic volatiles. Air was drawn through the metabolism cages at ~ 500 ml/minute.- Randomization and Identification: Animals were selected from those available having acceptable patent jugular veincannulae and randomly assigned to treatment groups using a computer-drivenrandomization procedure and identified by a uniquely assigned numbered metal eartag.- Feed and Water: Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum during the pre-exposure and study periods, except that feed was withdrawn from all orally dosed animals approximately 16 hours prior to the administration of 14C-CS-1135 and was returned about four hours post-dosing. Analysis of the feed was performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting.- Animal Welfare: In response to the Final Rules amending the U.S. Animal Welfare Act promulgated by the U.S. Department of Agriculture effective October 30, 1989, the Animal Care and Use Activities (ACUA) required for the conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC has assigned File Nos. Metabolism 01 and Animal ID 01 to these Animal Care and Use Activities.
Route of administration:
other: Oral and Dermal
Vehicle:
water
Duration and frequency of treatment / exposure:
Single oral or dermal exposure
Remarks:
Doses / Concentrations:Oral - 17.7 mg/kg bwDermal - 17.5 mg/kg bw
No. of animals per sex per dose:
4 males per dose route.
Control animals:
no
Positive control:
No
Details on study design:
This study consisted of a blood/plasma 14C concentration-time course (after oral and dermal application) to determine peak (Cmax) blood/plasma 14C concentrations and ADME experiments to determine absorption (including penetration of AMP from skin), distribution, metabolism, and elimination of AMP following either oral or dermal application. The study continued until ≥ 95% of the administered dose was recovered in the excreta and/or up to seven days post-dosing. Blood sampling from the dermally dosed animals was continued for 7 days regardless of the recovery of the administered dose in order to determine any post-application penetration of radioactivity through the skin at the application site.The rats dosed orally were fitted with indwelling jugular vein cannulae which were used to collect blood for the determination of time course 14C concentration of the test materials in plasma and red blood cell (RBC). The time course concentration data was used to determine peak (Cmax) and halfpeak (½ Cmax) plasma 14CAMP concentrations. Approximately 0.2-ml blood was collected at chosen times (0.25, 0.5, 1, 2, 4, 8, 12, 24 hours, and every 24 hours thereafter or until the jugular vein cannulae remained patent) and plasma prepared by centrifugation. Weighted aliquots (~25μl) of whole blood were placed in pre-weighed vials containing extraction and stabilizing solvents and stored at –80oC for the analysis of AMP.Aliquots of RBC were oxidized as described below and the RBC and aliquots of plasma analyzed for radioactivity by liquid scintillation spectrometry (LSS). Sampling of blood from the cannulae was stopped when radioactivity decreased to below the limit of detection, as described below, from all groups with the exception of dermally dosed animals. Samples collected from dermally dosed animal were counted for a longer period of time (i.e., 2 hours/sample) once the radioactivity became below twice the background levels in order to confirm that extremely low levels of the dermally applied radioactivity was not moving into the blood stream from the deeper layers of skin. This, along with collection of samples for 7 days instead of terminating the study upon recovery of ≥95% of the dermally applied dose, was done to confirm that any radioactivity associated with the skin was not becoming systemically bioavailable.
Details on dosing and sampling:
Oral and Dermal dosed rats:Time course concentration of AMP in whole blood (Plasma and RBC's) was assessed - blood taken via the cannula at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours, and every 24 hours thereafter or until the jugular vein cannulae remained patent. Dermal:Animals dosed dermally were anesthetized with isoflurane six hours post-dosing. The macroporous filter material covering was removed and the skin at the dose site was washed five times with cotton tip applicators dipped in an aqueous solution of detergent (i.e., ~2-4% Ivory dish washing liquid, Proctor and Gamble Co., Cincinnati, Ohio). The dose site was also rinsed several times with a gauze soaked with water and the area blotted dry with gauze squares. Following washing, the Teflon Spectra/Mesh covering was replaced and returned to their cages for the continuous collection of time-course blood, urine, feces, and terminal collection of tissues. Radioactivity was determined in the Teflon Spectra/Mesh coverings, the cotton tips, and gauze to determine the dose remained at the site six hours after the application. During the first 24 hours after the application of 14C-AMP to the skin, the air was drawn through a charcoal trap to trap the test material(s) volatilizing from the application site. This was conducted since the test material was found to be evaporating from the vehicle. Charcoal traps were used to trap the volatile test materials from the site for the first 24 hours as described above.UrineAll urine voided during the study was collected in dry-ice cooled traps. The urine traps were changed at 12 and 24 hours post-dosing, followed by 24-hour intervals for the remainder of the study. The cages were rinsed with water at the time the traps were changed and the rinse collected. Each urine specimen and urine/cage rinse was weighed, and a weighed aliquot of each sample was analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples from the 0–12 hour and 12–24 hour collection intervals was pooled and stored at -80ºC for chemical analysis.FecesFeces were collected in dry-ice chilled containers at 24-hour intervals. An aqueous homogenate (~ 25% w/w) was prepared and weighed aliquots of these homogenates oxidized (OxiMate 80 Sample Oxidizer, PerkinElmer Life Sciences, Inc., Boston, Massachusetts) and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates from each animal was taken from the 0–24 hour collection interval and pooled. These pooled samples were stored at –80ºC for possible chemical analysis.Expired VolatilesAir was drawn through the cages at approximately 500 ml/minute. Upon exiting each cage, the air was passed through charcoal to trap organic volatiles. The charcoal traps were changed at 24 hours. Radioactivity trapped on the charcoal was desorbed with weighed amounts of toluene. Weighed aliquots of the toluene were analyzed for radioactivity. Less than 1% of the administered dose was detected in the charcoal trap of the first orally dosed group during the first 24 hours of trapping. Therefore, the replacement traps were not analyzed for radioactivity and air was not trapped for any other groups. For animals that were exposed dermally to the test materials, a probe study was conducted to determine the volatility of the dermally applied 14C-AMP during the exposure. Radioactivity in the charcoal extracts was 3 to >70 fold higher than background; therefore, expired air was trapped for the first 24 hours post-dermal application animals.Expired CO2 Following the charcoal trap (described above) the expired air was passed through a solution of monoethanolamine:1-methoxy-2-propanol (3:7 v/v) to trap expired CO2 and analyzed for radioactivity. The CO2 trap was planned to change at 12- hour intervals through 48 hours followed by 24-hour intervals for the remainder of the study. However, < 1% of the administered dose was detected in the CO2 trap in the first orally dosed group during the first 12 hour interval therefore, CO2 traps were not used in the remainder of the study.
Statistics:
Descriptive statistics used (e.g. mean +/- SD)
Details on absorption:
AMP was rapidly absorbed from the Gastrointestinal tract following oral dosing, Cmax reached in less than 0.3 h. Almost the entire orally administered dose was absorbedDermal Dosing:Total recovery of applied radioactivity was approximately 82%. A total of approximately 40% of the administered dose was considered unabsorbed - skin wash 28%, application site covering 9% and charcoal trap 2% The total dermal absorption of AMP was approximately 43% of the administered dose.
Details on distribution in tissues:
Oral dose:at study termination, 2.2% of the dose was found inthe carcas, 0.8% in the liver and 0.3 in the skin. AMP partitioned equally between Red Blood Cells and Plasma (4.45 microg/g in each at Cmax)Dermal Dose:AMP in Red Blood Cells was 40% higher than inplasma (0.15 microg/g vs 0.09 microg/g)8% of the dose was found in the dose site skin at the end of the study (168 hours), of this, 0.4% of the radioactivity was found in the stratum corneum6% of the dose was found in tissues, of this about 5% was found in skin (not dose site) and 0.3% was found in the liver.
Details on excretion:
Oral Dose:Urinary elimintation of AMP accounted for approximately 91% of the adminsitered dose. Most of this dose (74%) was eliminated in the first 48 hours. Fecal elimination accounted for approximately 6% of the administered dose. At the end of the study (168 hours post dosing) only approximately 4% of the dose remained in tissues. There was a biphasic elimination of AMP. The first phase was rapid and lasted for about 6 hours post dosing, followed by the second phase which was slower with a half life of approximately 41 hours (plasma) and 69 hours (RBC) indicating that AMP appears to sequester into the RBC's. Dermal Dosing:. Urinary excretion appears to have accounted for approximately 56% of the absorbed dose, and fecal elimination accounting for approximately 2%. Of the dose excreted in the urine, the majority (approx 5%) was recovered within 48 hours of dosing.2% of the dose was found in the charcoal filter indicating some excretion via exhalation.
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.3 hours (RBC and Plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 4.45 microgram/g in plasma (oral dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 4.45 microgram/g in RBC (oral dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 41 hours (plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 69 hours (RBC, oral dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 29.7 microgram hours/g (plasma, oral dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 34.2 microgram hours/g (RBC, oral dose)
Test no.:
#1
Toxicokinetic parameters:
Tmax: 4 hours (RBC, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Tmax: 8 hours (Plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.09 microgram/g in plasma (dermal dose)
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.15 microgram/g in RBC (dermal dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 38 h (plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
half-life 2nd: 57 hours (RBC, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 3.7 microgram hours/g (plasma, dermal dose)
Test no.:
#1
Toxicokinetic parameters:
AUC: 5.4 microgram hours/g (RBC, dermal dose)
Metabolites identified:
no
Details on metabolites:
AMP does not appear to be metabolised.

Not applicable

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study resultsAMP is almost completely absorbed following adminstration of an oral bolus dose. Almost 100% of the dose had been excreted via the urine and feces within the course of the study (168 hours), the vast majority of the dose excreted within the first 24 hours. It is not metabolised prior to excretion and does not appear to accumulate in any tissues. Dermal dosing led to an absorption of approximately 40% of the dose, the majority of which was excreted in the urine. Of the absorbed dose , approximately a quarter remained in the skin at the dose site and it is not clear if this would subsequently have become systemically available.Based on this data there is no concern that AMP would accumulate in the body.
Executive summary:

Groups of rats were orally or dermally administered 14C-CS-1135 or 14C-AMP and absorption, distribution, metabolism and elimination determined. The orally administered CS-1135 was rapidly absorbed reaching Cmax within one hour and eliminated rapidly in urine. The rapid absorption was apparent from the drop in the dose from the GI tract within three hours between Cmax (56±10 to 68±9%) and ½Cmax (13±3 to 16±6%). Total urinary elimination accounted for 91-99% of the dose, most (77-92%) occurring within the first 48 hours. Fecal elimination accounted for only 4-8% of the dose. Absorption, distribution, and elimination of AMP was almost identical to CS-1135 with relatively faster absorption, reaching Cmax in <0.5 hour. Only 3-4% of the dose was found in tissues at the end of the study. Elimination of radioactivity from blood was biphasic with a rapid distributional (α) phase lasting for 24 hours for CS-1135 and 6 hours for AMP, followed by a slower elimination phase (β). The plasma t½ of the β phase of CS- 1135 was between 20 and 39 hr; females were slightly more efficient in elimination. The RBC t½ of the β phase of CS-1135 was slower (42-55 hours) indicating possible sequestration of radioactivity in RBCs. The administered CS-1135 was rapidly metabolized, apparently prior to reaching the systemic blood circulation. Out of 176 samples, parent CS-1135 was detected in only 10 samples without any specific pattern. AMP, one of the major metabolites of CS-1135, was detected in most of the blood samples. The AUC0→∝ of radioactivity in plasma and RBC was within dose proportionality between the low and high dose. The time-course decline of AMP derived radioactivity from plasma and RBC was almost identical to CS-1135 with slightly longer β elimination half-life. AMP was detected in the blood collected from the rats dosed with AMP at quite consistent concentrations found in rats dosed at an equimolar concentration of CS- 1135. Only the first three samples had ~2-fold higher AMP than was observed in CS-1135 dosed rats. There was an indication of sequestration of AMP to RBC. The majority (>80% in urine and ~8% in feces) of the orally administered radioactivity recovered in excreta from 14CCS- 1135 dosed rats was AMP and only one metabolite above 5% (6-7% in urine) of the administered dose. Urine of the 14C-AMP dosed rats contained only the parent compound.

Most of the dermally applied 14C-CS-1135 evaporated rapidly from the application site and recovered in the charcoal trap. Very little radioactivity was found in the dose site skin, averaging ≤1% of the dose with only ≤0.05% of the dose found in the stratum corneum. The total dermal absorption of 14C-CS-1135 was 13-15% of the dose. Out of which, ~11% was eliminated in urine, most (~8%) within 48 hours. Fecal elimination comprised <0.5% of the dose. The Cmax was reached within one hour post-dosing indicating rapid penetration. The difference in the AUC0→∝ between the two routes was 11 to 14-fold, consistent with the 13-15% dermal absorption compared to ~95% oral absorption. The AUC0→∝ of the dermally administered dose in RBC was 56-90% higher than plasma suggesting sequestration in RBCs. The elimination of radioactivity from blood was biphasic with a relatively slower α phase due to the presence of dose at the application site for six hours and its continuous penetration for some time after the removal/washing. The slope of the β phase was almost parallel to the oral group resulting in almost the same t½β for most of the rats. The total dermal absorption of 14C-AMP was ~43% of the dose. Twenty four percent of the total dose was eliminated in urine, most (~18%) within 48 hours. Fecal elimination was ~2%. Around 6% of the dose was found in tissues. It took much longer to reach Cmax after dermal application (~4 hours) than the oral dose (0.3 hour), indicating slower dermal penetration of AMP. The elimination of the radioactivity from blood was biphasic with relatively slower α phase. The t½β for most of the rats was similar between the two routes. The dermally applied radioactivity was eliminated slower (49% longer t½) from the RBCs than plasma, resulting in 44% higher AUC0→∝, an indication of sequestration of 14C-AMP derived radioactivity in RBCs, similar to that observed in the orally dosed group. Only AMP was detected in the urine of animals dosed dermally with 14C-CS-1135, accounted for 7-9% of the administered dose. Only AMP was detected in the urine of the rats dosed dermally with 14CAMP.

CS-1135 was rapidly hydrolyzed in whole blood, plasma and urine. The level of CS-1135 became undetectable in blood and plasma within 15 minutes after fortification to control samples. Similarly, CS-1135 was rapidly hydrolyzed in urine at room temperature.

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: review of literature
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Executive summary:

Following oral administration, p-toluene sulphonic acid is rapidly absorbed. In dogs were given 17.4 mg of radiolabelled 35S sodium tosylated/kg body weight, maximum radioactivity was measured in the blood after just 30 minutes (Dreifuss et al., 1971).

In rats and dogs which were given oral doses of p-toluene sulphonate as sultamicillin tosylate (100 mg/kg and 50 mg/kg body weight, respectively), maximum serum concentrations were measured 2 hours after administration (Kano et al., 1985)

In situ studies of intestinal absorbtion with Sprague-Dawley rats showed that at pH 7.5 /thus, in dissociated form) PTSA was not absorbed after 10 minutes (Ho et al., 1982)

The dispoersion of PTSA in the tissues also occurs rapidly. Its half life in the plasma of both dogs and rats was found to be 75 minutes (Dreufuss et al., 1971; Ho et al., 1982). Studies with sultamicillin tosylate produced a half life value in dogs of 60 minutes and in ratd of 100 minutes (Kano et al., 1985).

The distribution of PTSA in the tissues following intravenous injection (no information on dose size) has been studies.. The highest concentration after 35 minutes were found in urine and kidneys (Ho et al., 1982). Following adinistration as Sultamicillin tosylate to rats, its concentration was measured after 2 and 6 hours. After 6 hours, p-toluene sulphonate was no longer detectable in any tissues apart from the kidney (1.2 ug/g), heart (0.1 ug/g) and serum (0.3 ug/ml) (Kano et al., 1985)

As can be seen from these studies, elimination is very rapid and occurs mainly via the kidneys. Within four days, following oral administration of 34.8 mg of PTSA/kg body weight to rats, 82% was escreted in the urine and 13% in the faeces. For dogs, which were given 17.4 mg PTSA/kg body weight, the respective values were 84,5% and 17.5%. In both cases by far the greater part of the administerd dose was eliminated after just one day (Dreyfuss et al., 1971).

In rats, within 24 hours sultamicillin tosylate (200 mg/kg bw) was eliminated to 87% in the animal's urine and to 1.2% in the faeces (Kano et al., 1985). Analyses of urine and faeces showed that in both cases the substance had been eliminated unaltered. The excretion profiles for dogs which were administerd 17.4 mg sodium tosilate-35S/kg bw i.p. or p.o. and for rats which where administerd 34.8 mg/kg of the same substance p.o. were found to be very similar (Dreyfuss et al., 1971) . After 5 days, excretion of labelled sodium tosilate in the dogs was below the detection limit. In rats, too, even at a dose of 200 mg/kg, after 4 days 95% of the dose has been excreted (Dreyfuss et al., 1971).

Also when p-toluene sulphonate was administerd to rats in the form of sultamicillin tosylate in dayli doses of 100 mg/kg bw over a period of 21 days, no accumulation of p-toluene sulphonate in the animal was found. The highest p-toluene suklphonate concentrations were found in the kidneys (14 ug/g) and serum (2.9 ug/ml) 4 hours following administration of the last dose, while the concentration in other tissues was under 1 ug/g. These concentrations were of the same order of magnitude as those found 4 hours after administration of a single dose of the substance. 24 hours afteradministration of the final dose, p-toluene sulohinate was no longer detectable in the animals' organs (Kano et al., 1985)

Regarding dermal adsorption it can be evaluated the result of a model build to evaluate the dermal penetration for hydrotopes.

A multiple homogeneous layer model was used to derive an estimate of dermal penetration for hydrotropes. The mathematical model simulates the uptake of a chemical substance through the skin into a central sink compartment below the skin. The model uses the substance's diffusion and partitioning coefficients and calculates the total (cumulative) fraction of the substance that enters the stratum corneum for a specific exposure duration. The model does not include any metabolism and the model is believed to represent an upper bound estimate of the potential uptake of the substance through the skin.

Dermal penetration simulations based on a mechanistic model of the process of uptake of chemical substances in skin predicts that the dermal penetration of a generic hydrotrope is less than 0.6% of the applied amount (over a wide range of exposure scenarios). Simulations show that for an exposure extending to 23 hours, the dermal uptake does not exceed 2.8% of the applied amount, regardless of the applied amount (concentration) within the range of 0.0002% to 10%. 10% is considered an upper bound of the concentration of hydrotropes in consumer products.

 

Although polarity of the aromatic sulfonic acids is less than hydrotopes, the low Kow can help in supporting a very low penetration of acids too. The dermal pathway is in any case not included in the exposure scenarios because of the corrosivity of the substance.

 

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study follows the standard guidelines for a dermal penetration study, and was conducted according to GLP. However, the recovery of AMP at the end of the study was impacted by the poor containment of the more volatile base form of AMP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Remarks:
[14C]-aminomethylpropanol
Species:
other: rat and human skin
Strain:
Sprague-Dawley
Sex:
not specified
Details on test animals and environmental conditions:
Not applicable
Duration of exposure:
24 hours
No. of animals per group:
Not applicable
Details on study design:
The objectives of this study were to evaluate the rate and amount of [14C]-AMP absorption across fresh human and Sprague-Dawley rat skin after in vitro exposure, and to evaluate the disposition of [14C]-aminomethylpropanol ([14C]-AMP) in the various layers (stratum corneum, epidermis, and dermis) of fresh human and Sprague-Dawley rat skin after in vitro exposure.
Details on in vitro test system (if applicable):
See below.
Absorption in different matrices:
Not applicable
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 43 %
Remarks on result:
other: 24 hours
Remarks:
Rat skin, AMP Ultra PC 2000
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 50.9 %
Remarks on result:
other: 24 hours
Remarks:
Rat skin, 40% Aqueous AMP solution
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 30 %
Remarks on result:
other: 24 hours
Remarks:
Rat skin, Neat Control Lotion B, 4% AMP
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 16.7 %
Remarks on result:
other: 24 hours
Remarks:
Human skin, AMP Ultra PC 2000
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 14.1 %
Remarks on result:
other: 24 hours
Remarks:
Human skin, 40% Aqueous AMP solution
Dose:
20 μL/cm2
Parameter:
percentage
Absorption:
ca. 6.6 %
Remarks on result:
other: 24 hours
Remarks:
Human skin, Neat Control Lotion B, 4% AMP

Tables and Figures - see attached document

Rat

The barrier function tests showed that for full thickness Sprague-Dawley rat skin, less than 1% of the tritiated water applied on the epidermal surface was absorbed through the skin disks in 20 minutes (Table 1). These results indicate that all the skin disks used in the experiments possessed acceptable barrier function.

Flux rates associated with the percutaneous absorption of [14C]-AMP in Sprague-Dawley rat skin with the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations are presented in Tables 2 to 4, respectively. The flux rates are graphed for the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations in Figures 1 to 3, respectively, and a comparison of the absorption of the three formulations is presented in Figure 4.

Absorption of [14C]-AMP in the Ultra PC 2000™ formulation gradually increased throughout the incubation, and appeared to approach a plateau, with a maximum mean flux rate of 291 μg/cm2/hr at 24 hours. Absorption of [14C]-AMP in the 40% aqueous formulation increased progressively, but had not reached a plateau by 24 hours. The mean flux rate at 24 hours was 411 μg/cm2/hr. Absorption of [14C]-AMP in the Neat Control Lotion B formulation gradually increased throughout the incubation, and appeared to approach a plateau, with a maximum mean flux rate of 197 μg/cm2/hr at 24 hours. As summarized in Figure 4, the absorption of [14C]-AMP was comparable in the three formulations, although unlike the Ultra PC 2000™ and Neat Control Lotion B formulations, the 40% aqueous formulation does not appear to have reached a steady-state by 24 hours.

The cutaneous disposition of [14C]-AMP in Sprague-Dawley rat skin, expressed as μg/cm2, in the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations is presented in Tables 5 to 7, respectively. The cutaneous disposition of [14C]-AMP, expressed as percent of applied dose, in the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations is presented in Tables 8 to 10, respectively.

In general, the total recovered dose for all three formulations was relatively low. After 24 hours, the mean total absorbed dose of [14C]-AMP with the Ultra PC 2000™ formulation was 43.0%, and the mean total recovered dose was 59.6%. The dose was primarily recovered from the skin wipes (16.7%), the epidermis (14.3%), and the receptor fluid (21.2%). With the 40% aqueous formulation, the mean total absorbed dose of [14C]-AMP was 50.9%, and the mean total recovered dose was 60.6%. The dose was primarily recovered from the skin wipes (9.68%), the epidermis (18.9%), and the receptor fluid (19.7%). With the Neat Control Lotion B formulation, the mean total absorbed dose of [14C]-AMP was 30.0%, and the mean total recovered dose was 56.8%. The dose was primarily recovered from the skin wipes (26.8%) and the receptor fluid (17.3%), with smaller recoveries in the epidermis (5.33%) and the dermis (3.44%). No evidence of marked accumulation of [14C]-AMP in skin tissue was evident.

Human

Due to the low recovery of applied dose of the AMP Ultra PC 2000™ formulation with human skin, the Sponsor requested that this portion of the study be repeated with an additional donor. This additional donor is designated Donor 2. The barrier function tests showed that for human skin from Donors 1 and 2 used in this study, less than 0.3% of the tritiated water applied on the epidermal surface was absorbed through the skin disks in 20 minutes (Tables 11 and 12). These results indicate that all of the skin disks used in the study possessed acceptable barrier function.

Flux rates associated with the percutaneous absorption of [14C]-AMP in human skin with the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations are presented in Tables 13, 14, and 15, respectively. The flux rates are graphed for the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations in Figures 5 to 7 (donors 1 and 2), , 8, and 9, respectively, and a comparison of the absorption of the three formulations is presented in Figure 10.

Absorption of [14C]-AMP in the Ultra PC 2000™ formulation increased progressively throughout the incubation, and appeared to approach a plateau in donor 1 while it appeared continue upward in donor 2, with a maximum mean flux rate of 128 μg/cm2/hr at 24 hours. Absorption of [14C]-AMP in the 40% aqueous formulation also increased progressively, and appeared to approach a plateau, with a maximum mean flux rate of 111 μg/cm2/hr at 24 hours. The pattern of absorption of [14C]-AMP in the Neat Control Lotion B formulation was variable between the replicate skin disks (Figure 7). Two of the replicates (skin disks 13 and 14) showed much higher flux rates at 2 hours than the other 4 replicates. Reduced barrier function (Table 11) does not appear to adequately explain these observations, as no correlation between barrier function values and high flux rates is evident. Using all 6 replicate skin disks (skin disks 13–18), the mean maximal flux rate of 107 μg/cm2/hr occurred at 2 hours, followed by a gradual reduction in flux rates through 24 hours. As summarized in Figure 8, the absorption of [14C]-AMP is similar in the Ultra PC 2000™ and 40% aqueous formulations, gradually increasing or approaching a steady-state by 24 hours, while the Neat Control Lotion B formulation shows a rapid absorption phase followed by a gradual decline in flux rates through 24 hours. The cutaneous disposition of [14C]-AMP in human skin, expressed as μg/cm2, in the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations is presented in Tables 16, 17, and 18, respectively.

The cutaneous disposition of [14C]-AMP, expressed as percent of applied dose, in the Ultra PC 2000™, 40% aqueous, and Neat Control Lotion B formulations is presented in Tables 19, 20, and 21, respectively.

As with rat skin incubations, the total recovered dose for all three formulations was relatively low, particularly with the Ultra PC 2000™ formulation. After 24 hours, the mean total absorbed dose of [14C]-AMP with the Ultra PC 2000™ formulation was 16.7%, and the mean total recovered dose was 25.1%. The dose was primarily recovered from the skin wipes (8.32%) and the receptor fluid (8.30%), with lesser amounts recovered in the dermal and epidermal skin layers. With the 40% aqueous formulation, the mean total absorbed dose of [14C]-AMP was 14.1%, and the mean total recovered dose was 59.2%. The dose was primarily recovered from the skin wipes (45.1%) and the receptor fluid (6.48%). With the Neat Control Lotion B formulation, the mean total absorbed dose of [14C]-AMP was 6.65%, and the mean total recovered dose was 52.1%. The dose was primarily recovered from the skin wipes (45.5%), the dermis layer (2.26%), and the receptor fluid (2.71%). No evidence of excessive accumulation of [14C]-AMP in skin tissue was evident.

The reduced recoveries of dose with the three formulations may be explained by the results of control incubations in which the recovery of applied dose from PTFE disks was determined after a 24-hour incubation in diffusion cells. This control was intended to evaluate the evaporative loss of test article from the diffusion cell apparatus when no absorption could take place. Tables 22 and 23 present the results for the three formulations. The percent recovery of the applied dose was 26.0, 68.2, and 64.7 for the Ultra PC 2000™, 40% aqueous, and neat control lotion B, indicating that significant loss of the test article due to evaporation had occurred, and accounting for the low recovery of applied dose in the main study.

Conclusions:
The absorption and cutaneous disposition of [14C]-AMP in an Ultra PC 2000™ formulation, a 40% aqueous formulation, and a Neat Control Lotion B formulation were determined in rat and human skin. Rat skin showed gradually increasing flux rates for the three formulations, approaching a steady state through the 24-hour incubation. Maximal flux rates ranged from 197 to 411 μg/cm2/hr for the three formulations. The total absorbed dose as a percent of the applied dose ranged from 30.0 to 50.9 %. Human skin showed gradually increasing flux rates or rates approaching a steady state through the 24-hour incubation for the Ultra PC™ 2000 and 40% aqueous formulations. The Neat Control Lotion B formulation showed a rapid increase in flux rates through 2 hours, followed by a gradual decline through 24 hours. Maximal flux rates ranged from 107 to 128 μg/cm2/hr for the three formulations. The total absorbed dose as a percent of the applied dose was less than in rats, and ranged from 6.65 to 16.7 %. Recovery of the applied dose was low, however, evaporative loss of the test article from the test system could account for the low recovery of dose in both species. Rat skin had consistently higher flux rates than human skin, and the total absorbed dose was markedly higher in rat skin. This may explainthe higher recovery of the applied dose with the Ultra PC 2000™ formulation in rat skin relative to human skin (43.0% vs. 25.1%). That is, less evaporation of the dose could occur from rat skin because the dose was absorbed more rapidly and completely than human skin, so less test article was available for evaporation from rat skin. In both species, absorbed [14C]-AMP was primarily recovered from the receptor fluid. No evidence of marked accumulation of [14C]-AMP in skin tissue was evident.
Executive summary:

The objectives of this study were to evaluate the rate and amount of [14C]-aminomethylpropanol ([14C]-AMP) absorption across fresh human and Sprague-Dawley rat skin after in vitro exposure and to evaluate the disposition of [14C]-AMP in the various layers (stratum corneum, epidermis, and dermis) of fresh human and Sprague-Dawley rat skin after in vitro exposure.

Human and rat skin samples were incubated for 24 hours with the epidermal surface exposed to [14C]-AMP prepared in three formulations. The amount of [14C]-AMP absorbed across the skin and the disposition of [14C]-AMP in the various skin layers following this 24-hour incubation period were determined by liquid scintillation counting.

The absorption and cutaneous disposition of [14C]-AMP in an Ultra PC 2000™ formulation, a 40% aqueous formulation, and a Neat Control Lotion B formulation were determined in rat and human skin. Rat skin showed gradually increasing flux rates for the three formulations, approaching a steady state through the 24-hour incubation. Maximal flux rates ranged from 197 to 411 μg/cm2/hr for the three formulations. The total absorbed dose as a percent of the applied dose ranged from 30.0 to 50.9 %. Human skin showed gradually increasing flux rates or rates approaching a steady state through the 24-hour incubation for the Ultra PC 2000 and 40% aqueous formulations. The Neat Control Lotion B formulation showed a rapid increase in flux rates through 2 hours, followed by a gradual decline through 24 hours. Maximal flux rates ranged from 107 to 128 μg/cm2/hr for the three formulations. The total absorbed dose as a percent of the applied dose was less than in rats, and ranged from 6.65 to 16.7 %. Recovery of the applied dose was low, however, evaporative loss of the test article from the test system accounted for the low recovery of dose in both species. In both species, absorbed [14C]-AMP was primarily recovered from the receptor fluid. No evidence of marked accumulation of [14C]-AMP in skin tissue was evident.

Description of key information

No experimental data on absorption, distribution and excretion is available for the target substance PTSA Oxazolidine.

 

4,4-Dimethyloxazolidine

4,4-Dimethyloxazolidine hydrolyses so rapidly in water with pHs of 5, 7, or 9 that no rate or half-life could be determined. Only a small amount of parent 4,4-dimethyloxazolidine (2.7-3.6 % of applied amount) was present soon after solution preparation (time=0 samples) and throughout the hydrolysis study. The hydrolysis products are formaldehyde and 2-amino-2-methyl-1-propanol (AMP). AMP was stable for the length of the studies (30 days for hydrolysis and 6 days for photolysis). AMP was present at time=0 samples in concentrations of 96.4-97.3% of dosing rate (US-EPA RED Document). 

The toxicokinetics of AMP are therefore most relevant to be taken into consideration for the target compound. Reaction with water of the target has been found to produce primarily carbon dioxide, which is formed from the formaldehyde produced.

 

AMP

In vivo

In rats AMP was absorbed following administration of an oral bolus dose. Almost 100% of the dose is excreted via the urine (91-99%) and faeces (4-8%) within the course of the study (168 hours), the vast majority of the dose excreted within the first 24 hours. The substance is not metabolised prior to excretion and does not appear to accumulate in any tissues.

Dermal dosing in rats led to an absorption of approximately 40% of the dose, the majority of which was excreted in the urine. Of the absorbed dose, approximately a quarter remained in the skin at the dose site and it is not clear if this would subsequently have become systemically available. Based on this data there is no concern that AMP would accumulate in the body (Dow 2007).

 

Dermal absorptionin vitro

The absorption and cutaneous disposition of [14C]-AMP in an Ultra PC 2000™ formulation, a 40% aqueous formulation, and a Neat Control Lotion B formulation were determined in rat and human skin. In rat skin the total absorbed dose as a percent of the applied dose ranged from 30.0 to 50.9 %. In human skin the total absorbed dose as a percent of the applied dose was less than in rats, and ranged from 6.65 to 16.7 %. In both species, absorbed [14C]-AMP was primarily recovered from the receptor fluid. No evidence of marked accumulation of [14C]-AMP in skin tissue was evident (Dow 2007).

 

The findings of bothin vivo and in vitro studies indicate that via the oral route absorption is expected to be 100%, while a clear species difference is expected for dermal absorption. In general it is known that the rat skin absorbs substances more easily than does the human skin. This is here confirmed for the uptake of AMP, which is expected to be <20% in humans.

PTSA   

Following oral administration, p-toluene sulphonic acid is rapidly absorbed in rats and dogs. Plasma half-life was less than 2 hours with main concentrations found in kidneys. Elimination is very rapid and occurs mainly via urine. Within four days, following oral administration of 34.8 mg of PTSA/kg body weight to rats, 82% was excreted in the urine and 13% in the faeces. For dogs, which were given 17.4 mg PTSA/kg body weight, the respective values were 84.5% and 17.5%. In both cases the greater part of the administered dose was eliminated after just one day. Prolonged administration of PTSA to rats (21-d at 100 mg/kg bw) did not lead to accumulation. Concentrations in tissues in kidneys were comparable to those after administration of a single dose.

For dermal absorption limited modelled data are available for PTSA and analogues. These models show very limited uptake via the skin. This is in line with the expectations based on the low logKow and the high water solubility, which indicate that PTSA may be too hydrophilic to cross the lipid rich environment of the stratum corneum.

4,4-dimethyloxazolidinium toluenesulphonate

  

Based on the physico-chemical characteristics of the two parts of the compound, taking into account that the compound will readily dissolve into the gastrointestinal fluids and thus be present in its dissociated form[1], the absorption after oral administration of the substance can be attributed to the absorption of AMP and PTSA. In the case of PTSA oxazolidine, reaction with water has been found to produce primarily carbon dioxide, which is formed from the formaldehyde produced. As was found for the source substances, it is expected that absorption will be significant although the ionic nature of the parts will hamper absorption to a certain extent. The low vapour pressure indicates that PTSA oxazolidine is non-volatile at room temperature and thus the exposure of the substance via inhalation route is unlikely[2]. Therefore, absorption via inhalation is not further discussed. With dermal exposure the compound is expected to be present in its non-dissociated form and the uptake is expected to be very low based on high water solubility and low log Kow. PTSA oxazolidine may be too hydrophilic to cross the lipid rich environment of the stratum corneum.

Based on the low Log Kow and high water solubility, both parts of the substance are not expected to remain in the body fluids and may be rapidly excreted via urine, as is seen in the information available on the individual components. No bioaccumulation is expected.

Concluding the absorption of PTSA oxazolidine compound via the oral route is set at the default of 100%, while dermal absorption is set at the default value of 10%. This conclusion has been drawn in absence of data on toxicokinetics for the target substance. The metabolism and excretion data are based on the information on PTSA and oxazolidine/AMP. No other metabolic pathways are however expected for these kinds of substances.

Mode of Action 

For human toxicity the oxazolidine part of the compound is expected to be most relevant.

The effect of 4,4-dimethyl oxazolidine (and thus AMP) on the liver appears to be a result of interference with phospholipid synthesis in the hepatocytes. Various publications on AMP from the 1950's and 1960's have identified that it is capable of becoming incorporated into phospholipids in place of ethanolamine and/or choline and that it inhibits the uptake of choline by the liver cells. AMP also appears to inhibit the formation of choline in the liver via the conversion of ethanolamine to choline.

The transport of lipids from hepatocytes is fairly consistent throughout mammalian physiology, i.e. the use of a phospholipid based transport such as VLDL. Therefore the effects observed in rats and dogs in the studies on AMP are likely relevant to man. However the increase in hepatic lipids is only the first step in the toxicity to the liver, and further indicators of toxicity are only evident after some accumulation of lipids. There is no evidence of liver toxicity (e.g. increased liver weight, increase in liver enzymes in the blood etc.) at doses where lipid accumulation in hepatocytes is not apparent. It is also apparent that the liver toxicity is the most sensitive endpoint, occurring at doses lower than those causing other effects, such as increased post implantation loss.


[1]Aromatic sulphonic acids are almost completely ionized in watery environments even at low pH. In principle the salts of these acids get dissociated when in contact with water, so forming back to the acids. This is particularly relevant for studies that are conducted in water (e.g., ecotoxicity and biodegradation) as well as for mammalian toxicity studies where the relatively high acidity of the acid form has an immediate and harsh local effect, whereas the salt form provides an indication of potential systemic toxicity beyond the site of application or initial contact.

[2] [1]Although formaldehyde release during use in aqueous solutions cannot be fully excluded, the subsequent conversion to CO2 would minimize any toxicological effect. Theoretical release would be 4.4 w/w% of the substance and is expected to be below the OEL for formaldehyde.(SCOEL proposed 0.3 ppm (8 h TWA) with a STEL of 0.6 ppm, SCOEL/REC/125 draft 2016) . If necessary protective equipment will be used.

Key value for chemical safety assessment

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

Additional information