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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Simulated metabolism data from ECETOC recommended Metaprint2D-React tool (ALL metabolite model: 2010.2).
Objective of study:
metabolism
Principles of method if other than guideline:
MetaPrint2D-React is a tool that predicts xenobiotic metabolism through data-mining and statistical analysis of known metabolic transformations reported in scientific literature. It can make predictions concerning a wider range of reactions, and is able to predict the types of transformation that can take place at ease site of metabolism, and the likely metabolite formed. The modeling was conducted for the test substance, quaternary ammonium compounds, (quaternary ammonium compounds, benzyl-C12-C16-alkyldimethyl, chloride) using the SMILES notation as the input parameter.

Type:
other: Simulated metabolism (phase-I metabolism with (0.66 <= NOR <= 1.00)
Results:
As per the MetaPrint2D tool the reactions occuring at the terminal two carbon atoms of the longer alkyl chain are: oxidation at (=O-OH), single or double bond, ketone and di ketone group), alkylation, methylation, acylation, demethylation and dealkylation
Type:
other: Simulated metabolism (phase-I metabolism with (0.66 <= NOR <= 1.00)
Results:
As per the MetaPrint2D tool the reactions occuring at the para-position of the benzene ring include: hydroxylation, methoxylation and glutathionation.
Metabolites identified:
yes
Details on metabolites:
The most frequently reported site of phase-I metabolism (with 0.66 <= NOR <= 1.00) for C12-16 ADBAC is at the terminal two carbon atoms of the longer alkyl chain were: oxidation at (=O-OH), single or double bond, ketone and di ketone group), alkylation, methylation, acylation, demethylation and dealkylation

Metabolism transformation reactions with (0.66 <= NOR <= 1.00) for C12-16 ADBAC at the para-position of the benzene ring were: hydroxylation, methoxylation and glutathionation.

Refer to attached background material for details (types of transformation reactions and metabolites).
Conclusions:
- Interpretation of results: bioaccumulation potential cannot be judged based on study results
- The most frequently reported site of metabolism for the test substance is at the terminal two carbon atoms of the longer alkyl chain and the para-position of the benzene ring. The transformation reactions at these two sites include different types of phase-I metabolism reactions (including oxidation, hydroxylation, alkylation, methylation, acylation, demethylation, dealkylation),conjugation reactions (i.e., glutathionation and methoxylation.
Executive summary:

A study was conducted to determine and predict the most likely site of metabolism and the metabolites of the test substance using the ECETOC recommended Metaprint2D-React tool (ALL metabolite model: 2010.2). The modelling was conducted using SMILES, as the input parameters. The most frequently reported site of Phase I metabolism occurring at the terminal two carbon atoms of the longer alkyl chain were oxidation at (keto hydroxy, single or double bond, ketone and di ketone group), alkylation, methylation, acylation, demethylation and dealkylation. Also, the reactions occurring at the para-position of the benzene ring included hydroxylation, methoxylation and glutathionation (MetaPrint2D-React, 2013).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
other: Absorption, distribution, metabolism and excretion
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C-labelled test substance
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
- Source: Charles River
- Age at study initiation: 6 weeks
- Weight at study initiation: Average body weight: 268.6 g (male) and 167.0 g (female)

Details on exposure:
Preparation of dosing solutions:
Preliminary experiments: Oral gavage – single low dose
Experiment 1: Oral gavage – single low dose
Experiment 2: Dietary – repeated low dose
Experiment 3: Oral gavage – single high dose
Experiment 4: Intravenous


Vehicle:
Distilled water (Preliminary experiments and Experiments 1, 3 and 4)
Rodent diet/distilled water (Experiment 2)

Concentration in vehicle:
Preliminary experiments and Experiment 1: 1.0 mg/mL
Experiment 2: 100 ppm in diet for 14 d/1 mg/mL in water single oral dose
Experiment 3: 5.0 mg/mL
Experiment 4: 4 mg/mL


Homogeneicity and stability of the test material: Stable
Duration and frequency of treatment / exposure:
Preliminary experiments: Oral gavage – single low dose
Experiment 1: Oral gavage – single low dose
Experiment 2: Dietary – repeated low dose for 14 d
Experiment 3: Oral gavage – single high dose
Experiment 4: Intravenous
Remarks:
Doses / Concentrations:
Preliminary experiments and Experiment 1: 10 mg/kg
Experiment 2: 100 ppm non-radiolabelled substance for 14 d, followed by 10 mg/kg radiolabelled.
Experiment 3: 50 mg/kg
Experiment 4: 10 mg/kg
No. of animals per sex per dose:
Preliminary experiments: 2 per sex per group (Total 8 animals)
Main experiments:5 per sex per group (Total 40 animals)
Control animals:
no
Details on dosing and sampling:
Pharmacokinetic study (Absorption, distribution, excretion):
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes
- Time and frequency of sampling: Urine, faeces and urine/feces separator washing samples were collected at the following time intervals: 0-4, 4-8, 8-12, 12-24, 24-36, 36-48, 48-72, 72-96, 96-120, 120-144 and 144-168 h.

Metabolite characterisation studies:
- Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound.



Preliminary studies:
A preliminary study had indicated that insignificant 14CO2 was generated.
Details on absorption:
Percent Recovery:

Experiment 1:
Males: 5.77% urine; 98.61% faeces
Female: 6.88% urine; 91.20% faeces
Total Recovery: 104.54 ± 5.29% - males; 98.11 ± 3.25% females

Experiment 2:
Males: 4.76% urine; 95.12% faeces
Female: 5.80% urine; 97.22% faeces
Total Recovery: 100.19 ± 4.94% - males; 103.1 ± 5.18% females

Experiment 3:
Males: 7.75 % urine; 90.03% faeces
Female: 6.95% urine; 87.48% faeces
Total Recovery: 98.36 ± 2.42% - males; 94.58 ± 7.57% females

Experiment 4:
Males: 30.63% urine; 44.44% faeces
Female: 20.58% urine; 55.09% faeces
Total Recovery: 108.43 ± 5.56% - males; 111.45 ± 3.96% females

Less than 1% in tissues in all oral dosing experiments. Approximately 30-35% of the administered dose in tissues following i.v. dosing.
Details on distribution in tissues:
Residual 14C in tissues was negligible after administration of radiolabelled test substance by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. After i.v. administration a higher amount of radioactivity (30−35%) was found as residue in the tissues.
Details on excretion:
About 6−8% of orally administered test substance is excreted in the urine whereas, 87−98% was found in the faeces. Since no data on bile duct-cannulated rats are available, it is not possible to conclude if this radioactivity accounts exclusively for unabsorbed test substance or not. However, the i.v. experiment showed that 20−30% was excreted in the urine and 44-55% in the faeces, suggesting that both the kidney and liver are capable of excreting test substance once absorbed and that absorption is higher than the % found in the urine after oral administration.
Metabolites identified:
yes
Details on metabolites:
Over 50% of the faecal radioactivity was unchanged parent compound. Four major metabolites were identified, as oxidation products of the two decyl side chains to hydroxy and hydroxyketo derivatives. The only metabolism which occurred involved oxidation of the two decyl side chains to hydroxy and hydroxyketo derivatives. All were more polar and presumed less toxic than the parent compound. It is predicted that there is no major metabolite greater than 10% of the dosed radioactivity.
Conclusions:
- Interpretation of results: no bioaccumulation potential based on study results
- Following oral administration, 14C-radiolabelled test substance was rapidly absorbed, although in very limited amount as indicated by the low blood levels attained starting from 15 min after dosing and peaking between 3 and 8 h. 87 - 99% of the orally administered 14C-radiolabelled test substance was excreted in the faeces and 5 - 8% was excreted in the urine. No bioaccumulation was noted. Test substance was found to be widely distributed in tissues following intravenous administration with approximately 45 - 55% in the faeces, 20 - 30% in the urine and 30 - 35% associated with the tissues. Further, four major metabolites were identified, formed via oxidation of the alkyl chain.
Executive summary:

A study was conducted to determine the basic toxicokinetics of the test substance according to EPA OPP 85-1, in compliance with GLP. Sprague-Dawley rats (10 animals per sex per group) were treated with radiolabelled test substance. The study was conducted in four experiments: a single low dose (10 mg/kg); a single high dose (50 mg/kg); a14-day repeated dietary exposure with non-radiolabelled test substance (100 ppm) and single low dose of radiolabelled (14C) test substance (10 mg/kg); and a single intravenous dose (10 mg/kg). Following the single doses or the last dietary dose, urine and faeces were collected for 7 days. Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound. Following oral administration, radiolabelled test substance was rapidly absorbed, although in very limited amounts, consistent with its highly ionic nature. Residual 14C in tissues was negligible after administration by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. After i.v. administration, a higher amount of radioactivity (30−35%) was found as residue in the tissues. Approximately6−8% of orally administered test substance was excreted in the urine whereas 87−98% was found in the faeces. Since no data on bile duct-cannulated rats are available, it was not possible to conclude if this radioactivity accounted exclusively for unabsorbed test substance or not. However, the i.v. experiment showed that 20−30% was excreted in urine and 44-55% in faeces, suggesting that both the kidney and liver were capable of excreting test substance once absorbed and that absorption was higher than the % found in the urine after oral administration. Less than 50% of the orally administered test substance was metabolised to side-chain oxidation products. In view of the limited absorption of the test substance, the four major metabolites identified may be at least have been partially formed in the gut of rats, apparently by microflora. No significant difference in metabolism between male and female rats or amongst dosing regimens was observed. Repeated dosing did not alter uptake, distribution or metabolism of the test substance (Selim, 1987).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 06, 2004 to November 17, 2005.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
See 'Justification for type of information''
Justification for type of information:
Study was conducted according to the OECD 417 and in compliance with GLP with some acceptable deviations. However, the dermal application part suffered from design limitations, allowing for oral uptake from the skin after the 6h exposure, and therefore invalidating the results obtained for dermal uptake.
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
see 'Principles of method if other than guideline'
Principles of method if other than guideline:
Some deviations from study plan occurred which are not considered to have compromised the validity or integrity of the study:
- fluctuations of room temperature outside limits set
- Animals of group 7 were fed around 6 h after treatment instead of 4 h
- blood sample scheduled for D28709 (group 2) at 24 h was taken from D28704, as the earlier animal died during anesthesia prior to sampling
- Bile was collected into glass tubes instead of plastic tubes
- Animal D28763 (group 9, first treatment group - due to technical bile collection problems only second treatment group reported) was treated
according to the body weight of another animal by error.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
-Species: Sprague-Dawley rats
-Strain: Crl CD® (SD) IGS BR
-Source: Charles River Laboratories France, L’Arbresle, France. Caesarean Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
-Sex: 60 males and 60 females.
-Age/weight at study initiation: Young adults approximately 7 week old; for the bile collection group, animals were around 10 week old.
-Number of animals per group: Kinetics (5 groups): 9 males and 9 females (3/group/time/sex); Excretion balance: (3 groups): 5 males and 5 females
Bile collection (1 group): 4 males and 4 females
-Control animals: Yes: For the purposes of pre-dose sample analysis, plasma, blood and tissues will be collected from at least one untreated supplementary animal/sex using the above mentioned procedures.

Route of administration:
other: Gavage and topical
Vehicle:
water
Details on exposure:
-Specific activity of test substance 2.15 MBq/mg test item (58.2 µCi/mg)
-Volume applied: Oral: 10 mL/kg; Dermal: 1.5 mL/kg bw on approximately 10% body surface area (approximately 12 µL/cm2)
-Size of test site: Dermal: approximately 10% body surface area: 25 cm2 for 200 g rat, 30 cm2 for 250 g rat.
-Exposure period: Dermal: Treated area washed after 6 h.
-Sampling time: PK - oral: (3 animals/sex/group) post-gavage
⋅ first sampling set: 0.5, 4 and 24 h,
⋅ second sampling: 1, 8 and 48 h,
⋅ third sampling set: 2, 72 and 96 h.
PK - dermal (after initiation of skin contact):
⋅ first sampling set: 3, 8 and 24 h,
⋅ second sampling: 6, 10 and 48 h,
⋅ third sampling set: 7, 16 and 72 h.
MB oral and dermal:
Urine and faeces: 24 h before radioactive treatment, and during the periods 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 h after the radioactive gavage/dermal application.
Bile collection: 0-3, 3-6, 6-12 and 12-24 h post gavage.

-Samples: Blood/plasma, urine, faeces, bile, exhaled air, organs, carcass, skin with substance not removable, liquid used for washing the skin, protective appliances.
Duration and frequency of treatment / exposure:
6 h
Remarks:
Doses / Concentrations:
Males and females: Single and repeated oral low dose level: 50 mg/kg bw; high dose oral: 200 mg/kg bw; single dermal low dose: 1.5 mg/kg bw; igh dose dermal: 15 mg/kg bw.
No. of animals per sex per dose:
Males: 64
Females: 64
Details on dosing and sampling:
See 'Any other information on materials and methods incl. tables'
Type:
other: Absorption, distribution and excretion
Details on absorption:
-Percutaneous absorption:
Dose:
Radiochemical purity (99.5%) is sufficient (>98% ; OECD guidance), and formulated material was relevant in relation to possible human exposure: Concentrations for dermal application were 0.1 and 1% for 6h. This is based on already avaibale information on irritancy levels of the test substance: 4h, 5% on rabbit skin is mildly irritating; 24h occluded patches with 0.1% concentrations in water produced a well defined erythema and very slight oedema. The oral high dose of 200 mg a.i./kg bw could possibly lead to some toxic effects, as the acute LD50 by gavage is between 250 and 450 mg a.i./kg bw.
Recovery:
Total recovery for oral groups were suffficient with around 100% except for males of repeated dose group resulting to 95.3% recovery.
For dermal groups, total recovery was slightly low (87.2% males and 91.0% females). However, it should be remarked that probably the total radioactivity in the carcass samples with an avarage of about 5% was too low, as for all 4 animals for which no seperate internal organs were measured, total carcass levels were about 10%, compared to 1 to 3% for the 6 animals for which radioactivity in individual oragns were measured.

Oral absorption:
Most of the radioactivity were excreted via the faeces. About 3-4% left via urine, except for the males in the repeated dose groups showing a mean value of 8.31%. However, soft faeces were also observed in this group during the days of collection, possibly causing contamination of urine with faeces.
Also corresponding with high cage wash values. Elimination was quick, with 70-80% excreted within the first 24h. No radioactivity was left in the carcass. Bile examinations show that 4.58% (males) and 3.75% (females) of the total dose was recovered in the bile. Elimination was quick, with 25-30% already passed out in the bile in the 0-3 h period. The mean plasma and blood levels for males and females remained below quantifiable limits at all time points, except in the 50 mg/kg dose group for 0.5 to 2h time points for plasma (161 and 251 ng -eq/g for males at 1 and 2h respectively, and 109 ng-eq/g at 0.5 h, 212 ng-eq/g at 1h and 192 ng-eq/g at 2 h for females), and in blood only the 1 h time point in females (173 ng-eq/g). No plasma or blood levels could be determined for the 200 mg/kg dose group or the repeated 50 mg/kg dose group.
Following single oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 1, the mean radioactivity levels were below quantifiable limits in all tissues/organs at all time-points, except for the intestines and liver. Specifically, levels for males/females were 23.3/23.2% of the dose for the intestines and 0.087/0.039% of the dose for the liver at the 24h time-point. Levels decreased over time, and were all non-quantifiable by 168h.
Following single oral gavage at a nominal dose level of 200 mg/kg bw to rats of group 2, the mean radioactivity levels were above quantifiable limits in approximately half the analysed tissues and organs at 24 h. Specifically, high levels were present in the intestines (62.2/71.5% of the dose) for males/females, and trace levels were present in the abdominal fat, heart, kidneys, liver, lungs, lymph nodes and or pancreas (range 0.004 to 0.24% of the dose). As previous, levels decreased over time, and were all non-quantifiable by 96h with the exception of the intestines.
Following repeated oral gavage at a nominal dose-level of 50 mg/kg bw to rats of group 5, the mean radioactivity levels were below quantifiable limits in all tissues/organs, except for the intestines (e.g. levels for males/females of 15.9/36.9% of the dose at 24h) and the liver in males (0.072% of the dose at 24 h). Levels decreased over time and were non-quantifiable by 168h.

Dermal absorption:
The minimal percutaneous absorption (Faeces, urine and intestines) seemed to amount to 46.4% for males, and 47.4% for females. The maximum systemic absorption (faeces, urine, carcass and skin site) was 50.0% and 50.1% for males and females respectively. The data indicate that the skin application site is a reservoir for absorbed radioactivity in the animals. As the dermal application site was not protected from grooming after the 6h exposure period, test substance remained available on the skin for subsequent oral uptake from grooming.
Test substance was uniformly distributed in the stratum corneum. Cross-contamination to adjacent skin was observed. The mean plasma and blood levels for males and females for 1.5 mg/kg dose group remained below quantifiable limits at all time points, except for the 7 and 8 h time-points for blood (levels for males/females of 3.52/4.40 and 2.67/3.26 ng-eq/g, respectively). For the 15 mg/kg bw dose group only the 8 (levels of 70.2/68.6 ng-eq/g for males/females) and 24 (levels of 62.3/55.0 ng-eq/g for males/females) h time-points resulted in values above the quantifiable limits.
Following single dermal application at a nominal dose-level of 1.5 mg/kg to rats of group 3 and 15 mg/kg to rats of group 4, the mean radioactivity levels were below quantifiable limits in all tissues/organs at all time-points except for the intestines, "stripped" skin fiom the application site and adjacent site. Trace levels were found at 24 and 48h time-points in the eyes of the 1.5 mg/kg bw dose group.

No test substance-related mortality or morbidity were observed during the study. The only death observed (Male D28768, group 9, single oral dose at 50 mg/kg bw), was an isolated incident and not seen at the higher dose-level of 200 mg/kg bw, was considered to be due to complications following the bile duct cannulation. In terms of clinical signs, ptyalism was
seen in 1/9 males and 1/9 females of group 5 from Day 6 (repeat oral dose of 50 mg/kg bw) and soft faeces were noted in 4/5 males of group 8 from Day 6 (also, repeat oral dose of 50 mg/kg bw). As the ptyalism was of low incidence and is often seen in rats treated orally, this was not considered to be test substance-related. In contrast, as of high incidence, the
soft faeces were considered to be due to the repeated test substance treatment. No dermal irritation occurred.


Recovery: Total recovery for oral groups were sufficient with around 100% except for males of repeated dose group resulting to 95.3% recovery. For dermal groups, total recovery was slightly low (87.2% males and 91.0% females). However, it should be remarked that probably the total radioactivity in the carcass samples with an avarage of about 5% was too low, as for all 4 animals for which no separate internal organs were measured, total carcass levels were about 10%, compared to 1 to 3% for
the 6 animals for wich radioactivity in individual organs were measured.

-Oral application: 
Following single and/or repeated oral gavage at 50 and 200 mg/kg bw/day, the plasma, blood and organ radioactivity levels were essentially non-quantifiable indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was about 8% (urine and bile fractions); this was eliminated rapidly, essentially within a 48 to 72h period. The vast majority of the oral dose was excreted rapidly in the faeces. At the high oral dose-level only, quantifiable levels of radioactivity were found in some central organs at 8h post-dosing; otherwise, the vast majority of the dose was confined to the intestine and levels decreased over time. Only 0.62 to 8.15% of the oral
dose was eliminated in the bile in a 24h period.

-Dermal application:
Following single dermal application at 1.5 and 15 mg/kg bw, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2% and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48h period, suggesting that the dermal dose was highly absorbed via the skin. However, as the test site was not protected with an Elizabethan collar during the main part of the collection
period (the collar was worn during the 6h exposure period only), this may have been due to the animal licking the test site. This is also supported with the finding that after oral dosing only about 4% was excreted via bile back to intestines, and 4% excreted via urine. If similar routes of excretion are expected for dermal absorbed doses, it would
not be possible to find levels of 50% of applied doses in intestines with only 2% excreted via urine. This indicates that about 50% of the dermal applied dose was taken up orally after all, which following the same oral kinetics leads to the 2% excretion in urine as indeed was observed.

At 24 h post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestine for both dose-levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from
cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each
time-point. For all tissues/organs, the radioactivity levels essentially decreased over time.

Conclusions:
- Interpretation of results: no bioaccumulation potential based on study results
- Oral:
Following single and/or repeated oral administration at 50 and 200 mg/kg bw, the plasma, blood and organ radioactivity levels were essentially non-quantifiable indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was about 8% (urine and bile fractions); this was eliminated rapidly, essentially within a 48 to 72h period. The vast majority of the oral dose was excreted rapidly in the faeces. At the high oral dose-level only, quantifiable levels of radioactivity (2,386 to 23,442 ng-eq/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24h period of which about 30% during the first 3h.
- Dermal:
Following single dermal application at 1.5 and 15 mg/kg bw, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2% and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48h period, suggesting that the dermal dose was highly absorbed via the skin. However, as the test site was not protected with an Elizabethan collar during the main part of the collection period (the collar was worn during the 6h exposure period only), this may have been due to the animal licking the test site. This is also supported with the finding that after oral dosing only about 4% was excreted via bile back to intestine, and 4% excreted via urine. If similar routes of excretion are expected for dermal absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermal applied dose was taken up orally after all, which following the same oral kinetics leads to the 2% excretion in urine as indeed was observed.

At 24h post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose-levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels essentially decreased over time.
Executive summary:

A study was conducted to determine the basic toxicokinetics of the test substance according to OECD Guideline 417, in compliance with GLP. This toxicokinetic study was conducted using radiolabelled test substance. Rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose of 1.5 or 15 mg/kg bw. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was around 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 h period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24 h period, of which about 30% during the first 3 h. Following a single dermal application, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2 and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48 h period, suggesting that the dermal dose was highly absorbed via the skin. However, this apparent high absorption via the skin may have been due to the animal licking the test site. This is also supported with the finding that, after oral dosing, only about 4% was excreted via bile back to the intestine and 4% excreted via urine. If similar routes of excretion are expected for dermally absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken up orally after all. According to the same oral kinetics, this leads to the 2% excretion in urine as indeed was observed. At 24 h post-dosing, most of the radioactivity was in the "stripped" skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels decreased over time (Appelqvist, 2006).

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: Literature data
Adequacy of study:
supporting study
Study period:
1970
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Objective of study:
toxicokinetics
GLP compliance:
not specified
Conclusions:
- Interpretation of results: no bioaccumulation potential based on study results
- Depression in body weight gain was observed in rat receiving 100 mg/kg in water, but not in milk. Mortality occurred in dogs at 25 and 50 mg in water, but not in milk. The 12.5 mg/kg dose level was well tolerated.
Executive summary:

Studies conducted in rat and Beagle dogs compared the application of test substance in both milk and water as vehicle. Rats received 50 and 100 mg/kg bw/day for 12 weeks, and dogs 12.5, 25 and 50 mg/kg bw/day for 52 weeks. Depression in weight gain was observed in rat receiving 100 mg/kg bw/day in water, but not in milk. Mortality occurred in dogs at 25 and 50 mg/kg bw/day in water, but not in milk. The 12.5 mg/kg bw/day dose level was well tolerated (Cutler and Drobeck, 1970).

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: Literature data
Adequacy of study:
supporting study
Study period:
1971
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Objective of study:
distribution
Principles of method if other than guideline:
About 1 mL of 15% solution of the test substance in water/kg bw was administered by oral, rectal and intramuscular route, in order to evaluate body distribution. Distribution was measured after death of the animal, to quantify amount of the test substance in blood, liver, kidneys and area of application.
GLP compliance:
no
Species:
other: Rabbits, cats and dogs
Route of administration:
other: Oral, rectal and intramuscular
Vehicle:
water
Remarks:
Doses / Concentrations:
Males: 1 mL of 15% solution in water/kg bw for all routes in all animals
Type:
distribution
Results:
Less than 1.5% of the applied dose was found in organs investigated, of which the major part is in the liver.
Details on distribution in tissues:
Less than 1.5% of the applied dose was found in organs investigated, of which the major part is in the liver.

In a study to quantify the distribution of the test substance in body in rabbit, cat and dog (Bogs U and Lohse E, 1971), test substance had been administered by oral, rectal and intra-muscular route. Within few minutes animals died, and the test substance concentrations were measured locally at the sites of dosing and in blood, liver and kidneys. It was concluded that only little was absorbed and distributed over the body. Less than 1.5% of the applied dose was recovered from these organs, of which the major part is liver.


- Oral application:
                 
      Rabbit   Dog      Cat
Blood             0.3      0.05      0.21
liver                1.5     11.8      11.6
kidney              0.0      0.61      0.79
tongue            24       2.1       15
intestines        9.9      3.6       66.6
stomach         19.8    107      366
stomach content 131     860        567
dead in (min)       5       10         15

- Rectal application:
                  Rabbit   Dog      Cat
Blood               0.2      0.25       0.25
liver               1.5      7.85     15
kidney             0.2      0.7       0.1
colon             445      520        392
colon content     453      466        587
intestines          8.82     6.33       3.18
intest. content   0.29     0.31       2.28
dead in (min)    1.5      5          4

- Intramuscular application:
                Rabbit   Dog    Cat    rabbit
Blood              0.3     0.18    0.25    0.5
liver              1.3     3.23    3        0.3
kidney            0.0     0.8     0.1      0.41
injection
site     400     159     980     930

Conclusions:
Under the study conditions, only small fraction is absorbed and distributed over the body. Less than 1.5% of the applied dose was found in the organ investigated (i.e., sites of dosing, blood, liver and kidneys), of which the major part was found in the liver.
Executive summary:

A study was conducted to evaluate the distribution of the test substance inside the body of rabbits, cats and dogs. A single high dose (approximately 1 mL of 15% solution of the test substance in water/kg bw of animals, which is equal to about 10 times the lethal dose) was administered by oral, rectal and intramuscular route. Within a few minutes, the animals died and the test substance concentrations were measured locally at the sites of dosing, in blood, liver and kidneys. It was concluded that only a small fraction is absorbed and distributed in the body. Less than 1.5% of the applied dose was found in the organ investigated (sites of dosing, blood, liver and kidneys), of which the major part was found in the liver (Bogs, 1971).

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1964
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Based on literature reference. Exact source and size of skin and amount applied to the skin are not indicated.
Objective of study:
absorption
Principles of method if other than guideline:
The dermal penetration of the test substance was studied on normal, excised, human skin.
GLP compliance:
not specified
Specific details on test material used for the study:
- Aqueous solution of test substance 1.7 ppm (0.005 M having a molecular weight of 340. MW 240 means average chain length of 12).
Species:
human
Route of administration:
dermal
Vehicle:
water
Duration and frequency of treatment / exposure:
20h
Details on absorption:
From unbuffered aqueous solutions of the test substance ranging in concentration from 0.005 M (1.7 ppm) to 0.1 M (34 ppm), no measurable amount penetrates into the dermis of excised human skin within periods of 1 to 3d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 was of no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin. At these levels electrical conductivity indicated damage of the cutaneous barrier. Similarly, pretreatment of skin at that pH, caused damage of the skin and resulted in penetration of the test substance upon subsequent contact to test solutions. Also damaging the skin by repeated stripping of the stratum corneum with pressure-sensitive tape resulted penetration into the skin when stripped more than 10 times.
Key result
Toxicokinetic parameters:
other: see' details on absorption'
Conclusions:
- Interpretation of results: no bioaccumulation potential based on study results
- No measurable amount penetrates into the dermis of excised human skin within periods of 1 to 3 d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 was of no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin.
Executive summary:

A study was conducted to evaluate the dermal penetration of the test substance in normal excised human skin. From un-buffered aqueous solutions of the test substance ranging in concentration from 0.005 (1.7 ppm) to 0.1 M (34 ppm), no measurable amount penetrated into the dermis of excised human skin within periods of 1 to 3 d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 had no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin. At these levels, electrical conductivity indicated damage of the cutaneous barrier. Similarly, pre-treatment of skin at that pH caused damage of the skin and resulted in penetration of the test substance upon subsequent contact to test solutions. Also, damaging the skin by repeated stripping of the stratum corneum with pressure-sensitive tape resulted in penetration into the skin when stripped more than 10 times (Blank, 1964).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1986
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Objective of study:
absorption
Principles of method if other than guideline:
Single or multiple drops of radiolabelled test substance were applied on the cornea of rabbits to determine the corneal uptake.
Species:
rabbit
Conclusions:
- Interpretation of results: no bioaccumulation potential based on study results
- The lack of uptake has been demonstrated in a study in rabbits involving either single- or multiple-drop application of radiolabelled test substance on the cornea.
Executive summary:

A study was conducted with single or multiple drops of radiolabelled test substance on the cornea of rabbits to determine corneal penetration. The test substance was found in the palpebral and bulbar conjunctiva, corneal epithelium, stroma and endothelium. Single-drop administration resulted in high tissue levels in the anterior ocular tissues that were retained for up to 120 h. Multiple-drop administration led to accumulation in the epithelium to a greater degree than any other tissue. However, at no time did the test substance appear in the aqueous humour or any other tissue besides cornea and exposed conjunctivae (Green and Chapman, 1986).

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From February 18, 1991 to July 23, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-exposed skin was highly contaminated with the test substance, and likely grooming/licking caused oral uptake.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Deviations:
yes
Remarks:
See 'Principles of method if other than guidelines'
Principles of method if other than guideline:
Deviation: Multiple (3) groups (4 animals of one sex) are involved that are scheduled for sacrifice at different intervals after exposure (direct, 24 h and 72 h after exposure). In this study, only one group (6 males and 6 females) is used, with collection of excreta during different time intervals until 72 h after start of exposure.
Additionally, exposure period lasted the whole study period of 72 h, instead of washing after 6 or 24 h.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Benzalkonium chloride (Fractionated cocobenzyl dimethylammonium chloride, CAS no.: 61789-71-7)
- This substance is completely comparable to the test substance
- Non-radiolabelled material: E/0206 diluted with de-ionised water to an active ingredient content of 25.5%.
- Radiolabelled material: radiochemical purity > 98%.
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
water
Duration of exposure:
72 hours
Doses:
0.4 mL of a 0.77% w/w solution and about 5 MBq/mL (preliminary study) or 2 MBq/mL (Mass balance study).
No. of animals per group:
6 males and 6 females
Control animals:
no
Details on study design:
- Single application of radio-labelled test substance on the skin of rats. Absorption and distribution in skin, urine, faeces and carcass was measured.
- An area of approximately 20 cm2  on the back of rats (6 males and 6 females) was clipped free of hair 24 h prior to application of 0.4 mL of a 0.77% w/w solution and about 5 MBq/mL (preliminary study) or 2 MBq/mL (Mass balance study). Each rat was given a single topical application under a gauze patch. Immediately after administration the rats were individually housed in metabolism cages for the collection of urine and faeces. 
- Expired carbon dioxide was not collected as less than 0.1% was found in the preliminary study. Urine was collected and cages were washed at t = 8, 24, 48 and 72h. Faeces was collected at 24, 48 and 72h. Finally at the end of the observation period carcass and skin were collected.
Key result
Dose:
3 mg
Parameter:
percentage
Absorption:
16 %
Remarks on result:
other: 72 hours
Key result
Dose:
3
Parameter:
percentage
Absorption:
13.8 %
Remarks on result:
other: 72 hours
Conclusions:
Under the study conditions, the findings indicate that the dermal absorption of the test substance is limited and most of the absorbed test substance is excreted in the faeces.
Executive summary:

A study was conducted to determine the dermal absorption of the test substance a method comparable to OECD Guideline 427, in compliance with GLP. The dermal absorption and excretion study was conducted in rats following application of 0.4 mL of a 0.77% w/w solution of the test substance over approximately 20 cm2 of shaved skin, under a gauze patch for 72 h. After a single topical application of radio-labelled test substance, the total amount of radioactive substance was 16% in males (urine 0.8%, faeces about 9.9% and carcass about 5.3%) and 14% in females (urine about 0.7%, faeces about 6.1% and carcass about 7.0%). This was equivalent to a total mass of 24 µg equivalents (males) and 21 µg equivalents (females) absorbed per cm2(after a dose of approximately 3 mg). Most of the radio-labelled test substance (62.6%, males; 63.2%, females) was found in both the treated (48.0%, males; 45.1%, females) and the untreated (14.6%, males; 18.1%, females) skin after 72 h. The radioactive substance in the untreated skin may have been due to surface migration of the applied material from the perimeter of the treated area. The overall recoveries of radioactivity were acceptable for the experimental objectives of quantifying the absorption of radio-labelled test substance after a single dermal application. Under the study conditions, the findings indicate that the dermal absorption of the test substance is limited and most of the absorbed test substance is excreted in the faeces (Hallifax, 1991).

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 18, 2005 to August 17, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Species:
human
Details on test animals and environmental conditions:
Human skin membranes, in vitro
Type of coverage:
other: Automated flow-through diffusion cell system
Vehicle:
water
Doses:
0.03% (w/w) and 0.3% (w/w), in water
Details on in vitro test system (if applicable):
Skin preparation:
- Source of skin: Eight samples from Plastic Surgery unit, St. Johns Hospital, West Lothian NHS Trust, Livingston, UK
- Ethical approval if human skin: Yes
- Type of skin: Full-thickness human skin (1 upper arm, 2 abdomen and 5 breast)
- Preparative technique: Skin was transferred to Charles River Laboratories on ice and cleaned of subcutaneous fat and connective tissue using scalpel. Skin was washed in cold running tap water and dried using tissue paper. Each sample was then cut into smaller pieces, wrapped in aluminium foil, put into self sealing plastic bags and stored at -20°C until required. Split-thickness membranes were prepared by pinning the full thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200-400 µm depth using a Zimmer electric dermatome.
Principles of assay:
- Diffusion cell: Automated flow-through diffusion cell system (Scott/Dick, University of Newcastle-upon-Tyne, UK)
- Receptor fluid: Tissue culture medium containing approximately 5% (w/v) bovine serum albumin, 1% (w/v) streptomycin (approximately 0.1 mg/mL), and penicillin G (approximately 100 units/mL)
- Solubility of test substance in receptor fluid:
- Flow-through system: Automated
- Test temperature: The mean temperature 19°C (SD=0.8°C)
- Humidity: The mean relative humidity 35% (SD=0.6%)
Total recovery:
Refer to Table-1
Key result
Dose:
0.03%
Parameter:
percentage
Absorption:
0.05 %
Remarks on result:
other: 24 h
Remarks:
96.80% was not absorbed
Key result
Dose:
0.3%
Parameter:
percentage
Absorption:
0.03 %
Remarks on result:
other: 24 h
Remarks:
94.68% was not absorbed

- Low dose (0.030%):

The mean mass balance was 99.03% of the applied dose (3.09 μg equiv./cm2). The mean dislodgeable dose was 60.53% of the applied dose (1.89 μg equiv./cm2). The mean total unabsorbed dose was 96.80% of the applied dose (3.02 μg equiv./cm2). This consisted of the dislodgeable dose, unexposed skin (0.02%) and the radioactivity associated with the stratum corneum (36.25%). The stratum corneum acted as a good barrier to the test substance as the bulk of the radioactivity (30.26%) was recovered in the outermost 5 tape strips (tape strips 1-5). Considerably less radioactivity was recovered with each of the subsequent 3 groups of tape strips (3.20%, 1.82% and 0.97% in tape strips 6-10, 11-15 and 16-20 respectively), suggesting that the test substance would be sloughed off with the skin in the future. The absorbed dose (0.05%, or <0.01 μg equiv./cm2) was the sum of the receptor fluid (0.05%) and the receptor rinse (<0.01%). Dermal delivery (2.22%, or 0.07 μg equiv./cm2) was the sum of the absorbed dose and the exposed skin (2.18%). There was no apparent lag time and the fluxes ranges from 0.03 to 0.12 ng equiv./cm2/h over the 1 to 24-h exposure period.

 

- High dose (0.300%):

The mean mass balance was 96.84% of the applied dose (29.91 μg equiv./cm2). The dislodgeable dose was 77.87% of the applied dose (24.05 μg equiv./cm2). The mean total unabsorbed dose was 94.68% of the applied dose (29.24 μg equiv./cm2). This consisted of the dislodgeable dose, unexposed skin (0.17%) and the radioactivity associated with the stratum corneum (16.64%). The stratum corneum acted as a good barrier to the test item as the bulk of the radioactivity (10.86%) was recovered in the outermost 5 tape strips (tape strips 1-5). Considerably less radioactivity was recovered with each of the subsequent 3 groups of tape strips (3.11%, 1.71% and 0.96% in tape strips 6-10, 11-15 and 16-20, respectively), again suggesting that the test item would be sloughed off with the skin in the future. The absorbed dose (0.03%, or 0.01 μg equiv./cm2) was the sum of the receptor fluid (0.03%) and the receptor rinse (<0.01%). Dermal delivery (2.16%, or 0.67 μg equiv./cm2) was the sum of the absorbed dose and the exposed skin (2.13%). There was no apparent lag time. There was no apparent lag time and the fluxes ranges from 0.22 to 0.74 ng equiv./cm2/h over the 1 to 24-h exposure period.

Table 1. Summary of recoveries after 24 h

Test Preparation

Low Concentration

High Concentration

Target test substance concentration (%, w/w)

0.03

0.30

Test substance concentration by Radioactivity (%, w/w)

0.031

0.306

Test preparation application rate (mg/cm2)a

10.01

10.09

Test substance application Rate (μg equiv./cm2)

3.12

30.87

Dislodgeable Dose (% Applied Dose)

60.53

77.87

Unabsorbed Dose (% Applied Dose)

96.80

94.68

Absorbed Dose (% Applied Dose)

0.05

0.03

Dermal Delivery (% Applied Dose)

2.22

2.16

Mass Balance (% Applied Dose)

99.03

96.84

Dislodgeable Dose (μg equiv./cm2)

1.89

24.05

Unabsorbed Dose (μg equiv./cm2)

3.02

29.24

Absorbed Dose (μg equiv./cm2)

<0.01

0.01

Dermal Delivery (μg equiv./cm2)

0.07

0.67

Mass Balance (μg equiv./cm2)

3.09

29.91

a mg of test preparation per cm of skin

 

Conclusions:
Under the study conditions, following topical application of [14C]-radiolabelled test substance in low (0.03%, w/w) and high (0.3%, w/w) concentration, the mean absorbed dose and mean dermal delivery of [14C]- radiolabelled test substance were 0.05% (<0.01 μg equiv./cm2) and 2.22% (0.07 μg equiv./cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 μg equiv./cm2) and 2.16% (0.67 μg equiv./cm2) of the applied dose for the high concentration test preparation, respectively. The maximum fluxes for the low and high doses were 0.12 ng equiv./cm2/h and 0.74 ng equiv./cm2/h, respectively, at 2h.
Executive summary:

A study was conducted to determine the dermal absorption of the test substance according to OECD Guideline 428, in compliance with GLP. In the in vitro study, split-thickness human skin membranes were mounted into flow-through diffusion cells. Receptor fluid was pumped underneath the skin at a flow rate of 1.5 mL/h. The skin surface temperature was maintained at approximately 32°C. A barrier integrity test using tritiated water was performed and any skin sample exhibiting a permeability coefficient (kp) greater than 2.5 x 10-3cm/h was excluded from subsequent absorption measurements. Two test preparations containing (14C)-radiolabelled test substance (0.03 and 0.3%), were applied at an application rate of 10 mg/cm2. Absorption was assessed by collecting receptor fluid in hourly intervals from 0-6 h post dose and then in 2-hourly intervals from 6-24 h post dose. At 24 h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed from the skin by 20 successive tape strips. All samples were analysed by liquid scintillation counting. Under the study conditions, following topical application of (14C)-radiolabelled test substance in low (0.03%, w/w) and high (0.3%, w/w) concentration test preparations to human skin in vitro, the mean absorbed dose and mean dermal deliveries were 0.05% (0.01 ηg equiv. /cm2) and 2.22% (0.07 ηg equivalent/cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 ηg equivalent /cm2) and 2.16% (0.67 ηg equivalent/cm2) of the applied dose for the high concentration test preparation, respectively. The stratum corneum acted as a barrier to absorption, with the mean total unabsorbed doses (recovered in skin wash, tissue swabs, pipette tips, cell wash, stratum corneum and unexposed skin) of 96.80 and 94.68% of the applied dose for the low and high concentration test preparations, respectively. The maximum fluxes for the low and high doses were 0.12 ηg equivalent /cm2/h and 0.74 ηg equivalent /cm2/h, respectively, at 2 h (Roper, 2006).

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
10
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

Study 1. A study was conducted to determine the basic toxicokinetics of the test substance according to EPA OPP 85-1, in compliance with GLP. Sprague-Dawley rats (10 animals per sex per group) were treated with radiolabelled test substance. The study was conducted in four experiments: a single low dose (10 mg/kg); a single high dose (50 mg/kg); a 14-d repeated dietary exposure with non-radiolabelled test substance (100 ppm) and single low dose of radiolabelled (14C) test substance (10 mg/kg); and a single intravenous dose (10 mg/kg). Following the single doses or the last dietary dose, urine and faeces were collected for 7 d. Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound. Following oral administration, radiolabelled test substance was rapidly absorbed, although in very limited amounts, consistent with its highly ionic nature. Residual 14C in tissues was negligible after administration by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. After i.v. administration, a higher amount of radioactivity (30−35%) was found as residue in the tissues. Approximately 6−8% of orally administered test substance was excreted in the urine whereas 87−98% was found in the faeces. Since no data on bile duct-cannulated rats are available, it was not possible to conclude if this radioactivity accounted exclusively for unabsorbed test substance or not. However, the i.v. experiment showed that 20−30% was excreted in urine and 44-55% in faeces, suggesting that both the kidney and liver were capable of excreting test substance once absorbed and that absorption was higher than the % found in the urine after oral administration. Less than 50% of the orally administered test substance was metabolised to side-chain oxidation products. In view of the limited absorption of the test substance, the four major metabolites identified may be at least have been partially formed in the gut of rats, apparently by microflora. No significant difference in metabolism between male and female rats or amongst dosing regimens was observed. Repeated dosing did not alter uptake, distribution or metabolism of the test substance (Selim, 1987).

Study 2. A study was conducted to determine and predict the most likely site of metabolism and the metabolites of the test substance using the ECETOC recommended Metaprint2D-React tool (ALL metabolite model: 2010.2). The modelling was conducted using SMILES, as the input parameters. The most frequently reported site of Phase I metabolism occurring at the terminal two carbon atoms of the longer alkyl chain were oxidation at (keto hydroxy, single or double bond, ketone and di ketone group), alkylation, methylation, acylation, demethylation and dealkylation. Also, the reactions occurring at the para-position of the benzene ring included hydroxylation, methoxylation and glutathionation (MetaPrint2D-React, 2013).

Study 3. A study was conducted to determine the basic toxicokinetics of the test substance according to OECD Guideline 417, in compliance with GLP. This toxicokinetic study was conducted using radiolabelled test substance. Rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose of 1.5 or 15 mg/kg bw. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was around 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 h period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24 h period, of which about 30% during the first 3 h. Following a single dermal application, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2 and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48 h period, suggesting that the dermal dose was highly absorbed via the skin. However, this apparent high absorption via the skin may have been due to the animal licking the test site. This is also supported by the finding that, after oral dosing, only about 4% was excreted via bile back to the intestine and 4% excreted via urine. If similar routes of excretion are expected for dermally absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken up orally after all. According to the same oral kinetics, this leads to the 2% excretion in urine as indeed was observed. At 24 h post-dosing, most of the radioactivity was in the “stripped” skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels decreased over time (Appelqvist, 2006).

Study 4.Studies conducted in rat and Beagle dogs compared the application of test substance in both milk and water as vehicle. Rats received 50 and 100 mg/kg bw/d for 12 weeks, and dogs 12.5, 25 and 50 mg/kg bw/d for 52 weeks. Depression in weight gain was observed in rat receiving 100 mg/kg bw/d in water, but not in milk. Mortality occurred in dogs at 25 and 50 mg/kg bw/d in water, but not in milk. The 12.5 mg/kg bw/d dose level was well tolerated (Cutler and Drobeck, 1970).

Study 5. A study was conducted to evaluate the distribution of the test substance inside the body of rabbits, cats and dogs. A single high dose (approximately 1 mL of 15% solution of the test substance in water/kg bw of animals, which is equal to about 10 times the lethal dose) was administered by oral, rectal and intramuscular route. Within a few minutes, the animals died and the test substance concentrations were measured locally at the sites of dosing, in blood, liver and kidneys. It was concluded that only a small fraction is absorbed and distributed in the body. Less than 1.5% of the applied dose was found in the organ investigated (sites of dosing, blood, liver and kidneys), of which the major part was found in the liver (Bogs, 1971).

Dermal absorption

Study 6.A study was conducted to evaluate the dermal penetration of the test substance in normal excised human skin. From un-buffered aqueous solutions of the test substance ranging in concentration from 0.005 (1.7 ppm) to 0.1 M (34 ppm), no measurable amount penetrated into the dermis of excised human skin within periods of 1 to 3 d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 had no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin. At these levels, electrical conductivity indicated damage of the cutaneous barrier. Similarly, pre-treatment of skin at that pH caused damage of the skin and resulted in penetration of the test substance upon subsequent contact to test solutions. Also, damaging the skin by repeated stripping of the stratum corneum with pressure-sensitive tape resulted in penetration into the skin when stripped more than 10 times (Blank, 1964).

Study 7.A study was conducted with single or multiple drops of radiolabelled test substance on the cornea of rabbits to determine corneal penetration. The test substance was found in the palpebral and bulbar conjunctiva, corneal epithelium, stroma and endothelium. Single-drop administration resulted in high tissue levels in the anterior ocular tissues that were retained for up to 120 h. Multiple-drop administration led to accumulation in the epithelium to a greater degree than any other tissue. However, at no time did the test substance appear in the aqueous humour or any other tissue besides cornea and exposed conjunctivae (Green and Chapman, 1986).

Study 8. A study was conducted to determine the dermal absorption of the test substance according to OECD Guideline 428, in compliance with GLP. In thein vitrostudy, split-thickness human skin membranes were mounted into flow-through diffusion cells. Receptor fluid was pumped underneath the skin at a flow rate of 1.5 mL/h. The skin surface temperature was maintained at approximately 32°C. A barrier integrity test using tritiated water was performed and any skin sample exhibiting a permeability coefficient (kp) greater than 2.5 x 10-3cm/h was excluded from subsequent absorption measurements. Two test preparations containing (14C)-radiolabelled test substance (0.03 and 0.3%), were applied at an application rate of 10 mg/cm2. Absorption was assessed by collecting receptor fluid in hourly intervals from 0-6 h post dose and then in 2-hourly intervals from 6-24 h post dose. At 24 h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed from the skin by 20 successive tape strips. All samples were analysed by liquid scintillation counting. Under the study conditions, following topical application of (14C)-radiolabelled test substance in low (0.03%, w/w) and high (0.3%, w/w) concentration test preparations to human skinin vitro, the mean absorbed dose and mean dermal deliveries were 0.05% (0.01 ηg equiv. /cm2) and 2.22% (0.07 ηg equivalent/cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 ηg equivalent /cm2) and 2.16% (0.67 ηg equivalent/cm2) of the applied dose for the high concentration test preparation, respectively. The stratum corneum acted as a barrier to absorption, with the mean total unabsorbed doses (recovered in skin wash, tissue swabs, pipette tips, cell wash, stratum corneum and unexposed skin) of 96.80 and 94.68% of the applied dose for the low and high concentration test preparations, respectively. The maximum fluxes for the low and high doses were 0.12 ηg equivalent /cm2/h and 0.74 ηg equivalent /cm2/h, respectively, at 2 h (Roper, 2006).

Study 9.A study was conducted to determine the dermal absorption of the test substance a method comparable to OECD Guideline 427, in compliance with GLP. The dermal absorption and excretion study was conducted in rats following application of 0.4 mL of a 0.77% w/w solution of the test substance over approximately 20 cm2 of shaved skin, under a gauze patch for 72 h. After a single topical application of radio-labelled test substance, the total amount of radioactive substance was 16% in males (urine 0.8%, faeces about 9.9% and carcass about 5.3%) and 14% in females (urine about 0.7%, faeces about 6.1% and carcass about 7.0%). This was equivalent to a total mass of 24 µg equivalents (males) and 21 µg equivalents (females) absorbed per cm2(after a dose of approximately 3 mg). Most of the radio-labelled test substance (62.6%, males; 63.2%, females) was found in both the treated (48.0%, males; 45.1%, females) and the untreated (14.6%, males; 18.1%, females) skin after 72 h. The radioactive substance in the untreated skin may have been due to surface migration of the applied material from the perimeter of the treated area. The overall recoveries of radioactivity were acceptable for the experimental objectives of quantifying the absorption of radio-labelled test substance after a single dermal application. Under the study conditions, the findings indicate that the dermal absorption of the test substance is limited and most of the absorbed test substance is excreted in the faeces (Hallifax, 1991).