Registration Dossier
Registration Dossier
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EC number: 276-696-7 | CAS number: 72490-01-8
Endpoint summary
Administrative data
Link to relevant study record(s)
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2 Mar 1992 to 30 May 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Objective of study:
- absorption
- excretion
- metabolism
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 85-1 (Metabolism and Pharmacokinetics)
- Deviations:
- yes
- Remarks:
- The cage wipes of the control group were not analysed. Storage material of the test substance was at approximately -20°C. Some of the male rats were 27 days upon arrival. These deviations would not be expected to have an effect on the study
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 27 to 63 days old
- Weight at study initiation: 150 g to 174 g for males and 175 g and 199 g for females (arrival Feb 24, 1992) and 75 to 99 g and 150 to 174 g for the males and 125 to 149 g and 175 to 199 g for the females (arrival March 16, 1992)
- Housing: Individual metabolism cages for separation and collection of urine, faeces and expired volatile compounds. Individual, suspended, stainless steel wire-mesh cages in the acclimatisation period
- Diet: Rodent diet ad libitum
- Water: Ad libitum
- Acclimation period: At least 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 50 ±20
- Photoperiod (hrs dark / hrs light): 12/12
- Fasting period : Overnight and through 4 hours post dose (oral groups)
IN-LIFE DATES: 2 Mar 1992 to 30 May 1992
- Route of administration:
- other: Oral gavage and IV
- Vehicle:
- other: PEG 200
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The radiolabelled dosing solutions were prepared by transferring the radiolabelled test substance from its ampule to a serum vial. The ampule was rinsed with acetone and then with acetonitrile. Each rinse was transferred to the vial and solvent evaporated with nitrogen. The test substance was then diluted with a measured amount of polyethylene glycol 200 (PEG 200). The mixture was stirred for a minimum of 20 to 30 minutes, and aliquots were taken to determine the homogeneity and concentration. The nonradio labelled dosing solution was prepared by mixing known amounts of the test substance and PEG 200. The radiolabelled and non-labelled dosing solutions were prepared for 48 hours before the day of dosing and stored at or below 8 °C. - Duration and frequency of treatment / exposure:
- Once
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group D- P- H, oral gavage
- Dose / conc.:
- 1 mg/kg bw/day (actual dose received)
- Remarks:
- Group A, IV dose; Group B, Oral gavage; Group C, Oral gavage, 14 day preconditioned non-radiolabelled dose, followed by a single radiolabelled dose
- No. of animals per sex per dose / concentration:
- Low dose: 30
High dose: 10 - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Rationale for animal assignment: The rat is frequently used in metabolism studies as a representative rodent species
- Details on dosing and sampling:
- See ''Any other information on materials and methods incl. tables''.
- Details on absorption:
- After single dose absorption was 66.7-77.0% (females-males), but after repeated dosing 97.7% respectively 98.6% (females and males)
- Details on distribution in tissues:
- Very low amounts of radioactivity were found in tissues (individual tissues < 1% AR). Relatively high amounts were reported in fat (average: 1618-1795 ng eq./g), liver (average: 683-1140 ng eq./g) and kidneys (average: 282-335 ng eq./g) after a single dose at 300 mg/kg bw.
- Details on excretion:
- A single or repeated dose of 1.0 mg/kg bw 14C-test substance is excreted mainly via faeces (average per group 69%-82%). After 300 mg/kg bw the amount of radioactivity recovered from faeces is slightly lower (averages over males and females 56-60% AR), while urinary excretion is higher than in the low dose groups (average over males and females both 36% AR).
- Conclusions:
- After oral administration of 14C-test substance to male and female rats at the low dose level of approximately of 1.0 mg/kg, 66.7% to 98.6% of the radioactivity was absorbed from the intestinal tract into the general circulation. Negligible amounts of radioactivity were recovered in the expired air (CO2 traps and volatiles), for the preliminary group; therefore, the volatile organic compounds and expired carbon dioxide were not collected for the definitive phase groups. Total recoveries of radioactivity ranged from 90.0% to 105.5% of the total radioactivity administered for the IV and oral dose groups with 56.4% to 80.9% recovered in faeces, 16.6% to 36.8% in urine, not detected to 1.2% in carcass, and small amounts (less than 0.03%) in all other tissues combined. The majority of the radioactivity was eliminated in the urine and faeces within 48 hours after dosing. No apparent sex- related difference was noted for the absorption, elimination, and distribution of radioactivity for all dosing groups. The animals that received the multiple oral low dose show a higher percentage of absorption from the intestinal tract (71.9% for the single dose and 98.2% for the multiple dose). Animals that received a single oral high dose excreted higher amounts of radioactivity in urine (36.6%) compared to the amounts excreted from the single (16.8%) and multiple (23.2%) low dose group. Biliary excretion appeared to be an important elimination route for animals that received the IV dose. For the low-dose groups, at 7 days after dose administration, the mean concentration of radioactivity in the red blood cells and plasma were not detectable. The highest 14C- residue concentrations were detected in liver (0.003 to 0.006 ppm 14C-test substance equivalents), kidney (non-detectable to 0.001 ppm), and fat (0.001 to 0.003 pm), and lower residue concentrations were detected in all other tissues (not detectable to less than 0.001 ppm). At the high dose level, the residues were correspondingly higher.
- Executive summary:
The absorption, distribution and elimination of radioactivity were studies in male and female Sprague Dawley rats dosed with 14C-test substance according to EPA 85-1 and GLP principles. The 44 treated animals were divided into a preliminary group of 4 animals (2/sex/group) and 4 definitive groups of 10 animals (5/sex/group), which were dosed orally (gavage) or intravenously (IV) with 14C-test substance as follows: Group P-H (orally), at 300 mg/kg, Group A (IV) and B (orally) at 1.0 mg/kg, Group C received 14 consecutive daily oral doses of nonradio labelled test substance followed by a single radiolabelled oral dose at 1.0 mg/kg, Group D (orally), at 300 mg/kg. Organic volatiles and expired carbon dioxide were collected from the preliminary group (P-H). Based on the results of the preliminary group, organic volatiles and expired carbon dioxide were not collected from the definitive phase groups. Urine and faeces were collected from all treated and control animals at specified time intervals. Treated animals were sacrificed 7 days after administration of the radiolabelled dose. Selected tissue samples and the residual carcasses were collected for radio analysis.
Results showed, for oral dosed animals, faecal excretion was the primary elimination route. Total mean amounts of 75.5% (Group B), 80.6% (Group C), and 57.9% (Group D) were eliminated in faeces with the average majority (46.2% to 77.6%) excreted within 48 hours post dose. The total mean amounts excreted in urine were 16.8% (Group B), 23.2% (Group C), and 36.6% (Group D). A higher amount of radioactivity was excreted in urine from the high dose group than from the low dose group. For the IV dosed animals, 69.3% of the radioactivity was excreted in faeces, indicating biliary excretion was also an important elimination route. The test substance was well absorbed from the tract by rats dosed orally. No significant sex related differences were observed in the absorption, elimination, and/or distribution of radioactivity for any of the treated groups. Seven days after single oral administration at the low dose level, tissue residue levels were less than or equal to 0.006 ppm in all tissues with the exception of the liver (0.003 to 0.007 ppm). At the high dose level the resides were correspondingly higher (ranging from 0.683 to 1.14 ppm in liver). Total mean radioactive recoveries for groups A, B, C and F were 93%, 92.4%, 103.9% and 94.4%, respectively. Total mean radioactive recoveries for groups A, B, C and D were 93.0%, 82%, 103.9% and 94.9%, respectively.
In conclusion, after oral administration of 14C-test substance to male and female rats at the low dose level of approximately of 1.0 mg/kg, 66.7% to 98.6% of the radioactivity was absorbed from the intestinal tract into the general circulation. Negligible amounts of radioactivity were recovered in the expired air (CO2 traps and volatiles), for the preliminary group; therefore, the volatile organic compounds and expired carbon dioxide were not collected for the definitive phase groups. Total recoveries of radioactivity ranged from 90.0% to 105.5% of the total radioactivity administered for the IV and oral dose groups with 56.4% to 80.9% recovered in faeces, 16.6% to 36.8% in urine, not detected to 1.2% in carcass, and small amounts (less than 0.03%) in all other tissues combined. The majority of the radioactivity was eliminated in the urine and faeces within 48 hours after dosing. No apparent sex- related difference was noted for the absorption, elimination, and distribution of radioactivity for all dosing groups. The animals that received the multiple oral low dose show a higher percentage of absorption from the intestinal tract (71.9% for the single dose and 98.2% for the multiple dose). Animals that received a single oral high dose excreted higher amounts of radioactivity in urine (36.6%) compared to the amounts excreted from the single (16.8%) and multiple (23.2%) low dose group. Biliary excretion appeared to be an important elimination route for animals that received the IV dose. For the low-dose groups, at 7 days after dose administration, the mean concentration of radioactivity in the red blood cells and plasma were not detectable. The highest 14C- residue concentrations were detected in liver (0.003 to 0.006 ppm 14C-test substance equivalents), kidney (non-detectable to 0.001 ppm), and fat (0.001 to 0.003 pm), and lower residue concentrations were detected in all other tissues (not detectable to less than 0.001 ppm). At the high dose level, the residues were correspondingly higher.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 Jan 1986 to 01 Aug 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Objective of study:
- absorption
- metabolism
- tissue distribution
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The absorption, tissue distribution and excretion of 14C-test substance was investigated in 1 rat/sex after a single oral dose of 3000 mg/kg bw and in 3 males after a single and repeated dose of 50 mg/kg bw. In addition biliary excretion was investigated in bile cannulated rats after a single dose of 50 mg/kg bw. All animals received water and food ad libitum throughout the study, except for an overnight fast prior to treatment (groups 1 and 2) until 4 hours post-dose (group 1). Exposure and sampling of urine, faeces, cage wash, blood and tissues was collected. Metabolites were identified in pooled 24 hour urine and faeces samples of group 1 (and in one male at 48 and 72 hours), in 1-2, 12-13 and 21-22 hour bile samples of one male in group 2 and in group 3 in pooled samples of plasma, urine and faeces collected 24 hours after the first dose (day 1) and 24 hours after the dose on day 28. In addition in group 3 metabolites were identified in the liver of one male sacrificed on day 1 and one female sacrificed on day 28. Tissue samples with radioactivity levels below 1000 dpm/g were not analysed. Plasma, urine and bile samples were analysed by LSC (limit of reliable determination 30 dpm > background). Selected samples of urine and bile were reconstituted in methanol and subjected to metabolite identification by radio TLC (some of these after enzyme deconjugation with aryl sulphatase or glucuronidase). Radioactivity in faeces, tissues and blood was quantified by combustion/LSC (if required after homogenisation). Methanol extracts of plasma, faeces and liver were analysed by radio TLC.
- GLP compliance:
- yes
- Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 160- 240 g
- Diet: Ad libitum, rat diet
- Water: Ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 2
- Photoperiod (hrs dark / hrs light): 12/12
- Fasting period : Phase 1, for an overnight predose fast and for the first 4 h post dose. Animals for Phase 2 were deprived of food overnight prior to dosing, and were allowed free access to sucrose/saline solution (5% sucrose in 0.09% saline) immediately after dosing
IN-LIFE DATES: 01 Jan 1986 to 01 Aug 1986
- Route of administration:
- oral: gavage
- Vehicle:
- other: Rape seed oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Phase 1:
Non-radiolabelled test substance (10.923 g) was accurately weighed into a beaker and dissolved in acetonitrile. The solution was transferred to a pear-shaped flask and the solvent removed by rotary evaporation until the volume was ca. 15 mL (at 40 °C). Radiolabelled test substance (ca 0.25 mL stock solution) was added and the resultant solution mixed to ensure homogeneity. Following radiodilution excess the solvent was removed by rotary evaporation. The test substance was dissolved in the dose vehicle (rape seed oil), transferred to a 50 mL volumetric flask then made up to the correct volume. The check homogeneity and radioactivity content, 6 x 50 µL aliquots were removed for analysis by liquid scintillation counting (396963 dpm ± 3.4% cv). The resultant data was also used (in conjunction with the Standard Specific Activity) to calculate the new specific activity of the solution following radiodilution. A value of 0.016 µCi/mg was obtained and subsequently used in later calculations. Each animal in Phase 1 received a single 3 mL dose of the prepared dose solution by gastric gavage.
Phase 2 and 3
The low doses used in Phases 2 and 3 were prepared in 2 batches as follows:
1) The test substance (5.936 g) was accurately weighed into a round bottomed flask and dissolved in acetonitrile. The volume was reduced to ca 20 mL and ca 4.75 mL stock solution of [14C]-test substance was added. Complete dissolution was observed. Following radiodilution, the excess solvent was removed initially by rotary evaporation and then by nitrogen blown over the surface. The test substance was dissolved in rape seed oil and transferred to a 500 mL volumetric flask. After rinsing the round bottomed flask several times with rape seed oil, the washings were transferred to the volumetric flask and the volume made up to the 500 mL mark. To ensure homogeneity, the solution was subjected to ultrasonic agitation for 10 min. Aliquots (6 x 50 µL were then removed by Hamilton syringe and counted to confirm homogeneity and radioactive content (829515 dpm ±0.7% cv). Using the Standard Specific Activity, the specific activity of this batch of dose solution was calculated as being 0.62 µCi/mg.
2) The test substance (5.959 g) was accurately weighed into a round bottomed flask and dissolved in acetonitrile. The volume was reduced to ca 20 mL and ca 4 mL stock solution of [14C]- test substance was added. Complete dissolution was observed. Following radiodilution, the excess solvent was removed initially by rotary evaporation and then by nitrogen blown over the surface. The test substance was dissolved in rape seed oil and transferred to a 500 mL volumetric flask. After rinsing the round bottomed flask several times with rape seed oil, the washings were transferred to the volumetric flask and the volume made up to the 500 mL mark. To ensure homogeneity, the solution was subjected to ultrasonic agitation for 10 min. Aliquots (6 x 50 uL) were subsequently removed by Hamilton syringe and counted to confirm homogeneity and radioactive content (713545 dpm ±0.8% cv). Using the Standard Specific Activity, the specific activity of the second dose batch was calculated as being 0.53 µCi/mg.
Animals in Phase 2 each received a single oral dose, by gastric gavage, of Batch 1 dose solution (ca 1 mL, adjusted according to body weight).
Animals in Phase 3 each received a single oral dose daily, by gastric gavage, of either Batch 1 or Batch 2 dose solution (ca 1 mL, adjusted according to body weight). Doses 1-16 were taken from Batch 1 and doses 17-28 were taken from Batch 2. - Duration and frequency of treatment / exposure:
- Single or 28 days
- Dose / conc.:
- 3 000 mg/kg bw/day (actual dose received)
- Remarks:
- Phase 1, [dioxyphenyl-ring-14C]- test substance, single dose
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- Phase 2, [dioxyphenyl-ring-14C]- test substance, single dose
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- Phase 3, [dioxyphenyl-ring-14C]- test substance, repeated dose
- No. of animals per sex per dose / concentration:
- 3000 mg/kg (single dose): 5
50 mg/kg (single dose): 1
50 mg/kg (repeated dose): 18 - Control animals:
- no
- Details on dosing and sampling:
- TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Phase 1: 6, 24, 48, 72, 96 hours: urine, faeces (in 1/sex); 6, 24, 48 hours: expired CO2 (in 1/sex); 96 hours: cage wash
Phase 2: 6, 24 hours :urine, faeces; 24 hours: bile (hourly), cage wash, GI-tract, carcass
Phase 3: 3 males: 24 hours after dose 1: urine, faeces; 24, 48, 72, 96 hours after dose. Day 28: urine, faeces
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues and bile
- From how many animals:
Phase 1 (3000 mg/kg), 5 animals per sex:
Urine: Pooled 24 h sample; 24-48 h; 48-72h
Faeces: Pooled 24 h sample; 24-48 h; 48-72h
Tissue: Levels of radioactivity in the liver and kidney were ≤1000 dpm/mg and these samples were not analysed
Phase 2 (50 mg/kg), one animal per sex:
Bile: 1-2 h , 12-13h, 21-22 h for males and 0-1 h, 12-13 h and 21-22 h for females
Phase 3 (50 mg/kg), 18 animals per sex:
Plasma: For males, pooled samples from 24 h post first 14C dose and 24 h post twenty first dose. For females, pooled samples from 24 h post third 14C dose and 24 h post twenty eighth dose.
Urine: Pooled from 24 h post first 14C dose and 24 h post twenty eighth (final) dose.
Faeces: Pooled from 24 h post first 14C dose and 24 h post twenty eighth (final) dose.
Tissues: The livers with the highest concentration following acute and chronic administration. Levels of radioactivity were <1000 dpm/g in the kidney and therefore not analysed.
ANALYTICAL METHOD
Plasma, urine and bile samples were analysed by LSC (limit of reliable determination 30 dpm > background). Selected samples of urine and bile were reconstituted in methanol and subjected to metabolite identification by radio TLC (some of these after enzyme deconjugation with aryl sulphatase or glucuronidase). Radioactivity in faeces, tissues and blood was quantified by combustion/LSC (if required after homogenisation). Methanol extracts of plasma, faeces and liver were analysed by radio TLC. - Details on distribution in tissues:
- Very low amounts of radioactivity were recovered from blood, plasma, organs and tissues. The highest amounts were recovered in the intestines and the liver after a single dose of 3000 mg/kg bw. After repeated dosing the concentration of radioactivity in the tissues rose steadily and on day 28 the highest amounts were identified in liver and carcass. After cessation of treatment radioactivity decreased rapidly to 0.6 and 0.7 μg equiv/g in liver
and carcass, respectively, on day 41. Radioactivity in fat decreased more slowly than that in other tissues. - Details on excretion:
- In males up to 63% AR is excreted via the bile (in females 37% AR). Excretion is rapid and evenly divided between faeces and urine. Considering biliary excretion there seems to be a difference between the sexes, but as only one animal per sex was investigated the study does not allow definite conclusions.
- Metabolites identified:
- yes
- Details on metabolites:
- The number of metabolites identified in urine was limited to M12, M13 (single high dose only) and M14 (low dose) next to the parent and 1-2 unidentified. The bulk of the radioactivity was recovered from the origin (polar substances). Enzymatic hydrolysis with sulphatase increased the amount of all metabolites substantially indicating extensive conjugation. Treatment with glucuronidase was shown to increase the number of unconjugated metabolites at the high dose treatment. In faeces the parent was present in the highest amounts (84% of radioactivity on TLC plate at 3000 mg/kg bw, 36% at 50 mg/kg (single dose) and 8.4% at 50 mg/kg bw (repeated dose). Metabolites included M12 and M13 and 2 unknowns. The amount of polar substances in the origin was low (2.5%) after 3000 mg/kg and higher after 50 mg/kg bw (22-33%). In bile a similar pattern was seen and enzymatic treatment showed increased concentrations of unconjugated M12 and M13. In plasma and liver parent, M12 and M13 were identified.
- Conclusions:
- 14C-test substance is well absorbed after oral administration. In males up to 63% AR is excreted via the bile (in females 37% AR). Excretion is rapid and evenly divided between faeces and urine. This is indicative for an extensive entero-hepatic circulation. Considering biliary excretion there seems to be a difference between the sexes, but as only one animal per sex was investigated the study does not allow definite conclusions. Only a small part of 14C-test substance is excreted as the parent. Conjugated metabolites (mainly sulphates) form the major part of the metabolites. However, most radioactivity was collected from the origin of the TLC plates and was not further analysed.
- Executive summary:
The absorption, tissue distribution and excretion of 14C-test substance was investigated in 1 Sprague Dawley rat/sex after a single oral dose of 3000 mg/kg bw and in 3 males after a single and repeated dose of 50 mg/kg bw under GLP principles. In addition biliary excretion was investigated in bile cannulated rats after a single dose of 50 mg/kg bw. All animals received water and food ad libitum throughout the study, except for an overnight fast prior to treatment (groups 1 and 2) until 4 hours post-dose (group 1). Exposure and sampling of urine, faeces, cage wash, blood and tissues was collected. Metabolites were identified in pooled 24 hour urine and faeces samples of group 1 (and in one male at 48 and 72 hours), in 1-2, 12-13 and 21-22 hour bile samples of one male in group 2 and in group 3 in pooled samples of plasma, urine and faeces collected 24 hours after the first dose (day 1) and 24 hours after the dose on day 28. In addition in group 3 metabolites were identified in the liver of one male sacrificed on day 1 and one female sacrificed on day 28. Tissue samples with radioactivity levels below 1000 dpm/g were not analysed. Plasma, urine and bile samples were analysed by LSC (limit of reliable determination 30 dpm > background). Selected samples of urine and bile were reconstituted in methanol and subjected to metabolite identification by radio TLC (some of these after enzyme deconjugation with aryl sulphatase or glucuronidase). Radioactivity in faeces, tissues and blood was quantified by combustion/LSC (if required after homogenisation). Methanol extracts of plasma, faeces and liver were analysed by radio TLC.
Results showed, at 3000 mg/kg bw sedation (ataxia) and piloerection was observed, which lasted up to 7 hours post dose. One male died and necropsy revealed bleeding of the ileum. No other abnormalities were found. Radiochemical analysis of applied doses showed doses to be 96-116 % of nominal. After a single oral dose of 3000 mg/kg bw after 96 hours the majority of administered radioactivity was excreted in faeces (49-51% AR) and urine (37-44% AR). In a separate study with bile-duct cannulated rats receiving a single dose of 50 mg/kg bw, it was shown that 63% AR in the male rat and 37% AR in female rat was excreted via bile within 24 hours. In the male rat 11.3 and 12.1% AR was recovered in urine and faeces, while in the female rat 5.3 and 2.4% AR was recovered, respectively. In the GI tract of the female a substantial amount (51% AR) of radioactivity was recovered. Mass balances in both studies were >97% AR. After 24 hours in both studies 5-11% AR was excreted via urine. The high recovery of radioactivity in urine after 96 hours may be indicative of an extensive entero-hepatic circulation leading to a systemic absorption of above 50% (absorbed amounts reaching the liver may be up to 75%). It has to be noted that a single rat per sex was included in the mass balance studies. Very low amounts of radioactivity were recovered from blood, plasma, organs and tissues. The highest amounts were recovered in the intestines (198-345 μg eq./g) and the liver (32-42 μg eq./g) after a single dose of 3000 mg/kg bw. After repeated dosing the concentration of radioactivity in the tissues rose steadily and on day 28 the highest amounts were identified in liver (13 μg eq./g) and carcass (5.6 μg eq./g). After cessation of treatment radioactivity decreased rapidly to 0.6 and 0.7 μg equiv/g in liver and carcass, respectively, on day 41. Radioactivity in fat decreased more slowly than that in other tissues. The number of metabolites identified in urine was limited to M12, M13 (single high dose only) and M14 (low dose) next to the parent and 1-2 unidentified. The bulk of the radioactivity was recovered from the origin (polar substances). Enzymatic hydrolysis with sulphatase increased the amount of all metabolites substantially indicating extensive conjugation. Treatment with glucuronidase was shown to increase the number of unconjugated metabolites at the high dose treatment. In faeces the parent was present in the highest amounts (84% of radioactivity on TLC plate at 3000 mg/kg bw, 36% at 50 mg/kg (single dose) and 8.4% at 50 mg/kg bw (repeated dose). Metabolites included M12 and M13 and 2 unknowns. The amount of polar substances in the origin was low (2.5%) after 3000 mg/kg and higher after 50 mg/kg bw (22-33%). In bile a similar pattern was seen and enzymatic treatment showed increased concentrations of unconjugated M12 and M13. In plasma and liver parent, M12 and M13 were identified.
In conclusion, 14C-test substance is well absorbed after oral administration. In males up to 63% AR is excreted via the bile (in females 37% AR). Excretion is rapid and evenly divided between faeces and urine. This is indicative for an extensive entero-hepatic circulation. Considering biliary excretion there seems to be a difference between the sexes, but as only one animal per sex was investigated the study does not allow definite conclusions. Only a small part of 14C-test substance is excreted as the parent. Conjugated metabolites (mainly sulphates) form the major part of the metabolites. However, most radioactivity was collected from the origin of the TLC plates and was not further analysed.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Objective of study:
- absorption
- excretion
- tissue distribution
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The absorption, tissue distribution and excretion of 14C-test substance was investigated after a single oral dose of 50 mg/kg bw. All animals received water and food ad libitum throughout the study, except for an overnight fast prior to treatment until 4 hours post-dose. Exposure and sampling of urine, cage wash, faeces, blood and tissues was collected.
- GLP compliance:
- yes
- Radiolabelling:
- yes
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: ca 170- 250 g
- Housing: During Phase 1, the rats were housed singly in stainless steel and polypropylene cages with raised wire mesh floors to prevent coprophagy. During Phase 2, the rats were housed in Jencon's allglass metabolism cages specially designed for the separate, quantitative
collection of urine and faeces.
- Diet: Rat and mouse diet, ad libitum
- Water: Ad libitum
ENVIRONMENTAL CONDITIONS
- Fasting period : Overnight predose and 4 h post dose .
IN-LIFE DATES: not reported
- Route of administration:
- oral: gavage
- Vehicle:
- other: Rape seed oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The target dose level for the study was 50 mg/kg and represented a total radioactive dose of ca 0.44 MBq (12 µCi) per oral administration.
The test substance was prepared at the correct specific activity for dose administration by combining non-radiolabeled test substance (353.0 mg) and [14C]- test substance (ca 15.0 mg) in solution in acetonitrile (20 mL). The specific activity of the dose material was calculated to be 46.7 KBq/mg (1.26 µCi/mg).
On the day of dosing, the appropriate amount of the above stock solution was dispensed into a flask and the acetonitrile removed under N2. The dose material was then dissolved in the required amount of rape seed oil and a solution achieved by ultrasonication. - Duration and frequency of treatment / exposure:
- Single
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose / concentration:
- Phase 1: 3
Phase 2: 2 groups of 3 males and 2 groups of 3 females - Control animals:
- no
- Details on dosing and sampling:
- TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, liver, kidney, heart, lung, bone, brain, fat, skeletal muscle, testes/ovaries, spleen, intestinal tract (+contents), stomach (+contents), residual carcass.
- Time and frequency of sampling:
Blood, plasma: 0.25, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 72, 96 hours after dosing
Urine, faeces and cage wash: 0-6 h after dosing
ANALYTICAL METHOD
- Quantification of radioactivity: Radioactivity was analysed using a liquid scintillation analyser, with automatic quench correction by external standard-channels ratio. Each individual sample was counted for 5 min or the time taken to detect 900,000 counts. Where possible samples were measured at least in duplicate. Vials were allowed to heat and light stabilise overnight prior to analysis. Prior to calculation of each result, a background count rate was determined and subtracted from each sample count rate. A limit of reliable determination of 30 dpm above background count rate has been instituted in these laboratories. Where results have arisen from data below the limit of reliable determination the fact is so noted. Similarly, when a result has arisen from data less than 10 dpm above background the fact is noted.
- Sample preparation:
Liquid samples: Aliquots of urine, plasma and mock dose solutions, etc. were made up to 1.0 mL with distilled water if necessary and mixed with 'Unisolve' scintillator (10 mL).
Solid samples: Faeces samples were homogenised in acetone: 2% aqueous carboxymethylcellulose (CMC) (50:50 v/v). Stomach and intestine samples were homogenised in acetone. The residual carcasses were homogenised in water. Aliquots of homogenate (ca 0.2-0.5 g) were combusted using a sample oxidiser. The 14C-O2 resultant was collected by absorption in CarbosorbR to which Permafluor VR scintillation fluid was added. Combustion of standards showed recovery efficiencies to be in excess of 94% throughout. Tissue samples were finely chopped prior to combustion. - Details on distribution in tissues:
- Three hours post-dosing radioactivity was mainly recovered from stomach and gastro-intestinal tract. After 6 hours radioactivity in the stomach decreased and intestinal RA was increased in males and similar in females. Urinary excretion after 6 hours was higher than after 3 hours (3.5-4.0% AR to 9-12% AR), while excretion in faeces was found to be negligible in females and 2.8-3.4% AR in males. Radioactivity in organs was lower after 6 hours (3.5-3.8% AR) compared to after 3 hours (5.6-6.7% AR). Individual concentrations in urine and faeces differed 20-100-fold in males. Radioactivity in liver accounted for 5.2-6.4% AR after 3 hours and 3.2-3.7% AR after 6 hours. All other organ contained ≤2.0% AR.
- Conclusions:
- 14C-test substance is found mainly in the gastro-intestinal tract after oral administration (60-90% AR). In organs minor amounts (3.5-6.7% AR) were found. The highest concentrations were found in the liver. Maximum plasma concentrations were found after 4 hours.
- Executive summary:
The test substance was administered by oral gavage to 3 male and 3 female Sprague- Dawley rats at a target dose of 50 mg/kg according to GLP principles. Plasma levels of total radioactivity were determined at intervals up to 96 hours post dose. [14C]-test substance was administered orally to 2 groups of 3 male and 2 groups of 3 female rats at a target dose level of 50 mg/kg. One group of rats of each sex was sacrificed at each of 3 and 6 h post dose.
Results showed, peak plasma levels of total radioactivity were observed between 2 and 4 h post dose and represented peak mean values of 26.81 and 5.74 µg equiv./mL for male and female animals respectively. Thereafter mean plasma levels fell to 5.34 and 1.86 µg equiv./mL respectively at 8 h post dose. The half-life of elimination of total radioactivity from plasma during this period was ca 2 h. Plasma levels of total radioactivity were below the limit of reliable determination by 48 h post dose. Radioactivity was quantitively recovered in each group. The majority of the dose at sacrifice remained within the gastrointestinal tract. Excretion of total radioactivity prior to sacrifice accounted for means of and of the dose at 3 and 6 h respectively. Levels of total radioactivity in plasma at sacrifice were in the range 12-32 µg equiv./mL at 3 h and 2-15 µg equiv./mL at 6 h post dose. Levels of total radioactivity in liver were higher than those in plasma at each sacrifice time. Levels of total radioactivity in kidney were generally similar to those in plasma while levels in other organs and tissues were lower than those in plasma at each sacrifice time. Elimination of radioactivity from organs and tissues was generally at the same rate as elimination from plasma. Levels in the fat of male rats however were slightly higher in the 6 h sacrifice group than in the 3 h sacrifice group. Sex differences in levels of total radioactivity were most marked in plasma. In line with the plasma levels, levels of total radioactivity in the majority of cases in organs and tissues of male rats were slightly higher than in female rats. The exception to this was liver where levels in female animals were higher than in males at each sacrifice time. Levels of total radioactivity in liver accounted for of the administered dose. Levels of total radioactivity in plasma, calculated assuming a blood volume of 70 and a packed cell volume (PCV) of accounted for of the dose at 3 h and of the dose at 6 h post dose. The results show total radioactivity to be concentrated only in the organs of elimination (i.e. liver, kidney and gastro-intestinal tract) at the sacrifice times examined.
In conclusion, 14C-test substance is found mainly in the gastro-intestinal tract after oral administration (60-90% AR). In organs minor amounts (3.5-6.7% AR) were found. The highest concentrations were found in the liver. Maximum plasma concentrations were found after 4 hours.
- Endpoint:
- basic toxicokinetics in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 Jul 1981 to 30 Jul 1981
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Objective of study:
- absorption
- distribution
- excretion
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study investigated the absorption, distribution and excretion of 14C-test substance after a single oral dose of 49 mg/kg bw. Urine (including cage wash) and faeces were sampled over 0-6, 6-24, 24-48, 48-72 and 72-96 hours after dosing. After 96 hours blood was sampled and animals were sacrificed. At sacrifice, the following tissues and organs were taken from all animals: heart, liver, kidney, spleen, gonads, stomach (+ contents), intestines (+ contents), femur, muscle, fat and carcass. All animals received water and food ad libitum throughout the study. Radioactivity in urine and plasma was quantified by LSC. Blood cells and organs were combusted after lyophilisation. The residual carcass was solubilised in 1M KOH prior to LSC. Faeces were homogenised in acetonitrile-water (1:1) and submitted to LSC. Dried faecal residues and unextractable radioactivity was measured by combustion analysis. Liver and intestines (+ contents) were homogenised and lyophilised before combustion analysis.
- GLP compliance:
- no
- Radiolabelling:
- yes
- Species:
- rat
- Strain:
- other: Füllinsdorfer albino
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 174± 2 g for males and 154 ±4 g for females
- Housing: The animals were individually housed in metal metabolism cages allowing separate collection of urine and faeces.
- Diet: Ad libitum
- Water: Ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±1
- Humidity (%): 60 ±5
IN-LIFE DATES: 27 Jul 1981 to 30 Jul 1981 - Route of administration:
- oral: gavage
- Vehicle:
- other: rape oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Oral gavage with 8.5 mg 14C- test substance in 1.7 mL rape oil and 7.5 mg in 1.5 mL respectively. Total radioactivity was 9.65·10^7 dpm for males and 8.51· 10^7 dpm for females. The dosage amounted to 48.9 mg a.i./kg body weight for males and 48.7 mg a.i./kg body weight for females.
- Dosage solution: The solution was prepared by dissolving 14.18 mg of the 14-C test substance and 85.82 mg of the test substance (purity 99%) in 20 mL rape oil to give a specific activity of 5.11 µCi/mg (1 µg = 11 350 dpm) - Duration and frequency of treatment / exposure:
- Single dose
- Dose / conc.:
- 49 mg/kg bw/day (actual dose received)
- Remarks:
- 14C- radiolabelled in the dioxyphenyl ring, oral gavage
- No. of animals per sex per dose / concentration:
- 5
- Control animals:
- yes
- Details on dosing and sampling:
- TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, plasma, brain, liver, kidney, heart, spleen, gonads, stomach + contents, intestine + contents, 1 femur, 1 thigh muscle, and two fat samples which were removed from the kidneys and the gonads and residual carcass
- Time and frequency of sampling:
Urine and faeces: 0- 6, 6- 24, 24- 48, 48- 72, 72- 96 hours
Tissues: At the end of the collection period, 96 hours
ANALYTICAL METHOD
- Urine: All urine fractions were filtered through filterpaper and 2 mL aliquots were submitted to LSC. The filter paper containing the food residues was analysed by combustion analysis.
- Faeces: Faeces were homogenised in a blender with 35 mL acetonitrile – water (1:1). After centrifugation the residues were backwashed twice by shaking with acetonitrile. The volume of the combined extracts was made up to volume in a volumetric flask (250 mL) and aliquots submitted to LSC. The residues were dried in a vacuum-desiccator and unextractable radioactivity was measured by combustion analysis.
Tissues: Blood cells, brain, kidney, heart, muscle, spleen, bone, gonads, fat and stomach with contents were combusted after lyophilisation. The liver and the intestine plus contents were first homogenized in a blender with 9 mL of water, lyophilised and finally submitted to combustion analysis. Plasma aliquots (0.2 mL) were analysed directly by LSC. For the calculation of total radioactivity in plasma and blood cells a whole blood volume of 61.0 ±1.5 mL/kg body weight for male rats and 63.6 ± 1.1 mL/kg body weight for females were assumed (Schalm, 0.w., Jain, N.C. and Carrell, E.S., Veterinary Hematology, 3rd edition, Lea and Febiger, Philadelphia, USA). The volume of the blood cells was calculated as the difference of the blood and plasma volumes.
Residual carcass: The residual carcasses of 2 animals digested with l M KOH for 24 hours at 80°C. Aliquots were analysed by liquid scintillation counting (LSC). - Details on absorption:
- Absorption was between 84 and 96%
- Details on distribution in tissues:
- At 96 hours after oral administration 0.03 - 0.21% of the total dose was contained in the intestine. Average values in all other tissue and blood samples showed the presence of not more than 0.03% the total dose. Highest amounts were found in the liver where concentrations of 0.30 ppm and 0.20 ppm were determined in females and males respectively. Values in all other tissues including blood were below 0.20 ppm, this demonstrating that there is no bioretention. In the residual carcass of 2 animals, 0.15% and 0.32% of the administered dose were determined by alkaline digestion.
- Details on excretion:
- The overall excretion of radioactivity in urine plus faeces after 96 hours amounted to 90.95 ± 4.37 % (mean s.d., n=5) for males and 92.27 ± 3.02% for females, respectively. The excretion pattern shows that the test substance is well absorbed and rapidly excreted after oral administration to the rat.
- Conclusions:
- Absorption, distribution and excretion of 14C-test substance in rat was studied after a single oral dose of 49 mg/kg bw (dioxyphenyl ring-label). At 96 hours after dosing the average of radioactivity excreted in faeces (61-65% AR) was higher than that found in urine (27-30% AR, average). Very low amounts of radioactivity were recovered from tissues and the carcass (<0.35%, average). No information on radioactivity in expired air was available. Total recovery over the study period was 91-93% AR (average). No differences between males and females were noted, except that urinary excretion during the first 24 hours was lower in females
- Executive summary:
The study investigated the absorption, distribution and excretion of14C-test substance after a single dose by gavage of 49 mg/kg bw in Füllinsdorfer albino rats (5/sex/group). Rape oil was used as vehicle. Urine (including cage wash) and faeces were sampled over 0-6, 6-24, 24-48, 48-72 and 72-96 hours after dosing. After 96 hours blood was sampled and animals were sacrificed. At sacrifice, the following tissues and organs were taken from all animals: heart, liver, kidney, spleen, gonads, stomach (+ contents), intestines (+ contents), femur, muscle, fat and carcass. All animals received water and food ad libitum throughout the study. Radioactivity in urine and plasma was quantified by LSC. Blood cells and organs were combusted after lyophilisation. The residual carcass was solubilised in 1M KOH prior to LSC. Faeces were homogenised in acetonitrile-water (1:1) and submitted to LSC. Dried faecal residues and unextractable radioactivity was measured by combustion analysis. Liver and intestines (+ contents) were homogenised and lyophilised before combustion analysis.
Results showed, during the first 6 hours after dosing mean urinary excretion was 6.2% of applied radioactivity (AR). Thereafter (between 6 and 24 hours) 21.8% and 15.3% of AR was excreted by males and females, respectively. Over the whole study period urinary excretion was between 26.6% and 29.8% AR. Faecal excretion between 6 and 48 hours post dosing accounted for 60.1-61.7% of AR. During the 96 hours study period 61.1% and 65.5% AR was excreted via faeces for males and females, respectively. Tissue concentrations were very low accounting for <0.2% AR. Radioactivity in carcasses was assessed for one animal of each sex and was between 0.15% and 0.32% AR. Total recovery was 91.2-92.8%.
In conclusion, absorption, distribution and excretion of 14C-test substance in rat was studied after a single oral dose of 49 mg/kg bw (dioxyphenyl ring-label). At 96 hours after dosing the amount of radioactivity excreted in faeces (61 -65% AR, average ) was higher than that found in urine (27 -30% AR, average). Very low amounts of radioactivity were recovered from tissues and the carcass (<0.35%). No information on radioactivity in expired air was available. Total recovery over the study period was 91-93% AR. No differences between males and females were noted, except that urinary excretion during the first 24 hours was lower in females
- Endpoint:
- dermal absorption in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 Mar 2016 to 29 Jun 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 428 (Skin Absorption: In Vitro Method)
- Version / remarks:
- 2004
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- yes
- Species:
- other: Human skin
- Type of coverage:
- open
- Vehicle:
- other: physiological saline in a 1:1 (w/w) ratio (for the concentrate) and CIPAC D water (for the spray dilutions)
- Duration of exposure:
- The skin was exposed to the test preparations for 6 hours and receptor fluid samples were collected at 2, 4 and 6 hours post dose. To allow adequate characterisation of the absorption profile, receptor fluid samples were also collected at 8, 12 and 24 hours post dose.
- Doses:
- - 125 g test substance /kg Formulation Concentrate in saline
- 0.15 g test substance /L Spray Dilution 1
- 0.075 g test substance /L Spray Dilution 2 - Key result
- Time point:
- 24 h
- Dose:
- 125 g/kg
- Parameter:
- percentage
- Absorption:
- 0.44 %
- Remarks on result:
- other: Dermal absorption = (Sum of receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20) + k(n=8) x SD
- Key result
- Time point:
- 24 h
- Dose:
- 0.075 g/L
- Parameter:
- percentage
- Absorption:
- 19 %
- Remarks on result:
- other: Dermal absorption = (Sum of receptor fluid + receptor chamber wash + exposed skin + skin strips (3-20) + k(n=8) x SD
- Key result
- Time point:
- 24 h
- Dose:
- 0.15 g/L
- Parameter:
- percentage
- Absorption:
- 21 %
- Remarks on result:
- other: Dermal absorption = (Sum of receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20) + k(n=8) x SD
- Conclusions:
- The study demonstrated that the amount of the test substance absorbed through human split-thickness skin membranes over 24 h (following a 6 h exposure) from the Formulation Concentrate in Saline (125 g/kg) and the intended in-use concentrations (0.15 g/L and 0.075 g/L), was 0.19%, 11.18%, and 9.81% of the applied dose, respectively, as measured in the receptor fluid and receptor chamber wash. The recalculated values for sum of receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20 + k(n=8) x SD were 0.44%, 19% and 21% for 125 g/kg, 0.075 g/L and 0.15 g/L of the test substance, respectively.
- Executive summary:
The purpose of this study was to determine the rate and extent of the in vitro percutaneous absorption of test substance through human split-thickness skin (following 6 h exposure) over an experimental period of 24 h to aid the quantitative assessment of the risk arising from skin contact with the test substance according to OECD TG 428 and GLP principles. The Formulation Concentrate was mixed with physiological saline (1:1, w/w) to generate a paste. A paste was generated because wettable granules cannot be applied accurately to the skin. This is equivalent to an operator becoming exposed to the wettable granule which in turn mixes with sweat.
The active ingredient (the test substance) was assessed with the following applications: 125 g test substance /kg Formulation Concentrate in saline, 0.15 g test substance /L Spray Dilution 1 and 0.075 g test substance /L Spray Dilution 2. The doses were applied at 10 mg/cm2 for the formulation concentrate in saline and 10 μL/cm2 for spray dilution 1 and 2. Following dosing cells were left unoccluded for an experimental period of 24 h, with an interim wash at 6 h post-application. The absorption process was followed by taking samples of the receptor fluid (phosphate buffered saline containing polyoxyethylene 20 oleyl ether (PEG, ca 6%, w/v), sodium azide (ca 0.01%, w/v), streptomycin (ca 0.1 mg/mL) and penicillin (ca 100 units/mL)) at recorded intervals throughout the experimental period. The distribution of the test substance within the test system and a 24 h absorption profile were determined using liquid scintillation counting. Before conducting the main study stability and solubility assessments were carried out.
Results showed that the amount of test substance absorbed through human split-thickness skin membranes over 24 h (following a 6 h exposure) from the concentrate formulation in saline (125 g/kg) and the intended in-use concentrations (0.15 g/L and 0.075 g/L) in the test substance was 0.20%, 11.18% and 9.81% of the applied dose, respectively, as measured in the receptor fluid and receptor chamber wash. The recalculated values for sum of receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20 + k(n=8) x SD were 0.44%, 19% and 21% for 125 g/kg, 0.075 g/L and 0.15 g/L of the test substance, respectively.
- Endpoint:
- dermal absorption in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 Nov 2004 to 8 Dec 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 428 (Skin Absorption: In Vitro Method)
- Version / remarks:
- 2004
- GLP compliance:
- yes (incl. QA statement)
- Radiolabelling:
- yes
- Species:
- other: Human skin
- Type of coverage:
- open
- Vehicle:
- other: Emulsifiable concentrate formulation at 7.39% (w/w)
- Duration of exposure:
- After 6 and 24 h, mild skin washing removed the vast majority after exposure of the applied dose from the surface of human skin.
- Doses:
- 75 g/L
- Key result
- Time point:
- 24 h
- Dose:
- 75 g/L
- Parameter:
- percentage
- Remarks:
- Undiluted
- Absorption:
- 3.6 %
- Remarks on result:
- other: Recalculated from receptor fluid + epidermal tissue + stratum corneum
- Time point:
- 6 h
- Dose:
- 75 g/L
- Parameter:
- percentage
- Absorption:
- 0.36 %
- Remarks on result:
- other: Recalculated from receptor fluid + epidermal tissue + stratum corneum
- Conclusions:
- The results obtained in this study indicate that the test substance is absorbed through human epidermis from the concentrate formulation at a very slow rate. The majority of the applied dose (between 84.6 - 93.4%) was removed by mild skin washing at 6 and 24 hours. The recalculated values from receptor fluid+epidermal tissue+stratum corneum were 3.6 % and 0.36% at 24h and 6h, respectively.
- Executive summary:
The absorption of the test substance with a concentration 75 g/L has been performed with human epidermis to assess the in vitro percutaneous absorption according to OECD TG 428 and GLP principles. The absorption process was followed using the test substance which was added to the concentrated formulation prior to application. The distribution of the test substance within the test system (skin wash, donor chamber, stratum corneum and residual epidermal tissue) and an absorption profile over the 24 hour exposure period were determined. A 6 hour exposure was also included to investigate the amount of the test substance present in the each of the compartments (skin wash, donor chamber, stratum corneum and residual epidermal tissue) and the amount absorbed at the end of a typical ‘working day’ period. The absorbed (systemically available) dose is considered to be the test substance detected in the receptor fluid. Material removed from the surface of the epidermis by the washing procedure is regarded as unabsorbed. Test material recovered from the epidermis at the end of the exposure is also considered to be unabsorbed, although it is recognised that a proportion of this material may be absorbed beyond the duration of the exposure investigated in this study.
Results showed that the test substance absorption gradually increased over the 24 hour absorption period. Between 0- 4 h the rate of the test substance absorption was 0.30 µg/cm^2/h after which absorption increased to 0.75 µg/cm^2/h between 4- 12h. The test substance absorption was fastest between 12- 24h. Between 0-24 hours, the mean rate of absorption was 0.83 µg/cm^2/h. The amounts absorbed during working day periods of 6, 8 and 10 hours were 2.34 , 3.70 and 5.37 µg/cm^2, respectively. The respective amounts expressed as percentages of the applied dose were 0.31, 0.50 and 0.72%. The amount absorbed over the entire 24 hour exposure period was 19.7 µg/cm^2 (2.65% of the applied dose). Recovery of radiolabelled test substance in these experiments was very good, (means ranged between 95.3-97.9% of the applied dose). At 6 hours exposure, mild skin washing removed the vast majority (mean of 93. 1%) of the applied dose from the surface of human skin. The remaining proportion recovered from stratum corneum was 0.11% leaving a mean of 0. 14% of the applied dose in the remaining epidermal tissue. The amount (0.80 µg/cm^2) recovered from receptor fluid accounted for 0.11% of the applied dose. At 24 hours exposure, mild skin washing removed the vast majority (mean of 91 .7%) of the applied dose) from the surface of human skin. The remaining proportion recovered from stratum corneum was 0.20% leaving a mean of 0.73% of the applied dose in the remaining epidermal tissue. The amount (19.7 µg/cm^2) from receptor fluid accounted for 2.65% of the applied dose.
In conclusion, the results obtained in this study indicate that the test substance is absorbed through human epidermis from the concentrate formulation at a very slow rate. The majority of the applied dose (between 84.6 - 93.4%) was removed by mild skin washing at 6 and 24 hours. These data predict that the human dermal absorption of the test substance from potential exposure to this EC formulation as the concentrate formulation, would be low. The recalculated values from receptor fluid+epidermal tissue+stratum corneum were 3.6 % and 0.36% at 24h and 6h, respectively.
Referenceopen allclose all
Table 1. Excretion and retention of radioactivity (% AR) in rats after single and repeated exposure to 14C-test substance
sample |
P-H Single oral 300 mg/kgbw |
A Single iv 1.0 mg/kg bw |
B Single oral 1.0 mg/kg bw |
C Repeated oral 1.0 mg/kgbw |
D Single oral 300 mg/kg bw |
|||||
M |
F |
M |
F |
M |
F |
M |
F |
M |
F |
|
faeces 0-6 h 6-12 h |
6.7 |
0.6 |
0.01 1.1 |
0.3 1.6 |
NA 9.3 |
<0.01 22 |
NA 14 |
<0.01 23 |
NA 3.7 |
0.01 5.0 |
12-24 h |
21 |
25 |
39 |
30 |
41 |
38 |
47 |
38 |
13 |
10 |
24-48 h |
26 |
27 |
26 |
29 |
20 |
15 |
17 |
16 |
33 |
27 |
48-72 h |
2.8 |
3.8 |
3.7 |
6.5 |
2.9 |
2.5 |
2.7 |
2.4 |
8.8 |
12 |
72-96 h |
0.3 |
0.7 |
0.7 |
1.5 |
0.4 |
0.4 |
0.3 |
0.4 |
0.8 |
1.5 |
96-120 h |
0.09 |
0.08 |
0.2 |
0.3 |
0.1 |
0.1 |
0.1 |
0.09 |
0.1 |
0.3 |
120-144 h |
0.05 |
0.04 |
0.05 |
0.07 |
0.03 |
0.0 2 |
0.02 |
0.05 |
0.05 |
0.09 |
144-168 h |
0.05 |
0.03 |
<0.01 |
0.05 |
ND |
<0.01 |
ND |
<0.01 |
0.02 |
0.05 |
0-168 h |
55.8 |
57.9 |
70.0 |
68.6 |
73.4 |
77.7 |
80.9 |
80.4 |
59.6 |
56.4 |
urine 0-6 h 6-12 h |
11 |
15 |
5.2 4.2 |
6.5 5.0 |
4.1 3.3 |
5.5 3.4 |
5.4 5.6 |
8.2 5.2 |
2.3 3.7 |
4.1 3.5 |
12-24 h |
19 |
15 |
6.6 |
6.4 |
5.3 |
3.8 |
6.3 |
5.0 |
7.7 |
4.8 |
24-48 h |
7.6 |
8.0 |
3.6 |
4.3 |
2.8 |
2.8 |
2.9 |
3.0 |
20 |
20 |
48-72 h |
0.4 |
0.8 |
1.1 |
1.5 |
0.5 |
0.6 |
0.6 |
0.7 |
1.5 |
2.3 |
72-96 h |
0.2 |
0.2 |
0.2 |
0.6 |
0.1 |
0.2 |
0.2 |
0.3 |
0.5 |
0.5 |
96-120 h |
0.2 |
0.09 |
0.1 |
0.2 |
0.08 |
0.1 |
0.07 |
0.2 |
0.2 |
0.3 |
120-144 h |
0.08 |
0.04 |
0.06 |
0.1 |
0.06 |
0.07 |
0.06 |
0.09 |
0.1 |
0.1 |
144-168 h |
0.06 |
0.03 |
0.04 |
0.07 |
0.03 |
0.05 |
0.04 |
0.07 |
0.1 |
0.1 |
0-168 h |
37.8 |
38.7 |
21.0 |
24.7 |
16.3 |
16.5 |
21.0 |
22.7 |
36.0 |
35.2 |
expired air (CO2 0-168h and volatiles |
ND |
ND |
- |
- |
- |
- |
- |
- |
- |
- |
carcass 168 h |
0.2 |
0.3 |
0.3 |
0.7 |
0.02 |
0.2 |
ND |
0.2 |
0.2 |
0.3 |
cage wash/wipe 168 h |
0.8 |
0.5 |
0.3 |
0.5 |
0.3 |
0.5 |
0.3 |
2.3 |
0.6 |
0.9 |
Recovery 168 h |
94.6 |
97.4 |
91.6 |
94.5 |
90.0 |
94.9 |
102.2 |
105.6 |
96.4 |
92.8 |
ND not detectable NA not applicable
Table 2. Distribution of radioactivity in tissues and organs [ng test substance equivalents/g and % AR at 168 post dose 14C-test substance
sample |
A Single iv 1.0 mg/kg bw |
C Single oral 1.0 mg/kg bw |
C Repeated oral 1.0 mg/kg bw |
D-P-H Single oral 300 mg/kg bw |
||||||||||||
M |
F |
M |
F |
M |
F |
M |
F |
|||||||||
ng |
%AR |
ng |
%AR |
ng |
%AR |
ng |
%AR |
ng |
%AR |
ng |
%AR |
ng |
%AR |
ng |
%AR |
|
Bone |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
103 |
<0.01 |
205 |
<0.01 |
Brain |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
Carcass |
3 |
0.33 |
7 |
0.67 |
<1 |
0.02 |
2 |
0.15 |
ND |
ND |
1 |
0.17 |
430 |
0.17 |
839 |
0.29 |
Fat |
3 |
<0.01 |
2 |
<0.01 |
2 |
<0.01 |
2 |
<0.01 |
1 |
<0.01 |
2 |
<0.01 |
1618 |
<0.01 |
1795 |
<0.01 |
Heart |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
11 |
<0.01 |
15 |
<0.01 |
Kidneys |
<1 |
<0.01 |
ND |
ND |
<1 |
<0.01 |
ND |
ND |
1 |
<0.01 |
ND |
ND |
335 |
<0.01 |
282 |
<0.01 |
Liver |
3 |
0.02 |
5 |
0.02 |
4 |
0.03 |
6 |
0.03 |
4 |
0.03 |
6 |
0.03 |
683 |
0.01 |
1140 |
0.02 |
Lungs |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
83 |
<0.01 |
121 |
<0.01 |
Muscle |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
68 |
<0.01 |
66 |
<0.01 |
Ovaries |
NA |
NA |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
675 |
<0.01 |
Plasma |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
79 |
NA |
66 |
NA |
Red blood cells |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
23 |
NA |
93 |
NA |
Spleen |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
ND |
23 |
<0.01 |
31 |
<0.01 |
Testes |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
Uterus |
NA |
NA |
ND |
ND |
ND |
ND |
ND |
ND |
NA |
NA |
ND |
ND |
NA |
NA |
102 |
<0.01 |
Table 3. Absorption values
Percentage of dose |
|||||||
Group |
Sex |
Dose mg/kg |
Urine (0-168 h) b |
Tissue residues b |
Carcass residues b |
Total c |
Absorption % |
A (IV) |
M |
0.93 |
21.3 |
0.02 |
0.33 |
21.7 |
100 |
|
F |
0.94 |
25.1 |
0.02 |
0.67 |
25.8 |
100 |
B (oral) |
M |
1.00 |
16.6 |
0.03 |
16.7 |
77.0 |
|
|
F |
1.00 |
17.0 |
0.03 |
0.15 |
17.2 |
66.7 |
C (oral) |
M |
0.90 |
21.4 |
0.03 |
ND |
21.4 |
98.6 |
|
F |
0.92 |
25.0 |
0.03 |
0.17 |
25.2 |
97.7 |
Table 1. Excretion and retention of radioactivity (% AR) in rats after single oral exposure to 14C-test substance
sample |
3000 mg/kgbw 14C-test substance |
50 mg/kg bw 14C-test substance |
|||
M |
F |
M |
F |
||
urine |
0-12 h |
1.1 |
1.9 |
|
|
|
0-24 h |
7.8 |
6.7 |
11.3 |
5.3 |
|
0-48 h |
23.7 |
21.5 |
|
|
|
0-72 h |
42.3 |
34.8 |
|
|
|
0-96 h |
44.0 |
36.9 |
|
|
faeces |
0-12 h |
0.03 |
0.6 |
|
|
|
0-24 h |
13.3 |
12.3 |
12.1 |
2.4 |
|
0-48 h |
25.3 |
23.4 |
|
|
|
0-72 h |
43.7 |
43.5 |
|
|
|
0-96 h |
49.1 |
51.4 |
|
|
bile |
0-1 h |
|
|
0.8 |
1.0 |
|
0-2 h |
5.0 |
1.8 |
||
|
0-6 h |
23.8 |
2.7 |
||
|
0-12 h |
44.5 |
7.0 |
||
|
0-24 h |
62.8 |
36.9 |
||
GI tract |
96 h/24 h |
0.6 |
1.2 |
9.1 |
51.2 |
Carcass |
96 h/24 h |
0.8 |
2.6 |
1.5 |
1.5 |
expired air (CO2) |
0-48 h |
0.09 |
0.09 |
|
|
Cage wash |
0-24 h |
1.1 |
2.4 |
0.2 |
0.7 |
|
0-96 h |
3.3 |
5.6 |
|
|
Tissues/organs |
96 h/24 h |
0.08 |
0.07 |
|
|
Recovery |
96 h/24 h |
98.0 |
97.8 |
97.1 |
98.1 |
Table 2. Distribution of radioactivity in tissues and organs [% AR] at 96 hours post dose of 3000 mg/kg 14C-test substance
tissue |
3000 mg/kgbw 14C-test substance |
|||
male |
female |
|||
µg/g |
% AR |
µg/g |
% AR |
|
Bone |
9 |
- |
12 |
- |
Brain |
1 |
0.0 |
2 |
0.0 |
fat |
16 |
- |
34 |
- |
Heart |
4 |
0.0 |
5 |
0.0 |
Skeletal muscle |
5 |
- |
28 |
- |
Ovaries |
|
|
19 |
0.0 |
Testes |
3 |
0.0 |
|
|
Liver |
32 |
0.07 |
42 |
0.07 |
Lung |
6 |
0.0 |
7 |
0.0 |
Spleen |
4 |
0.0 |
4 |
0.0 |
Kidney |
17 |
0.0 |
13 |
0.0 |
Stomach (+contents) |
7 |
0.01 |
20 |
0.02 |
Intestines (+contents) |
198 |
0.58 |
345 |
1.17 |
Plasma |
10 |
- |
7 |
- |
Blood cells |
5 |
- |
5 |
- |
Table 3. Distribution of radioactivity in tissues and organs after repeated dose of 50 mg/kg 14C-test substance
Day/number of doses |
50 mg/kg bw 14C-test substance repeated dose |
|||||
Number-sex |
Plasma µg/g |
Carcass µg/g |
Liver µg/g |
Kidney µg/g |
Fat µg/g |
|
1/1 |
3M |
0.5 |
1.8 |
3.2 |
1.3 |
0.6 |
3/3 |
3F |
0.4 |
2.6 |
6.1 |
1.1 |
0.7 |
7/7 |
3M |
0.7 |
2.1 |
4.6 |
2.2 |
1.7 |
14/14 |
3F |
0.5 |
3.3 |
7.6 |
1.2 |
1.3 |
21/21 |
3M |
1.3 |
3.8 |
9.1 |
4.1 |
2.2 |
28/28 |
3F |
1.9 |
5.6 |
13.0 |
2.1 |
2.7 |
29/28 |
3M |
0.8 |
2.1 |
5.5 |
2.6 |
1.4 |
30/28 |
3F |
0.1 |
1.3 |
3.1 |
0.9 |
1.6 |
31/28 |
3M |
0.2 |
0.8 |
2.2 |
1.0 |
1.0 |
34/28 |
3F |
0.1 |
0.8 |
1.4 |
0.6 |
1.6 |
37/28 |
3M |
0.0 |
0.6 |
1.0 |
0.4 |
1.2 |
41/28 |
3F |
0.0 |
0.6 |
0.7 |
0.3 |
1.1 |
Table 4. Metabolite identification in 0-24 hours urine and faeces of male and/or female rats after single oral exposure to 3000 mg/kg and single and repeated exposure to 50 mg/kg bw 14C-test substance by radio-TLC (as % radioactivity on TLC plate)
Fraction (identity radio TLC) |
urine |
faeces |
||||||||||
3000 mg/kg |
50 mg/kg single |
50 mg/kg rep. |
3000 mg/kg |
50 mg/kg single |
50 mg/kg rep. |
|||||||
ut |
gt |
st |
ut |
gt |
st |
ut |
gt |
st |
|
|
|
|
Test substance |
0.8 |
3.4 |
4.2 |
0.1 |
0.2 |
2.3 |
0.0 |
0.2 |
1.9 |
84 |
36 |
8.4 |
M13 |
5.4 |
29 |
36 |
|
|
|
|
|
|
7.9 |
1.9 |
0.9 |
M12 |
0.1 |
0.9 |
17 |
0.2 |
0.8 |
9.8 |
- |
0.3 |
5.0 |
0.6 |
5.1 |
2.7 |
M14 |
|
|
|
0.1 |
0.6 |
4.1 |
0.3 |
0.3 |
3.2 |
|
|
|
Unknowns |
6.6 |
13 |
34 |
3.0 |
3.2 |
58 |
1.8 |
2.0 |
56 |
5.3 |
35 |
55 |
Origin |
87 |
54 |
8.6 |
97 |
95 |
26 |
97 |
97 |
33 |
2.5 |
22 |
33 |
% of total AR |
7.2 |
27 |
3(A) |
12.8 |
43 |
1(A) |
||||||
ut= untreated; gt = glucuronidase treated; st = sulfatase treated
(A) expressed as % of total radioactive dose received over 28 days
Table 5. Metabolite identification in 0-24 hours plasma, bile and liver of male and/or female rats after single and repeated oral exposure to 50 mg/kg bw 14C-test substance by radio-TLC (as % radioactivity on TLC plate)
Fraction (identity radio TLC) |
plasma |
bile |
liver |
||||||||
50 mg/kg single |
50 mg/kg rep. |
50 mg/kg single |
50 mg/kgsingle |
50 mg/kg rep. |
|||||||
1-2 ut |
1-2 gt |
1-2 st |
12-13 ut |
21-22 ut |
|||||||
M |
F |
M |
F |
M |
M |
M |
M |
M |
M |
F |
|
Test substance |
58 |
10 |
0 |
1 |
0.0 |
1.6 |
1.6 |
0.4 |
1.1 |
0 |
5 |
M13 |
|
|
|
|
0.3 |
15 |
14 |
3.4 |
9.5 |
3 |
6 |
M12 |
|
|
|
|
0.1 |
0.3 |
11 |
0.1 |
0.1 |
1 |
14 |
M14 |
|
|
|
|
|
|
|
|
|
|
|
Unknown |
16 |
38 |
10 |
19 |
0.4 |
1.9 |
52 |
1.1 |
1.2 |
34 |
57 |
Origin |
26 |
52 |
90 |
79 |
99 |
81 |
23 |
95 |
88 |
63 |
18 |
Total recovered (µg eq./mL or g) |
0.5 |
0.4 |
1.4 |
0.9 |
|
|
|
|
|
3.8 |
16 |
ut= untreated; gt = glucuronidase treated; st = sulfatase treated
Table 1. Pharmacokinetic parameters in plasma of male and female rats after single oral exposure to 14C-test substance at 50 mg/kg bw
|
single oral, 50 mg/kg bw |
|
M |
F |
|
Cmax (µg eq./kg) |
26.8 |
5.74 |
Tmax (h) |
4 |
4 (2-4) |
½ Cmax (µg eq./kg) |
11.7 |
2.1 |
½ Tmax (h) |
6 |
6 |
Table 2. Distribution of radioactivity (% AR) in rats after single oral exposure to 14C- test substance at 50 mg/kg bw
sample |
3 hours |
6 hours |
||
M |
F |
M |
F |
|
Urine |
4.0 |
3.5 |
9.1 |
12 |
Faeces |
2.8 |
0.04 |
3.4 |
0 |
Cage wash |
1.9 |
2.8 |
2.7 |
3.5 |
Stomach |
25 |
19 |
6.2 |
12 |
Intestine |
49 |
63 |
72 |
66 |
Organs |
5.6 |
6.7 |
3.5 |
3.8 |
Carcass |
7.3 |
3.9 |
3.2 |
2.0 |
Recovery |
96.1 |
99.0 |
100.1 |
98.8 |
Table 3. Distribution of radioactivity in tissues and organs [μg test substance equivalents/g and % AR] at 3 and 6 hours post dose of 50 mg/kg 14C-test substance
tissue |
3 hours |
6 hours |
||||||
male |
female |
male |
female |
|||||
µg/g |
% AR |
µg/g |
% AR |
µg/g |
% AR |
µg/g |
% AR |
|
Liver |
66 |
5.2 |
85 |
6.4 |
38 |
3.2 |
48 |
3.7 |
Kidney |
11 |
0.2 |
11 |
0.2 |
9.6 |
0.2 |
5.1 |
0.09 |
Spleen |
1.5 |
0.008 |
1.5 |
0.008 |
0.9 |
0.005 |
0.6 |
0.002 |
Heart |
3.5 |
0.03 |
2.0 |
0.02 |
1.6 |
0.02 |
0.7 |
0.006 |
Lungs |
3.9 |
0.08 |
3.7 |
0.04 |
2.8 |
0.04 |
1.4 |
0.02 |
Ovaries |
|
0.04 |
4.0 |
0.006 |
|
|
2.5 |
0.003 |
Testes |
1.7 |
0.01 |
|
|
1.9 |
0.05 |
|
|
Brain |
0.7 |
|
0.6 |
0.01 |
0.4 |
0.007 |
0.3 |
0.005 |
Bone |
2.1 |
0.02 |
1.4 |
0.02 |
1.0 |
0.01 |
0.5 |
0.005 |
Skeletal muscle |
1.8 |
0.02 |
1.3 |
0.01 |
1.2 |
0.01 |
0.5 |
0.006 |
Fat |
4.0 |
0.04 |
3.2 |
0.03 |
5.0 |
0.05 |
2.1 |
0.02 |
Plasma (µl-1) |
20 |
0.2 |
14 |
0.1 |
11 |
0.1 |
3.6 |
0.04 |
Blood cells |
1.6 |
0.02 |
1.6 |
0.02 |
1.0 |
0.01 |
0.5 |
0.005 |
Total |
5.7 |
6.8 |
3.6 |
3.9 |
||||
Table 1. Excretion and retention of radioactivity (% AR) in rats after single oral exposure to 14C-test substance. Valeus are averages.
Dose (mg/kg bw) |
|
||
Sex |
M |
F |
|
urine |
6-24 h |
21.8 |
15.3 |
urine |
0-96 h |
29.8 |
26.6 |
faeces |
6-48 h |
60.1 |
61.7 |
faeces |
0-96 h |
61.1 |
65.6 |
tissue (A) |
96 h |
0.081 |
0.17 |
carcass (B) |
96 h |
0.15 |
0.32 |
recovery |
0-96 |
91.2 |
92.8 |
(A) Predominantly in intestine (0.04-0.12%) and liver (0.02-0.03%)
(B) Carcass of 1/sex
Table 1. Summary of the test substance through Human Split-Thickness Membranes
Test Preparation |
Formulation Concentrate in Saline (125 g/kg) |
Spray Dilution 1 (0.15 g/L) (Normalised) |
Spray Dilution 2 (0.075 g/L) |
Test substance |
Test substance |
||
Distribution |
% Applied Dose |
||
Dislodgeable Dose 6 h* |
99.62 |
73.49 |
80.47 |
Total Dislodgeable Dose** |
100.44 |
82.33 |
85.55 |
Donor Chamber Wash |
0.39 |
0.64 |
0.40 |
Tape Strips 1-2 |
0.05 |
1.15 |
0.29 |
Tape Strips 3-20 |
0.08 |
1.33 |
0.54 |
Unexposed Skin |
<0.01 |
0.08 |
0.04 |
Exposed Skin |
0.06 |
3.93 |
2.45 |
Receptor Fluid |
0.18 |
10.69 |
9.38 |
Receptor Chamber Wash |
0.01 |
0.49 |
0.43 |
Mass Balance |
100.84 |
100.00 |
98.68 |
Distribution |
µg equiv./cm2 |
ng equiv./cm2 |
ng equiv./cm2 |
Dislodgeable Dose 6 h* |
1170.88 |
1098.20 |
604.66 |
Total Dislodgeable Dose** |
1180.54 |
1230.19 |
642.85 |
Donor Chamber Wash |
4.57 |
9.60 |
3.04 |
Tape Strips 1-2 |
0.63 |
17.15 |
2.20 |
Tape Strips 3-20 |
0.88 |
19.94 |
4.04 |
Unexposed Skin |
0.03 |
1.23 |
0.28 |
Exposed Skin |
0.72 |
58.73 |
18.40 |
Receptor Fluid |
2.17 |
159.78 |
70.47 |
Receptor Chamber Wash |
0.17 |
7.25 |
3.22 |
Mass Balance |
1185.14 |
1494.27 |
741.47 |
* Dislodgeable Dose 6 h = Skin Wash 6 h + Tissue Swab 6 h + Pipette Tip 6 h
** Total Dislodgeable Dose = Dislodgeable Dose 6 h + Skin Wash 24 h + Tissue Swab 24 h + Pipette Tip 24 h + Donor Chamber Wash
Table 2. Summary of test substance Absorption through Human Split-Thickness Membranes
Application of Test substance and Actual Concentration of Dose Preparation |
Mean Absorption Rates |
|
Time Period (h) |
Absorption Rate |
|
Formulation Concentrate in Saline |
|
µg equiv./cm2/h ± SEM |
(115 g/kg test substance) |
0-2 |
0.28 ± 0.04 |
10.25 µg/cm2(1175 µg ai/cm2) |
2-6 |
0.12 ± 0.02 |
Unoccluded |
6-24 |
0.06 ± 0.01 |
Duration of experiment: 24 h, n = 8 |
0-24 |
0.09 ± 0.01 |
Spray Dilution 1 |
|
ng equiv./cm2/h ± SEM |
(0.149 g/L test substance) |
0-2 |
1.90 ± 0.43 |
10.00 µL/cm2(1.49 µg ai/cm2) |
2-6 |
10.19 ± 2.06 |
Unoccluded |
6-24 |
6.40 ± 0.78 |
Duration of experiment: 24 h, n = 8 |
0-24 |
6.66 ± 0.85 |
Spray Dilution 2 |
|
ng equiv./cm2/h ± SEM |
(0.075 g/L test substance) |
0-2 |
0.57 ± 0.18 |
10.00 µL/cm2(0.751 µg ai/cm2) |
2-6 |
4.78 ± 0.23 |
Unoccluded |
6-24 |
2.79 ± 0.37 |
Duration of experiment: 24 h, n = 7 |
0-24 |
2.94 ± 0.28 |
Dermal absorption was calculated according to the Efsa guidance on dermal absorption (2017): receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20 + k (n=8) x SD. The following absorption values were calculated: 0.44 %, 19 % and 21 % for 125 g/kg, 0.075 g/L and 0.15 g/L, respectively at 24h. The k multiplication factor (n=8) was determined to be 0.84 and the following SD were calculated: 0.12, 5.76 and 5.38 for 125 g/kg, 0.075 g/L and 0.15 g/L, respectively.
Table 1. Summary of test substance absorption through human epidermis
Mean Absorption Rates |
Mean Amount and Percent of Dose Absorbed |
|||||
Time period |
Absorption rate (mg/cm2/h±SEM) |
Time (h) |
Amount (mg/cm2) |
Percent absorbed |
||
|
|
|
6 |
2.34 |
0.31 |
|
0-4 |
0.30 ± 0.13 |
8 |
3.70 |
0.50 |
||
4-12 |
0.75 ± 0.31 |
10 |
5.37 |
0.72 |
||
12-24 |
1.04 ± 0.42 |
24 |
19.7 |
2.65 |
||
0-24 |
0.83 ± 0.34 |
LOQ |
0.12 |
0.02 |
||
Table 2. Summary of test substance distribution from the concentrate formulation – 6 hour exposure
n = 6 |
mg of test substance per cm2 MeanSD |
% of applied dose Mean SD |
||
Donor chamber |
30.2 |
35.7 |
4.05 |
4.79 |
Skin wash |
694 |
32.7 |
93.1 |
4.38 |
Stratum Corneum |
0.82 |
0.38 |
0.11 |
0.05 |
Remaining Epidermis |
1.05 |
0.70 |
0.14 |
0.09 |
Absorbed |
0.80 |
0.74 |
0.11 |
0.10 |
TOTAL RECOVERED |
727 |
26.0 |
97.5 |
3.49 |
Table 3. Summary of test substance distribution from the concentrate formulation – 24 hour exposure
n = 6 |
mg of test substance per cm2 MeanSD |
% of applied dose Mean SD |
||
Donor chamber |
9.11 |
4.96 |
1.22 |
0.67 |
Skin wash |
684 |
39.7 |
91.7 |
5.32 |
Stratum Corneum |
1.52 |
1.44 |
0.20 |
0.19 |
Remaining Epidermis |
5.45 |
6.55 |
0.73 |
0.88 |
Absorbed |
19.7 |
21.5 |
2.65 |
2.89 |
TOTAL RECOVERED |
720 |
21.9 |
96.5 |
2.94 |
Description of key information
- Oral absorption: Rapid and well absorbed (100 %) ; EPA 85 -1, Cheng, 1993
- Dermal absorption: 21% (diluted product) , in vitro absorption study in human skin; OECD TG 428; Blackstock, Millican 2017
- Inhalation absorption: Based on the lack of information on inhalation absorption, the default absorption value from the REACH guidance (Chapter 8, R.8.4.2) is used for DNEL derivation, namely: 100%
Key value for chemical safety assessment
- Bioaccumulation potential:
- no bioaccumulation potential
- Absorption rate - oral (%):
- 100
- Absorption rate - dermal (%):
- 21
- Absorption rate - inhalation (%):
- 100
Additional information
Absorption:
Oral route: The test substance was rapid and well absorbed, 67-77% after low and high single oral dose and 98% following repeated dose. The oral absorption is estimated to be 100% (Dieterle 1981, Hassler 1971, Cheng 1993).
Dermal route:
Dermal absorption was calculated according to the EFSA guidance on dermal absorption (2017): receptor fluid + receptor chamber wash + exposed skin + skin strips 3-20 + k (n=8) x SD. The following absorption values were calculated: 0.44 %, 19 % and 21 % for 125 g/kg, 0.075 g/L and 0.15 g/L, respectively at 24h. The k multiplication factor (n=8) was determined to be 0.84 and the following SD were calculated: 0.12, 5.76 and 5.38 for 125 g/kg, 0.075 g/L and 0.15 g/L, respectively. The highest dermal absorption was 21% at a concentration of 0.15 g/L at 24h (Blackstock, Millican, 2017).
Distribution:
14C-test substance is found mainly in the gastro-intestinal tract after oral administration (60-90% AR). In organs minor amounts (3.5-6.7% AR) were found. The highest concentrations were found in the liver (Cameron 1988).
Metabolism:
Rapid and extensive, resulting in polar low molecular weight metabolites that are rapidly excreted in free and conjugated forms. The metabolism proceeds by hydroxylation and stepwise oxidations and conjugation with sulphates. The major metabolite in urine is 4-hydroxy-test substance (15%) mostly conjugated with sulphate. Further 17 minor metabolites were identified. In the faeces series of metabolites were identified along with small amounts of unchanged test substance (Cameron 1986, Hassler 1997, Pryde and Etterli 1983, Itterly 1995).
Excretion:
Rapid, mainly in the faeces (60-75%) with a high proportion eliminated via bile. 20-35% excreted in urine. At 96 hours after dosing the amount of radioactivity excreted in faeces (61-65% AR) was higher than that found in urine (27-30% AR) (Dieterle 1981). Considering biliary excretion there seems to be a difference between the sexes, but as only one animal per sex was investigated the study does not allow definite conclusions (Cameron 1986).
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