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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
toxicokinetics
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
(1984)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Version / remarks:
(1998)
Qualifier:
according to guideline
Guideline:
other: Japan/MAFF Guidelines on the Compiling of Test Results on Toxicity; Tests on In Vivo Fate In Animals (2001)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 954-1012

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 96.1%
- Specific activity: 69.2 MBq/mg
- Locations of the label: 1,2-C14

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at low temperatures and in the dark
- Solubility and stability of the test substance in the solvent/vehicle:The analytical investigations performed in the context of this study demonstrated the stability, homogeneity and correctness of the concentrations for the period of the test substance administration of 14C-Glyoxal in the vehicle for all performed experiments.

Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at start of acclimatization: 6 to 12 weeks
- Weight at study initiation: 250 - 350 g
- Housing: prior test initiation, in groups in Macrolon cages; during experiment, individually in all-glass metabolism cages type Metabowl (Jencons Leighton Buzzard, U.K.) labeled with the project number, animal number, dose and time of first application
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: planned, duration not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h
- Any deviations will be documented

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study will be assayed for chemical and microbial contaminants according to the Fed. Reg. Vol. 44, No. 91 of May. 09, 1979, p 27354 (EPA);
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants;
- The bedding (Type Lingocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany) is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–33, will serve as the guideline for maximum tolerable contaminants.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Radiolabelled and the non-radiolabelled test substance will be prepared in 0.5 % carboxymethylcellulose in tap water.
- For the balance experiments (experiments 4 and 5) and the bile excretion experiments (experiment 8 and 9), the non-radiolabelled test substance will be mixed with the 13C-labelled test substance in a ratio of 1:1 (w:w). In order to achieve the required specific activity, respective amounts of 14C-labelled compound will be added and the mixture will be filled up with 0.5 % carboxymethylcellulose in tap water.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The stability, homogeneity and correctness of the concentrations of the test item in the aqueous vehicle will be verified in all experiments by HPLC.
Dose / conc.:
25 mg/kg bw (total dose)
Remarks:
The required quantity of radioactivity per animal was about 0.2-2 MBq. About 10 mL/kg body weight of a preparation will be administered to rats by gavage. Doses were set to 25 and 250 mg/kg bw.
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
The required quantity of radioactivity per animal was about 0.2-2 MBq. About 10 mL/kg body weight of a preparation will be administered to rats by gavage. Doses were set to 25 and 250 mg/kg bw.
No. of animals per sex per dose / concentration:
Four males/group
Control animals:
no
Details on study design:
Following series of nine experiments was conducted with a total of 52 animals.

BLOOD AND PLASMA
Test #1: 4 animals dosed with 250 mg/kg bw, total radioactivity was measured in blood and plasma at different time points;
Test #2: 4 animals dosed with 25 mg/kg bw, total radioactivity was measured in blood and plasma at different time points;

BALANCE AND EXCRETION
Test #3: 4 animals dosed with 250 mg/kg bw, total radioactivity was measured in urine, feces, exhaled air (2 animals), and tissue distribution;
Test # 4: 4 animals dosed with 25 mg/kg bw, total radioactivity was measured in urine, feces, exhaled air (2 animals), and tissue distribution;
Test #5: 4 animals dosed with 250 mg/kg bw of non-labelled test material daily during 14 days, and with 250 mg/kg bw of labelled test material on day 15, total radioactivity was measured in urine, feces, exhaled air (2 animals), and tissue distribution;

TISSUE DISTRIBUTION
Test #6: 12 animals dosed with 250 mg/kg bw, sacrifice of 3 animals/time point at 4 different time points, total radioactivity was measured in different tissues;
Test #7: 12 animals dosed with 25 mg/kg bw, sacrifice of 3 animals/time point at 4 different time points, total radioactivity was measured in different tissues;

EXCRETION VIA BILE
Test #8: 4 animals dosed with 250 mg/kg bw, total radioactivity was measured in excreta and tissue;
Test #9: 4 animals dosed with 25 mg/kg bw, total radioactivity was measured in excreta and tissue.
Details on dosing and sampling:
COLLECTION OF SAMPLES
Sampling of blood and plasma:
Blood samples (100 – 200 μL) will be taken from the retroorbital sinus under isoflurane anaesthesia at the following time points or by exsanguination (under isoflurane anaesthesia) at the last time point: 1; 2; 4; 8; 24; 48; 72; 96; 120; 144; 168 hours

Sampling of urine, feces and exhaled air:
The dosed animals will be placed in metabolism cages in order to collect urine after 6, 12 and 24 hours and subsequently in time intervals of 24 hours up to 168 hours and feces in intervals of 24 hours up to 168 hours or until 90 % of the applied radioactivity is excreted, whatever occurs first. In the balance experiments 3 and 4, the first two male animals will be placed in closed metabolism cages in order to additionally collect exhaled air for 48 h. If more than 2 % of the total radioactive dose is detected in exhaled air, all experiments will be carried out in closed systems.After 168 hours, animals will be sacrificed and the following tissues will be checked for remaining radioactivity.

Sampling of organs and tissues:
After sacrifice following tissues will be checked for remaining radioactivity: heart, bone, blood cells and plasma, liver, muscle, pancreas, spleen, kidney, thyroid gland, brain, carcass, adrenal glands, skin, adipose tissue, gut and gut contents, lung, testes, stomach and stomach contents, uterus, ovaries, and bone marrow.

Sampling of bile:
Within experiment 8 and 9, the bile duct cannulated and dosed animals will be placed in metabolism cages in order to collect bile in three-hour time intervals.

Cage wash:
For balance estimates the cage wash will also be checked for radioactivity.

RADIOACTIVITY MEASUREMENT
Radioactivity in all samples of biological material will be counted in a liquid scintillation counter. The samples will be prepared for analysis using conventional methods described in standard operating procedures, any additional measures/deviations from standard practice will be detailed in the raw data and the study report. Unless stated otherwise in this protocol or in the study raw data, total radioactivity is measured.
Type:
distribution
Results:
highest tissue concentrations were found in the GI tract/GI-tract contents (25 and 250 mg/kg bw)
Type:
excretion
Results:
25 mg/kg bw: urine 13.2%, feces 24.3%, and CO2 about 32%
Type:
excretion
Results:
250 mg/kg bw: urine 12.2%, feces 27.3%, and CO2 about 30%
Type:
other: total recovery of radioactivity
Results:
about 110 and 92% of administered doses (25 and 250 mg/kg bw) were recovered
Details on distribution in tissues:
Following a single oral dose of [14C]-Glyoxal at a dose level of 250 mg/kg bw, tissue distribution was measured 24, 72, 120 and 168 hours post-dosing. At the low dose level of 25 mg/kg bw, the corresponding radioactivity measurements were performed 8, 36, 72 and 144 hours after administration. 24 hours after administration of 250 mg/kg bw [14C]-Glyoxal to male rats highest tissue concentrations (means) were found in the GI tractlGi-tract contents. With the exception of the GI-tract (including its content), highest residues (means) in male rats were found in thyroid, bonemarrow, kidney, liver and adrenal glands and lowest mean radioactive residues at this time point were measured in brain and adipose tissue. In male animals, radioactive residue concentrations generally declined in organs and tissues from the 24 h time point on. The residues in organs and tissues declined slower than the plasma concentration. Nearly no clearance could be observed for skin. Lower clearance than in plasma could be observed e.g. for thyroid and carcass. For other organs and tissues the clearance is observed to be about 50 to 75 % within the observation period. 8 hours after administration of 25 mg/kg bw [14C]-Glyoxal to male rats highest tissue concentrations (means) were found in the GI tract/GI-tract contents. With the exception of the GI-tract (including its content), highest residues (means) in male rats of this dose group were found in thyroid, liver, bonemarrow, pancreas and plasma. Lowest mean radioactive residues at this time point were measured in brain and adipose tissue. In male animals, radioactive residue concentrations declined generally in organs and tissues from the 8 h time point on. The residues in organs and tissues declined slower than the plasma concentration. Nearly no clearance could be observed for skin. For other organs and tissues the decrease in residue levels within the observation period was slower than for plama. Lower clearance than in plasma could be observed e.g. for adipose tissue and carcass.
The tissue distribution experiments showed a correlation between the radioactive residues in organs and tissues and the external dose more or less compareable to the Cmax values of plasma. However, the data indicate a slow partition of the radioactive residues between the compartments of the organism. This may be eventually due to the formation of bound residues in the respective matrices.
Details on excretion:
After a single oral administration of 250 mg/kg bw of [14C]-Glyoxal, total recoveries of radioactivity in the balance experiments were > 92 %. In exhaled air, about 30% of the administered radioactivity was excreted as CO2. After 168 hours the total amount of radioactivity excreted in urine was found to be 12.22%. Within 168 hours after single oral administration of 250 mg/kg bw to male rats, 27.30 % of the administered radioactivity were excreted via feces. The time course of the amount of radioactivity found in urine and feces indicates a slow excretion correlating to a residue in carcass after the observation period of one week of up to 30% of the dosed radioactivity. The pattern of excretion after repeated oral administration (14 oral applications with unlabeled Glyoxal at 250 mg/kg bw and one oral application with labeled [14C]-Glyoxal at 250 mg/kg bw) showed higher amounts of radioactivity excreted in urine than in the single dose experiment (21.36 versus 12.22% of dose), lower amounts of excreted radioactivity in feces and higher amounts of exhaled activity but comparable residues in carcass, giving an indication that changes in kinetics 1metabolism occur after multiple dosing of Glyoxal at a dose level of 250 mg/kg. After single oral administration of 25 mg/kg bw the mean total recovery of radioactivity was 109.79% of the administered dose. In exhaled air, about 32% ofthe administered radioactivity was excreted as C02. Within 168 hours after single oral administration of 25 mg/kg bw to male rats, 13.23% of the administered radioactivity were excreted in urine. After 168 hours the total amount of radioactivity excreted via feces was found to be 24.31%. The time course of the amount of radioactivity found in urine and feces indicates a slow excretion correlating to a residue in carcass after the observation period of one week of up to 30% of the dosed radioactivity.
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 63.5 h
Test no.:
#1
Toxicokinetic parameters:
Tmax: 24 h
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: 65.5 h
Test no.:
#2
Toxicokinetic parameters:
Tmax: 8 h
Metabolites identified:
not measured
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
metabolism
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 954-1012 (C14); 955-1011 (C13); B61 from 23.03.09 and from 01.09.09 (C12)

RADIOLABELLING INFORMATION
- Specific activity: 69.2 MBq/mg (C14)
- Locations of the label: 1,2-C14; 1,2-C13;
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Low temperature and dark; Ambient under N2 for 12C Glyoxal
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer and the manufacturer holds this responsibilty.

Radiolabelling:
yes
Remarks:
1,2-C14; radiochemical purity: 96.1%
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at start of acclimatization: 6 to 12 weeks
- Weight at study initiation: 250 - 350 g
- Housing: prior test initiation, in groups in Macrolon cages; during experiment, individually in all-glass metabolism cages type Metabowl (Jencons Leighton Buzzard, U.K.) labeled with the project number, animal number, dose and time of first application
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: planned, duration not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h
- Any deviations will be documented

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study will be assayed for chemical and microbial contaminants according to the Fed. Reg. Vol. 44, No. 91 of May. 09, 1979, p 27354 (EPA);
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants;
- The bedding (Type Lingocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany) is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–33, will serve as the guideline for maximum tolerable contaminants.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Radiolabelled and the non-radiolabelled test substance will be prepared in 0.5 % carboxymethylcellulose in tap water.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The stability, homogeneity and correctness of the concentrations of the test item in the aqueous vehicle will be verified in all experiments by HPLC.
Duration and frequency of treatment / exposure:
[14C]-Glyoxal was solved in acidified water and administered to rats either as a single 250 mg/kg or 25 mg/kg oral dose or daily administration of non-radiolabelled glyoxal (250 mg/kg dose) for 14 days followed by a single dose of radioabelled glyoxal (250 mg/kg dose) in a separate study (Study No. 02B0001/096001). Samples collected in study No. 02B0001/096001 were supplied by the Sponsor for analysis in this study.
Dose / conc.:
25 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
No. of animals per sex per dose / concentration:
3-5
Control animals:
no
Details on study design:
Tissues and faeces were extracted with organic and aqueous solvent mixtures. Each liver, kidney, muscle and faeces sample were homogenised with acetonitrile, acetonitrile:water (1:1 v/v) and acetonitrile:water (1:4 v/v) containing 1% formic acid. Fat was removed from adipose tissue by homogenising in dichloromethane then the remaining residues extracted using the acetonitrile and aqueous acetonitrile mixtures described for other tissues and faeces. Plasma was extracted with acetonitrile. Soluble residues in urine and bile were separated from insoluble residues by centrifugation. Other extraction solvents including tetrahydrofuran, acetone, ethyl acetate, diethyl ether, chloroform and cyclohexane were investigated but did not afford significant increases in extractable radioactive residues. Extractable residues from tissues, plasma and faeces and soluble residues in urine and bile were analysed by TLC. Normal phase HPLC methods were supplied by the Sponsor but did not afford sufficient retention of glyoxal and highly polar metabolites on the column and were not compatible with mass spectrometry. Glyoxal, due to its high polarity, was not sufficiently retained by typical reverse phase HPLC methods. However, analysis by TLC provided optimal metabolite profiles and was assessed to be the method of choice for metabolite profiling.
Type:
metabolism
Results:
Glyoxal was extensively metabolised with no glyoxal detected in excreta, tissues or plasma and residues were present as organic acids. Metabolism of glyoxal resulted in the incorporation of glyoxal residues into endogenous metabolic processes.
Metabolites identified:
yes
Details on metabolites:
Glyoxal was extensively metabolised in rats with no glyoxal detected in excreta, tissues or plasma. Metabolites with similar Rf values were observed across the range of excreta and tissues with the most extensive range of metabolites observed in urine and liver. A total of 9 metabolites were detected in urine, 5 in extracts of faeces and 5 in bile. A total of 7 metabolites were detected in extracts of liver, 6 in extracts of kidney while 2 components, one of which was only present following protease digestion, were present in extracts of muscle and of adipose tissue. A total of 4 components were detected following protease
digestion of plasma. In general, a component with Rf ca 0.15 was present across the range of tissues and excreta. The similar Rf values may indicate similar metabolites present in excreta and tissue. However, further chromatographic analysis by another method would be required to support that conclusion and despite extensive efforts it was not possible to develop an appropriate method. In addition to the discrete components observed following TLC extensive regions of diffuse, unassigned radioactivity and origin bound material were observed. Individual metabolites present in urine, extracts of faeces and bile each accounted for ca 0.01-6.7% dose with ca 0.2-6.0% dose present as diffuse unassigned material or origin bound material. Individual metabolites in extracts of liver and kidney from rats administered a 250 mg/kg dose accounted for ca 0.3-12% TRR (1-43 mg/kg) and ca 2.6-17% TRR (6-38 mg/kg), respectively while unassigned material and origin bound material in these tissue extracts accounted for ca 22-32% TRR (72-80 mg/kg). Extractable radioactivity in extracts of muscle was present as a single metabolite (28% TRR, 9 mg/kg) and unassigned and origin bound material which together accounted for ca 29% TRR (ca 9.5 mg/kg). The majority of radioactivity in extracts of adipose tissue was unassigned and accounted for ca 67% TRR (7.5 mg/kg) while ca 3.4% TRR (0.4 mg/kg) was present as a discrete metabolite and up to ca 7% TRR (ca 0.8 mg/kg) was origin bound. The % TRR recovered in extracts of tissues from rats administered a 25 mg/kg dose were similar while the concentrations were lower due to the lower dose rate. Individual metabolites in extracts of liver and kidney from rats administered a 25 mg/kg dose accounted for ca 0.3-11.5% TRR (0.2-8 mg/kg) and ca 5.2-14.4% TRR (1.6-4 mg/kg), respectively. Unassigned material and origin bound material in liver and kidney tissue extracts accounted for ca 17% TRR (12 mg/kg) and 38% TRR (12 mg/kg), respectivley. Extractable radioactivity in extracts of muscle was present as a single metabolite (ca 39% TRR, ca 1.8 mg/kg) while unassigned and origin bound material accounted for ca 32% TRR (1.4 mg/kg). The majority of radioactivity in extracts of adipose tissue was unassigned and accounted for ca 56% TRR (0.5 mg/kg) while ca 2.4-7.2% TRR (0.02-0.07 mg/kg) present as discrete metabolites. Indiviual radioactive residues of glyoxal were present at a concentration of 2.8-52 mg/kg in plasma from rats administered a 250 mg/kg and 0.3-12 mg/kg in plasma from rats administered a 25 mg/kg dose. Glyoxal residues in tissues, excreta and plasma were not readily extracted using organic and aqueous solvent mixtures which suggested that metabolites may become associated with insoluble fractions. This was investigated by protease digestion, solubilisation with surfactant and acid reflux. The chemical structure of glyoxal would also suggest that natural incorporation into endogenous material may occur. This may be inferred by the extensive regions of diffuse, unassigned radioactivity and origin bound material observed following TLC analysis. Individual radioactivity residues present following protease digestion of liver and kidney PES accounted for ca 2-10% (8-37 mg/kg) and ca 0.6-2.5% TRR (1.4-5.7 mg/kg), respectively for rats administered a 250 mg/kg dose. For rats administered a 25 mg/kg dose,
individual radioactivity residues present following protease digestion of liver and kidney PES accounted for ca 2-15% (1.2-11 mg/kg) and ca 1.2-5% TRR (0.4-1.5 mg/kg), respectively. Protease labile residues of glyoxal in plasma PES were present at a concentration of 3-11 mg/kg in plasma from rats administered a 250 mg/kg and 1.4-4 mg/kg in plasma from rats administered a 25 mg/kg dose. The profiling and identification of glyoxal metabolites was a significant technical challenge. Metabolites in urine, faeces, bile, tissue and plasma extracts were characterised by TLC. Attempts to identify metabolites involved several experimental strategies. These included
experiments to investigate the possibility of volatile metabolites and techniques to partially isolate and characterise metabolites. Both TLC and HPLC indicated that the metabolites present were highly polar in nature. In an attempt to alter the polarity of metabolites and facilitate mass spectral analysis several different derivatisation procedures were attempted. While some change in the polarity of radiolabelled components was achieved it was not sufficient to allow HPLC or LCMS analysis. In addition, HPLC methods designed to retain highly polar compounds and HPLC methods designed to retain metabolites predicted to be produced from glyoxal were investigated and glyoxal residues compared with reference standards for oxalate, citrate, succinate, lactate and pyruvate.
Extracts of all excreta and tissue samples were profiled within 6-8 months of sample receipt. At the end of the study repreparation and analysis of selected extracts using the same TLC conditions as the initial analysis indicated that there were no significant changes in the chromatographic profile during storage.
Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Version / remarks:
(2004)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7600 (Dermal Penetration)
Version / remarks:
(1998)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 954-1012

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 96.1%
- Specific activity: 69.2 MBq/mg
- Locations of the label: 1,2-14C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at low temperatures and the dark

Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at start of acclimatization: 6 to 12 weeks
- Weight at study initiation: 250 - 350 g
- Housing: prior test initiation, in groups in Macrolon cages; during experiment, individually in all-glass metabolism cages type Metabowl (Jencons Leighton Buzzard, U.K.) labeled with the project number, animal number, dose and time of first application
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: planned, duration not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h
- Any deviations will be documented

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study will be assayed for chemical and microbial contaminants according to the Fed. Reg. Vol. 44, No. 91 of May. 09, 1979, p 27354 (EPA);
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants;
- The bedding (Type Lingocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany) is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–33, will serve as the guideline for maximum tolerable contaminants.
Type of coverage:
semiocclusive
Vehicle:
water
Duration of exposure:
The complete exposure period was 8 hours.
Doses:
Nominal doses: 20 µg/cm2
The intended amount of radioactivity per animal was about 0.5 - 2 MBq/animal.
No. of animals per group:
Four animals/group
Control animals:
no
Details on study design:
DOSE PREPARATION
In order to achieve the intended activity in the solution, an appropriate amount of stock solution of the radiolabeled test substance will be evaporated to dryness. The dried residue will be mixed with an appropriate amount of Glyoxal solved in water. The final concentration of Glyoxal in water should be 0.2 % (w/w). The preparation will be stirred and sonicated in order to produce homogeneous preparation.

APPLICATION OF DOSE:
24 hours before treatment, the animal will be thoroughly clipped and then cleaned with acetone. Animals showing skin damage at the time of dosing will not be used. On the day of dosing, a spacer will be glued to the skin with tissue glue. The test item preparation will be administered to the application site (dosing volume: 10 μl/cm²; treated area: about 10 cm²). The treated skin area will be covered with a permeable (gauze) dressing and the whole further protected beneath a semi occlusive adhesive bandage.

REMOVAL OF TEST SUBSTANCE
After exposure, the gauze and bandage will be removed from the animals. The skin of the animals will be washed with a mild soap solution and rinsed with warm water. For animals with a post-observation period, a new gauze and bandage will be applied and an additional skin wash will be done before sacrifice.

SAMPLING
Excreta (urine, feces), blood (blood cells and plasma), application site, skin surrounding the application site, remaining carcass, and skin wash(es) will be sampled. For balance estimates the cage wash, the gauze with bandage and the glas ring also will be assayed for radioactivity.

ANALYSIS
Radioactivity in all samples of biological material will be counted in a liquid scintillation counter. The samples will be prepared for analysis using conventional methods described in standard operating procedures, any additional measures/deviations from standard practice will be detailed in the raw data and the study report. Unless stated otherwise in this protocol or in the study raw data, total radioactivity is measured.

Total recovery:
Following a single dermal administration [14C]-glyoxal, the mean recovery of radioaetivity in the different groups was between 102% and 107% of the applied radioactivity.
Dose:
0.68 mg/kg bw
Parameter:
percentage
Absorption:
ca. 4.6 %
Remarks on result:
other: 8 h
Dose:
0.68 mg/kg bw
Parameter:
percentage
Absorption:
ca. 6.4 %
Remarks on result:
other: 24 h
Dose:
0.68 mg/kg bw
Parameter:
percentage
Absorption:
ca. 5.4 %
Remarks on result:
other: 120 h

Description of key information

Toxicokinetics, metabolism and distribution of glyoxal was investigated in accordance to OECD TG 417. The bioavailability of glyoxal in male rats was calculated to range between 60-75%, showing an extensive metabolism. It was concluded, that metabolism of Glyoxal resulted in the incorporation of Glyoxal residues into endogenous metabolic processes (BASF, 2011 and 2013).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
60
Absorption rate - dermal (%):
6.4
Absorption rate - inhalation (%):
100

Additional information

After single oral administration to rats, [14C]-Glyoxal was rapidly absorbed from the gastrointestinal tract. Based on balance experiments, the bioavailability of [14C]-Glyoxal in male rats was calculated to be about 60 and 75% of the administered dose for dose levels of 25 mg/kg bw and 250 mg/kg bw, respectively. The excretion of radioactivity occurred mainly within three days after dosing. However, up to 30% of the administered radioactivity was detected in carcass and were not excreted within one week after dosing. 14 days of treatment with unlabeled Glyoxal at a dose level of 250 mg/kg bw changed the excretion pattern to a higher urinary and a lower fecal excretion. Increasing the dose by a factor of 10 (from 25 to 250 mg/kg bw) resulted in an increase of the AUC-values by a factor of about 7, indicating a sublinear correlation of the internal exposure with the external dose [BASF, 2011].

Considering the metabolic pathways of Glyoxal, a scheme was reported in the CICAD (2004) referring to endogenous Glyoxal (see attached document). According to Thornalley et al. [1998], the very effective catalytic efficiency of the glyoxalase system, by which Glyoxal is assumed to be metabolised to oxalic acid via Glycolic and Glyoxylic acids, will lead to detoxification and elimination of Glyoxal.

After a single dermal administration of [14C]-Glyoxal for 8 h solved in water, mean dermal absorption in rats treated amounted up to 6.40% of the applied radioactivity. The highest tissue concentrations were found in blood cells. The amount of radioactivity absorbed was 4.6%, 6.4%, and 5.4% at 8 h, 24 h, and 120 h after beginning of the 8 h exposure period, respectively [BASF, 2011].

Furthermore, a study with glyoxal using samples from study No. 02B0001/096001 [BASF, 2011] that were analysed to investigate the nature and identity of metabolites of [14C]-Glyoxal in vivo in the rat was performed. Glyoxal was formulated and orally administered to rats as radiolabelled compound with sufficient specific activity and a stable isotope label [13C] to facilitate structural elucidation of metabolites using mass spectrometry. Excreta and tissue samples were prepared for analysis by centrifugation (urine, bile) and extraction (tissues, plasma and faeces) using acetonitrile and aqueous acetonitrile solvent mixtures. Residues that were not readily extracted using these solvent mixtures were characterised by protease digestion, solubilisation with surfactant and acid reflux techniques. Glyoxal was extensively metabolised in the rat and was not detected in any sample analysed by TLC. A number of metabolites were detected: at least 9 in urine, 5 in bile, 5 in faeces, 7 in liver, 6 in kidney, 4 in plasma, 2 in adipose tissue and 2 in muscle. Metabolites were shown to be non-volatile and highly polar in nature by extraction techniques and retention behaviour in both TLC and HPLC. Glyoxal residues were incorporated into endogenous metabolic pathways as indicated by the similarity of retention time of radioactive residues with the retention time of the organic acids citrate and succinate and the retention of radioactive residues using a method specifically designed to retain amino acids. The fate of Glyoxal following oral administration to rats was well characterised within the current study. The highly reactive nature and polarity of Glyoxal resulted in significant technical challenges in the identification of the residue. Structure elucidation was not possible because it was not possible to develop a chromatographic method that sufficiently retained the residues and that was compatible with mass spectrometry.

Taken together, the results indicated that Glyoxal was extensively metabolised in rats with no Glyoxal detected in excreta, tissues or plasma and residues were present as oxalate and other organic acids such as citrate and succinate. This suggests that metabolism of Glyoxal resulted in the incorporation of Glyoxal residues into endogenous metabolic processes [BASF, 2013].