Registration Dossier

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Objective of study:
excretion
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The investigation was undertaken to determine the metabolism and excretion of the tritium-labelled test material by rats. The test material was injected subcutaneously at 5 mg/kg. Animals were housed in metabolism cages and urine and faeces collected. Ninety two hours after injection the rats were sacrificed and the intestinal contents were removed and added to the cage collected faeces.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
Tritium-labelled test material was synthesised and purified. The radioactive drug was diluted with non-radioactive test material to provide a specific radioactivity of 70 µc/mg.
Radiolabelling:
yes
Species:
rat
Strain:
other: Holtzman
Details on species / strain selection:
None specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Male rats weighing 350 – 390 g and female rats weighing 230 – 270 g.
- Housing: Each group of animals was placed in an individual metabolism cage.
- Diet: Fasted during the first 24 hours, ad libitum thereafter.
- Water: Ad libitum
Route of administration:
subcutaneous
Vehicle:
not specified
Details on exposure:
Each animal was injected subcutaneously with tritium-labelled test material at a dose of 5 mg/kg (calculated as free base).
Duration and frequency of treatment / exposure:
Single dose
Dose / conc.:
5 other: mg/kg (calculated as free base)
No. of animals per sex per dose:
Six male rats were divided into two groups of three, and eight female rats were separated into two groups of four.
Control animals:
yes, concurrent no treatment
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: Urine, faeces. Ninety two hours after injection, the rats were sacrificed and the intestinal contents were removed at sacrifice and added to the cage-collected faeces.
- Time and frequency of sampling: Urine was collected beneath the cage under toluene for three time periods: 0 to 24, 24 to 48 and 48 to 92 hours. Faeces were accumulated for the full 92 hours on a screen beneath the cage floor. Faeces were combined for the two groups of the same sex.
Cage-collected urine was diluted with water in proportion to the anticipated amount of radioactivity in the sample. Wet-weighed faeces were homogenised in 0.5 N hydrochloric acid in a Waring Blender. Two- or 4-mL aliquots of the dilution or homogenate were analysed. A double purification procedure was necessary to eliminate quenching materials from the final extract. This was accomplished by extracting the original sample aliquot buffered at pH into 10 mL of ethylene dichloride containing 30 % n-amyl alcohol. An 8 mL aliquot of organic solvent extract was transferred to a glass-stoppered centrifuge tube containing 5 mL of 0.5 N HCl and the tube was shaken for 20 minutes. The mixture was centrifuged and a 4 mL aliquot of the aqueous layer (upper) was transferred to a clean centrifuge tube, neutralised and buffered at pH 9, and extracted with 10 mL of ethylene dichloride containing 30 % n-amyl alcohol as before. Eight mL of this final extract were placed in a scintillation-grade counting vial and evaporated to dryness at 65 °C. The sample was prepared for counting as described.
Total tritium-labelled test material (i.e. free plus conjugated) was determined by autoclaving liquid samples with 1/10th volume conc. HCl as described previously and subsequently analysing for tritium-labelled test material using the double purification procedure.
Recoveries of total amounts of tritium-labelled test material ranging from 12 to 4800 ng added to 1:5 aqueous dilutions of urine were 100.5 ± 31 (SD) %. Additions of 500 ng quantities of tritium-labelled test material to acid homogenates of faeces were recovered to the extent of 89.9 ± 1.4 (SD) %. This recovery figure was used to correct final values for analysis of tritium-labelled test material in faeces to 100 %. As little as 0.1 ng of tritium-labelled test material with a specific radioactivity of 0.2 curies per millimole could be determined using the double purification procedure with accuracy of ± 20 %; 0.4 ng was determined with an accuracy of ± 4.5 %. The above recovery figures have been corrected for aliquot volumes transferred at each step of the double purification procedure.
In order to evaluate the specificity of the method, aliquots of non-autoclaved and acid autoclaved urine from rats given the tritium-labelled test material were extracted as above. The concentrated extracts were analysed by paper chromatography using the technique described below. In each case there was a single area of radioactivity which corresponded in Rf to that of non-radioactive test material applied to the same chromatograms.
Since tritium-labelled test material might be expected to be N-demethylated to dihydronormorphine-H3 in vivo, it was necessary to estimate to what degree the metabolite would interfere in the analysis for the tritium-labelled test material. Analysis of aqueous solutions of dihydronormorphine using the double purification procedure for tritium-labelled test material showed that only 14 % of the dihydronormorphine could be recovered. Thus, for example if up to 20 % of the radioactivity in a sample was due to dihydronormorphine-H3, it would introduce an error of less than 3.5 % in the analysis for tritium-labelled test material.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces. Ninety two hours after injection, the rats were sacrificed and the intestinal contents were removed at sacrifice and added to the cage-collected faeces.
- Time and frequency of sampling: Faeces were accumulated for the full 92 hours on a screen beneath the cage floor.
- Number of animals: Faeces were combined for the two groups of the same sex.
- Method type for identification: Paper chromatographic methods for tritium-labelled test material:
Descending paper chromatography was done using non-buffered and buffered Whatman No. 1 or No. 3 chromatography paper. Buffered paper was prepared by dipping the paper in an aqueous solution of potassium phosphate (0.2 M) of the appropriate pH (6 – 10) and allowing it to dry in room air for 1 to 2 hours before applying solutions to be chromatographed. Buffered chromatograms were developed in tert.-amyl alcohol, n-butanol, n-butyl ether, water (80:7:13 v/v) which is designated as the “buffered solvent system”. Non-buffered chromatograms were developed in n-butanol, glacial acetic acid, water (4:1:2 v/v) which is referred to as the “non-buffered solvent system”. Occasionally paper was buffered with 0.2 M borate buffer instead of potassium phosphate and the chromatogram was developed in tert.-amyl alcohol saturated with the buffer (“borate-buffered system”).
Non-radioactive test material and dihydronormorphine were applied routinely to each chromatogram to serve as controls on technique. These compounds were localised on dry chromatograms by visualisation under UV light and by spraying with iodoplatinate reagent and / or with a freshly prepared solution of Diazoblue-B. Rf values were calculated using the midpoint of the coloured spot.
Scanning chromatograms for radioactivity involved cutting out the path of migration and dividing it lengthwise into 1 cm sections. Each section was placed in a scintillation-grade glass vial and 4 mL of eluting solvent were added. One of three eluting solvents was used: Absolute methanol; methanol containing 10 % concentrated hydrochloric acid; or aqueous 0.25 N hydrochloric acid. After 1 to 2 hours the section of paper was pressed against the vial to remove as much elute as possible and then discarded. The elute was evaporated at 60 °C. The residue was dissolved in 0.2 mL absolute methanol and 5 mL of phosphor solution were added. The radioactivity was determined in an automatic liquid scintillation spectrometer. The average counts per minute (cpm) determined from two or more counting periods were plotted against centimetres of migration from the origin. The Rf value for an area of radioactivity was calculated using the centimetre-area containing the highest number of absolute cpm (i.e. total cpm minus background cpm).


TREATMENT FOR CLEAVAGE OF CONJUGATES:
Faeces: Non-autoclaved and autoclaved portions of acid homogenates of faeces from male rats (groups I and II) and from female rats (groups III and IV) were neutralized to pH 7 and centrifuged. Aliquots of the supernatants were chromatographed directly on non-buffered and buffered paper (pH 7, 8, 9, 10).
Urine:
1. To a 56 mL portion of male rat urine (15 % of the total collection for group 1) 100 ng of non-radioactive DNM were added and the solution was autoclaved in 1.2 N hydrochloric acid for 30 minutes Throughout the entire procedure, two controls, one consisting of tritium-labelled test material and non-radioactive DNM in aqueous solution, and the other of tritium-labelled test material and DNM in urine from non-injected male rats were handled in a fashion identical to that for urine from tritium-labelled test material-injected rats.
The pH of the autoclaved urine was adjusted to approximately 10 and then buffered at pH 10.35 with 40 % potassium phosphate. The total volume was divided into suitable aliquots and each aliquot was extracted three times. Less than 1 % of the total radioactivity remained in the aqueous phase.
The coloured extracts were pooled and concentrated to dryness at 60 °C. The brown residue was dissolved in methanol and chromatographed using the non-buffered system. Scanning a strip of the large chromatogram showed that a single, sharp peak of radioactivity migrated coincidentally with non-radioactive, standard test material, Rf 0.58.
The radioactive area (Rf 0.58) from the remainder of the chromatogram was eluted with 3- to 300-mL portions of methanol and the eluates were pooled and evaporated to dryness at 60 °C. Suitable aliquots of a methanol solution of the residue were chromatographed at various pH’s using buffered systems. A portion of each chromatogram was scanned for radioactivity. Attempts to elute and re-chromatograph the radioactive material corresponding to DNM on pH 8 buffered paper were inconclusive probably as a result of decomposition of the material during manipulation.
2. Portions of urine from male rats (group I) and from female rats (group III) were neutralized to pH 7 or acid-autoclaved and then neutralized before they were applied to non-buffered and buffered papers for chromatography.
3. Aliquots of urine from each of the four groups of animals were diluted 1:10 in 0.2 M sodium acetate (pH 6.9). Duplicate 2-mL volumes of each dilution were incubated with 25 mg (approximately 872 Fishman units) of β-glucuronidase for 24 hours at 37 °C. Similar volumes of each dilution were: (a) incubated without enzyme, (b) acid-autoclaved, (c) and left untreated. All samples were analysed for free tritium-labelled test material.
Type:
excretion
Results:
Males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.
Details on excretion:
Urinary and faecal excretion of free and conjugated tritium-labelled test material.
Rats of either sex excreted about the same percentage of the dose as unchanged (free) tritium-labelled test material in urine. Female rats excreted about twice as much conjugated tritium-labelled test material; the difference is significant (P < .05). Analysis of faeces revealed that male rats excreted approximately 1.5 times as much free tritium-labelled test material as the females; the difference cannot be evaluated statistically because of the pooling of the faeces for each sex. Both sexes excreted small amounts of conjugated tritium-labelled test material in faeces. The combined total excretion of tritium-labelled test material in urine and faeces in the 92-hour period following injection was almost identical for the two sexes: Males, 84.1 % of the injected dose; females, 86.2 %.

Identification of tritium-labelled test material in faeces.
Non-autoclaved and acid-autoclaved aliquots of the supernatant from faecal homogenates were analysed chromatographically. In all instances there was a small, but definite amount of radioactivity associated with the migration of standard DNM. Approximately 7.5 % of the total tritium in male rat faeces was present as tritium-labelled test material.
No radioactivity was present at the Rf of DNM in control chromatograms of non-radioactive faeces to which tritium-labelled test material was added in vitro. Whenever tritium-labelled test material was mixed with urine or faeces in vitro or in vitro and then chromatographed, a small amount of radioactivity remained at the origin on buffered chromatograms. It would appear that the radioactivity retained at the origin of buffered paper represented an artefact of manipulation inasmuch as there was a single area of radioactivity on non-buffered chromatograms, which were able to separate all of the expected in vitro metabolites of tritium-labelled test material except the N-demethylated form.
Metabolites identified:
yes
Remarks:
N-demethylated metabolite
Details on metabolites:
Identification of tritium-labelled test material and its metabolites in, urine: The radioactive area designated by Rf 0.58 was eluted and portions of the eluate were chromatographed on buffered papers. In all cases the largest portion of tritium radioactivity migrated with standard test material. A small amount of radioactivity migrated with DNM on pH 8 buffered paper but not on papers buffered at a higher pH.
Radioactivity in male rat urine chromatographed directly (i.e., without extraction) on pH 8 buffered paper separated into two areas, one of which corresponded to tritium-labelled test material. After autoclaving the urine, almost all of the radioactivity migrated with standard test material. No radioactivity was detected at the Rf of tritium-labelled test material.
Non-autoclaved male rat urine produced two, possibly three, areas of radioactivity on non-buffered chromatograms. A single radioactive peak was present at the Rf of standard test material when acid-autoclaved urine was chromatographed similarly
Results similar to those just described for direct chromatography of male rat urine were obtained when female rat urine was chromatographed before and after acid-autoclaving. Free and conjugated tritium-labelled test material was identified but no suggestion of the N-demethylated metabolite was evident.
β-Glucuronidase hydrolysed one-third (males) to one-half (females) of the total conjugated drug in rat urine. The possibility of urinary constituents interfering with optimal β-glucuronidase activity was not ruled out as an explanation for the incomplete hydrolysis of conjugated tritium-labelled test material. However, the results were reproduced in repeated incubations of different dilutions of urine suggesting that urinary constituents were not preventing enzymatic hydrolysis.

Identification of metabolites in faeces.
The faeces from female rats contained only a trace of the N-demethylated drug (1.8 % of the radioactivity in the homogenate).
Conclusions:
Following subcutaneous injection of tritium-labelled test material in rats, males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.
Executive summary:

The investigation was undertaken to determine the metabolism and excretion of tritium-labelled test material by rats. The test material was injected subcutaneously at 5 mg/kg. Animals were housed individually in metabolism cages and urine and faeces collected. Ninety two hours after injection the rats were sacrificed and the intestinal contents were removed and added to the cage collected faeces.

Males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.

Free and conjugated tritium-labelled test material was identified in urine but no suggestion of the N-demethylated metabolite was evident.

Faeces from female rats contained only a trace of the N-demethylated drug (1.8 % of the radioactivity in the homogenate).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Objective of study:
distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
Tritium-labelled test material was administered subcutaneously to near-term pregnant Sprague-Dawley rats. One-half to 16 hours later the foetuses were removed and specimens were obtained for estimation of free and conjugated test material in maternal and foetal brains, kidneys, and plasma.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
Tritium-labelled test material was prepared by the catalytic reduction of the 7,8-double bond of morphine with tritium gas. The following procedure was followed to remove any labile tritium and to purify the product.
The labelled compound, dissolved in methanol, was diluted 1:10 with non-labelled test material, and the methanol was removed by evaporation. The residue was dissolved in 5 mL of 0.5 N sulphuric acid and refluxed 16 hours. The solution was transferred to a centrifuge tube and alkalinized with 40 % ammonium hydroxide to precipitate the test material. The mixture was refrigerated overnight, the liquid drawn off, and the crystals were dried in an evacuated desiccator.
The crystals were dissolved in methanol and the solution was passed successively through three 3-mm diameter columns, each packed with 2 g of aluminium oxide. After evaporation of the eluates to dryness, the residue was dissolved in 5 mL of 6 N hydrochloric acid. Upon alkalinisation with 40 % ammonium hydroxide, dense crystals formed. The mixture was refrigerated overnight, centrifuged, and the liquid was drawn off. The crystals were washed once with 1 mL of cold distilled water (5-10 °C), and again with 0.5 mL of cold distilled water. The purified compound was placed in an evacuated desiccator for drying and storage. The resulting product was a light tan, crystalline material with a specific activity of 1.87 mc/mg.
Radiochemical purity of the final product was confirmed by descending paper chromatography and scanning of the radioactivity on the chromatograms. Solvents used for chromatography were:
(1) tert.-amyl alcohol, n-butyl ether, and water (80:7:13), saturated with pH 7 phosphate buffer, and
(2) n-butanol, acetic acid, and water (4:1:2). Development with the first solvent was on Whatman No 1 paper which had been dipped in pH 7 phosphate buffer and dried 2 hours at room temperature. Development with the second solvent was on unbuffered Whatman No 1 paper. Single peaks of radioactivity, Rf 0.27 with the first system, and 0.53 with the second system, were produced. Both corresponded with RI values of parallel control non-labelled test material.
The radioactivity of all injection solutions was evaluated by evaporating measured amounts in counting vials at 55 °C adding n-amyl alcohol and phosphor solution, and counting in a liquid scintillation spectrometer. Minor variations in radioactivity among different solutions were corrected by applying factors to make each solution comparable to the solution used in determining the standard curves.
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Pregnant rats were used no earlier than 19 days after breeding. The normal gestation period is 21 to 23 days.
Route of administration:
subcutaneous
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions for injection contained 1 mg/mL, 2 mg/mL, and 4 mg/mL total test material free base. Each contained 0.1 mg/mL of the purified tritium-labelled free base plus the appropriate amount of non-labelled test material hydrochloride to make the specified concentration. Sufficient 0.1 N hydrochloric acid was added to dissolve the labelled free base, and the solution was buffered with 0.2 M sodium phosphate buffer, pH 5.5, and brought to volume with normal saline.
All doses of drug were administered subcutaneously.
Duration and frequency of treatment / exposure:
Single subcutaneous administration
Dose / conc.:
1 other: mg/mL
Dose / conc.:
2 other: mg/mL
Dose / conc.:
4 other: mg/mL
No. of animals per sex per dose:
Not specified
Control animals:
yes
Details on study design:
The amount of test material in tissue homogenates or plasma dilutions was estimated from curves established by 186 extractions of known concentrations of injection solution from various tissue and plasma preparations. Amounts of drug added to standard samples extended over the range extracted from equivalent experimental samples, and linear relationships between cpm and weight of drug were found.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Distribution)
At the time of sacrifice, the maternal rats were anesthetized with ether and the foetuses were delivered surgically. The foetal rats were removed to another work area to avoid cross-contamination of radioactivity in succeeding steps. Seven to 10 mL of maternal blood were withdrawn from the abdominal aorta within 5 minutes after beginning anesthetization. Simultaneously, the foetuses were decapitated and the foetal blood was collected. Blood from all foetuses of each litter was pooled. Glassware used for blood collection was moistened with 4 % sodium polyanhydromannuronic acid sulfate (Manuronate) solution to prevent clotting. Care was taken to limit the volume of this solution to insignificant amounts.
Foetal and maternal brains were removed within 10 minutes after anesthetization. When applicable, samples of maternal livers and kidneys were removed. The maternal brain was cut mid-sagittally to yield two identical halves weighing approximately 1 g each. One half was used for the determination of drug concentrations in whole brains. In some experiments the other half was frozen and dissected into gross anatomical parts consisting of medulla and pons, cerebellum, mesencephalon, diencephalon, and cerebral cortex. In these experiments a specimen of cervical spinal cord about 1 cm long was obtained. The adult liver specimen consisted of a cross-section weighing about 1 g from the centre of the median lobe. Foetal liver specimens consisted of samples taken from each foetus, pooled, and adjusted to a weight of about 1 g. The entire left kidney of adult rats, with capsule removed, was used. Foetal kidney specimens consisted of both kidneys from all foetuses of each litter. Each tissue specimen was rinsed briefly with 5 to 6 mL of normal saline and blotted dry. Wet weight, to the nearest mg was obtained on all tissue samples.

Specimens of whole blood, usually 0.3 mL, and the tissue specimens were homogenized with a teflon-pestle tissue grinder in sufficient 0.007 M ammonium hydroxide to make a total volume of 10 mL each. The remaining blood was centrifuged and a specimen of the plasma, usually 0.3 mL, was diluted with sufficient 0.007 M ammonium hydroxide to make 10 mL.
Four 2-mL aliquots were pipetted from each of these 10-mL preparations into 40-mL glass-stoppered centrifuge tubes and 0.5 mL of “cold carrier” solution, consisting of 1 mg of test material hydrochloride per mL of deionized water, was added to each tube. These “extraction samples” were frozen and stored at -20 °C for 1 to 3 days until the extraction procedure was carried out.
On the same day the samples were prepared, 1-mL portions of the remaining blood and tissue homogenates were diluted appropriately (usually 1:10) for the determination of the volume of entrapped blood in the tissue by spectrophotometric method. Free test material content was determined in 2 of each of the 4 “extraction samples” and 2 were subjected to acid hydrolysis for the determination of total (free plus conjugated) test material.
Acid hydrolysis was accomplished by the addition of 0.2 mL of concentrated hydrochloric acid to each sample and autoclaving at a pressure of 15 to 20 pounds per square inch for 20 minutes.
Extraction of the test material was by a previously published method for morphine, except that 40 % and 4 % solutions of dibasic potassium phosphate were used for buffering and washing, respectively, instead of sodium phosphate solutions. This change eliminated the necessity of warming the samples.
Four blanks and 8 known dilutions of the injection solution, four samples each of 2 different dilutions, were carried through the extraction procedure with each group of unknowns as checks on the consistency of the method. The hydrolysis procedure was performed on 2 samples of each set, and 2 were extracted without hydrolysis.
The radioactivity in blood contained in tissue homogenates was determined by multiplying the amount of blood previously estimated in the homogenates by the radioactivity extracted from an equivalent amount of whole blood. This value was subtracted from the radioactivity of the tissue homogenates to yield the activity contained in the tissue itself. The radioactivity derived from autoclaved blood samples was subtracted from that of autoclaved tissue samples, and the radioactivity of non-autoclaved blood was subtracted from non-autoclaved tissue samples.

Specificity of the extraction
Specificity of the extraction procedure was evaluated by subjecting pooled extracts of autoclaved and non-autoclaved plasma samples and non-autoclaved kidney samples to descending paper chromatography, using the same procedures that were used in the determination of radiochemical purity of the original labelled compound. Spots of radioactivity corresponded to those of non-labelled test material controls.

Extraction efficiencies were calculated after correction for efficiency in counting of the radioactivity. Efficiency of extraction from water was 81.7 ± 62 % (S.D.). Extraction efficiencies from all plasma samples, non-autoclaved kidney samples, autoclaved foetal kidney samples, and non-autoclaved liver and brain samples averaged 76.6 ± 6.6 % (S.D.) and did not vary significantly among these groups. Extraction efficiency from autoclaved brain samples was 76.9 ± 7.7 % (S.D.), but showed a lower counting efficiency than the former groups. Extraction efficiency from autoclaved adult kidney samples was 73.5 ± 0.9 % (SD.). Autoclaved liver samples gave an extraction efficiency of 72.4 ± 52 % (SD.) with a lower counting efficiency than autoclaved adult kidney samples.
Details on distribution in tissues:
Test material levels in plasma
At 0.5 h the mean concentrations in both maternal and foetal plasma were at their maximums. The mean maternal plasma level of 470 ng/g was approximately 2.5 times the mean foetal plasma level at this time. This same ratio held at 1 hour, but at 2 hours and after there appeared to be no difference.
Paired t-tests were calculated on these data, comparing values obtained from each maternal rat with those of her own foetuses. When the points are compared by this method, the difference observed between maternal and foetal plasma levels at 0.5 hour has a P value between 0.20 and 0.10, 0.50 it is not significant at the accepted level of 0.05. The difference observed at 1 hour, however, is highly significant with a P value less than 0.001. The statistical non-significance at 0.5 hour can be attributed largely to the very high values obtained from the foetal plasma of one litter. The plasma level from this litter was estimated to be 435 ng/g while the other values at this time were 87, 93, and 133 ng/g. This was the only litter out of 13 tested at times of 1 hour or less in which the foetal plasma concentration was higher than the maternal plasma concentration.
Test material content in maternal brains attained a maximum mean value of 55 ng/g at 1 hour while in foetal brains a peak concentration of 150 ng/g was reached at 2 hours. Therefore, the maximum average concentration found in foetal brains was 2.7 times the maternal brain maximum. The paired t-test shows no significant difference between these levels at 0.5 h, but at 1, 2, and 4 hours the foetal levels were significantly higher than maternal levels with P values less than 0.001 in each case. At 8 hours, where only 2 animals were used, the trend continues, but the difference is not significant at the 0.05 level. At 16 hours the maternal brain concentration was down to trace amounts while 10 ng/g was still detectable in foetal brains.
Maternal plasma concentrations were much higher than brain concentrations at early times, but there was less difference at later times. The differences were statistically significant at 0.5, 1, 2, and 4 hours. At 8 hours the difference was not significant, and at 16 hours only trace amounts of drug were detectable in both maternal plasma and brain tissue. At no time was the mean maternal brain concentration higher than the mean plasma concentration.
A different relationship exists between foetal brain and plasma concentrations of free drug than between those of the maternal rat. In the foetuses there was no significant difference between brain and plasma levels at any time period up through 2 hours, but at 4 hours the foetal brain concentration was significantly greater than the plasma concentration with a P value between 0.01 and 0.001. The difference at 8 hours was not significant with only 2 litters. At 16 hours the foetal plasma showed only trace amounts of drug while 10 ng/g was still detectable in foetal brains.
Significant amounts of conjugated test material were not found in maternal brains at any time period. No conjugated drug was found in foetal brains at the 0.5 or 1 hour periods, but small amounts were recovered at the 2-, 4-, 8-, and 16-hour intervals.

Concentrations of drug found in maternal and foetal liver and kidney tissues.
When the free drug levels are compared to plasma levels, it is seen that the ratio of maternal liver to maternal plasma concentrations increased progressively from about 7 at 0.5 hour to 50 at 8 hours.
Maternal kidney concentrations of free drug were appreciably higher than those of maternal liver at early time periods, but they fell to levels equal to, or below liver levels at later times. A kidney-plasma ratio of approximately 25 was maintained at all time periods. In contrast to the maternal ratios, the ratios of free test material found in foetal livers to those in foetal plasma averaged 2.7. The foetal kidney-plasma ratios of free drug averaged 3.8. In general, higher levels of conjugated drug In general, higher levels of conjugated drug were found in livers and kidneys than in plasma of adult rats. Foetal liver-plasma relationships were variable for conjugated drug, with higher concentrations in liver than in plasma at 0.5 hour and 1 hour, equal at 2 hours, and lower at 4, 8, and 16 hours. Conjugated test material was detected in foetal kidneys only at the 4- and 8-hour time periods, and the mean levels at these times were less than mean conjugated drug levels in foetal plasma.

Effect of altering the dose.
Doses of 1 mg/kg and 4 mg/kg were administered, each to 2 pregnant rats, and drug concentrations were determined after 1 hour. These are compared with concentrations found 1 hour after maternal administration of 2 mg/kg. There was no significant difference between concentrations found after 2 mg/kg and 2 times the concentrations following 1 mg/kg or half the concentrations following 4 mg/kg. Two trends are suggested by these data, however. First, there was a trend for the ratio of conjugated to free drug in maternal plasma to decrease with increasing dose, although the total amount of conjugate increased. After 1 mg/kg the conjugated- free ratio was 2.6, after 2 mg/kg it was 1.8, and it was 1.0 after 4 mg/kg. Second, no more than trace amounts of conjugate were detectable in either maternal or foetal brain tissue at this time after the lower doses, but calculable amounts were found in both tissues after the 4-mg/kg dose.

Concentrations in gross CNS areas.
Concentrations of test material recovered from various gross anatomical areas of the rat central nervous system 1 hour after the administration of 2 mg/kg of the tritiated drug are surprisingly constant in view of the fact that, in many cases, the radioactivity was at the lower limit of detectability due to the small sample sizes. Higher concentrations were found in the cerebellum and cerebral cortex than in the deeper structures of the brain, the brain stem, and the spinal cord. No gross accumulation of drug was apparent in any of these areas. The concentrations observed were in the same range as those for whole brain at the same time after administration.

Concentrations in non-pregnant rats.
Statistical comparison of these values by means of the t-test with those of pregnant rats does not show a significant difference between these 2 groups.

Concentrations after natural delivery.
Concentrations of test material recovered from plasma and brain tissue of one maternal rat and her litter which was delivered naturally between 7 and 8 hours after the injection of 2 mg/kg of labelled test material. They were sacrificed 8 hours after drug administration. Only the maternal rat was anesthetized.
Levels of free drug in both mother and new-born rats coincide closely with those found in experiments in which the pregnant rat was anesthetized and the foetuses removed surgically.
The level of conjugated drug in foetal plasma, however, was considerably higher (90 ng/g compared to 25 ± 4 ng/g) in this group than in the surgically delivered rats.
Metabolites identified:
not measured
Conclusions:
Distribution of the drug did not differ significantly between pregnant rats and adult non-pregnant females. 
Executive summary:

Tritium-labelled test material was administered subcutaneously to near-term pregnant Sprague-Dawley rats. One-half to 16 hours later the foetuses were removed and specimens were obtained for estimation of free and conjugated test material in maternal and foetal brains, kidneys, and plasma.

Despite a lower maximum plasma level in the foetuses, the foetal brain level reached a peak which was 2.7 times the highest maternal brain level. The peak concentration in maternal brain was only about one eighth that of maternal plasma, but foetal brain levels followed plasma levels quite closely at early time periods. At later times foetal brain concentrations were higher than plasma concentrations; this relationship was not seen in maternal rats.

Conjugated test material was found in maternal plasma, livers, and kidneys, and in foetal plasma and livers at all time periods. Significant amounts of conjugate were not found in maternal brains after doses of 1 mg/kg or 2 mg/kg, but a small quantity was detected after 4 mg/kg. Low concentrations of conjugate were found in foetal brains 2 hours and more after drug administration, but not before. The ratio of conjugated-free drug was lower in foetal than in maternal specimens.

Distribution of free and conjugated test material did not appear to be greatly influenced by dose of the drug except that the proportion of conjugate in maternal plasma tended to decrease with increasing dose. Distribution of the drug did not differ significantly between pregnant rats and adult non-pregnant females. 

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Objective of study:
distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
Pregnant Sprague-Dawley female rats were made tolerant to the test material by initiating s.c. injections of 2 mg/kg (base) of drug, twice daily, 30 hours after breeding and increasing the dosage to 15 mg/kg. Control non-tolerant pregnant animals were treated in an identical manner except that they were injected with saline. Foetuses were removed by caesarean section at intervals of one-half to four hours after s.c. injection of 7 mg/kg (base) of H3- test material to the maternal rat on the 21st day of gestation. Free and conjugated-levels of test material were assessed in maternal and foetal tissues and plasma.
GLP compliance:
no
Test material information:
Composition 1
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Weighing 250 to 300 g.
- Diet: Ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Maintained under constant environmental conditions.
Route of administration:
subcutaneous
Vehicle:
physiological saline
Details on exposure:
Males were mixed with females in a ratio of 1:3 in the evening, and the next morning the pregnant animals, confirmed by the vaginal smear test, were separated. About 30 hours after breeding, the pregnant female rats were begun on s.c. injections (gluteal region), twice daily, of non-labelled test material HCl in saline. Two dosage regimens of test material were used. The first was 4 mg/kg (base) initially, increased by 4 mg/kg every 2 days to a final dosage of 28 mg/kg for the last 8 days. Because this regimen gave a small number of full-term pregnancies, a lower dosage of test material was used, initially 2 mg/kg and increased at 2-day intervals to 4, 7, 10 and 15 mg/kg, which was maintained for the last 12 days. The injection volume was 2 mL/kg. Control non-tolerant pregnant rats were injected with equal volumes of saline.
Analgesia after injection of 7 mg/kg of drug in both the control and tolerant pregnant animal groups on the 18th day of gestation was tested with the hot plate method and compared with the effect in non-pregnant female rats. The pregnant rats were injected s.c. with 7 mg/kg (base) of tritium-labelled test material between 12 and 16 hours after the last injection of test material on the 21st day of gestation.
Duration and frequency of treatment / exposure:
4 mg/kg (base) initially, increased by 4 mg/kg every 2 days to a final dosage of 28 mg/kg for the last 8 days.
2 mg/kg (base) initially, increased at 2-day intervals to 4, 7, 10 and 15 mg/kg, which was maintained for the last 12 days.
Dose / conc.:
28 other: mg/kg
Remarks:
4 mg/kg (base) initially, increased by 4 mg/kg every 2 days to a final dosage of 28 mg/kg for the last 8 days.
Dose / conc.:
15 other: mg/kg
Remarks:
2 mg/kg (base) initially, increased at 2-day intervals to 4, 7, 10 and 15 mg/kg, which was maintained for the last 12 days.
No. of animals per sex per dose:
Not specified
Control animals:
yes, concurrent vehicle
Details on study design:
None specified.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Distribution)
At the time of sacrifice the animals were anesthetized with ether and the foetuses were delivered by caesarean section with extreme care to avoid contamination by radioactive fluids from the mother. The foetuses were wiped, counted and weighed. The blood of foetuses was collected through decapitation, with heparin being used as an anticoagulant, and the livers, kidneys and brains were removed and pooled for each litter. Simultaneously, maternal blood was withdrawn from the abdominal aorta, and the maternal brain, liver, kidneys and about 4 square inches of skin of the injection site (including muscle) were removed, blotted dry, wrapped in aluminium foil and frozen. All samples were collected within 10 minutes after anesthetization.

At the time of drug determination each sample of organ tissue was thawed and 1 g homogenized with a Teflon pestle grinder in sufficient 0.007 M skin and muscle of injection site (5-8 g) was homogenized with an Omni-Mixer to make 50 mL. The amount of entrapped blood in the tissue was estimated.

Free and conjugated tritium-labelled test material in the biologic samples were determined. Free tritium-labelled test material was determined by buffering the sample with K2HPO4 solution and extracting with ethylene chloride containing 30 % n-butanol. Total (free plus conjugated) tritium-labelled test material was determined by autoclaving the sample in 8 % (by volume) of concentrated HCl in a portable sterilizer at 15 to 18 pounds pressure for 20 minutes, cooling, adjusting the pH to 10 by adding 0.4 mL of NH4OH (30 % NH1), buffering with K2HPO4 and extracting with solvent as mentioned. Conjugated tritium-labelled test material concentrations were estimated from the difference in results between the autoclaved and the non-autoclaved samples. The data of this report were corrected for the incomplete hydrolysis and extraction to give the true values.
Statistics:
Two-tailed t test was used to compare the mean concentration of the drug in the tissue at P < 0.05 level.
Details on distribution in tissues:
Plasma levels of free and conjugated test material.
Maximum plasma concentrations of the free drug were observed 30 minutes after injection in both groups of rats. Although the mean level appeared somewhat lower in tolerant animals, the difference was not statistically significant. Four hours after injection, the plasma concentration of free tritium-labelled test material in tolerant maternal rats was significantly lower than in control animals. At that time, free tritium-labelled test material in the plasma of tolerant and control maternal rats was 1/16 and 1/8, respectively, of the concentrations at 30 minutes. The plasma levels of conjugated tritium-labelled test material in tolerant maternal rats were significantly lower at three and four hours than in controls.
The maximum concentrations of free drug in the plasma of the tolerant and control foetuses were observed at one hour and two hours, respectively. The foetal plasma level of free tritium-labelled test material at one hour is significantly higher in the tolerant than in the control groups. At four hours, the conjugated tritium-labelled test material in foetal plasma of the tolerant animals is significantly lower than in control foetuses.

Test material levels in tissues.
The early samples (one-half hour) show a trend toward higher levels of tritium-labelled test material in the tolerant groups than in controls, and the opposite in the late samples (three and four hours). Some differences are statistically significant. Similar results were observed for free and conjugated tritium-labelled test material in liver and kidney.

Test material in the skin and muscle of injection site.
Percent recoveries of drug from skin and muscle of the injection site after one-half, one, two, three and four hours are: In tolerant pregnant rats, 24.7 %, 12.5 %, 3.9 %, 0.55 % and 0.55 %; in control pregnant rats, 36.5 %, 26.8 %, 6.6 %, 2.6 % and 1.1 %. There was more rapid disappearance of the drug in tolerant rats, the differences being statistically significant at one half to three hours. The regression lines of disappearance of free tritium-labelled test material in the injection site of tolerant and control rats are y = 0.41 - 0.541x, and y = 0.8- 0.414x, respectively. The 95 % fiducial limit on the slope of the regression line for the tolerant rats is - 0.543 to -0.539, which is significantly steeper than that for control animals, -0.425 to -0.403.
Metabolites identified:
not measured

The Effect of Test Material on Reproduction

The larger dose of test material administered chronically reduces the number of litters and gain of maternal body weight but has no effect on the foetal body weight and the number of foetuses per litter.

Conclusions:
Maternal plasma levels of free H3-test material were at maximum in the one-half-hour samples of both tolerant and control animals. Although the mean level appeared somewhat lower in tolerant animals, the difference was not statistically significant. The three- and four-hour maternal plasma levels in tolerant animals were significantly lower than in the control group. Foetal plasma concentrations in tolerant animals were significantly lower than in control animals at one hour, but the relationship was reversed at three and four hours. Both maternal and foetal tissue samples showed similar trends, but few gave differences of statistical significance. Recovery of drug from the skin and muscle of the injection site in tolerant rats was always lower than in control animals.
Executive summary:

Pregnant Sprague-Dawley female rats were made tolerant to the test material by initiating s.c. injections of 2 mg/kg (base) of drug, twice daily, 30 hours after breeding and increasing the dosage to 15 mg/kg. Control non-tolerant pregnant animals were treated in an identical manner except that they were injected with saline. Foetuses were removed by caesarean section at intervals of one-half to four hours after s.c. injection of 7 mg/kg (base) of H3-test material to the maternal rat on the 21st day of gestation. Maternal plasma levels of free H3-test material were at maximum in the one-half-hour samples of both tolerant and control animals. Although the mean level appeared somewhat lower in tolerant animals, the difference was not statistically significant. The three- and four-hour maternal plasma levels in tolerant animals were significantly lower than in the control group. Foetal plasma concentrations in tolerant animals were significantly lower than in control animals at one hour, but the relationship was reversed at three and four hours. Both maternal and foetal tissue samples showed similar trends, but few gave differences of statistical significance. Recovery of drug from the skin and muscle of the injection site in tolerant rats was always lower than in control animals.

Description of key information

Hugg and Mellett (1965)

Following subcutaneous injection of tritium-labelled test material in rats, males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.

 

Yeh & Woods (1970)

Maternal plasma levels of free H3-test material were at maximum in the one-half-hour samples of both tolerant and control animals. Although the mean level appeared somewhat lower in tolerant animals, the difference was not statistically significant. The three- and four-hour maternal plasma levels in tolerant animals were significantly lower than in the control group. Foetal plasma concentrations in tolerant animals were significantly lower than in control animals at one hour, but the relationship was reversed at three and four hours. Both maternal and foetal tissue samples showed similar trends, but few gave differences of statistical significance. Recovery of drug from the skin and muscle of the injection site in tolerant rats was always lower than in control animals.

 

Sanner & Woods (1965)

Distribution of the drug did not differ significantly between pregnant rats and adult non-pregnant females. 

Key value for chemical safety assessment

Additional information

Hugg and Mellett (1965)

The investigation was undertaken to determine the metabolism and excretion of tritium-labelled test material by rats. The test material was injected subcutaneously at 5 mg/kg. Animals were housed individually in metabolism cages and urine and faeces collected. Ninety two hours after injection the rats were sacrificed and the intestinal contents were removed and added to the cage collected faeces.

Males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.

Free and conjugated tritium-labelled test material was identified in urine but no suggestion of the N-demethylated metabolite was evident.

Faeces from female rats contained only a trace of the N-demethylated drug (1.8 % of the radioactivity in the homogenate).

 

Yeh & Woods (1970)

Pregnant Sprague-Dawley female rats were made tolerant to the test material by initiating s.c. injections of 2 mg/kg (base) of drug, twice daily, 30 hours after breeding and increasing the dosage to 15 mg/kg. Control non-tolerant pregnant animals were treated in an identical manner except that they were injected with saline. Foetuses were removed by caesarean section at intervals of one-half to four hours after s.c. injection of 7 mg/kg (base) of H3-test material to the maternal rat on the 21st day of gestation. Maternal plasma levels of free H3-test material were at maximum in the one-half-hour samples of both tolerant and control animals. Although the mean level appeared somewhat lower in tolerant animals, the difference was not statistically significant. The three- and four-hour maternal plasma levels in tolerant animals were significantly lower than in the control group. Foetal plasma concentrations in tolerant animals were significantly lower than in control animals at one hour, but the relationship was reversed at three and four hours. Both maternal and foetal tissue samples showed similar trends, but few gave differences of statistical significance. Recovery of drug from the skin and muscle of the injection site in tolerant rats was always lower than in control animals.

 

Sanner and Woods (1965)

Tritium-labelled test material was administered subcutaneously to near-term pregnant Sprague-Dawley rats. One-half to 16 hours later the foetuses were removed and specimens were obtained for estimation of free and conjugated test material in maternal and foetal brains, kidneys, and plasma.

Despite a lower maximum plasma level in the foetuses, the foetal brain level reached a peak which was 2.7 times the highest maternal brain level. The peak concentration in maternal brain was only about one eighth that of maternal plasma, but foetal brain levels followed plasma levels quite closely at early time periods. At later times foetal brain concentrations were higher than plasma concentrations; this relationship was not seen in maternal rats.

Conjugated test material was found in maternal plasma, livers, and kidneys, and in foetal plasma and livers at all time periods. Significant amounts of conjugate were not found in maternal brains after doses of 1 mg/kg or 2 mg/kg, but a small quantity was detected after 4 mg/kg. Low concentrations of conjugate were found in foetal brains 2 hours and more after drug administration, but not before. The ratio of conjugated-free drug was lower in foetal than in maternal specimens.

Distribution of free and conjugated test material did not appear to be greatly influenced by dose of the drug except that the proportion of conjugate in maternal plasma tended to decrease with increasing dose. Distribution of the drug did not differ significantly between pregnant rats and adult non-pregnant females.