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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
nephrotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity: 28/14-Day)
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrahydrofurane, Test substance No. 99/151-1- Physical state: colorless liquid, clear, homogeneous- Analytical purity: 99.9 %- Date of production: Apr. 28, 1999 - Lot/batch No.: from continuous production out of tank B566- Storage condition of test material: room temperature in closed containers under nitrogen, excluded from light

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Deutschland GmbH, Sulzfeld, Germany- Age at study initiation: about 10 weeks- Weight at study initiation: 234-248 g- Fasting period before study: none- Housing: individual- Diet: mouse/rat/hamster diet, Provimi Kliba, SA, Kaiseraugst, Switzerland ad libitum- Water: Tap water ad libitum- Acclimation period: 14 to 15 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-24- Humidity (%): 30-70- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: conditioned air
Details on exposure:
Type of inhalation exposure: nose/head only Generation of the inhalation atmospheres:Generator systems:- Piston metering pumps (DESAGA)- Glass vaporizers with thermostat (BASF Aktiengesellschaft)- Two-component atomizers (Beckmann)Generation procedure:For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer (about 30°C) at a constant rate by means of the metering pump. The vapor / air mixture was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50 % +/- 20 % relative humidity, 22°C+/- 2°C). Thereafter it was further mixed with conditioned supply air passed into the inhalation system.Exposures:In order to acclimatize the animals to the exposure conditions they were exposed to supply air in whole-body exposure systems for 2 days before the start of the exposure period (preflow period). Then the main groups were exposed for 6 hours' on workdays for 28 days (28-day test). The number of exposure days was 20 and the animals were sacrificed on the day following the last exposure. The satellite groups 1 were exposed for 6 hours/day on 5 consecutive days and sacrificed directly after the last exposure. The satellite groups 2 were exposed for 6 hours/day on 5 consecutive days and sacrificed after a 21 -day post-exposure observation period. The animals did not have access to water or feed during the exposures.Preflow period (chamber adaptation):A positive pressure was chosen for all test groups during the preflow period.Exposure period:Test group 0: A positive pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that no laboratory air reached the control animals. Test groups 1 - 3: A negative pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that the laboratory was not contaminated as the result of any leakage from the inhalation chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gas chromatography
Duration of treatment / exposure:
up to 20 exposures
Frequency of treatment:
6h/day, 5 days/week
Post exposure period:
3 weeks following 5 consecutive exposures
Doses / concentrations
Remarks:
Doses / Concentrations:0, 600, 1.800, 5.400 mg/l (0, 200, 600, 1800 ppm)Basis:analytical conc.
No. of animals per sex per dose:
Satellite groups (5 consecutive exposures):0 mg/l: 11 animals; 600 mg/l: 6 animals; 1800 mg/l: 6 animals; 5400 mg/l: 11 animalsSatellite recovery groups (5 consecutive exposures and 3 weeks recovery): 0 mg/l: 6 animals; 600 mg/l: 6 animals; 1800 mg/l: 6 animals; 5400 mg/l: 6 animalsMain groups (necropsy after 20 exposures):0 mg/l: 6 animals; 600 mg/l: 6 animals; 1800 mg/l: 6 animals; 5400 mg/l: 6 animals
Control animals:
yes
Details on study design:
There were no positive control groups used.

Examinations

Examinations:
CLlNlCAL EXAMINATIONSClinical observationsAn observation of the general state of health of the animals as well as a check for dead or moribund animals was performed twice a day on working days or once a day on weekends or public holidays, respectively.Clinical examinations of the test animals were carried out on workdays at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure day or observation period. During exposure only a group wise examination was possible.Body weight dataThe body weight of the animals was determined at the start of the preflow, at the start of the exposure period (day 0) and then, as a rule, about once a week. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the start of exposure. Group means were derived from the individual differences. Sacrifice and pathology Implantation of minipumpsMeasurements of cell proliferation were performed using the BrdU technique, which determines the rate of DNA synthesis. Osmotic minipumps (Alzet osmotic pump, rat: model2ML1, containing the marker (about 20mglml BrdU =5-Bromo-2'-deoxyuridine in saline) were prepared 2 to 3 hours before implantation. The pumps were implanted under Metofane (Janssen, Germany) anesthesia subcutaneously in the back region of the animals and stayed there for 7 days.Gross pathological assessmentAll animals - except those used for biochemical examinations - were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were then necropsied and subjected to a gross-pathological assessment. Weight assessmentThe following weight parameters from all animals sacrificed as scheduled were determined:1. anesthetized animals2. kidneys (male rats)Organ/tissue preservation listThe following organs or tissues were fixed in 4 % formaldehyde solution:1. brain2. pituitary gland3. thyroid gland4. parathyroid gland5. thymus6. trachea7. lungs8. heart9. aorta10. salivary glands (mandibular and sublingual glands)11. liver12. spleen13. kidneys14. adrenal glands15. skin16. pancreas17. testeslovaries18. accessory genital organs (epididymides, prostate, seminal vesicle)19. esophagus20. stomach (glandular and non-glandular stomachs)21 .duodenum, jejunum, ileum22. cecum, colon, rectum23. urinary bladder24. lymph nodes (Ln. mandibularis and Ln. mesenterialis)25. skeletal muscle26. sciatic nerve27. sternum28. bone marrow (femur with bone marrow)29. eyes30. femur with knee joint31. spinal cord (cervical, thoracic and lumbar cords)32. uterusloviductAfter the organs were fixed, histotechnical processing, the examination by light microscopy, immunohistology and the evaluation of findings were performed according to the following sections. The organs which were not processed histotechnically will be stored in 4 % formaldehyde solution for possible further histopathological evaluation. Other examinationsBiochemical examinationsFor the detection of possible enzyme induction the amounts of total Cytochrome P450 (Cyt.P450), Ethoxyresorufin-0-deethylase (EROD), Pentoxyresorufin-0-depentylase (PROD) and Nitrophenolhydroxylase (NPH) were determined in the kidneys of 5 rats of control and high concentration in the satellite animals.HistopathologyKidneys and jejunum of male rats were fixed in 4 % formaldehyde for 24 hours, followed by 70 % ethanol, and they were embedded in paraplast, thereafter.Immunohistology (IH)Using the same block as mentioned above (histopathology), in case of the liver, four slides of each specimen were prepared: two for S-phase response (BrdU) - one serving as the test slide, the other as a negative control slide - and two for apoptosis (TUNEL: one was the test slide, and one was the negative control). In case of kidney, two additional slides (from a total of six) were processed for the immunohistological evaluation of alpha-2u-globulin. Four slides were processed of the uterus, two for the determination of S-phase response one serving as the test slide, the other as a negative control slide - and two for the determination of the apoptotic index (TUNEL: one was the test slide, and one was the negative control).

Results and discussion

Details on results:
NOAEC (males) 600 mg/m3 airSummary:Kidneys of male F344 ratsGroup 1 (600 mg/m³):- no significant changes in the absolute or relative weight parameters, no treatment related gross lesions or microscopic findings in hematoxylin and eosin stained slides and no remarkable deviations in immuno-histological investigations.Group 2 (1,800 mg/m³):- hot spots with increased cell proliferation in the subcapsular proximal tubules ("cortex 1 ") after 20 exposures - increased apoptotic index (Al) after 20 exposures and after 5 exposures and a subsequent 3-week recovery period- increased alpha-2u-globulin accumulation in the proximal tubules of cortex 1 and cortex 2 after 20 exposures and in cortex I after 5 exposures with a 3-week recovery period, thereafterGroup 3 (5,400 mg/m³):- hot spots with increased cell proliferation, about 3-fold in the subcapsular proximal tubules ("cortex 1") and about 2-fold in the proximal tubules between outer stripe of 1 outer medulla (OSOM) and the subcapsular layer ("cortex 2") after 20 exposures - increased apoptotic index (Al) after 20 exposures and after 5 exposures and a subsequent 3-week recovery period - increased alpha-2u-globulin accumulation in the proximal tubules of cortex 1 and cortex 2 after 20 exposures and after 5 exposures with a 3-week recovery period, thereafter. Details:CLINICAL SIGNS AND MORTALITYNo substance related clinical effects were observed in rats. No deaths were recorded.BODY WEIGHT AND WEIGHT GAINThe mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.ORGAN WEIGHTSAnimals of groups I1 (5 exposures)Absolute weightsThere were no remarkable differences between treated and control animals.Relative weights (related to terminal body weight)There were no remarkable differences between treated and control animals.Animals of groups R1 (5 exposures + 3-week recovery period)Absolute weightsThere were no remarkable differences between treated and control animals.Relative weights (related to terminal body weight)There were no remarkable differences between treated and control animals.Animals of groups F1 (20 exposures)Absolute weightsThere were no remarkable differences between treated and control animals.Relative weights (related to terminal body weight)There were no remarkable differences between treated and control animals.GROSS PATHOLOGYAnimals of groups I1 (5 exposures)The only gross lesion noted was a focal constriction in the left medial lobe of the liver as the consequence of diaphragmic herniation of a mid concentration animal. This was not treatment related.Animals of groups R1 (5 exposures + 3-week recovery period)The only gross lesion noted was a focal thickened margo plicatus (limiting ridge) in the forestomach of a high concentration group animal. This was regarded to be an incidental finding.Animals of groups F1 (20 exposures)Gross lesions were noted in the liver (focal constriction due to diaphragmic herniation in one animal of the high concentration group), the urinary bladder (concretion - white, diameter 4 millimeters - in one animal of the high concentration group), the testes (focal, unilateral, thread like calcification and slightly reduced unilateral organ size / with loss of turgor in one animal of the high concentration group), the epididymides (unilateral white focus with a diameter of 2 millimeters in one animal of the high concentration group) and in the renal lymph nodes (red discoloration in 2 animals of the high concentration group). These lesions were regarded as spontaneous and not treatment related.HISTOPATHOLOGY:Animals of groups I1 (5 exposures)H. & E. histopathology of the kidneys revealed very few (grade 1) basophilic tubulialways unilateral and located directly subcapsular - in a control male, and in one male each of the mid and high concentration groups. These were not regarded to be treatment related. Hyalin or granular casts were not seen in the kidneys of any of the animals.Animals of groups R1 (5 exposures + 3-week recovery period)H. & E. histopathology of the kidneys revealed very few (grade 1) or few (grade 2) basophilic tubuli - always unilateral and located directly subcapsular - in two control males, a low concentration group male (No. 108) and in two males of the high concentration group. Further, focal, minimal (grade 1) unilateral lymphoid cell infiltration was recorded in a low and mid concentration rat and in two high concentration group animals. Finally, a unilateral cortical cyst was noted in a mid concentration animal, and minimal, unilateral, focal calcification in the cortico-medullary area occurred in a control animal. All these findings were not regarded as treatment related. Hyalin or granular casts were not seen in the kidneys of any of the animals.Animals of groups F1 (20 exposures)H. & E. histopathology of the kidneys revealed very few (grade 1) basophilic tubulialways unilateral and located directly subcapsular - in three control males, and one animal each of the low, mid, and high concentration groups. Further, focal, minimal (grade 1) unilateral lymphoid cell infiltration was recorded in three control animals, three low , two mid and three high concentration group rats. Finally, a minimal, unilateral, focal calcification in the papilla occurred in a low concentration group animal.IMMUNOHISTOLOGYCell proliferation (BrdU)Measurement of the renal cortex Labeling lndex (LI) according to the method described by LARSON et al. was performed on proximal tubuli, only (Table IC 13). Due to a rather low LI, the number of counted tubular cells was increased from about 1000/animal to about 2000/animal. Nevertheless, the LI did not show remarkable differences between control and treated animals, neither after 5 exposures (100% / 81 % / 91 % / 86% for groups 0, 1, 2, and 3, respectively), nor after 5 exposures followed by a 3-week recovery period ( 100 % / 120 % / 108 % / 70 % for groups 0, 1, 2, and 3, respectively), nor after 20 exposures (100 % / 132 % / 107 % / 140 % for groups 0, 1, 2, and 3, respectively). However, as focal "hot spots" of BrdU-labeled cells were noted during the Larson measurement in the area directly under the renal capsule and in areas of the outer cortex, these were measured separately as described above. In "cortexl", cell proliferation was slightly but significantly increased in the high concentration group after 5 exposures (100% / 95 % /109 % / 153 % for groups 0, I, 2, and 3, respectively).After 20 exposures, the LI of the high concentration group was about three times as high as in the respective control group (100 % /119 % / 159 % / 298 % for groups 0, 1, 2, and 3, respectively). A slight but significant increase was also noted in the mid concentration group. After 5 exposures followed by a 3-week recovery period, an increased cell proliferation was not seen (100 % / 78 % / 88 % / 110 % for groups 0, I, 2, and 3, respectively). In "cortex 2", after 20 exposures, the LI of the high concentration group was about twice as high as in the respective control group (100 % / 101 % / 113 % / 186 % for groups 0, 1, 2, and 3, respectively). This was statistically significant. After 5 exposures, cell proliferation was so marginally increased in the high concentration group (100 % / 102 % / 99 % / 125 % for groups 0, 1, 2, and 3, respectively) that a treatment related effect seemed unlikely. After 5 exposures followed by a 3-week recovery period, the LI did not differ remarkably from the control value (100 % / 86 % /86 % / 105 % for groups 0, 1, 2, and 3, respectively). Measurement of the Labeling lndex (LI) in the outer stripe of the outer medulla (OSOM) did not result in remarkable differences between control and high concentration animals, neither after 5 exposures (100 % in the control group versus 77 % in the high concentration group), nor after 5 exposures followed by a 3-week recovery period (100 % in the control group versus 104 % in the high concentration group), nor after 20 exposures (100 % in the control group versus 88 % in the high concentration group).Therefore, the intermediate groups were not assessed. As the "hot spots" in the cortex indicated the area of replicative activities and as the inner stripe of the outer medulla (ISOM) and the inner medulla (IM) did not show remarkable labeling activities, the assessment was cancelled.Measurement of apoptosis using TUNELDue to the low numbers of labeled cells, apoptosis was counted in the proximal tubuli of the whole cortex of the kidney cut longitudinally, giving no special consideration to the hot spot areas or zonation according to Larson et al. After 5 exposures, the LI for apoptosis showed no difference between control and treated animals (100 % / 115 % / 107 % / 92 % for groups 0, 1, 2, and 3, respectively). However, after 20 exposures, the number of apoptotic cells was nearly 2.5-fold higher in the high concentration group than in the concurrent control group (100 % / 74 % / 157 % / 234 % for groups 0, 1, 2, and 3, respectively). This was statistically significant. A slight increase (157 %) was also noted in the mid concentration group. After 5 exposures followed by a 3-week recovery period, the LI for apoptosis was significantly higher (more than 4.5folds) in the high concentration group than in the control group (100 % / 45 % / 145 % / 478 % for groups 0, 1, 2, and 3, respectively), indicative for a counter regulation to an increased BrdU-labeling index. A slight increase (145 %) was also noted in the mid concentration group, however, this was not statistically significant. As apoptosis (or necrosis) was not identified in the hematoxylin and eosin stained kidney slides, a specific evaluation could not be performed. The same applied to the mitosis counts, as only three mitosis were observed in two animals of the 11-Groups (one of the control group and one of the high concentration group) and in one male rat of the high concentration group after 20 exposures.alpha-2u-globulin Alpha-2u-globulin was measured in a first attempt in the renal cortex, giving no special consideration to the "hot spots". While the amount of alpha-2u-globulin was slightly although significantly increased in treated animals after 5 exposures (136 % / 171 % / 178%, in groups 1, 2 or 3, respectively), it was significantly increased after 20 exposures in all treatment groups (149 % / 221% / 259 % for groups 1, 2 or 3, respectively). After 5 exposures followed by a 3-week recovery period, the amount of alpha-2u-globulin significantly increased in mid and high concentration group (150 % / 212 %/ 299 %, in groups 1, 2 or 3, respectively). In the latter it was even higher as compared to the animals receiving 20 exposures (259 %).Thus, significantly increased amounts of alpha-2u-globulin were recorded in all treatment groups after 5 exposures and after 20 exposures. In the recovery animals, the amount of alpha2-µ-globulin was also increased in all treatment groups, however, significant only in the mid and high concentration groups.Following the considerations given to the "hot spots" under BrdU-labeling of cortical cells in the proximal tubuli, in a second attempt, the evaluation of the distribution of the alpha-2u-globulin in the proximal tubuli of the cortex was performed accordingly. While the amount of alpha-2u-globulin was only slightly increased in treated animals after 5 exposures in either "cortexl"- (125 % / 167 % / 175 %, in groups 1, 2 or 3, respectively) or "cortex2"-areas (131 % / 1 76 % / 188 % ), being significant in mid and high concentration groups, only, it was significantly increased after 20 exposures in all treatment groups in "cortexl" (149 % / 212 % / 253 % for groups 1,2 or 3, respectively) and in "cortex2" (149 % / 236 % / 265 % in groups I , 2 or 3, respectively). In "cortex 1", after 5 exposures followed by a 3-week recovery period, the amount of alpha-2u-globulin significantly increased in mid and high concentration groups (154 % / 213 % / 280 %, in groups 1, 2 or 3, respectively). In the latter it was even higher as compared to the animals receiving 20 exposures (253 %). In "cortex 2", after 5 exposures followed by a 3-week recovery period, the amount of alpha2-µ-globulin was significantly increased in mid and high concentration groups (141 % / 205 % / 324 %, in groups 1, 2 or 3, respectively), thus comparable to the values measured in "cortex 1 ". Thus, as well in "cortexl" as in "cortex2", significantly increased amounts of alpha-2u-globulin were recorded in mid and high concentration groups in all three time intervals, and in all treatment groups after 20 exposures. Using the Mallory-Heidenhain stain in control and high concentration animals of the F1 groups (20 exposures), in a blinded evaluation (i.e. evaluation was performed without knowing the treatment group of the investigated animal), a slightly (grade 2) increased amount of hyalin droplets (putatively alpha-2u-globulin) in cells of the proximal tubulus was recorded in five of six high concentration animals. In the control group, only one male gave the impression of an increased amount of the alpha-2u-globulin. This also points into the direction of an accumulation of the alpha-2u-globulin in the proximal tubuli of the renal cortex. However, hyalin or granular cast could not be detected in the kidneys of treated or of control animals.

Any other information on results incl. tables

The results of the study indicate, that in male rats tetrahydrofuran induced alpha-2u-globulin deposition in the cortex of kidneys accompanied by increased cell proliferation in cortical proximal tubules after exposure to concentrations of 1,800 and 5,400 mg/m³. These changes were accompanied by an increased apoptotic index. These effects in the target organ in male rats are considered to constitute the mechanisms of tumor formation.

The No Observed Adverse Effect Concentration (NOAEC) under the condition of this study was 600 mg/m³ (200 ppm).

Applicant's summary and conclusion