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Endpoint summary
Administrative data
Description of key information
Repeated Dose Oral 90d – NOAEL (systemic) = 40 mg/kg bw for rats
Repeated Dose Inhalation 90d – LOAEC (systemic) = 1760 mg/m3 for rats (females)
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 30/08/1988 - 01/02/1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- This data is being read across from the source study that tested technical hexane based on analogue read across.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- Minor deviations from the protocol are not considered to have influenced the integrity or outcome of the study
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Shell, Amsterdam, The Netherlands
- Purity: n-hexane 58.0% weight; benzene 0.060% weight (0.045% vol.) - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar derived, Bor:WISW (SPF Cpb))
- Details on species / strain selection:
- The rat was used because this species is considered the most suitable for this type of study and is usually required by regulatory agencies.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, F.R.G.
- Age at study initiation: 3-4 weeks old upon arrival
- Weight at study initiation: 66.6 g (males)/ 63.0 g (females)
- Housing: groups of 5 separated by sex, in suspended, stainless steel cages, fitted with wire screen bottom and front
- Diet (e.g. ad libitum): cereal-based open-formula stock diet for rats provided ad libitum
- Water (e.g. ad libitum): tap water provided ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 50-70 %
- Air changes (per hr): 10 air changes/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light
IN-LIFE DATES: From: 1988-08-30 To: 1988-12-02 (main study); 1989-02-01 (recovery study) - Route of administration:
- oral: gavage
- Vehicle:
- soya oil
- Remarks:
- soybean oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance in soybean oil containing 0.4, 2, 10 or 50% (w/ v) were freshly prepared once every week. The test solutions were stored in closed glass bottles in a refrigerator at about 5°C. Each of the rats was given daily a single dose (10 ml/kg body weight) of the appropriate solution into the stomach by means of a syringe. The controls (groups A and F) received 10 ml soybean oil/kg body weight/day. The amount of test substance administered to each rat was adapted once a week to the change in body weight.
VEHICLE
- Concentration in vehicle: 0.4, 2, 10, or 50% (w/v)
- Amount of vehicle (if gavage): 10 mL/kg body weight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- One sample of each of the test solutions prepared each week was analysed to verify the concentration of the test substance in the solutions.
The stability of the test substance was examined before the start of the study (see CIVO-report V 88.265, February 1989) and in the solutions prepared on September 20, 1988 after storage for 12 days in a refrigerator at about 5°C.
The homogeneity of the test substance was examined in the solutions prepared on September 20, 1988. Five samples of each solution were taken and analysed to verify the concentration of the test substance in these solutions .
The analyses were carried out by TNO-CIVO Analysis Institute, Zeist, The Netherlands - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once daily, 7 days/week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control - Soybean Oil (Group A)
- Dose / conc.:
- 40 mg/kg bw/day (nominal)
- Remarks:
- Group B
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Remarks:
- Group C
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Group D
- Dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- Remarks:
- Group E
- No. of animals per sex per dose:
- 20/sex/dose (Main study)
10/sex/dose (Recovery study - control and high-dose only) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
Doses were selected based on a range-finding toxicity study (Short-term (14-day) oral toxicity study with light petroleum solvent (technical hexane) for oil seed extraction in rats; Report number: V 88.265))
- Rationale for animal assignment (if not random): On the starting day of the administration period, the rats were weighed, checked for normal growth and allocated to the various groups, matched by weight, using a stratified randomization program.
- Fasting period before blood sampling for clinical biochemistry: Yes, deprivation of water for 24 hours and of food for 16 hours
- Other: A recovery study was conducted with two groups of 10 rats/sex which received the test substance simultaneously at dosage levels of 0 or 5000 mg/kg body weight/day. Because of the high mortality in the high-dose females, the planned recovery period was performed with males only. These male rats were withheld the test substance for 9 weeks after termination of the treatment period. In view of the known effects from hexane on the peripheral nerves, groups of 5 rats of both sexes were added for each dose level including the control, especially for neurohistopathological examination. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations were conducted to detect signs of ill health and reaction to treatment including neurological signs such as changes in posture and gait, hind limb weakness or paralysis ("dragging" one hind foot or "foot drop" while lifting the hind limb)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily working days/ Once daily weekends and public holidays
BODY WEIGHT: Yes
- Time schedule for examinations: recorded initially, weekly during the study, and on the day of scheduled sacrifice
FOOD CONSUMPTION: Yes
- recorded weekly for each cage (5 animals). Mean weekly intake calculated separately for males and females
FOOD EFFICIENCY: Yes
- calculated and expressed as gram weight gain per gram food consumed
WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual inspection of the water bottles was done throughout the study. Water consumptios was measured daily for control and high-dose groups in week 5, then measured daily for all groups in weeks 6, 11 and 12
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: examined prior to test substance administration and at the end of the study
- Dose groups that were examined: control and high-dose groups
HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 84 (males) and day 85 (females)/ days 113 and 150 (recovery group)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all surviving
- Parameters examined: haemoglobin concentration, packed cell volume, red and white blood cell counts, differential white blood cell count, thrombocyte count (main study only), reticulocyte count, prothrombin time (main study only), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 86 (Glucose), 91, 92 (males); day 87 (Glucose), 93, 94 (females)/ day 155 (recovery group)
- Animals fasted: Yes, deprivation of water for 24 hours and of food for 16 hours
- How many animals: all surviving
- Parameters examined: glucose, alkaline phosphatase activity, aspartate aminotransferase activity, gamma glutamyl transferase activity, lactate dehydrogenase activity, alpha-hydroxybutyrate dehydrogenase activity, cholinesterase activity, creatinine kinase activity, total protein, albumin, urea, creatinine, total bilirubin, cholesterol, triglycerides, sodium, potassium, calcium, chloride, inorganic phosphate
- Parameters examined (recovery): alkaline phosphatase activity, aspartate aminotransferase activity, gamma glutamyl transferase activity, cholinesterase activity, total protein, urea, cholesterol, triglycerides, sodium, calcium, chloride, inorganic phosphate
URINALYSIS: Yes
- Time schedule for collection of urine: day 86 (males) and day 87 (females)/ day 115 (recovery group)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, deprivation of water for 24 hours and of food for 16 hours
- Parameters examined: volume, apperance, protein, ketones, uribilinogen, glucose, density, pH, occult blood, bilirubin, microscopic examination of sediment
- Parameters examined (recovery): volume, density, ketones - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, Animals were killed by exsanguination from the abdominal aorta under ether anaesthesia (males on day 91 and 92, females on day 93 and 94), and then examined grossly for pathological changes. All surviving animals were subjected to a full detailed gross necropsy.
All superficial tissues, including the urogenital orifices and tail, each pinna, eye and external auditory meatus, were examined visually. Similar attention was given to the mammary glands, subcutaneous structures, the external nares, buccal cavity and tongue. After ventral midline incision
and skin deflection, subcutaneous tissues were examined, including regional lymph nodes and mammary, salivary and thyroid glands. The thoracic and the abdominal viscera were examined before and after removal. The stomach and intestine including caecum were incised and examined. The lungs were removed and all pleural surfaces examined. The cranial roof was removed to allow observation of the brain.
A thorough autopsy was also performed on animals that died intercurrently or that had been killed because they were moribund.
Samples of the following tissues and organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4 per cent solution of formaldehyde: adrenals; aorta; auxillary lymph nodes; brain (brain stern, cerebrum and cerebellum); caecum; colon; coagulating glands; epididymides; exorbital lachrymal glands; eyes; femur with joint; Harderian glands; heart; kidneys; liver (two lobes); lungs (all lobes, mainstern and bronchi); mammary gland (females); rnesenteric lymph nodes; oesophagus; ovaries (Fallopian tube); pancreas; parotid salivary glands; pituitary; prostrate; rectum; sciatic nerve; seminal vesicles; skeletal muscle (thigh); skin; small intestine (duodenum, ileum and jejunum); spinal cord (at three levels); spleen; sternum (with bone marrow); stomach (forestomach, glandular stomach); sublingual salivary glands; submaxillary salivary glands; testes; thymus; thyroid with parathyroid; trachea and bronchi; tongue; urinary bladder; uterus (with cervix); vagina; all gross lesions.
The following organs of all surviving animals of the main study killed at the end of the exposure period were weighed: adrenals; brain (brain stern, cerebrum and cerebellum); heart; kidneys; liver (two lobes); ovaries (Fallopian tube); pituitary; spleen; testes; thymus; thyroid with parathyroid; uterus (with cervix).
HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the tissues listed above for all animals of the control and high-dose groups. Additionally, the adrenals, kidneys, liver, lungs, peripheral nerve, spleen, testes and thymus were examined in animals of the 1000 and 200 mg/kg groups because organ weights or histopathology of the 5000 mg/kg rats indicated possible treatment-related effects.
The surviving animals of the 5 males and 5 females of each test and control group designated for neuropathological examination were anaesthetised [with Nembutal (40 mg/kg) containing 1% heparin (50 units/ ml)] and then subjected to total body perfusion via the aorta with phosphate-buffered paraformaldehyde/glutaraldehyde fixative. The peripheral nerve of 2 high dose males was fixed by immersion fixation after being carefully removed, since these animals were also designated for complete histopathological examination. The tibial nerve together with its branches (if possible) and the sciatic nerve were removed and post-fixed in Dalton's chrome-osmium solution, dehydrated in ascending concentrations of ethanol, cleared with propylene oxide solution and embedded in Epon. Cross sections and longitudinal sections (1 μm) were prepared of various parts of the nerves, including the proximal, mid and distal parts (8 to 10 slides per animal). The sections were stained with toluidine blue and examined light microscopically. Histopathological examination was carried out on all animals designated for neuropathological examination of the 5 g/kg group and the controls. The nerves of the animals, both males and females, of the 1 g/kg group were processed, cut at 1 μm and examined microscopically in view of treatment-related changes in rats of the 5 g/kg group, also.
Recovery Study: In the recovery group; the surviving rats were killed by exsanguination from the abdominal aorta, under ether anaesthesia on day 155, i.e. on day 64 after withdrawal of the test substance. The organs which were weighed in the main study were also weighed in all rats at the end of the recovery period. Histopathological examination was performed on the tissues and organs identified as showing effects in the main study, viz. the adrenals, kidneys, liver, lungs, peripheral nerve, spleen, testes and thymus. - Statistics:
- Body weight: evaluated by one-way analysis of covariance, followed by Dunnett's multiple comparison test.
Food and water intake: evaluated by analysis of variance, followed by least significant difference tests.
Red blood cell characteristics, total white blood cells, clinical chemistry values, urine volume and density and organ weights: evaluated by one-way analysis of variane (ANOVA), followed by Dunnett's multiple comparison tests.
Differential white blood cell counts, reticulocyte counts and urine analysis: analysed by the Mann-Whitney U-test.
Ophthalmoscopic changes: evaluated by the chi-square test.
Mortality data and histopathologcial changes: evaluated by Fisher's exact probability test. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- All animals in the 5000 mg/kg group showed sluggishness and piloerection soon after dosing each day that disappeared within one hour. Emaciation, weakness and lethargy, hunched posture, swollen belly and focal alopecia were also frequently seen in the 5000 mg/kg group. There were no noticeable differences in appearance or behaviour between the treated rats and the controls.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Mortality occured in all test groups, but was statistically increased in the 5000 mg/kg group (Table 1). Mortality was partly due to faulty dosing because of incorrect technical administration of the test solution and partly because of regurgitation and aspiration of the test solution. Mortality in the lower dose groups did not show a dose-response.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant decreases in mean body weights were observed in the 5000 mg/kg group at all weighings for males and on days 7 to 49 in females (Table 3). Body weights of the 0, 200, and 1000 mg/kg male rats were also lower than in controls, the differences being statistically significant in the 1000 mg/kg group from day 35 onward. In females growth depression only occurred at 5000 mg/kg.
During the recovery period the test rats gained weight much faster than the control animals. By the end of the recovery period the differences in body weight were no longer statistically significant, although the mean body weights in the test group were still lower than in controls (Table 4). - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food intake was decreased in the 5000 mg/kg group males during the first 5 weeks and for females during the first 2 weeks (Table 5). Thereafter, food intake in this group was comparable with or higher than in controls in both sexes. Food intake in the other test groups was comparable to the controls.
During the recovery period food intake was increased in the test group (Table 6). - Food efficiency:
- effects observed, treatment-related
- Description (incidence and severity):
- Food conversion efficiency was markedly decreased in males of the 5000 mg/kg goup throughout the treatment period and also to a much lesser extent in males of the 1000 mg/kg group (Table 7). In females of the 5000 mg/kg group food efficiency was occasionally decreased.
During the recovery period food efficiency was better (sometimes significantly) in rats previously treated with technical hexane than in controls (Table 8). - Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- Water intake was generally higher in all test groups, especially in the 5000 mg/kg group (Table 9).
During the recovery period, water intake of the test group was comparable to controls (Table 10). - Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Red blood cell count, haemoglobin concentration and packed cell volume were increased in males and females of the 5000 mg/kg group and in males of the 1000 mg/kg group (Table 11). These changes were associated with an increase in reticulocyte count and in the 5000 mg/kg males with increases in mean corpuscular haemoglobin and mean corpuscular haemoglobin volume. In the recovery group, decreased red blood cell count, increased reticulocyte count, increased mean corpuscular haemoglobin volume and mean corpuscular haemoglobin values and decreased white blood cell count (males) were still present after a recovery period of 22 days, but had returned to normal after a recovery period of 59 days (Table 12).
White blood cell count was decreased in males of the 5000 mg/kg group due to decreased lymphocyte count (Table 13). This effect was still present after a recovery period of 22 days, but had disappeared after a recovery period of 59 days (Table 14).
An isolated decrease in prothrombin time occurred in females of the 1000 mg/kg group. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A number of changes in the 5000 mg/kg group were observed (Table 15). In females increased alanine aminotransferase activity, decreased aspartate aminotransferase activity, decreased cholinesterase activity (not statistically significant), increased calcium level and increased sodium level were observed. In both sexes increased gamma glutamyl transferase activity, decreased fasting blood glucose level, increased total protein level, increased urea level (statistically significant only in males), increased cholesterol concentration, increased triglyceride concentration (statistically significant only in males), decreased chloride level and increased inorganic phosphate level were observed.
Increases in urea concentration in males and in cholesterol concentration in females were also present in the 1000 mg/kg group. An isolated decrease in fasting blood glucose level occurred in low-dose females.
Increases in plasma urea and inorganic phosphate were still present at the end of the recovery period (Table 16). A decrease in gamma glutamyl transferase activity and an increase in aspartase aminotransferase activity was also observed at the end of the recovery period, although these changes weren't seen in males at the end of the treatment period. - Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Urine volume was increased in males and females of the 5000 mg/kg group and in males of the 1000 mg/kg group (Table 17). No consistent differences in density were observed between the groups.
Ketones occurred in the urine of males and females of the 5000 mg/kg group and in the urine of males in the 1000 mg/kg group. The amount of crystals in the urine was decreased in males of the 1000 and 5000 mg/kg groups. The occurrence of epithelial cells in the urine was higher in males of the 200 and 1000 mg/kg groups (Table 18).
The above changes had disappeared after a recovery period of 24 days (Table 19). - Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 5000 mg/kg group the absolute weights of the thymus, brain, spleen and heart were decreased in both sexes and the testes in males, and those of the adrenals (both sexes) and liver (females) were increased (Table 20). Absolute brain weight was also decreased in males of the 1000 mg/kg group.
The relative weights of the gonads, adrenals, kidneys and liver were increased in the 5000 mg/kg group in both sexes, while kidney weights were also increased in the 200 and 1000 mg/kg groups in males and liver weights in the 1000 mg/kg group in both sexes and in the 200 mg/kg group in males only. Furthermore, the relative weight of the pituitary, thyroid, brain and heart in males were increased in the 5000 mg/kg group. The relative weight of the thymus in both sexes and that of the spleen in females were was decreased in the 5000 mg/kg group (Table 21).
At the end of the recovery period the absolute weight of testes and brain were still decreased in males (Table 22), while the relative weights of the kidneys, heart and liver were still increased (Table 23). - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Gross necropsy revealed evidence of inflammation of the thoracic organs in all animals of all dose groups that included adhesions of thoracic tissues, presence of whitish or turbid liquid in the thoracic cavity, hydrothorax, haemothorax and evidence of pneumonia. The changes were considered to be partly due to the instillation procedure and partly due to the physicochemical properties of the administered material.
In animals of the main study autopsied terminally, large adrenals were observed in 10 out of 15 females and 2 out of 15 males of the 5000 mg/kg group but not in the other groups (Table 24). Bilateral small testes were observed in 3 out of 15 males of the 5000 mg/kg group (still present in 2 out of 6 males of the 5000 mg/kg group at the end of the recovery period (Table 24)) and not in the other groups. The other changes were randomly distributed among the test groups and controls, or they occurred in one or two rats only. - Neuropathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In the 5000 mg/kg group, changes characterized by the presence of scattered nerve fibres showing swollen enlarged axons with relatively rounded contours were observed in the 1 um slides. The myelin sheaths appeared to be intact. The axonal swelling was restricted to one or a few fibres per cross section of the peripheral nerve. The axonal swelling was distinctly more pronounced in males than in females. It occurred to a slight degree in 5 out of 5 males, and occurred to a minimal degree in 3 out 6 females (Table 25).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Main Study:
Treatment-related changes were observed in the adrenals, kidneys, liver, lungs, peripheral nerve, spleen, testes and thymus (Table 25).
In the 5000 mg/kg males and females, the cortex/medulla ratio in the adrenals was significantly increased (this correlated with the increased weight and the gross enlargement of the adrenals).
The proximal convoluted tubules of the kidneys showed accumulation of brown granules or droplets in rats of both sexes in the 5000 mg/kg group only. The droplets were negative upon PAS and Perl's staining. While the nature of these granules could not be established, they may represent proteinaceous material. The incidence of basophilic proximal tubules was statistically significantly increased in males as well as in females of the 1000 mg/kg group, but not in the 5000 mg/kg group. The toxicological significance of this finding is questionable since a consistent dose-response relationship was absent and, moreover, this change may also occur in untreated controls. The corticomedullary mineral deposits that normally occur in females of this strain of rat, were absent in the 5000 mg/kg females of this study.
The liver showed a significantly increased incidence of centrilobular hepatocellular hypertrophy in the 5000 mg/kg group in both sexes and in males of the 1000 mg/kg group. The glycogen content of the liver was significantly decreased in males of the 5000 mg/kg group. These findings were consistent with the increased relative liver weights in the 1000 and 5000 mg/kg groups.
Inflammatory changes in the lungs, e.g. pleuritis, pneumonitis and focal pleural thickening were observed in the study. These changes occurred in all groups (controls included), without a dose-response relationship, and were relatively little pronounced in males and females of the 5000 mg/kg group and in control males. These inflammatory changes were considered to be due to the instillation procedure and not treatment-related. The low incidence in 5000 mg/kg rats may have resulted from the high acute mortality in this group, following the gavage administration. Inflammatory changes, seen as mononuclear cell aggregates, were also present in the mediastinum of one or more males or females in all groups. A considerable variation in the incidence of mineralisation of the arterial walls in the lungs was observed. The difference with the controls reached the level of statistical significance in the 40 and 1000 mg/kg group in males and the 200 and 1000 mg/kg group in females. Since a dose-response relationship was absent its toxicological significance was considered to be doubtful.
The incidence of axonal swelling of the peripheral nerve was significantly increased in males of the 5000 mg/kg group only. In females both the control group and the 5000 mg/kg group showed a considerable incidence of axonal swelling. The microscopic picture was similar to that in males. Although in males the increase in axonal swelling is ascribed to the administration of hexane, the high background incidence of this change indicates that at least part of the axonal alterations, in males as well as in females, might be artificial. Therefore, the neuropathological effects were evaluated using 1 μm sections derived from perfused animals. Microscopic examination of 1 μm plastic embedded slides of the peripheral nerve revealed treatment-related changes in males and females of the 5000 mg/kg group. The changes were characterised by the presence of scattered nerve fibres showing swollen enlarged axons with relatively rounded contours. The myelin sheaths appeared to be intact. The axonal swelling was restricted to one or a few fibres per cross section of the peripheral nerve. Although in all groups, including the controls, there was some variation in the diameters of the nerve fibres, the typical picture of scattered distinctly swollen fibres, among otherwise normally sized and relatively uniform fibres, was only seen in the 5000 mg/kg group. The axonal swelling was distinctly more pronounced in males than in females. It occurred to a slight degree in 5 out of 5 males, whereas in females it was restricted to 3 out of 6 animals in which it occurred only to a minimal degree. There was no distinct or consistent trend of the changes to be more pronounced in the proximal than in the distal parts of the nerve. Occasionally, the changes appeared to be slightly more pronounced in the smaller branches. However, also in controls, the smaller branches showed more variation in the size of the fibres. The intra-axonal myelin deposits, seen as small irregular protrusions of the myelin into the axon, were observed in all groups, including the controls. Therefore, they are considered to represent fortuitous, perhaps artificial, findings, without toxicological importance.
In the spleen, a shift was noticed towards increased brown pigment accumulation in males and females of the 5000 mg/kg group.
Tubular atrophy of the testes was significant in males of the 5000 mg/kg group. Thymic involution was significantly increased in males of the 5000 mg/kg group and higher (although not significantly) in females of this group. These changes were consistent with the decreased thymus weight in males and females of the 5000 mg/kg group.
Mild inflammatory changes that occurred in the controls were occasionally decreased in incidence in the treated groups. The incidence of the normally occurring RES cell aggregates, accompanied by some necrotic hepatocytes was statistically significantly decreased in males of the 1000 and 5000 mg/kg group. In the prostate the incidence of focal interstitial mononuclear-cell infiltrates was significantly decreased in the 5000 mg/kg group. Focal interstitial mononuclear cell infiltrates in the epididymides were significantly decreased in 5000 mg/kg males. Since an increase rather than a decrease of these types of lesions is considered to represent a toxic effect, no toxicological significance was attached to these findings.
Incidental findings such as focal epithelial necrosis of the forestomach in a 5000 mg/kg male, squamous metaplasia of the trachea/bronchi of a single male and a single female of the 5000 mg/kg group, pericarditis in one control male and 2 control females, inflammatory cell infiltrate and cellular debris in the oesophagus of a 1000 mg/kg female, may be due to the instillation procedure since such changes are unusual for rats of the strain and age used.
Recovery study:
Tubular atrophy of the testes was still present after withdrawal of the test substance for a period of 64 days (Table 26). The treatment-related changes observed in the kidneys, liver, spleen, adrenals and thymus of animals of the main groups were absent in the recovery groups (only males available). In the lungs and the thoracic cavity there were still signs of inflammation, probably due to the instillation procedure, however, the incidence in the test group was comparable to that in controls.
Intercurrent deaths:
Lungs of the animals which died or were killed in extremis during the experimental period, showed pleuritis, pneumonitis, hyperaemia and/or presence of droplets of unknown material in about half of the cases. Effects of toxicological importance which could be ascribed to test material administration were observed in various organs in both males and females of the 5000 mg/kg group. In the 1000 mg/kg group the effect was restricted to centrilobular hepatocellular hypertrophy in males. After a recovery period of 64 days the testicular atrophy persisted whereas the other effects had disappeared (males only). - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 40 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: body weight gain/ haematopoetic effects/ organ weight changes/ liver and kidney pathological effects/ testicular atrophy/ neurotoxic effects
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 200 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: increased liver and kidney weights (males only)
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 200 mg/kg bw/day (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 200 mg/kg bw/day (nominal)
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- System:
- male reproductive system
- Organ:
- testes
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- System:
- peripheral nervous system
- Organ:
- neurons
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- System:
- immune system
- Organ:
- spleen
- thymus
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The no-effect level of technical hexane is 40 mg/kg bw/day, but treatment related effects at the next higher dose level of 200 mg/kg bw/day were slight and occurred in male rats only.
- Executive summary:
In a key OECD Guideline 408 sub-chronic repeated dose toxicity study, the test material (Technical hexane (hexane food grade) containing 58% n-hexane) was administered once daily to Wistar derived (Bor: WISW (SPF Cpb)) rats (15/sex/dose) via oral gavage at doses of 0, 40, 200, 1000 or 5000 mg/kg/bw/day in soya bean oil for 13 weeks. Two additional groups (10/sex/dose) received either 0 or 5000 mg technical hexane/kg/bw/day via oral gavage for 13 weeks and were held for a 9-week recovery period. However, due to high mortality observed in the high-dose females, only males were used in the recovery study.
All animals were observed for general condition, behaviour and neurological signs. Individual body weights of all animals were recorded initially (Day 0), weekly and at termination. Food consumption was measured per cage (5 animals) weekly and food utilisation efficiency was calculated and expressed as gram weight gain per gram food consumed. Water consumption was measured daily in week 5 per cage (5 animals) for the control and high-dose groups, then extended to all groups in week 6, 11 and 12 after water intake appeared to be higher in the high-dose group in week 5. Ophthalmoscopic examinations were conducted on the control and high-dose groups prior to treatment and at the end of the study. Haematology was conducted in week 13 in all animals of the main study. Clinical chemistry parameters were measured from blood samples taken at autopsy. Blood glucose measurements and urinalysis were conducted on all animals of the main study in week 13 after deprivation from food and water. Selected haematological, clinical chemistry and urinalysis measurements were conducted in the recovery group.
At autopsy, all animals were sacrificed by exsanguination under ether anaesthesia and examined for macroscopic pathological changes. Major organs were weighed, and the organs, tissues and gross lesions were preserved. Tissues required for microscopic evaluation were embedded in paraffin wax, sectioned at 5 µm, and stained with haematoxylin and eosin. Microscopic evaluation of required organs was conducted on all animals of the control and high dose groups and in all dose groups where organ weights or histopathology of the high-dose group indicated possible treatment-related effects. Animals of the recovery group were sacrificed by exsanguination under ether anaesthesia 64 days after withdrawal from the test substance. Organs weighed in the main study were weighed at the end of the recovery period. Microscopic examinations were conducted on tissues and organs showing an effect in the main study. Animals designated for neuropathological examination were anaesthetised and subjected to total body perfusion with paraformaldehyde/glutaraldehyde fixative. The peripheral nerve of high dose males was fixed, and cross and longitudinal sections (1 µm) were prepared of the proximal, mid and distal parts of the nerve, and stained with toluidine blue. Histopathological examination was performed on all animals of the control and 5000 mg/kg/day group. Additionally, animals of the 1000 mg/kg/day group were examined microscopically because of treatment-related changes observed in the 5000 mg/kg/day group.
Mortality was observed in all test groups but was statistically significant in the high dose group (5000 mg/kg). Mortality in the high dose group was determined to be a result of faulty dosing due to incorrect technical administration of the test solution and from regurgitation and aspiration of the test substance. Mortality in the lower dose groups did not show a dose response. Animals in the 5000 mg/kg dose group displayed sluggishness and piloerection shortly after dosing that disappeared within one hour. Emaciation, weakness and lethargy, hunched posture, swollen belly and focal alopecia were also frequently observed in this group, but not in the other treatment groups.
Mean body weights in male rats were lower than controls in all treatment groups, but statistically significant from day 35 onward in the 1000 mg/kg group and at all time points in the 5000 mg/kg group. Mean body weights in female rats were only lower than controls in the 5000 mg/kg group and statistically significant in this group from day 7 to 49. Food intake was decreased for males and females of the 5000 mg/kg group in the first 5 weeks and first 2 weeks of the study, respectively. No difference between the other treatment groups and controls were observed. Food conversion efficiency was statistically significantly decreased for males of the 5000 mg/kg group throughout the study and at some time points for the 1000 mg/kg group. Food conversion efficiency was lower for females of the 5000 mg/kg group at some time points. Water intake was increased for all treatment groups. In the recovery period food intake and food conversion efficiency were increased in the test group while water intake was comparable to controls. Body weights remained lower than controls but were no longer statistically significant.
Red blood cell count, haemoglobin concentration and packed cell volume were increased in the 5000 and 1000 mg/kg groups and for males and in the 5000 mg/kg group for females. Increases in reticulocyte count, MCH and MCHC and decreases in white blood cell count and lymphocyte were also observed in 5000 mg/kg group males. These effects were still present 22 days into the recovery period but returned to normal by day 59 of the recovery period. In the 5000 mg/kg group, both sexes exhibited increased gamma glutamyl transferase, decreased fasting blood glucose, increased total protein, increased urea (statistically significant in males only), increased cholesterol, increased triglycerides (statistically significant in males only), decreased chloride and increased inorganic phosphate. Females of this group also exhibited increased alanine aminotransferase, decreased aspartate aminotransferase, decreased cholinesterase (though not statistically significant), increased calcium, and increased sodium. In the 1000 mg/kg group increased urea concentration in males and increased cholesterol concentration in females was also observed. Increased plasma urea and inorganic phosphate remained elevated at the end of the recovery period. In both sexes of the 5000 mg/kg group and in males of the 1000 mg/kg group urine volume was increased, ketones were observed in the urine, and the number of crystals in the urine were decreased. An increase in epithelial cells in the urine was observed in males of the 200 and 1000 mg/kg groups. All these changes had returned to normal in the recovery period.
In both sexes of the 5000 mg/kg group absolute organ weights of the thymus, brain, spleen and heart were decreased and the absolute weight of the adrenals were increased. Increased absolute liver weights in females and decreased absolute testes weights in males were also observed for this treatment group. In the 1000 mg/kg group males decreased absolute brain weights were observed. In the 5000 mg/kg group relative weights of the gonads, adrenals, kidneys and liver were increased and relative weights of the thymus were decreased in both sexes. Males in this group also exhibited increased relative weights of the pituitary, thyroid, brain and heart while females of this group also had decreased relative spleen weights. Relative kidney weights were also increased in 200 and 1000 mg/kg group males and relative liver weights were increased in 200 mg/kg group males and both sexes of the 1000 mg/kg group. By the end of the recovery period, absolute brain and testes weights remained lower than controls, while relative kidney, heart and liver weights remained higher than controls.
At gross necropsy, animals in all treatment groups showed evidence of inflammation of thoracic tissues. This was due to the instillation procedure and the physicochemical properties of the test substance. In the 5000 mg/kg group, large adrenals were observed in males and females, and bilateral small testes were observed in males. Bilateral small testes were still observed in the 5000 mg/kg group at the end of the recovery period.
Treatment-related changes were observed in the microscopic examination of the adrenals, kidneys, liver, lungs peripheral nerve, spleen, testes, and thymus. In the 5000 mg/kg group, the cortex/medulla ratio of the adrenals was significantly increased in both sexes. These changes agree with the enlargement and increased weight observed in the adrenals. In the kidneys an accumulation of brown granules or droplets in the proximal convoluted tubules was observed only in both sexes of the 5000 mg/kg group. The incidence of basophilic proximal tubules was also increased in both sexes of the 1000 mg/kg group, but not the high-dose group; therefore, the toxicological significance of this finding is questionable since there is no clear dose-response relationship. In the liver, the incidence of centrilobular hypertrophy was significantly increased in both sexes of the 5000 mg/kg group and in males of the 1000 mg/kg group. The 5000 mg/kg group males also exhibited a significant decrease in glycogen content. These changes are consistent with the increased liver weights observed in the 1000 and 5000 mg/kg groups. Inflammatory changes in the lung were present in all dose groups without a dose-response relationship and were due to the instillation procedure. Inflammatory changes in the mediastinum were also observed in all dose groups, without a dose-response relationship, so the toxicological significance of these changes is doubtful. Both sexes of the 5000 mg/kg group exhibited increased brown pigment accumulation in the spleen. Tubular atrophy of the testes was increased in males of the 5000 mg/kg group. Thymic involution was increased in males and females of the 5000 mg/kg group (though only significant in males). These changes are consistent with the decreased thymus weights observed in this group. Axonal swelling was observed in males and females of the 5000 mg/kg group. By the end of the recovery period, the only treatment-related change still observed was tubular atrophy in the testes.
In conclusion, the 5000 mg/kg group rats exhibited treatment-related changes including reduced body weight gain, haematopoietic effects, organ weight changes, pathological changes in the liver and kidneys, testicular atrophy, and neurotoxic effects. Treatment-related changes in the 1000 mg/kg group rats included reduced weight gain, increased liver and kidney weights and pathological changes in the liver. Treatment-related changes in the 200 mg/kg group were observed only in males and included increased liver and kidney weights without microscopic evidence of damage. No treatment-related changes were observed in the 40 mg/kg group rats. Testicular atrophy was the only adverse effect still present after the 64-day recovery period. The no-effect level was concluded to be 40 mg/kg bw/day, but treatment-related effects at the next higher dose level of 200 mg/kg bw/day were slight and occurred in male rats only.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.
- Justification for type of information:
- This data is being read across from the source study that tested technical hexane based on analogue read across.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- 5 male rats were exposed to concentrations of 6.60, 13.2, and 46.2 mmol/kg bw (568, 1135, 3973 mg/kg) by oral gavage for 90 to 120 days. During the exposure, the rats were examined for body weight, clinical signs, mortality, and neurological effects. Animals were sacrificed after exhibiting hindlimb paralysis, or the end of the exposure period. After sacrifice, a histopathological examination was done on the testes, epididymis, and nerve tissue of the animals.
- GLP compliance:
- no
- Species:
- rat
- Strain:
- other: CD (SD) BR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River COBS
- Weight at study initiation: 214.5 +/- 17.1 g
- Housing: singly in wire cages
- Diet (e.g. ad libitum): Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum): ad libitum - Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- once daily for 90 days, except for animals at the 46.2 mmol/kg dose which were treated for 120 days
- Frequency of treatment:
- 5 days/week
- Remarks:
- Doses / Concentrations:
6.6, 13.2, and 46.2 mmol/kg (568, 1135, 3973 mg/kg)
Basis: - No. of animals per sex per dose:
- 5 males
- Control animals:
- yes, sham-exposed
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: toxicity and general condition changes in posture, gait, and toe pinch
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:
BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly
FOOD CONSUMPTION:
- Food consumption: Yes
- Time schedule for examinations: twice weekly
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Battery of functions tested: changes in posture, gait, and toe pinch
- Sacrifice and pathology:
- HISTOPATHOLOGY: Yes, testes, epididymis, and nerve tissue were examined.
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Two rats in the 13.2 mmol/kg group and one in the 46.2 mmol/kg group died immediately after intubation. Only the 46.2 mmol/kg dose produced hindlimb paralysis in 90 days.
BODY WEIGHT AND WEIGHT GAIN
Body weight gain was reduced after 3 weeks of exposure at all dose levels. This reduction in body weight followed a reduction in food consumption. Significant and dose dependant weight reduction was seen in the 13.2 and 46.2 mmol/kg groups.
NEUROBEHAVIOUR
Hindlimb paralysis was seen in the 46.2 mmol/kg dose animals an average of 101.3 +/- 9. 4 days after start of exposure.
HISTOPATHOLOGY: NON-NEOPLASTIC
The 46.2 mmol/kg dose produced multifocal axonal swellings, adaxonal myelin infolding, and paranodal myelin retraction. Atrophy of the germinal epithelium was also seen in the testes of animals at this dose level. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 6.6 other: mmol/kg bw
- Sex:
- male
- Basis for effect level:
- other: reduced body weight gain
- Dose descriptor:
- NOAEL
- Effect level:
- 13.2 other: mmol/kg bw
- Sex:
- male
- Basis for effect level:
- other: neurological effects
- Dose descriptor:
- LOAEL
- Effect level:
- 46.2 other: mmol/kg bw
- Sex:
- male
- Basis for effect level:
- other: neurological effects
- Critical effects observed:
- not specified
- Conclusions:
- Neurological effects were only seen at the highest dose level after an average of 101.3 days of exposure. The LOAEL for neurological effects is 46.2 mmol/kg bw (37973 mg/kg), and the NOAEL is 13.2 mmol/kg bw (1135 mg/kg). Reduced body weight gain was seen at all three dose levels, however was only considered treatment related in the 13.2 and 46.2 mmol/kg bw groups. The NOAEL is therefore 6.60 mmol/kg bw.
- Executive summary:
This study examined the effect of oral exposure to the test substance n-hexane. 5 male rats were exposed to concentrations of 6.60, 13.2, and 46.2 mmol/kg bw (568, 1135, 3973 mg/kg) by oral gavage for 90 to 120 days. During the exposure, the rats were examined for body weight, clinical signs, mortality, and neurological effects. Animals were sacrificed after exhibiting hindlimb paralysis, or the end of the exposure period. After sacrifice, a histopathological examination was done on the testes, epididymis, and nerve tissue of the animals. Neurological effects were only seen at the highest dose level after an average of 101.3 days of exposure. The LOAEL for neurological effects is 46.2 mmol/kg bw (37973 mg/kg), and the NOAEL is 13.2 mmol/kg bw (1135 mg/kg). Reduced body weight gain was seen at all three dose levels, however was only considered treatment related in the 13.2 and 46.2 mmol/kg bw groups. The NOAEL is therefore 6.60 mmol/kg bw.
- Endpoint:
- sub-chronic toxicity: oral
- Data waiving:
- other justification
- Justification for data waiving:
- other:
- Justification for type of information:
- The ‘justification for the read across’ is provided in the ‘Attached justification’ section below.
- Species:
- rat
Referenceopen allclose all
Homogeneity analysis on five samples taken from each test solution indicated that the test substance was uniformly distributed in the solution. Two stability studies indicated that the test substance was stable in the test solutions for at least 7 days storage at +5°C.
Table 1: Cumulative number of animals found dead or killed in extremis
Males |
||||||||||
Day |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|||||
n |
% |
n |
% |
n |
% |
n |
% |
n |
% |
|
6 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
13 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
20 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
27 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
34 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
10** |
25% |
41 |
0 |
0% |
0 |
0% |
0 |
0% |
1 |
3% |
11*** |
28% |
48 |
0 |
0% |
0 |
0% |
0 |
0% |
2 |
7% |
11*** |
28% |
55 |
0 |
0% |
0 |
0% |
2 |
7% |
2 |
7% |
11*** |
28% |
62 |
0 |
0% |
0 |
0% |
2 |
7% |
2 |
7% |
11*** |
28% |
69 |
0 |
0% |
0 |
0% |
2 |
7% |
2 |
7% |
11*** |
28% |
76 |
0 |
0% |
0 |
0% |
2 |
7% |
2 |
7% |
12*** |
30% |
83 |
0 |
0% |
0 |
0% |
2 |
7% |
2 |
7% |
12*** |
30% |
90 |
0 |
0% |
0 |
0% |
3 |
10% |
2 |
7% |
13*** |
33% |
Females |
||||||||||
Day |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|||||
n |
% |
n |
% |
n |
% |
n |
% |
n |
% |
|
6 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
13 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
20 |
0 |
0% |
0 |
0% |
0 |
0% |
0 |
0% |
3 |
8% |
27 |
2 |
5% |
0 |
0% |
0 |
0% |
1 |
3% |
6 |
15% |
34 |
4 |
10% |
0 |
0% |
1 |
3% |
1 |
3% |
17** |
43% |
41 |
5 |
13% |
0 |
0% |
1 |
3% |
1 |
3% |
19** |
48% |
48 |
5 |
13% |
0 |
0% |
1 |
3% |
1 |
3% |
19** |
48% |
55 |
5 |
13% |
0 |
0% |
1 |
3% |
2 |
7% |
20*** |
50% |
62 |
5 |
13% |
0 |
0% |
1 |
3% |
2 |
7% |
29*** |
73% |
69 |
5 |
13% |
0 |
0% |
1 |
3% |
2 |
7% |
30*** |
75% |
76 |
5 |
13% |
1 |
3% |
1 |
3% |
2 |
7% |
30*** |
75% |
83 |
5 |
13% |
1 |
3% |
1 |
3% |
2 |
7% |
30*** |
75% |
90 |
5 |
13% |
1 |
3% |
1 |
3% |
2 |
7% |
30*** |
75% |
Table 2: Mean body weights
Males |
|||||||||||||||
Day |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||||||||||
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
|
0 |
66.7 |
0.7 |
40 |
66.6 |
1.0 |
30 |
66.6 |
0.9 |
30 |
66.7 |
0.8 |
30 |
66.6 |
0.9 |
40 |
7 |
96.6 |
1.0 |
40 |
96.2 |
1.6 |
30 |
96.7 |
1.2 |
30 |
94.0 |
1.2 |
30 |
86.2* |
1.2 |
40 |
14 |
130.1 |
1.6 |
40 |
131.4 |
2.2 |
30 |
127.5 |
2.5 |
30 |
125.7 |
1.8 |
30 |
106.0** |
1.6 |
40 |
21 |
164.5 |
2.2 |
40 |
165.9 |
3.1 |
30 |
159.8 |
3.3 |
30 |
158.0 |
2.7 |
30 |
125.8** |
2.0 |
40 |
28 |
191.2 |
2.8 |
40 |
195.4 |
3.9 |
30 |
187.9 |
4.0 |
30 |
183.4 |
3.4 |
30 |
143.9** |
2.6 |
40 |
35 |
210.1 |
3.4 |
40 |
212.6 |
4.9 |
30 |
203.1 |
5.3 |
30 |
195.7* |
3.9 |
30 |
155.5** |
2.9 |
30 |
42 |
225.3 |
4.2 |
40 |
230.4 |
5.3 |
30 |
218.9 |
5.6 |
30 |
206.4* |
4.7 |
29 |
159.4** |
3.2 |
29 |
49 |
240.9 |
4.6 |
40 |
247.7 |
5.8 |
30 |
234.8 |
6.0 |
30 |
225.7 |
4.2 |
28 |
170.2** |
3.1 |
29 |
56 |
255.1 |
4.7 |
40 |
261.4 |
6.1 |
30 |
250.5 |
5.2 |
28 |
236.2* |
4.3 |
28 |
175.5** |
3.3 |
29 |
63 |
267.5 |
4.7 |
40 |
273.5 |
6.5 |
30 |
261.0 |
5.2 |
28 |
245.3** |
4.5 |
28 |
178.3** |
3.6 |
29 |
70 |
276.7 |
5.0 |
40 |
282.2 |
6.8 |
30 |
270.9 |
5.5 |
28 |
253.4** |
4.8 |
28 |
182.0** |
3.7 |
29 |
77 |
287.2 |
5.3 |
40 |
293.3 |
7.1 |
30 |
278.3 |
5.5 |
28 |
262.4** |
5.1 |
28 |
183.6** |
3.9 |
28 |
84 |
295.5 |
5.5 |
40 |
300.5 |
7.2 |
30 |
284.9 |
5.6 |
28 |
267.4** |
5.3 |
28 |
184.0** |
4.2 |
28 |
91 |
303.8 |
5.7 |
40 |
308.1 |
7.6 |
30 |
293.5 |
6.0 |
27 |
274.3** |
5.5 |
28 |
187.7** |
4.3 |
27 |
Grand means |
148.5 |
|
150.5 |
|
149.4 |
|
149.3 |
|
122.9 |
|
|||||
Females |
|||||||||||||||
Day |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||||||||||
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
|
0 |
63.0 |
0.7 |
40 |
63.0 |
0.9 |
30 |
63.0 |
1.0 |
30 |
63.1 |
0.8 |
30 |
63.1 |
0.7 |
40 |
7 |
89.5 |
1.0 |
40 |
89.6 |
1.1 |
30 |
88.4 |
1.2 |
30 |
89.3 |
1.2 |
30 |
84.0** |
1.1 |
40 |
14 |
109.8 |
1.5 |
40 |
110.8 |
1.5 |
30 |
108.3 |
1.6 |
30 |
109.9 |
2.0 |
30 |
101.5** |
1.5 |
40 |
21 |
128.2 |
1.8 |
40 |
128.0 |
1.7 |
30 |
127.2 |
2.4 |
30 |
128.5 |
2.4 |
30 |
117.1** |
2.4 |
37 |
28 |
142.4 |
2.1 |
38 |
142.4 |
1.9 |
30 |
141.4 |
2.5 |
30 |
142.4 |
2.6 |
29 |
133.2** |
2.7 |
34 |
35 |
150.0 |
2.7 |
35 |
150.0 |
2.0 |
30 |
149.8 |
2.8 |
29 |
151.1 |
3.0 |
29 |
143.2 |
4.2 |
23 |
42 |
159.1 |
2.4 |
35 |
159.2 |
2.3 |
30 |
159.0 |
3.0 |
29 |
158.8 |
3.2 |
29 |
145.9** |
4.7 |
21 |
49 |
167.6 |
2.7 |
35 |
167.2 |
2.2 |
30 |
166.1 |
3.2 |
29 |
166.3 |
3.3 |
29 |
154.2** |
5.0 |
20 |
56 |
173.9 |
2.8 |
35 |
172.9 |
2.5 |
30 |
173.2 |
3.5 |
29 |
172.8 |
3.6 |
28 |
162.1 |
4.9 |
10 |
63 |
179.2 |
2.8 |
35 |
178.4 |
2.6 |
30 |
177.9 |
3.9 |
29 |
177.3 |
3.6 |
28 |
161.5 |
7.3 |
10 |
70 |
180.0 |
3.3 |
35 |
182.0 |
3.0 |
29 |
180.1 |
3.7 |
29 |
181.3 |
3.4 |
28 |
168.6 |
8.2 |
10 |
77 |
183.9 |
3.1 |
35 |
186.0 |
2.7 |
29 |
184.3 |
3.9 |
29 |
184.0 |
3.6 |
28 |
167.2 |
7.0 |
10 |
84 |
190.5 |
3.0 |
35 |
190.1 |
2.9 |
29 |
189.2 |
4.0 |
29 |
188.7 |
4.0 |
28 |
171.4 |
8.0 |
10 |
91 |
192.2 |
3.0 |
35 |
192.7 |
3.0 |
29 |
190.6 |
4.2 |
29 |
190.7 |
3.9 |
28 |
172.1 |
8.1 |
10 |
Grand means |
148.5 |
|
150.5 |
|
149.4 |
|
149.3 |
|
122.9 |
|
Statistics: Covar + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01
Table 3: Mean body weights (g) – Recovery Period (males only)
Day |
Mean |
|
Control |
5000 mg/kg |
|
98 |
305.0 |
204.8*** |
105 |
310.3 |
215.7*** |
112 |
317.5 |
231.4*** |
119 |
321.5 |
242.4*** |
Grand means |
313.6 |
223.5 |
|
||
126 |
324.4 |
255.0*** |
133 |
328.3 |
266.2** |
140 |
330.4 |
277.5* |
147 |
332.7 |
285.5 |
154 |
334.6 |
294.5 |
Grand means |
330.1 |
275.7 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p<0.001
Table 4: Mean food intake (g/rat/day) over the week preceding the day indicated
Day |
Males |
Females |
||||||||
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
7 |
11.7 |
11.7 |
11.5 |
11.3 |
9.1*** |
10.7 |
11.0 |
10.7 |
10.9 |
9.3*** |
14 |
14.3 |
14.5 |
13.7 |
14.0 |
10.4*** |
11.5 |
11.9 |
11.6 |
11.9 |
10.5** |
21 |
14.2 |
14.6 |
14.2 |
14.2 |
11.3*** |
11.0 |
10.9 |
10.7 |
11.1 |
11.0 |
28 |
14.7 |
14.9 |
14.9 |
15.5 |
11.9*** |
11.0 |
11.2 |
11.3 |
11.2 |
11.4 |
35 |
13.6 |
13.5 |
13.5 |
12.7 |
11.7*** |
10.0 |
10.7 |
10.1 |
11.0 |
11.4 |
42 |
12.3 |
12.6 |
12.4 |
11.9 |
11.6 |
10.3 |
10.3 |
10.4 |
11.1 |
11.7 |
49 |
11.7 |
12.2 |
12.1 |
11.8 |
11.8 |
10.0 |
9.6 |
9.8 |
10.5 |
11.9*** |
56 |
11.2 |
11.5 |
11.1 |
11.6 |
11.6 |
9.1 |
8.7 |
9.1 |
10.0 |
11.7*** |
63 |
11.2 |
11.1 |
11.4 |
11.4 |
12.3 |
8.6 |
8.5 |
8.3 |
9.3 |
12.1*** |
70 |
10.4 |
10.4 |
10.6 |
10.5 |
11.6** |
7.8 |
7.8 |
7.5 |
8.6 |
12.1*** |
77 |
10.4 |
10.3 |
9.9 |
10.0 |
10.8 |
8.3 |
8.1 |
7.7 |
8.4 |
11.4*** |
84 |
9.8 |
9.8 |
9.5 |
9.7 |
10.8** |
7.9 |
7.3 |
7.4 |
8.2 |
11.5*** |
91 |
9.1 |
8.7 |
8.9 |
8.9 |
10.5** |
7.1 |
6.9 |
6.5 |
7.4 |
10.9*** |
Grand means |
11.9 |
12.0 |
11.9 |
11.9 |
11.1 |
9.5 |
9.5 |
9.3 |
10.0 |
11.0 |
Statistics: Anova + L.S.D. tests (two-sided): * p<0.05; ** p<0.01; *** p <0.0001
Table 5: Mean food intake (g/rat/day) over the week preceding the day indicated – Recovery Period (males only)
Day |
Mean |
|
Control |
5000 mg/kg |
|
98 |
14.6 |
16.5*** |
105 |
15.3 |
14.8 |
112 |
15.8 |
16.3* |
119 |
14.6 |
15.9*** |
Grand means |
15.1 |
15.9 |
|
||
126 |
15.8 |
17.8** |
133 |
15.6 |
17.8*** |
140 |
15.2 |
18.0** |
147 |
15.3 |
17.6** |
154 |
15.5 |
18.2*** |
Grand means |
15.5 |
17.9 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p <0.0001
Table 6: Mean food conversion efficiency (g weight gain/g food consumed) over the week preceding the day indicated
Day |
Males |
Females |
||||||||
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
7 |
0.37 |
0.36 |
0.37 |
0.34 |
0.30*** |
0.35 |
0.35 |
0.34 |
0.34 |
0.32** |
14 |
0.33 |
0.35 |
0.32 |
0.32 |
0.27** |
0.25 |
0.25 |
0.24 |
0.25 |
0.24 |
21 |
0.35 |
0.34 |
0.33 |
0.33 |
0.25*** |
0.24 |
0.23 |
0.25 |
00.24 |
0.20 |
28 |
0.26 |
0.28 |
0.27 |
0.23 |
0.22** |
0.19 |
0.18 |
0.18 |
0.17 |
0.18 |
35 |
0.20 |
0.18 |
0.16* |
0.14** |
0.15* |
0.12 |
0.10 |
0.11 |
0.11 |
0.09 |
42 |
0.17 |
0.20 |
0.18 |
0.14 |
0.05*** |
0.13 |
0.13 |
0.13 |
0.10 |
0.04*** |
49 |
0.19 |
0.20 |
0.19 |
0.20 |
0.13*** |
0.12 |
0.12 |
0.10 |
0.10 |
0.10 |
56 |
0.18 |
0.17 |
0.15 |
0.13* |
0.06*** |
0.10 |
0.09 |
0.11 |
0.09 |
0.08 |
63 |
0.16 |
0.16 |
0.13 |
0.11** |
0.03*** |
0.09 |
0.09 |
0.08 |
0.07 |
0.00*** |
70 |
0.13 |
0.12 |
0.13 |
0.11 |
0.04*** |
0.01 |
0.06 |
0.04 |
0.07 |
0.08 |
77 |
0.14 |
0.15 |
0.11* |
0.13 |
0.02*** |
0.07 |
0.07 |
0.08 |
0.05 |
-0.01** |
84 |
0.12 |
0.11 |
0.10 |
0.07* |
0.00*** |
0.12 |
0.08 |
0.10 |
0.08 |
0.05 |
91 |
0.13 |
0.13 |
0.13 |
0.11 |
0.04*** |
0.03 |
0.05 |
0.03 |
0.04 |
0.01 |
Grand means |
0.21 |
0.21 |
0.20 |
0.18 |
0.12 |
0.14 |
0.14 |
0.14 |
0.13 |
0.11 |
Statistics: Anova + L.S.D. tests (two-sided): * p<0.05; ** p<0.01; *** p <0.0001
Table 7: Mean food conversion efficiency (g weight gain/g food consumed) over the week preceding the day indicated – Recovery Period (males only)
Day |
Mean |
|
Control |
5000 mg/kg |
|
98 |
0.04 |
0.09 |
105 |
0.05 |
0.10* |
112 |
0.07 |
0.14*** |
119 |
0.04 |
0.10* |
Grand means |
0.05 |
0.11 |
|
||
126 |
0.02 |
0.10 |
133 |
0.03 |
0.09 |
140 |
0.01 |
0.09 |
147 |
0.02 |
0.06 |
154 |
0.02 |
0.07** |
Grand means |
0.02 |
0.08 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p <0.001
Table 8: Mean water consumption (g/rat/day) over the week preceding the day indicated
Day |
Males |
Females |
||||||||
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
35 |
18.0 |
-- |
-- |
-- |
20.4 |
13.0 |
-- |
-- |
-- |
18.2 |
42 |
17.7 |
21.0 |
20.0 |
19.1 |
22.8 |
14.5 |
16.0 |
16.7 |
18.0 |
20.4 |
77 |
15.9 |
17.0 |
17.7 |
19.3 |
22.7 |
13.6 |
14.4 |
14.9 |
15.8 |
20.4 |
84 |
15.9 |
18.8 |
18.2 |
19.8 |
23.7 |
13.9 |
15.0 |
15.3 |
16.8 |
21.4 |
-- not determined
Table 9: Mean water consumption (g/rat/day) over the week preceding the day indicated – Recovery Period (males only)
Day |
Mean |
|
Control |
5000 mg/kg |
|
98 |
18.3 |
19.0 |
105 |
19.2 |
18.0 |
112 |
19.9 |
20.0 |
119 |
21.0 |
23.0 |
Table 10: Mean haematological findings in blood collected on day 84 (males)/85 (females)
Males |
|||||||||||
Dosage |
Haematological parameter |
||||||||||
RBCa |
HBa |
PCVa |
Throma |
PTTa |
Reticulb |
MCVa |
MCHa |
MCHCa |
|||
Control |
Mean |
7.0 |
8.7 |
0.453 |
925 |
38.1 |
5.6 |
64.8 |
1.24 |
19.2 |
|
sem |
0.1 |
0.1 |
0.004 |
22 |
0.6 |
1.0 |
0.9 |
0.01 |
0.1 |
||
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
||
40 mg/kg |
Mean |
7.3 |
8.8 |
0.459 |
909 |
37.0 |
6.1 |
62.5 |
1.20 |
19.2 |
|
sem |
0.1 |
0.1 |
0.004 |
20 |
0.3 |
1.0 |
0.5 |
0.01 |
0.1 |
||
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
||
200 mg/kg |
Mean |
7.3 |
8.9 |
0.464 |
935 |
37.6 |
5.4 |
63.8 |
1.22 |
19.2 |
|
sem |
0.1 |
0.1 |
0.005 |
24 |
0.6 |
1.0 |
1.0 |
0.01 |
0.2 |
||
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
||
1000 mg/kg |
Mean |
7.7** |
9.2** |
0.482** |
920 |
36.4 |
8.8* |
62.5 |
1.20 |
19.1 |
|
sem |
0.1 |
0.1 |
0.005 |
18 |
0.4 |
0.9 |
0.9 |
0.01 |
0.1 |
||
n |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
||
5000 mg/kg |
Mean |
7.5** |
9.7** |
0.491** |
943 |
36.5 |
10.3* |
66.1 |
1.30* |
19.6* |
|
sem |
0.1 |
0.1 |
0.005 |
26 |
0.6 |
1.6 |
0.9 |
0.02 |
0.1 |
||
n |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
||
Females |
|||||||||||
Dosage |
Haematological parameter |
||||||||||
RBCa |
HBa |
PCVa |
Throma |
PTTa |
Reticulb |
MCVa |
MCHa |
MCHCa |
|||
Control |
Mean |
7.3 |
8.9 |
0.454 |
983 |
36.1 |
8.6 |
62.0 |
1.22 |
19.6 |
|
sem |
0.1 |
0.1 |
0.004 |
39 |
0.6 |
1.0 |
0.7 |
0.01 |
0.1 |
||
n |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
||
40 mg/kg |
Mean |
7.5 |
8.9 |
0.459 |
956 |
35.1 |
8.3 |
61.5 |
1.20 |
19.5 |
|
sem |
0.0 |
0.1 |
0.003 |
28 |
0.6 |
1.2 |
0.4 |
0.01 |
0.1 |
||
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
||
200 mg/kg |
Mean |
7.6 |
9.0 |
0.460 |
970 |
34.7 |
8.3 |
60.5 |
1.18 |
19.5 |
|
sem |
0.0 |
0.1 |
0.003 |
31 |
0.4 |
1.0 |
0.4 |
0.01 |
0.1 |
||
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
||
1000 mg/kg |
Mean |
7.5 |
9.1 |
0.464 |
946 |
34.0* |
6.7 |
62.0 |
1.22 |
19.7 |
|
sem |
0.1 |
0.1 |
0.005 |
21 |
0.3 |
0.9 |
0.7 |
0.01 |
0.1 |
||
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
||
5000 mg/kg |
Mean |
7.6 |
9.5** |
0.482** |
975 |
36.2 |
11.0 |
63.4 |
1.24 |
19.6 |
|
sem |
0.1 |
0.1 |
0.005 |
42 |
0.8 |
1.8 |
0.8 |
0.02 |
0.1 |
||
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01
b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002
RBC = red blood cells (10E12/l)
HB = haemoglobin (mmol/l)
PCV = packed cell volume (l/l)
Throm = thrombocytes (10E9/l)
PTT = prothrombin time (sec)
Reticul = reticulocytes (/1000)
MCV = mean corpuscular volume (fl)
MCH = mean corpuscular haemoglobin (fmol)
MCHC = mean corpuscular haemoglobin concentration (mmol/l)
Table 11: Mean haematological findings in blood collected on days 113 and 150 (22 and 59 days, respectively after withdrawal of the test substance) - Males only
113 days (22 days after withdrawal) |
|||||||||
Dosage |
Haematological parameter |
||||||||
RBCa |
HBa |
PCVa |
Reticulb |
MCVa |
MCHa |
MCHCa |
|||
Control |
Mean |
7.9 |
9.1 |
0.455 |
4.6 |
57.8 |
1.16 |
20.0 |
|
sem |
0.1 |
0.1 |
0.005 |
1.0 |
0.7 |
0.01 |
0.2 |
||
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
||
5000 mg/kg |
Mean |
7.4*** |
9.1 |
0.456 |
10.0** |
61.3* |
1.22** |
19.9 |
|
sem |
0.1 |
0.1 |
0.008 |
1.5 |
1.1 |
0.01 |
02 |
||
n |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
||
150 days (59 days after withdrawal) |
|||||||||
Dosage |
Haematological parameter |
||||||||
RBCa |
HBa |
PCVa |
Reticulb |
MCVa |
MCHa |
MCHCa |
|||
Control |
Mean |
7.4 |
9.0 |
0.461 |
15.6 |
62.4 |
1.21 |
19.4 |
|
sem |
0.3 |
0.3 |
0.013 |
9.6 |
1.1 |
0.02 |
0.2 |
||
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
||
5000 mg/kg |
Mean |
7.8 |
9.6 |
0.480 |
4.3 |
61.6 |
1.23 |
20.0 |
|
sem |
0.1 |
0.1 |
0.006 |
1.1 |
1.1 |
0.01 |
0.3 |
||
n |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01
b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002
RBC = red blood cells (10E12/l)
HB = haemoglobin (mmol/l)
PCV = packed cell volume (l/l)
Reticul = reticulocytes (/1000)
MCV = mean corpuscular volume (fl)
MCH = mean corpuscular haemoglobin (fmol)
MCHC = mean corpuscular haemoglobin concentration (mmol/l)
Table 12: Mean total and differential white blood cell counts in blood collected on day 84 (males)/85 (females)
Males |
|||||||||
Dosage |
|
||||||||
WBCa |
Neutroa |
Lymphoa |
Eosinophb |
Neutrophb |
Lymphocb |
Monocytb |
Basophilb |
||
Control |
Mean |
13.1 |
1.7 |
11.3 |
1.1 |
12.7 |
85.8 |
0.4 |
0.0 |
sem |
0.5 |
0.1 |
0.5 |
0.4 |
0.9 |
1.1 |
0.2 |
0.0 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
40 mg/kg |
Mean |
12.6 |
1.4 |
11.0 |
0.9 |
10.7 |
87.6 |
0.7 |
0.1 |
sem |
0.5 |
0.1 |
0.4 |
0.3 |
0.5 |
0.7 |
0.3 |
0.1 |
|
n |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
|
200 mg/kg |
Mean |
13.8 |
1.4 |
12.1 |
0.7 |
10.5 |
87.7 |
1.0* |
001 |
sem |
0.6 |
0.2 |
0.6 |
0.2 |
1.1 |
1.2 |
0.2 |
0.1 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
1000 mg/kg |
Mean |
12.9 |
2.0 |
10.7 |
0.8 |
14.9 |
83.4 |
0.8 |
0.1 |
sem |
0.8 |
0.3 |
0.5 |
0.2 |
1.5 |
1.6 |
0.2 |
0.1 |
|
n |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
|
5000 mg/kg |
Mean |
10.5** |
2.0 |
8.2** |
1.7 |
18.7** |
78.8*** |
0.9 |
0.0 |
sem |
0.5 |
0.3 |
0.3 |
0.4 |
1.8 |
1.7 |
0.3 |
0.0 |
|
n |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
|
Females |
|||||||||
Dosage |
|
||||||||
WBCa |
Neutroa |
Lymphoa |
Eosinophb |
Neutrophb |
Lymphocb |
Monocytb |
Basophilb |
||
Control |
Mean |
12.2 |
2.2 |
9.8 |
1.1 |
13.2 |
85.2 |
0.3 |
0.2 |
sem |
1.3 |
1.2 |
0.5 |
0.2 |
4.0 |
4.1 |
0.1 |
0.1 |
|
n |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
13 |
|
40 mg/kg |
Mean |
11.7 |
1.2 |
10.2 |
1.8 |
10.7 |
87.2 |
0.3 |
0.0 |
sem |
0.7 |
0.2 |
0.7 |
0.4 |
1.3 |
1.6 |
0.1 |
0.0 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
200 mg/kg |
Mean |
11.6 |
1.3 |
10.5 |
1.6 |
11.2 |
86.7 |
0.4 |
0.0 |
sem |
0.6 |
0.2 |
0.5 |
0.3 |
1.4 |
1.4 |
0.2 |
0.0 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
1000 mg/kg |
Mean |
11.3 |
1.2 |
10.0 |
1.1 |
10.5 |
88.0 |
0.4 |
0.1 |
sem |
0.5 |
0.1 |
0.5 |
0.2 |
1.1 |
1.3 |
0.2 |
0.1 |
|
n |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
14 |
|
5000 mg/kg |
Mean |
11.3 |
1.4 |
9.7 |
1.6 |
13.3 |
84.6 |
0.5 |
0.0 |
sem |
0.6 |
0.2 |
0.7 |
0.4 |
2.6 |
3.0 |
0.3 |
0.0 |
|
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01
b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002
WBC = White blood cells (10E9/l)
Neutro = Absolute number of neutrophils (10E9/l)
Lympho = Absolute number of lymphocytes (10E9/l)
Eosinoph = Eosinophils (%)
Neutroph = Neutrophils (%)
Lymphoc = Lymphocytes (%)
Monocyt = Monocytes (%)
Basophil = Basophils (%)
Table 13: Mean total and differential white blood cell counts in blood collected on days 113 and 150 (22 and 59 days, respectively after withdrawal of the test substance) - Males only
113 days (22 days after withdrawal) |
|||||||||
Dosage |
|
||||||||
WBCa |
Neutroa |
Lymphoa |
Eosinophb |
Neutrophb |
Lymphocb |
Monocytb |
Basophilb |
||
Control |
Mean |
14.4 |
1.4 |
12.7 |
1.2 |
9.6 |
88.7 |
0.5 |
0.0 |
sem |
0.7 |
0.1 |
0.6 |
0.3 |
1.1 |
1.0 |
0.2 |
0.0 |
|
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
5000 mg/kg |
Mean |
11.7* |
1.9 |
9.4** |
2.0 |
16.3* |
80.5** |
0.8 |
0.3 |
sem |
0.8 |
0.4 |
0.6 |
0.4 |
2.4 |
2.4 |
0.3 |
0.2 |
|
n |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
|
150 days (59 days after withdrawal) |
|||||||||
Dosage |
|
||||||||
WBCa |
Neutroa |
Lymphoa |
Eosinophb |
Neutrophb |
Lymphocb |
Monocytb |
Basophilb |
||
Control |
Mean |
17.8 |
3.5 |
13.9 |
1.4 |
16.0 |
82.0 |
0.6 |
0.0 |
sem |
2.0 |
1.4 |
0.8 |
0.3 |
4.4 |
4.7 |
0.2 |
0.0 |
|
n |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
5000 mg/kg |
Mean |
13.5 |
1.6 |
11.6 |
2.3 |
11.3 |
86.3 |
0.0 |
0.0 |
sem |
1.0 |
0.3 |
0.8 |
0.6 |
1.3 |
1.1 |
0.0 |
0.0 |
|
n |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
6 |
a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01
b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002
WBC = White blood cells (10E9/l)
Neutro = Absolute number of neutrophils (10E9/l)
Lympho = Absolute number of lymphocytes (10E9/l)
Eosinoph = Eosinophils (%)
Neutroph = Neutrophils (%)
Lymphoc = Lymphocytes (%)
Monocyt = Monocytes (%)
Basophil = Basophils (%)
Table 14: Mean results of clinical chemistry of blood collected on day 86 (males)/87 (females) or terminally
Males |
|||||||||||||||
|
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||||||||||
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
|
ALP |
231.5 |
10.4 |
15 |
226.9 |
11.1 |
15 |
246.3 |
21.7 |
13 |
219.9 |
10.2 |
13 |
240.9 |
16.0 |
11 |
ALAT |
41.3 |
2.1 |
15 |
44.9 |
4.7 |
15 |
42.2 |
2.3 |
13 |
44.0 |
2.9 |
13 |
48.8 |
3.2 |
11 |
ASAT |
57.1 |
1.8 |
15 |
60.0 |
4.5 |
15 |
54.3 |
1.8 |
13 |
50.5 |
1.2 |
13 |
49.4 |
1.9 |
11 |
GGT |
3.2 |
0.3 |
15 |
3.0 |
0.5 |
15 |
2.8 |
0.6 |
13 |
3.3 |
0.4 |
13 |
8.8** |
0.8 |
11 |
CreatKin |
64.9 |
11.2 |
15 |
52.8 |
4.1 |
15 |
54.0 |
3.2 |
13 |
49.5 |
1.7 |
13 |
65.1 |
9.9 |
11 |
CHE |
419 |
24 |
15 |
400 |
16 |
15 |
462 |
30 |
13 |
502 |
35 |
13 |
389 |
19 |
11 |
LDH |
141.1 |
13.4 |
15 |
164.7 |
42.9 |
15 |
140.4 |
10.2 |
13 |
119.5 |
7.9 |
13 |
195.4 |
43.9 |
11 |
a-HBDH |
25.4 |
2.7 |
15 |
28.6 |
6.2 |
15 |
24.8 |
1.7 |
13 |
22.6 |
1.7 |
13 |
31.5 |
6.3 |
11 |
Glucose |
3.6 |
0.1 |
15 |
3.7 |
0.1 |
15 |
3.7 |
0.1 |
13 |
3.7 |
0.1 |
13 |
3.0** |
0.2 |
11 |
TP |
58.2 |
0.2 |
15 |
58.8 |
0.7 |
15 |
59.3 |
0.6 |
13 |
58.3 |
0.6 |
13 |
61.1** |
0.5 |
11 |
Albumin |
35.8 |
0.3 |
15 |
36.2 |
0.4 |
15 |
36.3 |
0.6 |
13 |
34.8 |
0.4 |
13 |
37.5 |
0.7 |
11 |
Urea |
4.31 |
0.15 |
15 |
4.51 |
0.32 |
15 |
4.55 |
0.12 |
13 |
5.06* |
0.19 |
13 |
6.11** |
0.19 |
11 |
Creatin |
67.9 |
1.5 |
15 |
64.4 |
1.2 |
15 |
64.6 |
1.5 |
13 |
67.9 |
1.6 |
13 |
62.4 |
2.1 |
11 |
Bili-Tot |
1.36 |
0.06 |
15 |
1.39 |
0.06 |
15 |
1.27 |
0.10 |
13 |
1.30 |
0.04 |
13 |
1.47 |
0.16 |
11 |
Cholest |
2.02 |
0.07 |
15 |
2.06 |
0.08 |
15 |
2.12 |
0.11 |
13 |
2.12 |
0.09 |
13 |
2.43* |
0.12 |
11 |
Triglyc |
0.69 |
0.04 |
15 |
0.71 |
0.05 |
15 |
0.69 |
0.06 |
13 |
0.73 |
0.05 |
13 |
0.98** |
0.11 |
11 |
Ca |
2.48 |
0.03 |
15 |
2.45 |
0.02 |
15 |
2.45 |
0.03 |
13 |
2.42 |
0.02 |
13 |
2.49 |
0.02 |
11 |
K |
3.63 |
0.07 |
15 |
3.49 |
0.06 |
15 |
3.68 |
0.06 |
13 |
3.68 |
0.10 |
13 |
3.42 |
0.10 |
11 |
Na |
145.9 |
0.3 |
15 |
145.4 |
0.3 |
15 |
145.8 |
0.3 |
13 |
146.5 |
0.3 |
13 |
146.3 |
0.3 |
11 |
Cl |
105.9 |
0.6 |
15 |
105.7 |
0.5 |
15 |
105.5 |
0.4 |
13 |
104.7 |
0.4 |
13 |
102.3** |
0.4 |
11 |
Inorg-P |
1.91 |
0.09 |
15 |
1.98 |
0.07 |
15 |
1.81 |
0.08 |
13 |
1.99 |
0.10 |
13 |
2.27** |
0.07 |
11 |
Females |
|||||||||||||||
Day |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||||||||||
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
Mean |
sem |
n |
|
ALP |
182.3 |
25.5 |
13 |
183.1 |
8.3 |
14 |
177.0 |
8.2 |
14 |
135.8 |
7.7 |
14 |
167.8 |
11.5 |
10 |
ALAT |
32.8 |
1.2 |
13 |
33.3 |
1.2 |
14 |
34.3 |
1.2 |
14 |
32.5 |
2.1 |
14 |
41.4** |
2.5 |
10 |
ASAT |
57.0 |
3.9 |
13 |
51.3 |
1.1 |
14 |
55.8 |
1.8 |
14 |
51.0 |
1.6 |
14 |
46.6* |
1.9 |
10 |
GGT |
2.1 |
0.2 |
13 |
1.8 |
0.3 |
14 |
2.0 |
0.3 |
14 |
1.7 |
0.2 |
14 |
5.7** |
0.8 |
10 |
CreatKin |
50.3 |
3.1 |
13 |
50.6 |
2.3 |
14 |
53.1 |
2.7 |
14 |
50.1 |
3.2 |
14 |
56.2 |
2.2 |
10 |
CHE |
1160 |
181 |
13 |
1188 |
106 |
14 |
1228 |
143 |
14 |
1283 |
86 |
14 |
750 |
86 |
10 |
LDH |
125.8 |
5.5 |
13 |
124.8 |
7.2 |
14 |
148.3 |
9.8 |
14 |
125.5 |
8.5 |
14 |
118.3 |
8.7 |
10 |
a-HBDH |
23.3 |
0.8 |
13 |
22.9 |
1.0 |
14 |
27.3 |
1.8 |
14 |
22.2 |
1.6 |
14 |
21.9 |
1.5 |
10 |
Glucose |
3.8 |
0.1 |
13 |
3.3** |
0.1 |
14 |
3.4 |
0.1 |
14 |
3.5 |
0.1 |
14 |
2.6** |
0.1 |
10 |
TP |
60.0 |
0.8 |
13 |
58.5 |
0.5 |
14 |
59.4 |
0.4 |
14 |
59.9 |
0.4 |
14 |
62.6* |
0.6 |
10 |
Albumin |
36.8 |
0.7 |
13 |
36.6 |
0.5 |
14 |
36.5 |
0.4 |
14 |
35.6 |
0.8 |
14 |
36.0 |
1.3 |
10 |
Urea |
5.44 |
0.27 |
13 |
5.31 |
0.26 |
14 |
5.66 |
0.25 |
14 |
4.97 |
0.26 |
14 |
6.19 |
0.28 |
10 |
Creatin |
67.4 |
1.7 |
13 |
67.9 |
1.3 |
14 |
70.4 |
2.2 |
14 |
66.8 |
1.5 |
14 |
67.9 |
2.0 |
10 |
Bili-Tot |
1.25 |
0.12 |
13 |
1.17 |
0.10 |
14 |
1.27 |
0.08 |
14 |
1.18 |
0.06 |
14 |
1.51 |
0.05 |
10 |
Cholest |
1.84 |
0.08 |
13 |
1.77 |
0.06 |
14 |
1.83 |
0.06 |
14 |
2.16* |
0.08 |
14 |
2.86** |
0.11 |
10 |
Triglyc |
0.50 |
0.02 |
13 |
0.54 |
0.03 |
14 |
0.62 |
0.06 |
14 |
0.56 |
0.06 |
14 |
0.70 |
0.05 |
10 |
Ca |
2.38 |
0.03 |
13 |
2.38 |
0.01 |
14 |
2.37 |
0.01 |
14 |
2.39 |
0.01 |
14 |
2.49** |
0.02 |
10 |
K |
3.31 |
0.14 |
13 |
3.19 |
0.08 |
14 |
3.37 |
0.10 |
14 |
3.20 |
0.07 |
14 |
3.00 |
0.07 |
10 |
Na |
143.6 |
0.6 |
13 |
143.7 |
0.4 |
14 |
143.3 |
0.6 |
14 |
144.8 |
0.3 |
14 |
145.6* |
0.6 |
10 |
Cl |
107.9 |
0.5 |
13 |
107.5 |
0.6 |
14 |
107.3 |
0.3 |
14 |
108.3 |
0.04 |
14 |
105.1** |
0.7 |
10 |
Inorg-P |
1.46 |
0.09 |
13 |
1.52 |
0.09 |
14 |
1.42 |
0.08 |
14 |
1.50 |
0.11 |
14 |
2.10** |
0.09 |
10 |
Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01
ALP = Alkaline Phosphatase (U/l) – collected day 91 (males)/93 (females)
ALAT = Alanine Aminotransferase (GPT) (U/l) – collected day 91 (males)/93 (females)
ASAT = Aspartate Aminotransferase (GOT) (U/l) – collected day 91 (males)/93 (females)
GGT = Gamma Glutamyl Transferase (U/l) – collected day 91 (males)/93 (females)
CreatKin = Creatinine Kinase (U/l) – collected day 91 (males)/93 (females)
CHE = Cholinesterase in Plasma (U/l) – collected day 91 (males)/93 (females)
LDH = Lactate Dehydrogenase (U/l) – collected day 91 (males)/93 (females)
a-HBDH = a-Hydroxy Butericacid Dehydrogenase (U/l) – collected day 91 (males)/93 (females)
Glucose (mmol/l) – collected day 86 (males)/87 (females)
TP = Total Protein (g/l) – collected day 91 (males)/93 (females)
Albumin (g/l) – collected day 91 (males)/93 (females)
Urea (mmol/l) – collected day 91 (males)/93 (females)
Creatin = Creatinine (umol/l) – collected day 91 (males)/93 (females)
Bili-Tot = Bilirubin (total) (umol/l) – collected day 91 (males)/93 (females)
Cholest = Cholesterol (mmol/l) – collected day 91 (males)/93 (females)
Triglyc = Triglycerides (mmol/l) – collected day 91 (males)/93 (females)
Ca = Calcium (mmol/l) – collected day 91 (males)/93 (females)
K = Potassium (mmol/l) – collected day 91 (males)/93 (females)
Na = Sodium (mmol/l) – collected day 91 (males)/93 (females)
Cl = Chloride (mmol/l) – collected day 91 (males)/93 (females)
Inorg-P = Inorganic Phosphate (mmol/l) – collected day 91 (males)/93 (females)
Table 15: Mean results of clinical chemistry of blood collected on day 155 (64 days after withdrawal of the test substance) – Males only
|
Control |
5000 mg/kg |
||||
Mean |
sem |
n |
Mean |
sem |
n |
|
ASAT |
54.2 |
1.6 |
10 |
64.1* |
4.4 |
6 |
ALAT |
44.3 |
1.7 |
10 |
49.1 |
2.4 |
6 |
GGT |
10.2 |
1.1 |
10 |
6.5* |
1.2 |
6 |
CHE |
335 |
24 |
10 |
310 |
12 |
6 |
TP |
61.0 |
0.7 |
10 |
60.0 |
0.9 |
6 |
Urea |
6.34 |
0.22 |
10 |
7.48* |
0.44 |
6 |
Cholest |
2.09 |
0.08 |
10 |
1.87 |
0.07 |
6 |
Triglyc |
1.36 |
0.19 |
10 |
1.24 |
0.19 |
6 |
Ca |
2.57 |
0.03 |
10 |
2.55 |
0.02 |
6 |
Na |
144.4 |
0.5 |
10 |
144.9 |
0.4 |
6 |
Cl |
103.9 |
0.6 |
10 |
104.1 |
1.0 |
6 |
Inorg-P |
1.55 |
0.02 |
10 |
1.82*** |
0.07 |
6 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p<0.001
ASAT = Aspartate Aminotransferase (GOT) (U/l)
ALAT = Alanine Aminotransferase (GPT) (U/l)
GGT = Gamma Glutamyl Transferase (U/l)
CHE = Cholinesterase in Plasma (U/l)
TP = Total Protein (g/l)
Urea (mmol/l)
Cholest = Cholesterol (mmol/l)
Triglyc = Triglycerides (mmol/l)
Ca = Calcium (mmol/l)
Na = Sodium (mmol/l)
Cl = Chloride (mmol/l)
Inorg-P = Inorganic Phosphate (mmol/l)
Table 16: Mean volume and density of the urine collected on day 85-86 (Males) or day 86-87 (Females)
Dosage |
Males |
Females |
|||
Volume (mL) |
Density (kg/L) |
Volume (mL) |
Density (kg/L) |
||
Control |
Mean |
1.1 |
1.077 |
1.2 |
1.069 |
sem |
0.1 |
0.002 |
0.2 |
0.005 |
|
n |
15 |
15 |
13 |
13 |
|
40 mg/kg |
Mean |
1.0 |
1.081 |
1.1 |
1.066 |
sem |
0.1 |
0.003 |
0.1 |
0.004 |
|
n |
15 |
15 |
14 |
14 |
|
200 mg/kg |
Mean |
1.2 |
1.089** |
1.2 |
1.066 |
sem |
0.1 |
0.001 |
0.1 |
0.004 |
|
n |
14 |
14 |
14 |
14 |
|
1000 mg/kg |
Mean |
2.1** |
1.080 |
1.2 |
1.077 |
sem |
0.1 |
0.002 |
0.1 |
0.003 |
|
n |
13 |
13 |
14 |
14 |
|
5000 mg/kg |
Mean |
2.7** |
1.071 |
2.9** |
1.065 |
sem |
0.1 |
0.001 |
0.2 |
0.004 |
|
n |
11 |
11 |
10 |
10 |
Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01
Table 17: Selected mean semi-quantitative observations in the urine collected on day 85-86 (Males) or day 86-87 (Females)
Dosage |
Males |
Females |
|||||
Keton-U (0-3) |
Epith-U (0-5) |
Cryst-U (0-5) |
Keton-U (0-3) |
Epith-U (0-5) |
Cryst-U (0-5) |
||
Control |
Mean |
0 |
1 |
2 |
0 |
1 |
1 |
n |
15 |
15 |
15 |
13 |
13 |
13 |
|
40 mg/kg |
Mean |
0 |
1 |
2 |
0 |
1 |
1 |
n |
15 |
15 |
15 |
14 |
14 |
14 |
|
200 mg/kg |
Mean |
0 |
2* |
2 |
0 |
1 |
1 |
n |
14 |
14 |
14 |
14 |
14 |
14 |
|
1000 mg/kg |
Mean |
1*** |
2*** |
1** |
0 |
1 |
1 |
n |
13 |
13 |
13 |
14 |
14 |
14 |
|
5000 mg/kg |
Mean |
2*** |
1 |
1** |
1* |
1 |
0 |
n |
11 |
11 |
11 |
10 |
10 |
10 |
Statistics: Mann/Whitney U-test (two-sided): *p<0.05; ** p<0.02; *** p<0.002
Keton-U = Ketones in Urine
Epith-U = Epithelial Cells in Urine
Cryst-U = Crystals in Urine
Table 18: Mean volume, density, semi-quantitative- and microscopic observations in the urine collected on day 114-115 (23-24 days after withdrawal of the test substance) – Males only
Dosage |
Volumea (mL) |
Densitya (kg/L) |
Keton-Ub (0-3) |
Epith-Ub (0-5) |
Cryst-Ub (0-5) |
|
Control |
Mean |
2.0 |
1.071 |
0 |
1 |
2 |
sem |
0.1 |
0.002 |
|
|||
n |
10 |
10 |
10 |
10 |
10 |
|
5000 mg/kg |
Mean |
1.9 |
1.074 |
0 |
1 |
2 |
sem |
0.1 |
0.002 |
|
|||
n |
6 |
6 |
6 |
6 |
6 |
a. Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p<0.001
b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002
Keton-U = Ketones in Urine
Epith-U = Epithelial Cells in Urine
Cryst-U = Crystals in Urine
Table 19: Mean terminal body weights (g) and absolute organ weights (g) recorded terminally
Organ |
Males |
Females |
|||||||||
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||
Body wt |
Mean |
303.9 |
308.7 |
293.6 |
274.2** |
188.1** |
193.5 |
187.0 |
184.0 |
187.6 |
172.5 |
sem |
5.7 |
7.6 |
6.0 |
5.5 |
4.2 |
5.7 |
4.0 |
4.8 |
3.4 |
7.8 |
|
n |
40 |
30 |
27 |
28 |
27 |
13 |
14 |
14 |
14 |
10 |
|
Pituitary |
Mean |
0.012 |
0.011 |
0.012 |
0.011 |
0.011 |
0.015 |
0.014 |
0.014 |
0.015 |
0.014 |
sem |
0.000 |
0.000 |
0.000 |
0.000 |
0.000 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Thyroid |
Mean |
0.019 |
0.018 |
0.018 |
0.017 |
0.017 |
0.018 |
0.017 |
0.016 |
0.016 |
0.018 |
sem |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
0.001 |
|
n |
15 |
15 |
13 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Adrenals |
Mean |
0.043 |
0.046 |
0.044 |
0.046 |
0.070** |
0.057 |
0.055 |
0.053 |
0.051 |
0.097** |
sem |
0.001 |
0.001 |
0.001 |
0.001 |
0.003 |
0.002 |
0.002 |
0.001 |
0.002 |
0.006 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Kidneys |
Mean |
1.64 |
1.72 |
1.69 |
1.75 |
1.51 |
1.22 |
1.18 |
1.16 |
1.22 |
1.33 |
sem |
0.06 |
0.05 |
0.05 |
0.04 |
0.04 |
0.03 |
0.03 |
0.04 |
0.03 |
0.06 |
|
n |
15 |
15 |
14 |
13 |
11 |
14 |
14 |
14 |
14 |
10 |
|
Thymus |
Mean |
0.262 |
0.270 |
0.290 |
0.267 |
0.097** |
0.252 |
0.241 |
0.258 |
0.228 |
0.156** |
sem |
0.019 |
0.024 |
0.024 |
0.017 |
0.007 |
0.013 |
0.012 |
0.025 |
0.014 |
0.015 |
|
n |
15 |
15 |
15 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Brain |
Mean |
1.80 |
1.80 |
1.75 |
1.70** |
1.43** |
1.69 |
1.71 |
1.68 |
1.67 |
1.46** |
sem |
0.02 |
0.02 |
0.02 |
0.02 |
0.02 |
0.02 |
0.01 |
0.01 |
0.02 |
0.02 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Spleen |
Mean |
0.438 |
0.428 |
0.431 |
0.400 |
0.229** |
0.365 |
0.341 |
0.367 |
0.341 |
0.275** |
sem |
0.021 |
0.021 |
0.018 |
0.017 |
0.010 |
0.017 |
0.012 |
0.015 |
0.014 |
0.016 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Heart |
Mean |
0.94 |
0.95 |
0.92 |
0.87 |
0.61** |
0.70 |
0.67 |
0.68 |
0.67 |
0.61** |
sem |
0.03 |
0.02 |
0.03 |
0.03 |
0.02 |
0.02 |
0.02 |
0.01 |
0.01 |
0.02 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Liver |
Mean |
9.72 |
10.12 |
9.88 |
10.41 |
9.78 |
6.62 |
6.49 |
6.45 |
6.82 |
9.12** |
sem |
0.39 |
0.36 |
0.36 |
0.32 |
0.27 |
0.29 |
0.16 |
0.15 |
0.16 |
0.39 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Testes |
Mean |
3.00 |
2.96 |
2.88 |
2.87 |
2.04** |
|
||||
sem |
0.08 |
0.07 |
0.09 |
0.05 |
0.25 |
||||||
n |
15 |
15 |
14 |
13 |
11 |
||||||
Ovaries |
Mean |
|
0.097 |
0.098 |
0.093 |
0.093 |
0.108 |
||||
sem |
0.004 |
0.004 |
0.004 |
0.004 |
0.007 |
||||||
n |
13 |
14 |
14 |
14 |
10 |
||||||
Uterus |
Mean |
0.619 |
0.637 |
0.566 |
0.612 |
0.344 |
|||||
sem |
0.083 |
0.075 |
0.074 |
0.093 |
0.064 |
||||||
n |
13 |
14 |
14 |
14 |
10 |
Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01
Table 20: Mean relative organ weights (g/kg body weight) recorded terminally
Organ |
Males |
Females |
|||||||||
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
Control |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
||
Pituitary |
Mean |
0.040 |
0.039 |
0.042 |
0.042 |
0.060** |
0.078 |
0.073 |
0.077 |
0.079 |
0.079 |
sem |
0.001 |
0.001 |
0.002 |
0.001 |
0.003 |
0.003 |
0.003 |
0.003 |
0.003 |
0.04 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Thyroid |
Mean |
0.065 |
0.061 |
0.065 |
0.065 |
0.097** |
0.092 |
0.093 |
0.090 |
0.085 |
0.106 |
sem |
0.002 |
0.002 |
0.003 |
0.002 |
0.007 |
0.005 |
0.003 |
0.005 |
0.004 |
0.005 |
|
n |
15 |
15 |
13 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Adrenals |
Mean |
0.145 |
0.157 |
0.159 |
0.173 |
0.401** |
0.293 |
0.296 |
0.288 |
0.274 |
0.561** |
sem |
0.004 |
0.005 |
0.006 |
0.008 |
0.024 |
0.010 |
0.006 |
0.009 |
0.010 |
0.026 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Kidneys |
Mean |
5.51 |
5.79 |
6.08** |
6.58** |
8.60** |
6.33 |
6.33 |
6.31 |
6.54 |
7.70** |
sem |
0.07 |
0.13 |
0.08 |
0.17 |
0.16 |
0.13 |
0.09 |
0.19 |
0.12 |
0.18 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Thymus |
Mean |
0.87 |
0.91 |
1.04 |
0.99 |
0.55** |
1.31 |
1.28 |
1.39 |
1.22 |
0.90** |
sem |
0.05 |
0.07 |
0.07 |
0.05 |
0.04 |
0.07 |
0.05 |
0.11 |
0.07 |
0.07 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Brain |
Mean |
6.13 |
6.09 |
6.32 |
6.40 |
8.15** |
8.77 |
9.21 |
9.18 |
8.95 |
8.56 |
sem |
0.19 |
0.14 |
0.14 |
0.12 |
0.24 |
0.17 |
0.19 |
0.22 |
0.17 |
0.31 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Spleen |
Mean |
1.47 |
1.43 |
1.54 |
1.50 |
1.30 |
1.89 |
1.82 |
2.00 |
1.82 |
1.59* |
sem |
0.06 |
0.05 |
0.04 |
0.05 |
0.05 |
0.09 |
0.05 |
0.07 |
0.06 |
0.06 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Heart |
Mean |
3.16 |
3.21 |
3.30 |
3.24 |
3.47** |
3.64 |
3.61 |
3.71 |
3.59 |
3.52 |
sem |
0.04 |
0.05 |
0.05 |
0.05 |
0.06 |
0.06 |
0.05 |
0.06 |
0.05 |
0.06 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Liver |
Mean |
32.5 |
33.9 |
35.4* |
39.0** |
55.7** |
34.1 |
34.7 |
35.1 |
36.4* |
53.0** |
sem |
0.04 |
0.4 |
0.6 |
0.7 |
1.7 |
0.6 |
0.4 |
0.4 |
0.6 |
1.1 |
|
n |
15 |
15 |
14 |
13 |
11 |
13 |
14 |
14 |
14 |
10 |
|
Testes |
Mean |
10.13 |
10.01 |
10.34 |
10.80 |
11.48 |
|
||||
sem |
0.24 |
0.26 |
0.23 |
0.30 |
1.32 |
||||||
n |
15 |
15 |
14 |
13 |
11 |
||||||
Ovaries |
Mean |
|
0.505 |
0.516 |
0.504 |
0.497 |
0.622* |
||||
sem |
0.28 |
0.028 |
0.017 |
0.023 |
0.031 |
||||||
n |
13 |
14 |
14 |
14 |
10 |
||||||
Uterus |
Mean |
3.20 |
3.44 |
3.12 |
3.27 |
2.02 |
|||||
sem |
0.43 |
0.43 |
0.44 |
0.50 |
0.42 |
||||||
n |
13 |
14 |
14 |
14 |
10 |
Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01
Table 21: Mean terminal body weight (g) and absolute organ weights (g) recorded on day 155 (64 days after withdrawal of the test substance) – Males only
Organ |
Control |
5000 mg/kg |
||||
Mean |
sem |
n |
Mean |
sem |
n |
|
Body weight |
337.4 |
17.5 |
10 |
296.7 |
12.2 |
6 |
Tests |
3.12 |
0.11 |
10 |
2.13* |
0.48 |
6 |
Pituitary |
0.013 |
0.000 |
10 |
0.013 |
0.001 |
6 |
Thyroid |
0.023 |
0.001 |
10 |
0.020 |
0.001 |
6 |
Adrenals |
0.042 |
0.002 |
10 |
0.045 |
0.002 |
6 |
Kidneys |
1.87 |
0.07 |
10 |
1.98 |
0.10 |
6 |
Thymus |
0.250 |
0.033 |
10 |
0.229 |
0.018 |
6 |
Brain |
1.88 |
0.02 |
10 |
1.64*** |
0.02 |
6 |
Spleen |
0.538 |
0.031 |
10 |
0.476 |
0.024 |
6 |
Heart |
1.03 |
0.04 |
10 |
1.04 |
0.03 |
6 |
Liver |
10.72 |
0.59 |
10 |
10.64 |
0.60 |
6 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p< 0.001
Table 22: Mean relative organ weights (g/kg bodyweight) recorded on day 155 (64 days after withdrawal of the test substance) – Males only
Organ |
Control |
5000 mg/kg |
||||
Mean |
sem |
n |
Mean |
sem |
n |
|
Tests |
9.38 |
0.31 |
10 |
6.94 |
1.46 |
6 |
Pituitary |
0.040 |
0.003 |
10 |
0.046 |
0.004 |
6 |
Thyroid |
0.069 |
0.003 |
10 |
0.069 |
0.004 |
6 |
Adrenals |
0.132 |
0.017 |
10 |
0.151 |
0.006 |
6 |
Kidneys |
5.61 |
0.16 |
10 |
6.69*** |
0.15 |
6 |
Thymus |
0.72 |
0.09 |
10 |
0.78 |
0.07 |
6 |
Brain |
5.73 |
0.37 |
10 |
5.58 |
0.22 |
6 |
Spleen |
1.62 |
0.09 |
10 |
1.60 |
0.04 |
6 |
Heart |
3.08 |
0.08 |
10 |
3.53* |
0.15 |
6 |
Liver |
31.8 |
0.6 |
10 |
35.9** |
1.2 |
6 |
Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p< 0.001
Table 23: Summary of selected macroscopic changes
Changes |
Incidence of Gross Lesions (numeric) |
|||||||||
Males |
Females |
|||||||||
0 mg/kg |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
0 mg/kg |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
Lungs: |
||||||||||
Spotted surface |
0 |
1 |
0 |
0 |
2 |
1 |
0 |
0 |
0 |
0 |
Discoloured |
0 |
0 |
0 |
1 |
2 |
0 |
1 |
0 |
0 |
4 |
Evidence of inflammation |
0 |
1 |
2 |
0 |
0 |
1 |
0 |
4 |
1 |
0 |
Focal foamy appearance |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
Thoracic cavity: |
||||||||||
Hydrothorax |
0 |
0 |
0 |
2 |
1 |
0 |
1 |
0 |
0 |
2 |
Haemothorax |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
Filled with whitish liquid |
0 |
4 |
4 |
2 |
0 |
0 |
1 |
6 |
3 |
0 |
Turbid liquid |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
Evidence of inflammation |
1 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Thickening of the mediastinum |
0 |
0 |
0 |
0 |
0 |
2 |
1 |
1 |
1 |
0 |
Adhesions of thoracic tissues |
1 |
1 |
4 |
3 |
0 |
2 |
2 |
7 |
2 |
0 |
Adrenals: |
||||||||||
Enlarged |
0 |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
10 |
Testes: |
||||||||||
Bi-lateral small |
0 |
0 |
0 |
0 |
3 |
|
Table 24: Summary of selected macroscopic changes – Recovery period (males only)
Changes |
Incidence of Gross Lesions (numeric) |
|
Males |
||
0 mg/kg |
5000 mg/kg |
|
Lungs: |
||
Evidence of inflammation |
1 |
0 |
Thoracic cavity: |
||
Hydrothorax |
2 |
0 |
Adhesion of lung-lobes and thoracic wall |
2 |
0 |
Testes: |
||
Soft tissue |
0 |
1 |
Bi-lateral small |
0 |
2 |
Table 25: Summary of selected microscopic changes
Changes |
Incidence of Lesions (numeric) |
|||||||||
Males |
Females |
|||||||||
0 mg/kg |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
0 mg/kg |
40 mg/kg |
200 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
Adrenals: |
(15) |
(15) |
(15) |
(15) |
(14) |
(15) |
(15) |
(13) |
(15) |
(15) |
Increased ratio cortex/medulla |
0 |
0 |
0 |
0 |
9*** |
0 |
0 |
1 |
0 |
12*** |
Focal increased cortical vacuolation |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
Diffuse increased cortical vacuolation |
0 |
2 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
Focal medullary mononuclear-cell infiltrate |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Kidneys: |
(15) |
(15) |
(15) |
(15) |
(15) |
(14) |
(15) |
(15) |
(15) |
(15) |
Cortical proximal tubular brown pigment accumulation: |
|
|||||||||
Very slight |
0 |
0 |
0 |
0 |
7** |
0 |
0 |
0 |
0 |
6* |
slight |
0 |
0 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
2 |
Total incidence for score expanded finding |
0 |
0 |
0 |
0 |
10*** |
0 |
0 |
0 |
0 |
8** |
Basophilic tubules: |
|
|||||||||
Very slight |
6 |
9 |
7 |
5 |
2 |
0 |
3 |
0 |
5* |
0 |
Slight |
0 |
0 |
4 |
9*** |
2 |
0 |
0 |
0 |
0 |
0 |
Total incidence for score expanded finding |
6 |
9 |
11 |
14** |
4 |
0 |
3 |
0 |
5* |
0 |
Uni-lateral hydronephrosis |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
Focal mononuclear-cell infiltrate |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
Medullary mineralisation |
1 |
1 |
0 |
1 |
0 |
0 |
3 |
1 |
1 |
0 |
Corticomedullary mineralisation |
0 |
0 |
0 |
0 |
0 |
9 |
7 |
8 |
9 |
0*** |
Cortical mineralisation |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
Liver: |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
Centrilobular hepatocellular hypertrophy: |
|
|||||||||
Slight |
1 |
0 |
0 |
9** |
12*** |
0 |
0 |
1 |
2 |
12*** |
Total incidence for score expanded finding |
1 |
0 |
0 |
9** |
12*** |
0 |
0 |
1 |
2 |
12*** |
Decreased glycogen content |
1 |
0 |
0 |
0 |
12*** |
1 |
0 |
1 |
0 |
0 |
Increased glycogen content |
0 |
3 |
3 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
Aggregates of res cells and necrotic hepatocytes: |
|
|||||||||
Very slight |
7 |
5 |
4 |
0** |
3 |
4 |
4 |
6 |
4 |
5 |
Slight |
3 |
3 |
5 |
0 |
0 |
3 |
2 |
1 |
2 |
0 |
Total incidence for score expanded finding |
10 |
8 |
9 |
0*** |
3* |
7 |
6 |
7 |
6 |
5 |
Periportal mononuclear-cell infiltrate |
1 |
0 |
1 |
0 |
1 |
2 |
1 |
3 |
1 |
0 |
Centrilobular mononuclear-cell infiltrate |
0 |
1 |
0 |
0 |
0 |
2 |
0 |
0 |
1 |
0 |
Focal hepatocellular necrosis |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
Hepatocellular vacuolation |
2 |
0 |
0 |
0 |
1 |
4 |
0 |
0 |
0 |
0 |
Focal subcapsular mineralisation |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Lungs: |
(15) |
(15) |
(15) |
(15) |
(14) |
(15) |
(15) |
(15) |
(15) |
(15) |
Fibrotic activation |
1 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
Pleuritis |
1 |
1 |
2 |
3 |
1 |
3 |
1 |
1 |
2 |
0 |
Focal pleural thickening |
0 |
1 |
2 |
3 |
0 |
3 |
1 |
6 |
1 |
0 |
Focal bronchiolar epithelial hyperplasia |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
Focal increased septal cellularity |
0 |
0 |
0 |
2 |
0 |
2 |
2 |
3 |
0 |
1 |
Mineralisation of arterial wall |
10 |
3* |
6 |
3 |
7 |
9 |
6 |
0*** |
1** |
4 |
Focal bronchiolar cellular debris |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Focal pneumonitis |
0 |
0 |
3 |
0 |
0 |
1 |
0 |
3 |
0 |
0 |
Brown pigment accumulation |
1 |
0 |
1 |
0 |
1 |
2 |
1 |
2 |
0 |
2 |
Nerve - Peripheral: |
(14) |
(15) |
(14) |
(15) |
(15) |
(15) |
(15) |
(15) |
(15) |
(14) |
Blue axonal swelling |
4 |
5 |
9 |
7 |
11* |
5 |
6 |
11 |
6 |
7 |
Spleen: |
(15) |
(15) |
(15) |
(15) |
(14) |
(13) |
(15) |
(10) |
(15) |
(15) |
Brown pigment accumulation |
|
|||||||||
Very slight |
10 |
13 |
11 |
14 |
1** |
0 |
0 |
1 |
1 |
0 |
Slight |
0 |
0 |
4 |
1 |
11*** |
13 |
13 |
8 |
12 |
6*** |
Moderate |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
1 |
2 |
9*** |
Total incidence for score expanded finding |
10 |
13 |
15* |
15* |
12 |
13 |
15 |
10 |
15 |
15 |
Extra medullary haematopoiesis |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
White-pulp depletion |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
Thymus: |
(15) |
(14) |
(14) |
(14) |
(13) |
(14) |
(15) |
(14) |
(14) |
(15) |
Involution: |
|
|||||||||
Very slight |
3 |
3 |
5 |
5 |
5 |
4 |
7 |
1 |
4 |
6 |
Slight |
0 |
0 |
0 |
0 |
4* |
0 |
0 |
0 |
0 |
4 |
Moderate |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
Total incidence for score expanded finding |
3 |
3 |
5 |
5 |
9* |
5 |
7 |
2 |
4 |
10 |
Focal haemorrhage |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
3 |
Starry sky appearance |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
3 |
Thoracic cavitya: |
(1) |
(3) |
(6) |
(4) |
(1) |
(6) |
(2) |
(7) |
(4) |
(2) |
In mediastinum inflammatory cell infiltrate |
1 |
1 |
5 |
3 |
1 |
4 |
1 |
6 |
3 |
2 |
Distended lymph vessels |
1 |
3 |
2 |
1 |
0 |
4 |
1 |
6 |
3 |
0 |
Testes: |
(15) |
(15) |
(15) |
(14) |
(14) |
|
||||
Tubular atrophy: |
|
|||||||||
Very slight |
0 |
1 |
1 |
1 |
1 |
|||||
Slight |
0 |
1 |
0 |
0 |
0 |
|||||
Severe |
0 |
0 |
0 |
0 |
2 |
|||||
Very severe |
0 |
0 |
0 |
0 |
1 |
|||||
Total incidence for score expanded finding |
0 |
2 |
1 |
1 |
4* |
|||||
Epididymides: |
(15) |
|
|
|
(14) |
|||||
Focal interstitial mononuclear-cell infiltrate |
5 |
0* |
||||||||
Prostate: |
(15) |
|
|
|
(14) |
|||||
|
|
|
||||||||
|
|
|
||||||||
Focal interstitial mononuclear-cell infiltrate |
5 |
0* |
Figures in brackets represent the number of animals examined microscopically
a. Microscopic verification of gross observations only, no statistical analysis performed
Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01
Table 26: Summary of selected microscopic changes – Recovery period (males only)
Changes |
Incidence of Lesions (numeric) |
|
Males |
||
0 mg/kg |
5000 mg/kg |
|
Adrenals: |
(10) |
(6) |
Diffuse increased cortical vacuolation |
6 |
1 |
Kidneys: |
(10) |
(6) |
Proximal tubular brown pigment accumulation |
7 |
6 |
Basophilic tubules |
4 |
0 |
Medullary mineralisation |
1 |
0 |
Liver: |
(10) |
(6) |
Aggregates of res cells and necrotic hepatocytes |
4 |
3 |
Periportal mononuclear-cell infiltrate |
0 |
1 |
Centrilobular mononuclear-cell infiltrate |
0 |
1 |
Lungs: |
(10) |
(6) |
Focal pleuritis |
1 |
0 |
Focal pleural thickening |
2 |
0 |
Focal increased septal cellularity |
1 |
2 |
Focal accumulation of alveolar macrophages |
0 |
2 |
Single granuloma |
1 |
0 |
Mineralisation of arterial wall |
7 |
1 |
Increased perivascular lymphoid aggregates |
0 |
2 |
Nerve-Peripheral: |
(10) |
(6) |
No abnormality detected |
10 |
6 |
Spleen: |
(10) |
(6) |
Brown pigment accumulation: |
|
|
Slight |
7 |
3 |
Moderate |
3 |
3 |
Total incidence for score expanded finding |
10 |
6 |
Extra medullary haematopoiesis |
1 |
2 |
Testes: |
(9) |
(6) |
Tubular atrophy: |
|
|
Very slight |
0 |
1 |
Slight |
1 |
3 |
Very severe |
0 |
2 |
Total incidence for score expanded finding |
1 |
6** |
Tubular mineralisation |
1 |
4 |
Thymus: |
(9) |
(6) |
No abnormality detected |
9 |
6 |
Figures in brackets represent the number of animals examined microscopically
Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01
Table 27: Summary of selected microscopic changes – Neuro-histology
Changes |
Incidence of Lesions (numeric) |
|||||
Males |
Females |
|||||
0 mg/kg |
1000 mg/kg |
5000 mg/kg |
0 mg/kg |
1000 mg/kg |
5000 mg/kg |
|
Nerve-Peripheral: |
(5) |
(5) |
(5) |
(5) |
(5) |
(6) |
Axonal distention: |
|
|
|
|
|
|
Very slight |
0 |
0 |
0 |
0 |
0 |
3 |
Slight |
0 |
0 |
5** |
0 |
0 |
0 |
Total incidence for score expanded finding |
0 |
0 |
5** |
0 |
0 |
3 |
Minimal intra-axonal myelin deposits |
5 |
3 |
4 |
3 |
4 |
3 |
Slight intra-axonal myelin deposits |
0 |
2 |
0 |
1 |
1 |
2 |
Moderate intra-axonal myelin deposits |
0 |
0 |
1 |
1 |
0 |
1 |
Figures in brackets represent the number of animals examined microscopically
Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 40 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- 1 supporting short-term and 2 key sub-chronic read across studies from a structural analogue available for assessment.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Male and nulliparous, nonpregnant female Sprague-Dawley rats (VAF/ Plus Crl:CD BR)
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approximately 5 wk old when purchased, acclimated for approximately 2 wk prior to the initiation of the study
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages in air-conditioned rooms
- Diet (e.g. ad libitum): Certified rodent diet 5002 (PMI Feeds, Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water from an automated watering system was available ad libitum
- Acclimation period: 2 weeks. All animals were assigned a temporary identification number at receipt and examined by the staff veterinarian during the acclimation period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12 during the acclimation and all nonexposure periods - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- other: nitrogen
- Details on inhalation exposure:
- Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Exposure levels were determined three times daily by gas chromatography. The highest concentration was approximately 75% of the lower explosive limit. Animals¿ positions in the cages were rotated for each exposure to ensure uniform exposure of every animal.
Chamber Operation: Animals were housed individually in wire mesh, stainless steel cages within a 1000-L glass and stainless steel exposure chamber. Chamber temperature and humidity were monitored every half hour during exposure and maintained, to the extent possible, within the ranges of 20¿24°C temperature and 40¿60% relative humidity. Animals did not receive food or water during the exposure period. Exposure chambers were operated dynamically at a calibrated airflow rate of 200 L/min (lpm). Recordings of airflow and static pressure were made every half hour. All animals remained in the chamber for a minimum of 30 min at the end of exposure while the chamber was operated using clean air only. Chambers were exhausted through a system of coarse filter, HEPA filter, and charcoal filter.
Atmosphere Generation: LAND-2 was pumped directly from the 5-gal container, housed within a freezer constantly flushed with nitrogen, using a laboratory pump, equipped with a piston, that was insulated within a styrofoam container. An ice bag was placed on top of the piston to keep the piston chamber cold to inhibit volatilization of LAND-2 in the pump and delivery lines. LAND-2 was delivered onto the central glass helix of a countercurrent volatilization chamber (one generator per chamber). The glass helix was heated by an internal nichrome wire inserted in the center of the glass tube that supported the helix (external to the volatilization chamber) and was controlled by a variable autotransformer. House-line nitrogen delivered from a regulator with a backpressure gauge was divided with a stainless steel T into the generation flow system and a purge flow system. Purge nitrogen was delivered to the bottom of the tube containing the nichrome wire to continuously purge the area surrounding the wire, protecting it from oxidation. Nitrogen for the generation system was directed through a flowmeter to the ball-and-socket joint at the bottom of the volatilization chamber, flowed up the chamber, passed over the coil, and volatilized the test material. The LAND-2-laden nitrogen flowed through a T tube at the top of the volatilization chamber into the turret of a 1-m3 glass and stainless steel exposure chamber, where it mixed with room air to appropriate exposure concentrations as it was drawn into the chamber (flow rate of 200 lpm). Control animals were sham-exposed to nitrogen alone introduced into the turret and mixed with air in the chamber.
Exposure Chamber Monitoring: Samples for determination of analytical exposure levels and the major components of LAND-2 vapor were withdrawn by vacuum pump from the breathing zone in the exposure chambers three times per exposure for treated groups and once per exposure for controls. Samples were pulled through Teflon lines into the multipositional control module and directed to a Hewlett Packard 5890II gas chromatograph, equipped with a flame ionization detector for analysis by ASTM method D5134-92 (ASTM, 1992). Composition and stability of the test material were evaluated by characterizing neat LAND-2 and comparing the major components in the neat and generated atmospheres at the beginning and end of the study. Particle size distribution measurements of any background aerosol were performed once during each exposure for chambers and room air using a TSI Aerodynamic Particle Sizer. Samples were drawn for 20 s at a rate of 5 L/min. Mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD), and total mass concentration (TMC) were calculated. Nominal concentrations (mg/m3) were calculated from the loss of weight from the generation apparatus divided by the total air flow through the chamber during exposure. This value was converted to parts per million (ppm) using an average molecular weight for this mixture of 89.2. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Determined three times daily by gas chromatography.
Composition and Uniformity Chamber Gas Chromatographic Results (% Weight): n-Butane: 3.21; iso-Pentane: 34.343; 2,3-Dimethylbutane: 12.977, 2-Methylpentane: 4.096; 2,4-Dimethylpentane: 5.663; 2,3-Dimethylpentane: 2.680; 2,2,4-Trimethylpentane: 16.885; 2,3,4-Trimethylpentane: 3.578; 2,3,3 ¿Trimethylpentane: 4.505; 2,2,5-Trimethylhexane: 2.499. - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hours/day, 5 days/week
- Remarks:
- Doses / Concentrations:
0, 668, 2220, and 6646 ppm (0, 2.4, 8.1, and 24.3 g/m3)
Basis:
analytical conc. - No. of animals per sex per dose:
- 12
- Control animals:
- yes, sham-exposed
- Details on study design:
- Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures.
Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically. - Observations and examinations performed and frequency:
- Animals were evaluated twice daily for mortality and gross signs of toxicological or pharmacological effects. During each exposure, rats were observed as a group once for abnormal behavior. Detailed physical examinations were performed twice pretest and weekly during the study. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at wk 14 (terminal) and 18 (recovery).
Body Weights and Food Consumption. All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period.
Animals were transported from the room in which they were housed during non-exposure hours to the neurotoxicity laboratories. Behaviors were evaluated pretest and during wk 5, 9, 14, and 18 (recovery groups). Temperature, humidity, and illumination were measured and recorded to minimize variation in environmental conditions during evaluations. Noise levels were not recorded. Testing was staggered over 8 sessions within a 4-d period, each session consisting of approximately 2 rats/sex/treatment group.
Motor Activity. Locomotor activity was monitored as a function of the number of beam breaks in an activity box, using an automated photo- beam activity system. Sessions were 60 mm in length divided into twelve 5-mm intervals. Rats were evaluated at pretest, at 3 time points during the treatment period (wk 5, 9, and 1 4), and at the end of the recovery period. Treatment groups were counter balanced across test times. Three sets of analyses were performed. The first analysis was conducted using the pre-dose data with animals nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals. The second analysis used data from the 3 treatment time points, and animals were nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals nested within the 3 time periods. The third analysis was similar to the first, using the recovery data. All three analyses tested for treatment effects, sex differences, treatment group by sex interactions, and these effects crossed with periods (second analysis only) and intervals. Analyses were repeated using transformed rank data to achieve a normal distribution of the residuals. Residuals from the models were tested for normality by the Shapiro-Wilk W-test or the Kolomogorov D-test.
Functional Operational Battery. The battery was comprised of home- cage evaluations (posture, vocalizations, and palpebral closure), handling evaluations (reactivity to general stimuli, signs of autonomic function), open-field behavior (arousal level and gait, urination and defecation frequency, convulsions, tremor, abnormal behaviors, piloerection, and exophthalmos), and reflex assessments (response to visual and auditory stimuli, tail pinch, pupillary function). Animals were also evaluated for forelimb and hindlimb grip strength, landing foot splay, and air righting ability. For landing foot splay, a small dot of paint was applied to each hindpaw. The rat was dropped from a height of 2 ft above a flat surface and the distance between the marks left by the hindpaws was measured in centimeters. To evaluate air righting reflex ability, the rat was held upside down, dropped from a height of 2 ft above a container of bedding, and the landing position was observed. Treatment groups were counterbalanced across test times. The observer performing the evaluation did not know the identity of the animal¿s dose group. - Sacrifice and pathology:
- All animals were sacrificed by intraperitoneal injection of sodium pentobarbital, and tissues were preserved in situ by transcardial perfusion with phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde/1 % glutaraldehyde in the same buffer. Animals were killed at termination of 13 wk of exposure (week 13 terminal sacrifices performed during week 14) or at the completion of the 4-wk recovery period (control and high-dose groups only). A complete macroscopic examination was performed on all animals and 12 organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus, and uterus. The length and width of the brain of each rat was measured.
Thirty-nine tissues were preserved from all animals in all dose groups. Tissues from all animals in the control and high-dose groups were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological examination. The kidneys of selected animals were also stained with Mallory-Heidenhain stain. In addition, tissues of the nervous system were fixed for all animals. Brain, spinal cord, ganglia, and spinal nerve roots were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin-eosin, Luxol fast blue, and Sevier-Munger stains. Peripheral nerve sections (sciatic, tibial, sural, and optic) were embedded in glycol methacrylate and stained with toluidine blue. Slides of nervous system tissues were examined from animals (6/sex/group) designated through random selection for neuropathology, in the control and high-dose groups sacrificed at the end of 13 weeks of exposure. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons, and medulla. - Statistics:
- Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett¿s test at 1 % significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1 % significance, two- sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett¿s test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn¿s summed rank test was used to determine differences from controls.
A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal orJonckheere¿s test for monotonic trend in nonparametric cases. - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- All animals survived the treatment period and were sacrificed according to study design at the end of 13 wk or at 18 wk (recovery groups). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. No LAND-2-related ocular disease was observed. All groups showed similar mean body weights, body weight gains, and food consumption.
At wk 13 terminal sacrifice, there was a statistically significant decrease, relative to control values, in hemoglobin (5%), hematocrit (5%), and erythrocytes (7%) in blood of high dose males (data not shown). The hemoglobin was still decreased (4%) after the 28-d recovery period. However, because these differences were small and within the historical range for control animals in this laboratory, they are not considered toxicologically relevant. Clinical chemistry results showed a statistically significant decrease in aspartate aminotransferase (AST 32%) and alanine aminotransferase (ALT 46%) in females of the high-dose group (data not shown) compared to controls. However, several control female rats had elevated AST and ALT relative to the other nine female rats in the group and historical controls. Comparison of ALT and AST values from high-dose females with these elevated concurrent control values produced a statistical significance that is not toxicologically relevant. These results are not considered LAND-2 related.
NEUROBEHAVIORAL STUDIES
Motor Activity. Shapiro-Wilk analysis of data from the predose period indicated that the only statistically significant effects on response pattern occurred in the low-dose group due to inactivity among females for intervals 6¿8, and increased activity of males during interval 10 (data not shown). Data from the treatment intervals and the recovery period were analyzed based on the Blom transformed data because the residuals from the model were not normally distributed by the Shapiro¿Wilk statistic at the .01 level. There were statistically significant differences in the number (ct < .04) and relative pattern (cx. < .02) of beam breaks among the dose groups over the treatment testing periods. There were expected differences between sexes, and pattern differences across the 1 2 measuring intervals. In the recovery period in which only the room air controls and the high-dose animals were evaluated, there were no dose-group-related differences in response. Overall, dose-group differences did not occur in a dose-related pattern. Although statistically significant, the magnitudes of the differences were not large, and none of the treatment-group differences were larger than differences seen during the pre-dose period.
Functional Operational Battery. No differences were detected in the distance between foot splay for male or female rats in any dose group over any time interval evaluated. Grip strength of both fore- and hindlimbs in general increased from pretest through wk 14 for both sexes in all treatment groups. Values for control and high-dose recovery animals were lower at wk 18 than in previous treatment weeks but similar between the groups. There was no test-material-related effect on any endpoint evaluated within the functional operational battery of tests.
Pathology. At the wk 13 terminal sacrifice there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex, based on positive staining reaction by the Mallory-Heidenhain method, and increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of `light hydrocarbon nephropathy¿ also known as hyaline droplet nephropathy and are male rat specific and are not considered relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at wk 13 sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Lung and brain weights were comparable to controls. Lungs were macroscopically and microscopically comparable to controls. Brain length and width measurements showed no test-material-related effects. There were no microscopic findings in the brain, spinal cord, or peripheral nerves that could be attributable to exposure to LAND-2. - Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 24 300 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no effects except adaptive response of liver weight to exposure equivalent to 6646 ppm
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
- Executive summary:
The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: This study is classified as reliable with restrictions because no GLP statement was included, but the study was otherwise well-documented and closely followed OECD guideline 413.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Frederick Cancer Research Facility
- Age at study initiation: 7 weeks
- Weight at study initiation: mean weights: 23.3-23.8 g male, 18.3-20.0 female
- Housing: individually
- Diet (e.g. ad libitum): NIH 07 Rat and Mouse Ration, ad libitum overnight during exposure
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-24.8
- Humidity (%): 40-84
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- not specified
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 0.5 m3 Hinners-type chambers
- Source and rate of air: Air was passed through a 2 l reserve of n-hexane at 50 degree C with a fritted bubbler. Separate generators were used for each chamber.
- Temperature, humidity, pressure in air chamber: 21.0-24.8 degree C, and 40-84% humidity
TEST ATMOSPHERE
- Brief description of analytical method used: The 10,000 ppm chamber was monitored with a Miran 1A IR. The other chambers were monitored with a Miran 980 IR. Chambers were sampled once per hour. Biweekly samples were also analyzed using GC with flame ionization detection. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The 10,000 ppm chamber was monitored with a Miran 1A IR. The other chambers were monitored with a Miran 980 IR. Chambers were sampled once per hour. Biweekly samples were also analyzed using GC with flame ionization detection.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hrs/day, 5 days per week except for one 1000 ppm group that was exposed 22 hrs/day for 5 days/week
- Remarks:
- Doses / Concentrations:
0, 500, 1000, 4000, 10,000 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
1000 ppm
Basis:
nominal conc. - No. of animals per sex per dose:
- 18 mice per sex
- Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: not provided
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
HAEMATOLOGY: Yes
- Parameters checked: leukocytes, lymphocytes, monocytes, eosinophils, segmented neutrophils, hematocrit, hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, erythrocytes, reticulocytes, platelets
NEUROBEHAVIOURAL EXAMINATION: Yes
- Dose groups that were examined: 0, 1000 (22 hr exposure), 10,000 ppm
- Battery of functions tested: startle response, foot splay, analgesia response, grip strength, locomotor activity, exploratory behaviour - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, brain, heart, right kidney, liver, lung, spleen, right testis, thymus
HISTOPATHOLOGY: Yes, adrenal glands, brain, bronchial lymph nodes, cecum, colon, duodenum, esophagus, gallbladder, gross lesions, tissue masses, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mainstream bronchi, mammary glands, mandibular lymph nodes, mesenteric lymph nodes, mediastinal lymph nodes, nasal cavity, turbinates, pancreas, parathyroid glands, pituitary gland, rectum, salivary glands, sciatic nerves, spinal cord, spleen, sternum including marrow, stomach, testes, ovaries, uterus, thymus, thyroid gland, trachea, urinary bladder - Statistics:
- Multiple comparison methods of Dunnett.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
All animals survived to the end of the study. Clinical signs were limited to sneezing in the 10,000 ppm group.
BODY WEIGHT AND WEIGHT GAIN
Mean body weights of males in the 1000 ppm, 22 hr, exposure group and 10,000 ppm group were significantly reduced. The mean body weight of females in the 10,000 ppm group were also signficantly reduced.
HAEMATOLOGY
Segmented neutrophils were significantly increased in male mice exposed to 10,000 ppm.
NEUROBEHAVIOUR
Female mice in the 1000 ppm, 22 hr, exposure group and 10,000 ppm showed decreased locomotor activity.
ORGAN WEIGHTS
Liver, kidney, and heart weights were increased in exposed female mice.
HISTOPATHOLOGY: NON-NEOPLASTIC
Paranodal swellings in the tibial nerve were observed in 10,000 ppm exposed males and females, and the 1000 ppm, 22 hr exposure females. Inflammation and regeneration of the respiratory epithelium and olfactory epithelium, and metaplasia of olfactory epithelium to olfactory epithelium was in mice exposed to 10,000 ppm. Similar lesions, but of less severity were also seen in females in the 4,000 ppm group and 1000, 22 hr exposure, group females. Females in the 1000 and 500 ppm group showed minimal olfactory epithelium changes. Males in the 1000 ppm, 22 hr exposure group, and 1000 ppm group, had minimal lesions. Males in the 4000 ppm group and 500 ppm group did not show nasal lesions.
- Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 500 ppm
- Sex:
- female
- Basis for effect level:
- other: nasal lesions
- Dose descriptor:
- LOAEC
- Effect level:
- 1 000 ppm
- Sex:
- male
- Basis for effect level:
- other: nasal lesions
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 500 ppm
- Sex:
- male
- Basis for effect level:
- other: nasal lesions
- Critical effects observed:
- not specified
- Conclusions:
- The LOAEC for female mice was 500 ppm based on nasal lesions. No NOAEC was found for female mice. The LOAEC for male mice was 1000 ppm based on nasal lesions, and the NOAEC was 500 ppm.
- Executive summary:
This study examined the effect of 13 weeks inhalation exposure of n-hexane to mice. Groups of 18 female and 18 male mice were exposed to concentrations of 0, 500, 1000, 4000, or 10,000 ppm of test substance for 6 hrs/day, 5 days/week, for 13 weeks. An additional group was exposed to 1000 ppm of test substance for 22 hrs/day, 5 days/week, for 13 weeks. Clinical examinations were done twice daily, and body weights taken weekly. After sacrifice, animals were examined for histopathological parameters, and organ weights. 8 mice of each sex in each exposure group were examined for neurobehaviour. No animals died during the study, and the only neurological effects were decreased locomotion in females exposed to 10000 ppm. Nasal lesions were seen in females in all exposure groups, and in males exposed to 1000 ppm of test substance. The LOAEC for females was therefore 500 ppm (1760 mg/m3), with no NOAEC found. The NOAEC for males was 500 ppm (1760 mg/m3), with an LOAEC of 1000 ppm (3520 mg/m3) based on nasal lesions.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- April 10, 1989-July 12, 1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- yes
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: approx. 181 g male, approx. 123 g female
- Housing: individually in stainless steel wire mesh cages, identified by tail tattoo
- Diet (e.g. ad libitum): Purina Rodent Chow Brand Animal Diet#5002, ad libitum
- Water (e.g. ad libitum): ad libitum, Elizabethtown Water Company
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-75 degree F
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
IN-LIFE DATES: From: April 10, 1989 To: July 12, 1989 - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Remarks on MMAD:
- MMAD / GSD: Particle size measurements showed that there was no measurable amount of test substance present as aerosol.
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 l glass and stainless steel exposure chamber
- Method of holding animals in test chamber: individual cages
- Source and rate of air: chamber supplied air, 200-216 lpm
- Method of conditioning air: Test substance in a 5-gallon drum passed through a fluid metering pump into teflon tubing to a coiled glass rod in the volatilization chamber. Nitrogen was also fed into the volitization chamber. A heating element was positioned in the center of the glass coil to aid volatilization. The nitrogen and test substance mixture, then entered the exposure chamber.
- Air flow rate: 200-216 lpm
- Air change rate: 4.6-5.0 min.
TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes, once per week - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Air samples were drawn from the chamber via teflon tubing into charcoal tubes.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hrs/day, 5 days/week
- Remarks:
- Doses / Concentrations:
0, 900, 3,000, 9000 ppm
Basis:
other: target concentrations - Remarks:
- Doses / Concentrations:
904, 2984, 8992 ppm
Basis:
analytical conc. - No. of animals per sex per dose:
- 10 animals of each sex per dose
- Control animals:
- yes, sham-exposed
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, toxicological and pharmacological effects
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to test and week during the test
BODY WEIGHT: Yes
- Time schedule for examinations: twice prior to test, weekly during test and at termination
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly beginning one week prior to test
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to test and prior to sacrifice
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to test on 10 animals per sex, and at sacrifice
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes, ether
- Parameters checked: erythrocyte count, hemoglobin count, hematocrit, total and differential leucocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to test on 10 animals per sex, and at sacrifice
- Animals fasted: Yes
- Parameters checked: glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, creatinine, blood urea nitrogen, fasting glucose, total protein, alkaline phosphatase, albumin, potassium, sodium, calcium, chloride, inorganic phophorus, gamma glutamyl transpeptidase, total bilirubin, creatine phosphokinase, lactic acid dehydrogenase - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
all orifices, cranial cavity, brain, spinal cord, nasal cavity, thoracic, abdominal, and pelvic cavities
ORGAN WEIGHTS: Yes
adrenals, ovaries, testes with epididymides, kidneys, liver, brain, lungs, heart, spleen
HISTOPATHOLOGY: Yes
abdominal aorta, adrenals, bone, bone marrow, brain, esophagus, eyes, optic nerve, larynx, ovaries, testes with epididymus, heart, kidneys, liver, intestine, gall bladder, lungs, lymph nodes, nerve, skeletal muscle, trachea, nasopharyngeal tissues, pancreas, pituitary, prostate, salivary gland, thymus, spinal cord, seminal vesicles, spleen, skin, stomach, thyroid, urinary bladder, uterus, exorbital lacrimal glands, Zymbal gland - Statistics:
- Statistical analysis was done on body weight, body weight gain, change in body weight, food consumption, change in food consumption, hematology, clinical chemistry, organ weights, organ/body and organ/brain weight ratios.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Clinical biochemistry findings:
- effects observed, treatment-related
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No rats died during the study. Transient excess lacrimation was observed in the female rats. No other effects attributable to exposure were noted.
BODY WEIGHT AND WEIGHT GAIN
Females in the high exposure group had sporadic reduced weight gain. As there was no consistant overall effect, this was not considered treatment related.
FOOD CONSUMPTION
There was no effect on food consumption.
OPHTHALMOSCOPIC EXAMINATION
Two males in the high exposure group showed corneal dystrophy. As this is not uncommon in males of this strain of rat, it is not considered treatment related.
HAEMATOLOGY
There were increased platelets in high exposure males. High and medium exposure males also had increased mean corpuscular volume. The significance of these changes is uncertain.
CLINICAL CHEMISTRY
High exposure males had increased creatinine, total protein, and albumin. They had decreased serum chloride. The significance of these changes is also uncertain.
ORGAN WEIGHTS
High exposure males had increased organ/body and organ/brain weight ratios. High exposure males and females had increased relative spleen weights. Liver weights were also increased in high exposure males. The liver effects appeared to be treatment related.
GROSS PATHOLOGY
No treatment related effects were noted.
HISTOPATHOLOGY: NON-NEOPLASTIC
There was hemorrhage and inflammation in male rat livers at the high dose level. There was also inflammation in the kidneys of males in the high and middle exposure groups. The significance of these effects is uncertain.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 5 469 mg/m³ air (analytical)
- Sex:
- male
- Basis for effect level:
- other: The value was re-calculated from the analytical dose of 2984 ppm.
- Dose descriptor:
- LOAEC
- Effect level:
- 16 479 mg/m³ air (analytical)
- Sex:
- male
- Basis for effect level:
- other: liver and kidney effects The value was re-calculated from the analytical dose of 8992 ppm.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 16 479 mg/m³ air (analytical)
- Sex:
- female
- Basis for effect level:
- other: The value was re-calculated from the analytical dose of 8992 ppm.
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEC for male rats exposed via inhalation was 5469 mg/m3 based on liver and kidney effects. The NOAEC for female animals was 16479 mg/m3.
- Executive summary:
The NOAEC for male rats exposed via inhalation was 5469 mg/m3 based on liver and kidney effects. The NOAEC for female animals was 16479 mg/m3.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- April 11, 1989-July 13, 1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- other: B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: 8 weeks
- Weight at study initiation: approx. 25 g male, approx. 19 g female
- Housing: individually in stainless steel wire mesh cages, identified by tail tattoo or cage card
- Diet (e.g. ad libitum): Purina Rodent Chow Brand Animal Diet#5002, ad libitum
- Water (e.g. ad libitum): ad libitum, Elizabethtown Water Company
- Acclimation period: 22 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-75 degree F
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
IN-LIFE DATES: From: April 11, 1989 To: July 13, 1989 - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Remarks on MMAD:
- MMAD / GSD: Particle size measurements showed that there was no measurable amount of test substance present as aerosol.
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 l glass and stainless steel exposure chamber
- Method of holding animals in test chamber: individual cages
- Source and rate of air: chamber supplied air, 200-216 lpm
- Method of conditioning air: Test substance in a 5-gallon drum passed through a fluid metering pump into teflon tubing to a coiled glass rod in the volatilization chamber. Nitrogen was also fed into the volitization chamber. A heating element was positioned in the center of the glass coil to aid volatilization. The nitrogen and test substance mixture, then entered the exposure chamber.
- Air flow rate: 200-216 lpm
- Air change rate: 4.6-5.0 min.
TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes, once per week - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Air samples were drawn from the chamber via teflon tubing into charcoal tubes.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hrs/day, 5 days/week
- Remarks:
- Doses / Concentrations:
0, 900, 3,000, 9000 ppm
Basis:
other: target concentrations - Remarks:
- Doses / Concentrations:
904, 2984, 8992 ppm
Basis:
analytical conc. - No. of animals per sex per dose:
- 10 animals of each sex per dose
- Control animals:
- yes, sham-exposed
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, toxicological and pharmacological effects
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to test and week during the test
BODY WEIGHT: Yes
- Time schedule for examinations: twice prior to test, weekly during test and at termination
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly beginning one week prior to test
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to test and prior to sacrifice
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to test on 10 animals per sex, and at sacrifice
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes, ether
- Parameters checked: erythrocyte count, hemoglobin count, hematocrit, total and differential leucocyte count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to test on 10 animals per sex, and at sacrifice
- Animals fasted: Yes
- Parameters checked: glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, creatinine, blood urea nitrogen, fasting glucose, total protein, alkaline phosphatase, albumin, potassium, sodium, calcium, chloride, inorganic phophorus, gamma glutamyl transpeptidase, total bilirubin, creatine phosphokinase, lactice acid dehydrogenase - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
all orifices, cranial cavity, brain, spinal cord, nasal cavity, thoracic, abdominal, and pelvic cavities
ORGAN WEIGHTS: Yes
adrenals, ovaries, testes with epididymides, kidneys, liver, brain, lungs, heart, spleen
HISTOPATHOLOGY: Yes
abdominal aorta, adrenals, bone, bone marrow, brain, esophagus, eyes, optic nerve, larynx, ovaries, testes with epididymus, heart, kidneys, liver, intestine, gall bladder, lungs, lymph nodes, nerve, skeletal muscle, trachea, nasopharyngeal tissues, pancreas, pituitary, prostate, salivary gland, thymus, spinal cord, seminal vesicles, spleen, skin, stomach, thyroid, urinary bladder, uterus, exorbital lacrimal glands, Zymbal gland - Statistics:
- Statistical analysis was done on body weight, body weight gain, change in body weight, food consumption, change in food consumption, hematology, clinical chemistry, organ weights, organ/body and organ/brain weight ratios.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Seven mice died during the study, 3 from drawing blood, and 4 others died accidently. None of these deaths were attributed to treatment. There was sporadic excessive lacrimation due to exposure, no other clinical signs were considered treatment related.
BODY WEIGHT AND WEIGHT GAIN
There was no effect on body weight observed.
FOOD CONSUMPTION
There was no effect on food consumption.
OPHTHALMOSCOPIC EXAMINATION
No effects were observed.
HAEMATOLOGY
High exposure males had increased mean corpuscular volume. The significance of these changes are uncertain.
CLINICAL CHEMISTRY
No treatment related effects were observed.
ORGAN WEIGHTS
No effects to organ weights were noted.
GROSS PATHOLOGY
No treatment related effects were noted.
HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related effects were noted.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 16 479 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: The value was re-calculated based on the analytical dose of 8992 ppm.
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEC for male and female mice exposed via inhalation is 16479 mg/m3.
- Executive summary:
The NOAEC for male and female mice exposed via inhalation is 16479 mg/m3.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: This study is classified as reliable with restrictions because there was no GLP statement provided, and limited data on methods were reported, but the study seemed to be well-conducted.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- 7 rats were exposed to 3000 ppm of hexane vapors for 12 hrs a day for 16 weeks. Body weights and conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks. At the end of the exposure period, two animals were sacrificed and the nerve tissue examined.
- GLP compliance:
- not specified
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 308 +/- 18 g
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.5-24.5 - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- not specified
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Temperature, humidity, pressure in air chamber: 23.5-24.5 degree C, 41-61% humidity
TEST ATMOSPHERE
- Brief description of analytical method used: gas detector measurements were taken daily, liquid chromatography measurements were taken twice weekly - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- gas detector measurements were taken daily, liquid chromatography measurements were taken twice weekly
- Duration of treatment / exposure:
- 16 weeks
- Frequency of treatment:
- daily, 12 hrs per day
- Remarks:
- Doses / Concentrations:
3000 ppm
Basis:
no data - No. of animals per sex per dose:
- 7
- Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: not provided
- Rationale for animal assignment (if not random): not provided
- Rationale for selecting satellite groups: not provided
- Post-exposure recovery period in satellite groups: not provided
- Section schedule rationale (if not random): not provided - Observations and examinations performed and frequency:
- BODY WEIGHT: Yes
- Time schedule for examinations: 0, 4, 8, 12, and 16 weeks after start of exposure
OTHER: The conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks - Sacrifice and pathology:
HISTOPATHOLOGY: Yes, 2 rats were sacrificed at the end of 16 weeks, and their gastrocnemius and soleus muscles, dorsal trunk of the tail nerve, and the tibial nerve examined.- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Two animals died during the study. One animal died 1 day before the end of the exposure period, and one animal died three days before the end of the exposure period.
BODY WEIGHT AND WEIGHT GAIN
Body weight gain was significantly reduced at 4 weeks after start of exposure, and remained depressed for the rest of the experiment.
NEUROBEHAVIOUR
Unsteady gait was observed in one animal at 10 weeks exposure, and in 4 animals at 12 weeks. 2 animals at this time point also showed foot drop. At 16 weeks exposure, the five surviving rats had unsteady gait, and two had foot drop. All animals had muscular atrophy at this time point.
HISTOPATHOLOGY: NON-NEOPLASTIC
There were paranodal swellings in the myelinated fibers of the tibial nerve and dorsal trunk of the tail nerve. There were an excessive number of neurofilaments, vesicles, multivesicular bodies, mitochondria, myelin figures, and dense bodies in the paranodal axoplasm and no neurotubules. Denervated neuromuscular junctions in the muscles were observed. Muscle fibers were of irregular shape and size, and had an increased number of nuclei, and had disordered myofilaments, zig-zagging of the z-band, and invaginations of the plasma membrane.
OTHER FINDINGS
The motor nerve conduction velocity (MCV) was significantly less than controls by 4 weeks of exposure. MCVs could not be measured in 2 animals after 16 weeks due to nerve damage. Distal latencies (DL) were signficantly prolonged after 4 weeks of exposure, and could not be measured in 2 aminals at 16 weeks of exposure. - Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 3 000 ppm
- Sex:
- male
- Basis for effect level:
- other: neurotoxicology
- Critical effects observed:
- not specified
- Conclusions:
- The LOAEC for sub-chronic exposure to hexane vapors was 3000 ppm based on reduced body weight gain, mortality and neurological effects.
- Executive summary:
In this study, 7 rats were exposed to 3000 ppm of hexane vapors for 12 hrs a day for 16 weeks. Body weights and conduction velocity of the peripheral nerve of the tail was measured at 0, 4, 8, 12 and 16 weeks. At the end of the exposure period, two animals were sacrificed and the nerve tissue examined. Two animals died before the end of the exposure period. All animals showed reduced weight gain after 4 weeks of exposure. Neurological effects were seen beginning at 10 weeks exposure. Motor nerve conduction velocity and distal latency were significantly affected after 4 weeks exposure. Examination of neural tissue showed damage to the tibial nerve and dorsal trunk of the tail nerve. The LOAEC for sub-chronic exposure was 3000 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 17 April 1978 - 30 March 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions. Limited documentation on animal housing, only 2 concentrations tested, exposure duration 84 days, no ophthalmological examination.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass. 01887
- Age at study initiation: males 6 wks, females 7 wks
- Weight at study initiation: males 185 g mean (range 165-217 g); females 162 g mean (range 138-189)
- Fasting period before study: no
- Housing: paired in chamber, individual out of chamber
- Diet (e.g. ad libitum): Standard laboratory pellet diet (Purina Laboratory Chow) ad libitum (out of chamber only)
- Water (e.g. ad libitum): ad libitum (out of chamber only)
- Acclimation period: 13 days - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- other: unchanged (no vehicle)
- Remarks on MMAD:
- MMAD / GSD: not applicable, vapour
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass chambers with 1 cubic meter total volume (760 L effective volume)
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 134 L/min
- Air change rate: 8 per hour
- Method of particle size determination: not applicable, vapour
TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric sampling was performed using a Wilks Scientific Corp., Miran 1A Ambient Air Analyzer (long pathlength infrared). A calibration curve relating the absorption to the airborne concentration of the test material was prepared. On each exposure day, three samples were drawn from each exposure chamber (at about 1, 3, and 5 hours) and the exposure concentration calculated by comparing the absorption of this sample to the standard curve.
In addition, the composition of the test atmosphere was analyzed for homogeneity by gas chromatographic analysis of several charcoal-trapped vapour samples collected from each chamber during the 12-wk exposure period - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test atmosphere was analysed for concentration and homogeneity by measurement of the infrared spectrum and by gas chromatographic analysis, respectively. Based on the infrared analysis the animals were exposed to cumulative mean concentrations of 385 and 1200 ppm, respectively. Gas chromatographic analysis of the chamber atmosphere demonstrated that the test material composition was representative of the initial sample.
- Duration of treatment / exposure:
- 12 weeks
- Frequency of treatment:
- 6 hours/day, 5 days/week
- Remarks:
- Doses / Concentrations:
400, 1200 ppm
Basis:
nominal conc. - No. of animals per sex per dose:
- 35
- Control animals:
- yes, sham-exposed
- Details on study design:
- - Rationale for animal assignment (if not random): assigned to group by weight
- Positive control:
- none
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: incidence of abnormal signs
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (full recorded physical assessment)
BODY WEIGHT: Yes
- Time schedule for examinations: weekly, from 5 days prior to exposure through termination
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes (retro-orbital sinus)
- Time schedule for collection of blood: 4, 8, 12 weeks
- Anaesthetic used for blood collection: Yes (exsanguination under ether anesthesia)
- Animals fasted: Yes (fasted overnight prior to bleeding)
- How many animals: 10/sex/group (4 and 8 weeks), 15/sex/group (12 weeks, all survivors)
- Parameters examined: hemoblobin, hematocrit, erythrocyte count, clotting time, total and differential leukocytes
CLINICAL CHEMISTRY: Yes (retro-orbital sinus)
- Time schedule for collection of blood: 4, 8, 12 weeks
- Animals fasted: Yes (exsanguination under ether anesthesia)
- How many animals: 10/sex/group (4 and 8 weeks), 15/sex/group (12 weeks, all survivors)
- Parameters examined: blood urea nitrogen, serum glutamic pyruvic transaminase (SGPT), glucose, alkaline phosphatase
OTHER:
Organ weights and organ/body weight ratios determined in animals sacrificed at 4, 8 and 12 weeks (adrenals, brain (sans pituitary), gonads, kidneys, liver, lungs) - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes: adrenals, brain (without pituitary), gonads, kidneys, liver, lungs
HISTOPATHOLOGY: Yes (control and 1200 ppm group): adrenals (2), bone marrow (sternal), brain (2 sections), eye, gonad, heart (with coronary vessels) intestine, colon, duodenum, ileum, kidneys (2), liver (2 sections), lung (2 sections), lymph node (mesenteric), mammary gland, pancreas, pituitary, salivary gland, skeletal muscle, skin, spinal cord (cervical), spleen, stomach, thyroid, trachea, urinary bladder, uterus/prostate, gross lesions, tissue masses - Statistics:
- Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were statistically evaluated. Mean values for all treatment groups were compared to the control group at each time interval (4, 8, and 12 weeks). Hematology and clinical chemistry parameters were compared by the F-test and Student's t-test. When variances differed significantly (F-test), Student's t-test was appropriately modified using Cochran's approximation (t'). Body weight, organ weight and organ/body weight ratios were compared to control according to Dunnett.
- Clinical signs:
- effects observed, treatment-related
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY
No treatment-related mortality occured (1 male of the 1200 ppm group was accidentally killed).
Several animals in all groups exhibited dry rales and red and mucoid nasal discharge (more numerous in the treated groups, but not clearly treatment-related), moist rales, excessive lacrimation, hair loss and chromodacryorrhea were found in a limited number of animals in all groups (not treatment-related)
1200 ppm: singular occurrences of excessive salivation, laboured, irregular breathing; yellow staining of the anogenital fur in 6 males and 35 females from wk 3 through 12
400 ppm: yellow staining of the anogenital fur in 2 females
Control: singular occurrences of excessive salivation and bleeding inside the ear; a limited number of animals with brown staining of the ano-genital region and soft stool; three observations (in one animal) of an abnormally dark red or red and yellow eye
BODY WEIGHT AND WEIGHT GAIN
1200 ppm: mean body weights in males significantly higher at wk 2 and significantly lower (p?0.05) from wk 8 through 11 than in controls
400 ppm: mean body weight and weight gains in males similar to control throughout the study, except wk 2 (significantly higher, p?0.01), in females mean body weights significantly depressed (p?0.01 and p?0.05) at wk 5 through 8.
HAEMATOLOGY
Several statistically significant (p < 0.05 and p < 0.01) decreases in mean hematocrit values of males and females of both treated groups at wk 4 and 8, statistically significant decreases (p?0.05) in mean hemoglobin values at wk 8 in the males of both treated groups and the females of the 400 ppm group at wk 4. Mean red blood cell values were significantly decreased in 1200 ppm males at wk 8 and 400 ppm females at wk 12. Since all values were within normal biological limits, these findings were not considered to be treatment-related.
CLINICAL CHEMISTRY
Mean SGPT levels were significantly (p?0.01) depressed in 1200 ppm males at wk 4, 400 and 1200 ppm males at wk 8, and in 1200 ppm females at wk 12. Mean blood urea nitrogen levels were significantly increased in the males of both treated groups at wk 8. Mean glucose levels were significantly (p?0.01 or p?0.05) increased in 400 ppm males at wk 8, decreased in 1200 ppm males at wk 12, and decreased in 1200 ppm females at wk 4 and 12. The observed effects were not considered to be treatment-related.
ORGAN WEIGHTS
Mean kidney weights and kidney/body weight ratios were significantly (p?0.05) higher in the 1200 ppm males at wk 8. In the 400 ppm males these values were also elevated, but not statistically significant. At wk 12, mean kidney weights and kidney/body weight ratios for 400 and 1200 ppm males were significantly (p?0.01) elevated, indicating a treatment-related response. The only other statistically significant (p?0.05) findings were elevated mean adrenal/body weight ratios for the 1200 ppm males at wk 4 and the 400 ppm females at wk 12.
GROSS PATHOLOGY
Microscopic evaluation of organs and tissues from the control and high level exposure groups revealed a mild tubular injury in the kidneys of some exposed male rats sacrificed after exposure for 8 and 12 wk. Other changes were unrelated to group or sex and were considered to be spontaneous.
HISTOPATHOLOGY: NON-NEOPLASTIC
See Gross Pathology - Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 1 200 ppm (nominal)
- Sex:
- male
- Basis for effect level:
- other: overall effects
- Critical effects observed:
- not specified
- Conclusions:
- In a 12 -week inhalation study with rats the test substance hydrocarbons, C7 -C9, isoalkanes was tested. Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study.
The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males.
The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of ?2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC.
Renal effects were strictly limited to males, therefore the authors concluded an ?2u-globulin-related mechanism for the observed nephropathy. The observation was not considered for determination of the NOAEC. - Executive summary:
In a 12 -week inhalation study with rats the test substance hydrocarbons, C7 -C9, isoalkanes was tested. Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study. The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males. The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of ?2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC. Renal effects were strictly limited to males, therefore the authors concluded an ?2u-globulin-related mechanism for the observed nephropathy. The observation was not considered for determination of the NOAEC.
Referenceopen allclose all
Significant results of mouse repeated dose inhalation study
Concentration |
Control |
500 ppm |
1000 ppm |
4000 ppm |
10,000 ppm |
1000 ppm (22 hr) |
Male body weight (g) |
33.4 ± 0.55 |
32.3 ± 0.97 |
31.2 ± 0.57 |
31.2 ± 0.75 |
27.8 ± 0.50 |
30.0 ± 0.60 |
Female body weight (g) |
25.3 ± 0.49 |
25.1 ± 0.62 |
26.4 ± 0.66 |
25.6 ± 0.75 |
23.8 ± 0.57 |
25.4 ± 0.88 |
Male segmented neutrophils (103/microliter) |
0.31 ± 0.054 |
0.40 ± 0.068 |
0.46 ± 0.108 |
0.25 ± 0.036 |
1.16 ± 0.363 |
0.55 ± 0.079 |
Locomotor activity in female mice - week 13 |
170 ± 9.1 |
168 ± 8.7 |
166 ± 9.2 |
152 ± 6.6 |
141 ± 10.6 |
126 ± 10.5 |
Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study.
The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males.
The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of alpha-2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 1 760 mg/m³
- Study duration:
- subchronic
- Experimental exposure time per week (hours/week):
- 30
- Species:
- mouse
- Quality of whole database:
- 6 Key and 1 supporting read acros sub-chronic along with 2 supporting chronic read across studies from structural analogues available for assessment.
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
- Critical effects observed:
- not specified
- Endpoint:
- sub-chronic toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Critical effects observed:
- not specified
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
There are no repeated dose toxicity data available on Hydrocarbons, C6-C10 (even numbered), n-alkanes, isoalkanes, >5% n-hexane. However, there is reliable data available for structural analogues, hexane, Naphtha (petroleum), light alkylate, Hydrcarbons, C7-C9, isoalkanes and Light catalytic reformed naphtha distillate and presented in the dossier. This data is read across to Hydrocarbons, C6 -C10 (even numbered), n-alkanes, isoalkanes, >5% n-hexane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.
Oral
n-hexane/ Technical hexane
In a sub-chronic repeated dose toxicity study (Til et al., 1989) performed to OECD Guideline 408, the test material (Technical hexane (hexane food grade) containing 58% n-hexane) was administered once daily to Wistar derived (Bor: WISW (SPF Cpb)) rats (20/sex/dose) via oral gavage at doses of 0, 40, 200, 1000 or 5000 mg/kg/bw/day in soya bean oil for 13 weeks. Two additional groups (10/sex/dose) receiving either 0 or 5000 mg technical hexane/kg/bw/day via oral gavage for 13 weeks, were held for a 9-week recovery period. However, due to high mortality in the high-dose females, only males were used in the recovery study.
All animals were observed for general condition, behaviour and neurological signs. Individual body weights of all animals were recorded initially (Day 0), weekly and at termination. Food consumption was measured per cage (5 animals) weekly and food utilisation efficiency was calculated and expressed as gram weight gain per gram food consumed. Water consumption was measured daily in week 5 per cage (5 animals) for the control and high-dose groups, then extended to all groups in week 6, 11 and 12 after water intake appeared to be higher in the high-dose group in week 5. Ophthalmoscopic examinations were conducted on the control and high-dose groups prior to treatment and at the end of the study. Haematology was conducted in week 13 in all animals of the main study. Clinical chemistry parameters were measured from blood samples taken at autopsy. Blood glucose measurements and urinalysis were conducted on all animals of the main study in week 13 after deprivation from food and water. Selected haematological, clinical chemistry and urinalysis measurements were conducted in the recovery group. At autopsy, all animals were sacrificed by exsanguination under ether anaesthesia and examined for macroscopic pathological changes. Major organs were weighed, and the organs, tissues and gross lesions were preserved. Tissues required for microscopic evaluation were embedded in paraffin wax, sectioned at 5 µm, and stained with haematoxylin and eosin. Microscopic evaluation of required organs was conducted on all animals of the control and high dose groups and in all dose groups where organ weights or histopathology of the high-dose group indicated possible treatment-related effects. Animals of the recovery group were sacrificed by exsanguination under ether anaesthesia 64 days after withdrawal from the test substance. Organs weighed in the main study were weighed at the end of the recovery period. Microscopic examinations were conducted on tissues and organs showing an effect in the main study. Animals designated for neuropathological examination were anaesthetised and subjected to total body perfusion with paraformaldehyde/glutaraldehyde fixative. The peripheral nerve of high dose males was fixed, and cross and longitudinal sections (1 µm) were prepared of the proximal, mid and distal parts of the nerve, and stained with toluidine blue. Histopathological examination was performed on all animals of the control and 5000 mg/kg/day group. Additionally, animals of the 1000 mg/kg/day group were examined microscopically because of treatment-related changes observed in the 5000 mg/kg/day group.
Mortality occurred in all test groups but was statistically significant in the 5000 mg/kg group. Mortality in the high dose group was determined to be a result of faulty dosing due to incorrect technical administration of the test solution and from regurgitation and aspiration of the test substance. Mortality in the lower dose groups did not show a dose response. Animals in the 5000 mg/kg group showed sluggishness and piloerection shortly after dosing that disappeared within one hour. Emaciation, weakness and lethargy, hunched posture, swollen belly and focal alopecia were also frequently observed in this group, but not the other treatment groups.
Mean body weights in male rats were lower than controls in all treatment groups, but statistically significant from day 35 onward in the 1000 mg/kg group and at all time points in the 5000 mg/kg group. Mean body weights in female rats were only lower than controls in the 5000 mg/kg group and statistically significant in this group from day 7 to 49. Food intake was decreased for males and females of the 5000 mg/kg group in the first 5 weeks and first 2 weeks of the study, respectively. No difference between the other treatment groups and controls were observed. Food conversion efficiency was statistically significantly decreased for males of the 5000 mg/kg group throughout the study and at some time points for the 1000 mg/kg group. Food conversion efficiency was lower for females of the 5000 mg/kg group at some time points. Water intake was increased for all treatment groups. In the recovery period food intake and food conversion efficiency were increased in the test group while water intake was comparable to controls. Body weights remained lower than controls but were no longer statistically significant.
Red blood cell count, haemoglobin concentration and packed cell volume were increased in the 5000 and 1000 mg/kg groups and for males and in the 5000 mg/kg group for females. Increases in reticulocyte count, MCH and MCHC and decreases in white blood cell count and lymphocyte were also observed in 5000 mg/kg group males. These effects were still present 22 days into the recovery period but returned to normal by day 59 of the recovery period. In the 5000 mg/kg group, both sexes exhibited increased gamma glutamyl transferase, decreased fasting blood glucose, increased total protein, increased urea (statistically significant in males only), increased cholesterol, increased triglycerides (statistically significant in males only), decreased chloride and increased inorganic phosphate. Females of this group also exhibited increased alanine aminotransferase, decreased aspartate aminotransferase, decreased cholinesterase (though not statistically significant), increased calcium, and increased sodium. In the 1000 mg/kg group increased urea concentration in males and increased cholesterol concentration in females was also observed. Increased plasma urea and inorganic phosphate remained elevated at the end of the recovery period. In both sexes of the 5000 mg/kg group and in males of the 1000 mg/kg group urine volume was increased, ketones were observed in the urine, and the number of crystals in the urine were decreased. An increase in epithelial cells in the urine was observed in males of the 200 and 1000 mg/kg groups. All these changes had returned to normal in the recovery period.
In both sexes of the 5000 mg/kg group absolute organ weights of the thymus, brain, spleen and heart were decreased and the absolute weight of the adrenals were increased. Increased absolute liver weights in females and decreased absolute testes weights in males were also observed for this treatment group. In the 1000 mg/kg group males decreased absolute brain weights were observed. In the 5000 mg/kg group relative weights of the gonads, adrenals, kidneys and liver were increased and relative weights of the thymus were decreased in both sexes. Males in this group also exhibited increased relative weights of the pituitary, thyroid, brain and heart while females of this group also had decreased relative spleen weights. Relative kidney weights were also increased in 200 and 1000 mg/kg group males and relative liver weights were increased in 200 mg/kg group males and both sexes of the 1000 mg/kg group. By the end of the recovery period, absolute brain and testes weights remained lower than controls, while relative kidney, heart and liver weights remained higher than controls.
At gross necropsy, animals in all treatment groups showed evidence of inflammation of thoracic tissues. This was due to the instillation procedure and the physicochemical properties of the test substance. In the 5000 mg/kg group, large adrenals were observed in males and females, and bilateral small testes were observed in males. Bilateral small testes were still observed in the 5000 mg/kg group at the end of the recovery period.
Treatment-related changes were observed in the microscopic examination of the adrenals, kidneys, liver, lungs peripheral nerve, spleen, testes, and thymus. In the 5000 mg/kg group, the cortex/medulla ratio of the adrenals was significantly increased in both sexes. These changes agree with the enlargement and increased weight observed in the adrenals. In the kidneys an accumulation of brown granules or droplets in the proximal convoluted tubules was observed only in both sexes of the 5000 mg/kg group. The incidence of basophilic proximal tubules was also increased in both sexes of the 1000 mg/kg group, but not the high-dose group; therefore, the toxicological significance of this finding is questionable since there is no clear dose-response relationship. In the liver, the incidence of centrilobular hypertrophy was significantly increased in both sexes of the 5000 mg/kg group and in males of the 1000 mg/kg group. The 5000 mg/kg group males also exhibited a significant decrease in glycogen content. These changes are consistent with the increased liver weights observed in the 1000 and 5000 mg/kg groups. Inflammatory changes in the lung were present in all dose groups without a dose-response relationship and were due to the instillation procedure. Inflammatory changes in the mediastinum were also observed in all dose groups, without a dose-response relationship, so the toxicological significance of these changes is doubtful. Both sexes of the 5000 mg/kg group exhibited increased brown pigment accumulation in the spleen. Tubular atrophy of the testes was increased in males of the 5000 mg/kg group. Thymic involution was increased in males and females of the 5000 mg/kg group (though only significant in males). These changes are consistent with the decreased thymus weights observed in this group. Axonal swelling was observed in males and females of the 5000 mg/kg group. By the end of the recovery period, the only treatment-related change still observed was tubular atrophy in the testes.
In conclusion, the 5000 mg/kg group rats exhibited treatment-related changes including reduced body weight gain, haematopoietic effects, organ weight changes, pathological changes in the liver and kidneys, testicular atrophy, and neurotoxic effects. Treatment-related changes in the 1000 mg/kg group rats included reduced weight gain, increased liver and kidney weights and pathological changes in the liver. Treatment-related changes in the 200 mg/kg group were observed only in males and included increased liver and kidney weights without microscopic evidence of damage. No treatment-related changes were observed in the 40 mg/kg group rats. Testicular atrophy was the only adverse effect still present after the 64-day recovery period. The no-effect level was concluded to be 40 mg/kg bw/day, but treatment-related effects at the next higher dose level of 200 mg/kg bw/day were slight and occurred in male rats only.
In another key repeat dose oral toxicity study (Krasavage, 1980) the effect of oral exposure to the test substance n-hexane was examined. 5 male rats were exposed to concentrations of 6.60, 13.2, and 46.2 mmol/kg bw (568, 1135, 3973 mg/kg) by oral gavage for 90 to 120 days. Neurological effects were only seen at the highest dose level after an average of 101.3 days of exposure. The LOAEL for neurological effects is 46.2 mmol/kg bw (37973 mg/kg), and the NOAEL was 13.2 mmol/kg bw (1135 mg/kg). Reduced body weight gain was seen at all three dose levels, however was only considered treatment related in the 13.2 and 46.2 mmol/kg bw groups. The NOAEL was therefore 6.60 mmol/kg bw (568 mg/kg).
In a short-term supporting oral toxicity study (Til et al., 1989), 5 groups of 5 male and 5 female Wistar rats were administered technical hexane by gavage at dose levels of 0 (control), 40, 200, 1000 or 5000 mg/kg body weight/day, once daily, 7 days a week for a period of 2 weeks. Adverse effects were seen only in the top-dose groups. These rats showed sluggishness and piloerection immediately after dosing which cleared within 1 hour. They had reduced body weights and slightly diminished food intake. Top-dose group males exhibited increased water intake and increased urine volume. Relative adrenal weights were increased and relative spleen weights decreased in both sexes. Relative liver weight was increased in males only. From the results obtained in the present study it is expected that the levels of technical hexane used in this study are also suitable to be used in the subsequent 90-day study.
Additionally, in order to comply with standard information requirements for Annex X substances, OECD Guideline 90-day sub-chronic (OECD 408) toxicity tests are proposed for structural analogue Hydrocarbons, C7-C9, isoalkanes, cyclics, <2% aromatics (EC# 921-728-3). The testing proposals for the same have been presented in the lead registrant dossiers for these substances already submitted to ECHA. These studies will be conducted subsequent to ECHA's approval and this endpoint will be updated upon completion of the above studies.
Inhalation
Light catalytic reformed naphtha distillate / naptha (petroleum), light alkylate
In a key sub-chronic inhalation toxicity study (Schreiner et al., 1998), rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures. Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically. The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.
In another study of Schreiner et al. (2000) conducted according to OECD 413, rats were exposed to light catalytic reformed naphtha distillate via whole body inhalation at 0, 750, 2490 or 7480 ppm (analytical conc.). Male and female rats were exposed for 6 hours/day, 5 days/week, for 13 weeks. All animals survived to study termination. The increased relative kidney weights and microscopically observed hyaline droplet formation and renal tubule dilation in high dose males reflected the occurrence of alpha-2µ-globulin-induced nephropathy, effects in male rats which have been determined to have no relevance to humans. The increased spleen weights and hematological changes had no microscopic correlate and appeared reversible with 4 weeks of recovery. There were no neurobehavioral effects observed during the study and the increased motor activity in recovery high dose males were not supported by other behavior changes or microscopic abnormalities in neural tissues, suggesting the observation may have been fortuitous. Therefore, the NOAEC was 2490 ppm.
Commercial hexane
An inhalation study was conducted with commercial hexane. Similar to OECD 413, groups of male/female rats and mice were exposed by whole body inhalation to 0, 904, 2984, 8992 ppmcommercial hexane (40-55% n-hexane, > 10% methylcyclopentane)for 6 hours/day, 5 days/week for 13 weeks (API,1990a,b).
Rats exposed to hexane: No rats died during the study. Transient excess lacrimation was observed in the female rats. No other effects attributable to exposure were noted. In high exposed males, increased platelets and mean corpuscular volume was observed. Clinical chemistry examinations showed increased creatinine, total protein, and albumin levels and decreased serum chloride contents for males at the high dose level. Increased relative spleen and liver weights were noted for high exposed males. The liver effects appeared to be treatment related. Females at the high dose level showed increased relative spleen weights. Histopathological examinations revealed hemorrhage and inflammation in the liver of the high exposed males. There was also inflammation in the kidneys of males in the high and medium exposure groups. Based on liver and kidney effects, the NOAEC for male rats was determined to be 5500 mg/m³ corresponding to 2984 ppm. The NOAEC for female rats was 16500 mg/m³ corresponding to 8992 ppm.
Mice exposed to hexane: Seven mice died during the study, 3 from drawing blood, and 4 others died accidently. None of these deaths were attributed to treatment. There was sporadic excessive lacrimation due to exposure, no other clinical signs were considered treatment related. Males at the high dose level showed increased mean corpuscular volume. No other treatment related effects were observed in male or female mice. The NOAEC for male and female mice was determined to be 16500 mg/m³ corresponding to 8992 ppm.
n-hexane
In a key repeat dose inhalation study (Takeuchi, 1980), 7 rats were exposed to 3000 ppm of hexane vapors for 12 hrs a day for 16 weeks. Two animals died before the end of the exposure period. All animals showed reduced weight gain after 4 weeks of exposure. Neurological effects were seen beginning at 10 weeks exposure. Motor nerve conduction velocity and distal latency were significantly affected after 4 weeks exposure. Examination of neural tissue showed damage to the tibial nerve and dorsal trunk of the tail nerve. The LOAEC for sub-chronic exposure was 3000 ppm.
In another key repeat dose inhalation study (Dunnick, 1991) the effect of 13 weeks inhalation exposure of n-hexane to mice was examined. Groups of 18 female and 18 male mice were exposed to concentrations of 0, 500, 1000, 4000, or 10,000 ppm of test substance for 6 hrs/day, 5 days/week, for 13 weeks. An additional group was exposed to 1000 ppm of test substance for 22 hrs/day, 5 days/week, for 13 weeks. 8 mice of each sex in each exposure group were examined for neurobehaviour. No animals died during the study, and the only neurological effects were decreased locomotion in females exposed to 10000 ppm. Nasal lesions were seen in females in all exposure groups, and in males exposed to 1000 ppm of test substance. The LOAEC for females was therefore 500 ppm (1760 mg/m3), with no NOAEC found. The NOAEC for males was 500 ppm (1760 mg/m3), with an LOAEC of 1000 ppm (3520 mg/m3) based on nasal lesions.
In a chronic inhalation toxicity study (API, 1983a,b), n-hexane was administered to 114 male, Sprague-Dawley rats by dynamic whole body exposure at concentrations of 0, 125, 250, 500, and 1500 ppm for 22 hours per day, 7 days/week for a total of 168 days. Clinical examinations were done once per week and body weights taken weekly. After sacrifice, animals were examined for histopathological parameters and organ weights. One animal died in the study, but from causes determined to be unrelated to exposure to the test material. The most significant pharmotoxic sign was the abnormal gait which was observed in Group VI (positive control group). This response appeared to be a specific response to that concentration of the n-hexane isomer. The average weekly body weights of group VI rats were significantly low. There were toxicologically and statistically significant differences in mean liver weight for animals in group V (1500 ppm exposure) and VI (positive control group). Significant differences in liver weight for rats in groups IV, V, and VI were observed when compared to the negative control, group I. One unscheduled death was observed in group III, however it was determined that death was not related to test-material exposure. One death occurred early in the study and did not appear to be related to exposure to test materials.
Hydrocarbons, C7 -C9, isoalkanes
In a 12 -week inhalation study (Exxon, 1979) with rats the test substance hydrocarbons, C7 -C9, isoalkanes was tested. Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study. The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males. The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of ?2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC. Renal effects were strictly limited to males, therefore the authors concluded an ?2u-globulin-related mechanism for the observed nephropathy. The observation was not considered for determination of the NOAEC.
Justification for classification or non-classification
Based on available read across data, Hydrocarbons, C6 -C10 (even numbered), n-alkanes, isoalkanes, >5% n-hexane meets the criteria for classification for repeated dose toxicity (STOT-RE 2: H373: May cause damage to organs) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).
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