N-hexane

Endpoint summary

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

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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

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

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
		RBC^a	HB^a	PCV^a	Throm^a		PTT^a	Reticul^b	MCV^a	MCH^a	MCHC^a
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
		RBC^a	HB^a	PCV^a	Throm^a	PTT^a		Reticul^b	MCV^a	MCH^a	MCHC^a
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
		RBC^a	HB^a	PCV^a	Reticul^b		MCV^a	MCH^a	MCHC^a
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
		RBC^a	HB^a	PCV^a	Reticul^b	MCV^a		MCH^a	MCHC^a
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
		WBC^a	Neutro^a	Lympho^a	Eosinoph^b	Neutroph^b	Lymphoc^b	Monocyt^b	Basophil^b
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
		WBC^a	Neutro^a	Lympho^a	Eosinoph^b	Neutroph^b	Lymphoc^b	Monocyt^b	Basophil^b
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
		WBC^a	Neutro^a	Lympho^a	Eosinoph^b	Neutroph^b	Lymphoc^b	Monocyt^b	Basophil^b
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
		WBC^a	Neutro^a	Lympho^a	Eosinoph^b	Neutroph^b	Lymphoc^b	Monocyt^b	Basophil^b
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		Volume^a (mL)	Density^a (kg/L)	Keton-U^b (0-3)	Epith-U^b (0-5)	Cryst-U^b (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 cavity^a:	(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

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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.

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 (3973 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 (3973 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 conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

2 key sub-chronic and 1 supporting study available for assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

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.

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:

NOAEC

Effect level:

500 ppm

Sex:

male

Basis for effect level:

other: nasal lesions

Key result

Dose descriptor:

LOAEC

Effect level:

1 000 ppm

Sex:

male

Basis for effect level:

other: nasal lesions

Key result

Dose descriptor:

LOAEC

Effect level:

500 ppm

Sex:

female

Basis for effect level:

other: nasal lesions

Critical effects observed:

not specified

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 (10^3/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

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.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

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.

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 conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

1 760 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

2 key sub-chronic and 4 supporting chronic studies 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

Reference

Reference 1

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

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose oral and inhalation data is available for n-hexane. Additionally, repeated dose inhalation data is available for structural analogue, mixed hexanes. This data is read across to 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 key repeat dose oral toxicity study (Krasavage, 1980; Klimisch score =2) 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 (3973 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.

 

In a short-term 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.

 

In a sub-chronic repeated dose toxicity study 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.

 

Inhalation:

 

n-hexane

In a key repeat dose inhalation study (Takeuchi, 1980; Klimisch score =2) 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; Klimisch score =2) 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.

 

Mixed hexanes

In a chronic inhalation toxicity study (API, 1983a,b), mixed hexanes was administered to 40 Sprague-Dawley, rats by dynamic whole body exposure at concentrations of 500 ppm for 22 hours per day, 7 days per week for a total of 168 days. The only significant pharmacotoxic sign was abnormal gait in the positive control group and in a group being exposed to 500 ppm of mixed hexanes. Abnormal gait increased in incidence and severity over time in these groups. The average body weights in these same groups were 25 to 30 percent less than that of the negative controls. Six months following continuous inhalation of either n-hexane, mixed hexanes, or a combination of both, all rats were sacrificed and necropsied. Tissues were weighed and a full set of tissues was preserved for future microscopic examination. No toxicologically significant lesions were observed macroscopically in mixed hexane treated and n-hexane treated rats, however, microscopic lesions were observed. A trace to mild level of atrophy was observed in skeletal muscle. A slight increase in the incidence and severity of chronic nephritis and kidney weight was observed. One death occurred early in the study and did not appear to be related to exposure to test materials.

Justification for classification or non-classification

Based on available data, n-hexane does meet the criteria for classification for repeated dose toxicity (STOT-RE 1: H372: May cause damage to organs) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).