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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
30/08/1988 - 01/02/1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data is being read across from the source study that tested technical hexane based on analogue read across.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
30/08/1988 - 01/02/1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This data is being read across from the source study that tested technical hexane based on analogue read across.
Reason / purpose for cross-reference:
read-across source
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

RBCa

HBa

PCVa

Throma

PTTa

Reticulb

MCVa

MCHa

MCHCa

Control

Mean

7.0

8.7

0.453

925

38.1

5.6

64.8

1.24

19.2

sem

0.1

0.1

0.004

22

0.6

1.0

0.9

0.01

0.1

n

15

15

15

15

15

15

15

15

15

40 mg/kg

Mean

7.3

8.8

0.459

909

37.0

6.1

62.5

1.20

19.2

sem

0.1

0.1

0.004

20

0.3

1.0

0.5

0.01

0.1

n

15

15

15

15

15

15

15

15

15

200 mg/kg

Mean

7.3

8.9

0.464

935

37.6

5.4

63.8

1.22

19.2

sem

0.1

0.1

0.005

24

0.6

1.0

1.0

0.01

0.2

n

14

14

14

14

14

14

14

14

14

1000 mg/kg

Mean

7.7**

9.2**

0.482**

920

36.4

8.8*

62.5

1.20

19.1

sem

0.1

0.1

0.005

18

0.4

0.9

0.9

0.01

0.1

n

13

13

13

13

13

13

13

13

13

5000 mg/kg

Mean

7.5**

9.7**

0.491**

943

36.5

10.3*

66.1

1.30*

19.6*

sem

0.1

0.1

0.005

26

0.6

1.6

0.9

0.02

0.1

n

12

12

12

12

12

12

12

12

12

Females

Dosage

Haematological parameter

RBCa

HBa

PCVa

Throma

PTTa

Reticulb

MCVa

MCHa

MCHCa

Control

Mean

7.3

8.9

0.454

983

36.1

8.6

62.0

1.22

19.6

sem

0.1

0.1

0.004

39

0.6

1.0

0.7

0.01

0.1

n

13

13

13

13

13

13

13

13

13

40 mg/kg

Mean

7.5

8.9

0.459

956

35.1

8.3

61.5

1.20

19.5

sem

0.0

0.1

0.003

28

0.6

1.2

0.4

0.01

0.1

n

14

14

14

14

14

14

14

14

14

200 mg/kg

Mean

7.6

9.0

0.460

970

34.7

8.3

60.5

1.18

19.5

sem

0.0

0.1

0.003

31

0.4

1.0

0.4

0.01

0.1

n

14

14

14

14

14

14

14

14

14

1000 mg/kg

Mean

7.5

9.1

0.464

946

34.0*

6.7

62.0

1.22

19.7

sem

0.1

0.1

0.005

21

0.3

0.9

0.7

0.01

0.1

n

14

14

14

14

14

14

14

14

14

5000 mg/kg

Mean

7.6

9.5**

0.482**

975

36.2

11.0

63.4

1.24

19.6

sem

0.1

0.1

0.005

42

0.8

1.8

0.8

0.02

0.1

n

10

10

10

10

10

10

10

10

10

a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01

b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002

RBC = red blood cells (10E12/l)

HB = haemoglobin (mmol/l)

PCV = packed cell volume (l/l)

Throm = thrombocytes (10E9/l)

PTT = prothrombin time (sec)

Reticul = reticulocytes (/1000)

MCV = mean corpuscular volume (fl)

MCH = mean corpuscular haemoglobin (fmol)

MCHC = mean corpuscular haemoglobin concentration (mmol/l)

 

Table 11: Mean haematological findings in blood collected on days 113 and 150 (22 and 59 days, respectively after withdrawal of the test substance) - Males only

113 days (22 days after withdrawal)

Dosage

Haematological parameter

RBCa

HBa

PCVa

Reticulb

MCVa

MCHa

MCHCa

Control

Mean

7.9

9.1

0.455

4.6

57.8

1.16

20.0

sem

0.1

0.1

0.005

1.0

0.7

0.01

0.2

n

10

10

10

10

10

10

10

5000 mg/kg

Mean

7.4***

9.1

0.456

10.0**

61.3*

1.22**

19.9

sem

0.1

0.1

0.008

1.5

1.1

0.01

02

n

6

6

6

6

6

6

6

150 days (59 days after withdrawal)

Dosage

Haematological parameter

RBCa

HBa

PCVa

Reticulb

MCVa

MCHa

MCHCa

Control

Mean

7.4

9.0

0.461

15.6

62.4

1.21

19.4

sem

0.3

0.3

0.013

9.6

1.1

0.02

0.2

n

10

10

10

10

10

10

10

5000 mg/kg

Mean

7.8

9.6

0.480

4.3

61.6

1.23

20.0

sem

0.1

0.1

0.006

1.1

1.1

0.01

0.3

n

6

6

6

6

6

6

6

a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01

b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002

RBC = red blood cells (10E12/l)

HB = haemoglobin (mmol/l)

PCV = packed cell volume (l/l)

Reticul = reticulocytes (/1000)

MCV = mean corpuscular volume (fl)

MCH = mean corpuscular haemoglobin (fmol)

MCHC = mean corpuscular haemoglobin concentration (mmol/l)

 

Table 12: Mean total and differential white blood cell counts in blood collected on day 84 (males)/85 (females)

Males

Dosage

 

WBCa

Neutroa

Lymphoa

Eosinophb

Neutrophb

Lymphocb

Monocytb

Basophilb

Control

Mean

13.1

1.7

11.3

1.1

12.7

85.8

0.4

0.0

sem

0.5

0.1

0.5

0.4

0.9

1.1

0.2

0.0

n

15

15

15

15

15

15

15

15

40 mg/kg

Mean

12.6

1.4

11.0

0.9

10.7

87.6

0.7

0.1

sem

0.5

0.1

0.4

0.3

0.5

0.7

0.3

0.1

n

15

15

15

15

15

15

15

15

200 mg/kg

Mean

13.8

1.4

12.1

0.7

10.5

87.7

1.0*

001

sem

0.6

0.2

0.6

0.2

1.1

1.2

0.2

0.1

n

14

14

14

14

14

14

14

14

1000 mg/kg

Mean

12.9

2.0

10.7

0.8

14.9

83.4

0.8

0.1

sem

0.8

0.3

0.5

0.2

1.5

1.6

0.2

0.1

n

13

13

13

13

13

13

13

13

5000 mg/kg

Mean

10.5**

2.0

8.2**

1.7

18.7**

78.8***

0.9

0.0

sem

0.5

0.3

0.3

0.4

1.8

1.7

0.3

0.0

n

12

12

12

12

12

12

12

12

Females

Dosage

 

WBCa

Neutroa

Lymphoa

Eosinophb

Neutrophb

Lymphocb

Monocytb

Basophilb

Control

Mean

12.2

2.2

9.8

1.1

13.2

85.2

0.3

0.2

sem

1.3

1.2

0.5

0.2

4.0

4.1

0.1

0.1

n

13

13

13

13

13

13

13

13

40 mg/kg

Mean

11.7

1.2

10.2

1.8

10.7

87.2

0.3

0.0

sem

0.7

0.2

0.7

0.4

1.3

1.6

0.1

0.0

n

14

14

14

14

14

14

14

14

200 mg/kg

Mean

11.6

1.3

10.5

1.6

11.2

86.7

0.4

0.0

sem

0.6

0.2

0.5

0.3

1.4

1.4

0.2

0.0

n

14

14

14

14

14

14

14

14

1000 mg/kg

Mean

11.3

1.2

10.0

1.1

10.5

88.0

0.4

0.1

sem

0.5

0.1

0.5

0.2

1.1

1.3

0.2

0.1

n

14

14

14

14

14

14

14

14

5000 mg/kg

Mean

11.3

1.4

9.7

1.6

13.3

84.6

0.5

0.0

sem

0.6

0.2

0.7

0.4

2.6

3.0

0.3

0.0

n

10

10

10

10

10

10

10

10

a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01

b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002

WBC = White blood cells (10E9/l)

Neutro = Absolute number of neutrophils (10E9/l)

Lympho = Absolute number of lymphocytes (10E9/l)

Eosinoph = Eosinophils (%)

Neutroph = Neutrophils (%)

Lymphoc = Lymphocytes (%)

Monocyt = Monocytes (%)

Basophil = Basophils (%)

 

Table 13: Mean total and differential white blood cell counts in blood collected on days 113 and 150 (22 and 59 days, respectively after withdrawal of the test substance) - Males only

113 days (22 days after withdrawal)

Dosage

 

WBCa

Neutroa

Lymphoa

Eosinophb

Neutrophb

Lymphocb

Monocytb

Basophilb

Control

Mean

14.4

1.4

12.7

1.2

9.6

88.7

0.5

0.0

sem

0.7

0.1

0.6

0.3

1.1

1.0

0.2

0.0

n

10

10

10

10

10

10

10

10

5000 mg/kg

Mean

11.7*

1.9

9.4**

2.0

16.3*

80.5**

0.8

0.3

sem

0.8

0.4

0.6

0.4

2.4

2.4

0.3

0.2

n

6

6

6

6

6

6

6

6

150 days (59 days after withdrawal)

Dosage

 

WBCa

Neutroa

Lymphoa

Eosinophb

Neutrophb

Lymphocb

Monocytb

Basophilb

Control

Mean

17.8

3.5

13.9

1.4

16.0

82.0

0.6

0.0

sem

2.0

1.4

0.8

0.3

4.4

4.7

0.2

0.0

n

10

10

10

10

10

10

10

10

5000 mg/kg

Mean

13.5

1.6

11.6

2.3

11.3

86.3

0.0

0.0

sem

1.0

0.3

0.8

0.6

1.3

1.1

0.0

0.0

n

6

6

6

6

6

6

6

6

a. Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p <0.01

b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002

WBC = White blood cells (10E9/l)

Neutro = Absolute number of neutrophils (10E9/l)

Lympho = Absolute number of lymphocytes (10E9/l)

Eosinoph = Eosinophils (%)

Neutroph = Neutrophils (%)

Lymphoc = Lymphocytes (%)

Monocyt = Monocytes (%)

Basophil = Basophils (%)

 

Table 14: Mean results of clinical chemistry of blood collected on day 86 (males)/87 (females) or terminally

Males

 

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Mean

sem

n

Mean

sem

n

Mean

sem

n

Mean

sem

n

Mean

sem

n

ALP

231.5

10.4

15

226.9

11.1

15

246.3

21.7

13

219.9

10.2

13

240.9

16.0

11

ALAT

41.3

2.1

15

44.9

4.7

15

42.2

2.3

13

44.0

2.9

13

48.8

3.2

11

ASAT

57.1

1.8

15

60.0

4.5

15

54.3

1.8

13

50.5

1.2

13

49.4

1.9

11

GGT

3.2

0.3

15

3.0

0.5

15

2.8

0.6

13

3.3

0.4

13

8.8**

0.8

11

CreatKin

64.9

11.2

15

52.8

4.1

15

54.0

3.2

13

49.5

1.7

13

65.1

9.9

11

CHE

419

24

15

400

16

15

462

30

13

502

35

13

389

19

11

LDH

141.1

13.4

15

164.7

42.9

15

140.4

10.2

13

119.5

7.9

13

195.4

43.9

11

a-HBDH

25.4

2.7

15

28.6

6.2

15

24.8

1.7

13

22.6

1.7

13

31.5

6.3

11

Glucose

3.6

0.1

15

3.7

0.1

15

3.7

0.1

13

3.7

0.1

13

3.0**

0.2

11

TP

58.2

0.2

15

58.8

0.7

15

59.3

0.6

13

58.3

0.6

13

61.1**

0.5

11

Albumin

35.8

0.3

15

36.2

0.4

15

36.3

0.6

13

34.8

0.4

13

37.5

0.7

11

Urea

4.31

0.15

15

4.51

0.32

15

4.55

0.12

13

5.06*

0.19

13

6.11**

0.19

11

Creatin

67.9

1.5

15

64.4

1.2

15

64.6

1.5

13

67.9

1.6

13

62.4

2.1

11

Bili-Tot

1.36

0.06

15

1.39

0.06

15

1.27

0.10

13

1.30

0.04

13

1.47

0.16

11

Cholest

2.02

0.07

15

2.06

0.08

15

2.12

0.11

13

2.12

0.09

13

2.43*

0.12

11

Triglyc

0.69

0.04

15

0.71

0.05

15

0.69

0.06

13

0.73

0.05

13

0.98**

0.11

11

Ca

2.48

0.03

15

2.45

0.02

15

2.45

0.03

13

2.42

0.02

13

2.49

0.02

11

K

3.63

0.07

15

3.49

0.06

15

3.68

0.06

13

3.68

0.10

13

3.42

0.10

11

Na

145.9

0.3

15

145.4

0.3

15

145.8

0.3

13

146.5

0.3

13

146.3

0.3

11

Cl

105.9

0.6

15

105.7

0.5

15

105.5

0.4

13

104.7

0.4

13

102.3**

0.4

11

Inorg-P

1.91

0.09

15

1.98

0.07

15

1.81

0.08

13

1.99

0.10

13

2.27**

0.07

11

Females

Day

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Mean

sem

n

Mean

sem

n

Mean

sem

n

Mean

sem

n

Mean

sem

n

ALP

182.3

25.5

13

183.1

8.3

14

177.0

8.2

14

135.8

7.7

14

167.8

11.5

10

ALAT

32.8

1.2

13

33.3

1.2

14

34.3

1.2

14

32.5

2.1

14

41.4**

2.5

10

ASAT

57.0

3.9

13

51.3

1.1

14

55.8

1.8

14

51.0

1.6

14

46.6*

1.9

10

GGT

2.1

0.2

13

1.8

0.3

14

2.0

0.3

14

1.7

0.2

14

5.7**

0.8

10

CreatKin

50.3

3.1

13

50.6

2.3

14

53.1

2.7

14

50.1

3.2

14

56.2

2.2

10

CHE

1160

181

13

1188

106

14

1228

143

14

1283

86

14

750

86

10

LDH

125.8

5.5

13

124.8

7.2

14

148.3

9.8

14

125.5

8.5

14

118.3

8.7

10

a-HBDH

23.3

0.8

13

22.9

1.0

14

27.3

1.8

14

22.2

1.6

14

21.9

1.5

10

Glucose

3.8

0.1

13

3.3**

0.1

14

3.4

0.1

14

3.5

0.1

14

2.6**

0.1

10

TP

60.0

0.8

13

58.5

0.5

14

59.4

0.4

14

59.9

0.4

14

62.6*

0.6

10

Albumin

36.8

0.7

13

36.6

0.5

14

36.5

0.4

14

35.6

0.8

14

36.0

1.3

10

Urea

5.44

0.27

13

5.31

0.26

14

5.66

0.25

14

4.97

0.26

14

6.19

0.28

10

Creatin

67.4

1.7

13

67.9

1.3

14

70.4

2.2

14

66.8

1.5

14

67.9

2.0

10

Bili-Tot

1.25

0.12

13

1.17

0.10

14

1.27

0.08

14

1.18

0.06

14

1.51

0.05

10

Cholest

1.84

0.08

13

1.77

0.06

14

1.83

0.06

14

2.16*

0.08

14

2.86**

0.11

10

Triglyc

0.50

0.02

13

0.54

0.03

14

0.62

0.06

14

0.56

0.06

14

0.70

0.05

10

Ca

2.38

0.03

13

2.38

0.01

14

2.37

0.01

14

2.39

0.01

14

2.49**

0.02

10

K

3.31

0.14

13

3.19

0.08

14

3.37

0.10

14

3.20

0.07

14

3.00

0.07

10

Na

143.6

0.6

13

143.7

0.4

14

143.3

0.6

14

144.8

0.3

14

145.6*

0.6

10

Cl

107.9

0.5

13

107.5

0.6

14

107.3

0.3

14

108.3

0.04

14

105.1**

0.7

10

Inorg-P

1.46

0.09

13

1.52

0.09

14

1.42

0.08

14

1.50

0.11

14

2.10**

0.09

10

Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01

ALP = Alkaline Phosphatase (U/l) – collected day 91 (males)/93 (females)

ALAT = Alanine Aminotransferase (GPT) (U/l) – collected day 91 (males)/93 (females)

ASAT = Aspartate Aminotransferase (GOT) (U/l) – collected day 91 (males)/93 (females)

GGT = Gamma Glutamyl Transferase (U/l) – collected day 91 (males)/93 (females)

CreatKin = Creatinine Kinase (U/l) – collected day 91 (males)/93 (females)

CHE = Cholinesterase in Plasma (U/l) – collected day 91 (males)/93 (females)

LDH = Lactate Dehydrogenase (U/l) – collected day 91 (males)/93 (females)

a-HBDH = a-Hydroxy Butericacid Dehydrogenase (U/l) – collected day 91 (males)/93 (females)

Glucose (mmol/l) – collected day 86 (males)/87 (females)

TP = Total Protein (g/l) – collected day 91 (males)/93 (females)

Albumin (g/l) – collected day 91 (males)/93 (females)

Urea (mmol/l) – collected day 91 (males)/93 (females)

Creatin = Creatinine (umol/l) – collected day 91 (males)/93 (females)

Bili-Tot = Bilirubin (total) (umol/l) – collected day 91 (males)/93 (females)

Cholest = Cholesterol (mmol/l) – collected day 91 (males)/93 (females)

Triglyc = Triglycerides (mmol/l) – collected day 91 (males)/93 (females)

Ca = Calcium (mmol/l) – collected day 91 (males)/93 (females)

K = Potassium (mmol/l) – collected day 91 (males)/93 (females)

Na = Sodium (mmol/l) – collected day 91 (males)/93 (females)

Cl = Chloride (mmol/l) – collected day 91 (males)/93 (females)

Inorg-P = Inorganic Phosphate (mmol/l) – collected day 91 (males)/93 (females)

 

Table 15: Mean results of clinical chemistry of blood collected on day 155 (64 days after withdrawal of the test substance) – Males only

 

Control

5000 mg/kg

Mean

sem

n

Mean

sem

n

ASAT

54.2

1.6

10

64.1*

4.4

6

ALAT

44.3

1.7

10

49.1

2.4

6

GGT

10.2

1.1

10

6.5*

1.2

6

CHE

335

24

10

310

12

6

TP

61.0

0.7

10

60.0

0.9

6

Urea

6.34

0.22

10

7.48*

0.44

6

Cholest

2.09

0.08

10

1.87

0.07

6

Triglyc

1.36

0.19

10

1.24

0.19

6

Ca

2.57

0.03

10

2.55

0.02

6

Na

144.4

0.5

10

144.9

0.4

6

Cl

103.9

0.6

10

104.1

1.0

6

Inorg-P

1.55

0.02

10

1.82***

0.07

6

Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

ASAT = Aspartate Aminotransferase (GOT) (U/l)

ALAT = Alanine Aminotransferase (GPT) (U/l)

GGT = Gamma Glutamyl Transferase (U/l)

CHE = Cholinesterase in Plasma (U/l)

TP = Total Protein (g/l)

Urea (mmol/l)

Cholest = Cholesterol (mmol/l)

Triglyc = Triglycerides (mmol/l)

Ca = Calcium (mmol/l)

Na = Sodium (mmol/l)

Cl = Chloride (mmol/l)

Inorg-P = Inorganic Phosphate (mmol/l)

 

Table 16: Mean volume and density of the urine collected on day 85-86 (Males) or day 86-87 (Females)

Dosage

Males

Females

Volume

(mL)

Density

(kg/L)

Volume

(mL)

Density

(kg/L)

Control

Mean

1.1

1.077

1.2

1.069

sem

0.1

0.002

0.2

0.005

n

15

15

13

13

40 mg/kg

Mean

1.0

1.081

1.1

1.066

sem

0.1

0.003

0.1

0.004

n

15

15

14

14

200 mg/kg

Mean

1.2

1.089**

1.2

1.066

sem

0.1

0.001

0.1

0.004

n

14

14

14

14

1000 mg/kg

Mean

2.1**

1.080

1.2

1.077

sem

0.1

0.002

0.1

0.003

n

13

13

14

14

5000 mg/kg

Mean

2.7**

1.071

2.9**

1.065

sem

0.1

0.001

0.2

0.004

n

11

11

10

10

Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01

 

Table 17: Selected mean semi-quantitative observations in the urine collected on day 85-86 (Males) or day 86-87 (Females)

Dosage

Males

Females

Keton-U

(0-3)

Epith-U

(0-5)

Cryst-U

(0-5)

Keton-U

(0-3)

Epith-U

(0-5)

Cryst-U

(0-5)

Control

Mean

0

1

2

0

1

1

n

15

15

15

13

13

13

40 mg/kg

Mean

0

1

2

0

1

1

n

15

15

15

14

14

14

200 mg/kg

Mean

0

2*

2

0

1

1

n

14

14

14

14

14

14

1000 mg/kg

Mean

1***

2***

1**

0

1

1

n

13

13

13

14

14

14

5000 mg/kg

Mean

2***

1

1**

1*

1

0

n

11

11

11

10

10

10

Statistics: Mann/Whitney U-test (two-sided): *p<0.05; ** p<0.02; *** p<0.002

Keton-U = Ketones in Urine

Epith-U = Epithelial Cells in Urine

Cryst-U = Crystals in Urine

 

Table 18: Mean volume, density, semi-quantitative- and microscopic observations in the urine collected on day 114-115 (23-24 days after withdrawal of the test substance) – Males only

Dosage

Volumea

(mL)

Densitya

(kg/L)

Keton-Ub

(0-3)

Epith-Ub

(0-5)

Cryst-Ub

(0-5)

Control

Mean

2.0

1.071

0

1

2

sem

0.1

0.002

 

n

10

10

10

10

10

5000 mg/kg

Mean

1.9

1.074

0

1

2

sem

0.1

0.002

 

n

6

6

6

6

6

a. Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p<0.001

b. Statistics: Mann/Whitney U-test (two-sided): * p<0.05; ** p<0.02; *** p<0.002

Keton-U = Ketones in Urine

Epith-U = Epithelial Cells in Urine

Cryst-U = Crystals in Urine

 

Table 19: Mean terminal body weights (g) and absolute organ weights (g) recorded terminally

Organ

Males

Females

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Body wt

Mean

303.9

308.7

293.6

274.2**

188.1**

193.5

187.0

184.0

187.6

172.5

sem

5.7

7.6

6.0

5.5

4.2

5.7

4.0

4.8

3.4

7.8

n

40

30

27

28

27

13

14

14

14

10

Pituitary

Mean

0.012

0.011

0.012

0.011

0.011

0.015

0.014

0.014

0.015

0.014

sem

0.000

0.000

0.000

0.000

0.000

0.001

0.001

0.001

0.001

0.001

n

15

15

14

13

11

13

14

14

14

10

Thyroid

Mean

0.019

0.018

0.018

0.017

0.017

0.018

0.017

0.016

0.016

0.018

sem

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

0.001

n

15

15

13

13

11

13

14

14

14

10

Adrenals

Mean

0.043

0.046

0.044

0.046

0.070**

0.057

0.055

0.053

0.051

0.097**

sem

0.001

0.001

0.001

0.001

0.003

0.002

0.002

0.001

0.002

0.006

n

15

15

14

13

11

13

14

14

14

10

Kidneys

Mean

1.64

1.72

1.69

1.75

1.51

1.22

1.18

1.16

1.22

1.33

sem

0.06

0.05

0.05

0.04

0.04

0.03

0.03

0.04

0.03

0.06

n

15

15

14

13

11

14

14

14

14

10

Thymus

Mean

0.262

0.270

0.290

0.267

0.097**

0.252

0.241

0.258

0.228

0.156**

sem

0.019

0.024

0.024

0.017

0.007

0.013

0.012

0.025

0.014

0.015

n

15

15

15

13

11

13

14

14

14

10

Brain

Mean

1.80

1.80

1.75

1.70**

1.43**

1.69

1.71

1.68

1.67

1.46**

sem

0.02

0.02

0.02

0.02

0.02

0.02

0.01

0.01

0.02

0.02

n

15

15

14

13

11

13

14

14

14

10

Spleen

Mean

0.438

0.428

0.431

0.400

0.229**

0.365

0.341

0.367

0.341

0.275**

sem

0.021

0.021

0.018

0.017

0.010

0.017

0.012

0.015

0.014

0.016

n

15

15

14

13

11

13

14

14

14

10

Heart

Mean

0.94

0.95

0.92

0.87

0.61**

0.70

0.67

0.68

0.67

0.61**

sem

0.03

0.02

0.03

0.03

0.02

0.02

0.02

0.01

0.01

0.02

n

15

15

14

13

11

13

14

14

14

10

Liver

Mean

9.72

10.12

9.88

10.41

9.78

6.62

6.49

6.45

6.82

9.12**

sem

0.39

0.36

0.36

0.32

0.27

0.29

0.16

0.15

0.16

0.39

n

15

15

14

13

11

13

14

14

14

10

Testes

Mean

3.00

2.96

2.88

2.87

2.04**

 

sem

0.08

0.07

0.09

0.05

0.25

n

15

15

14

13

11

Ovaries

Mean

 

0.097

0.098

0.093

0.093

0.108

sem

0.004

0.004

0.004

0.004

0.007

n

13

14

14

14

10

Uterus

Mean

0.619

0.637

0.566

0.612

0.344

sem

0.083

0.075

0.074

0.093

0.064

n

13

14

14

14

10

Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01

 

Table 20: Mean relative organ weights (g/kg body weight) recorded terminally

Organ

Males

Females

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Control

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Pituitary

Mean

0.040

0.039

0.042

0.042

0.060**

0.078

0.073

0.077

0.079

0.079

sem

0.001

0.001

0.002

0.001

0.003

0.003

0.003

0.003

0.003

0.04

n

15

15

14

13

11

13

14

14

14

10

Thyroid

Mean

0.065

0.061

0.065

0.065

0.097**

0.092

0.093

0.090

0.085

0.106

sem

0.002

0.002

0.003

0.002

0.007

0.005

0.003

0.005

0.004

0.005

n

15

15

13

13

11

13

14

14

14

10

Adrenals

Mean

0.145

0.157

0.159

0.173

0.401**

0.293

0.296

0.288

0.274

0.561**

sem

0.004

0.005

0.006

0.008

0.024

0.010

0.006

0.009

0.010

0.026

n

15

15

14

13

11

13

14

14

14

10

Kidneys

Mean

5.51

5.79

6.08**

6.58**

8.60**

6.33

6.33

6.31

6.54

7.70**

sem

0.07

0.13

0.08

0.17

0.16

0.13

0.09

0.19

0.12

0.18

n

15

15

14

13

11

13

14

14

14

10

Thymus

Mean

0.87

0.91

1.04

0.99

0.55**

1.31

1.28

1.39

1.22

0.90**

sem

0.05

0.07

0.07

0.05

0.04

0.07

0.05

0.11

0.07

0.07

n

15

15

14

13

11

13

14

14

14

10

Brain

Mean

6.13

6.09

6.32

6.40

8.15**

8.77

9.21

9.18

8.95

8.56

sem

0.19

0.14

0.14

0.12

0.24

0.17

0.19

0.22

0.17

0.31

n

15

15

14

13

11

13

14

14

14

10

Spleen

Mean

1.47

1.43

1.54

1.50

1.30

1.89

1.82

2.00

1.82

1.59*

sem

0.06

0.05

0.04

0.05

0.05

0.09

0.05

0.07

0.06

0.06

n

15

15

14

13

11

13

14

14

14

10

Heart

Mean

3.16

3.21

3.30

3.24

3.47**

3.64

3.61

3.71

3.59

3.52

sem

0.04

0.05

0.05

0.05

0.06

0.06

0.05

0.06

0.05

0.06

n

15

15

14

13

11

13

14

14

14

10

Liver

Mean

32.5

33.9

35.4*

39.0**

55.7**

34.1

34.7

35.1

36.4*

53.0**

sem

0.04

0.4

0.6

0.7

1.7

0.6

0.4

0.4

0.6

1.1

n

15

15

14

13

11

13

14

14

14

10

Testes

Mean

10.13

10.01

10.34

10.80

11.48

 

sem

0.24

0.26

0.23

0.30

1.32

n

15

15

14

13

11

Ovaries

Mean

 

0.505

0.516

0.504

0.497

0.622*

sem

0.28

0.028

0.017

0.023

0.031

n

13

14

14

14

10

Uterus

Mean

3.20

3.44

3.12

3.27

2.02

sem

0.43

0.43

0.44

0.50

0.42

n

13

14

14

14

10

Statistics: Anova + Dunnett’s tests (two-sided): * p<0.05; ** p<0.01

 

Table 21: Mean terminal body weight (g) and absolute organ weights (g) recorded on day 155 (64 days after withdrawal of the test substance) – Males only

Organ

Control

5000 mg/kg

Mean

sem

n

Mean

sem

n

Body weight

337.4

17.5

10

296.7

12.2

6

Tests

3.12

0.11

10

2.13*

0.48

6

Pituitary

0.013

0.000

10

0.013

0.001

6

Thyroid

0.023

0.001

10

0.020

0.001

6

Adrenals

0.042

0.002

10

0.045

0.002

6

Kidneys

1.87

0.07

10

1.98

0.10

6

Thymus

0.250

0.033

10

0.229

0.018

6

Brain

1.88

0.02

10

1.64***

0.02

6

Spleen

0.538

0.031

10

0.476

0.024

6

Heart

1.03

0.04

10

1.04

0.03

6

Liver

10.72

0.59

10

10.64

0.60

6

Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p< 0.001

 

Table 22: Mean relative organ weights (g/kg bodyweight) recorded on day 155 (64 days after withdrawal of the test substance) – Males only

Organ

Control

5000 mg/kg

Mean

sem

n

Mean

sem

n

Tests

9.38

0.31

10

6.94

1.46

6

Pituitary

0.040

0.003

10

0.046

0.004

6

Thyroid

0.069

0.003

10

0.069

0.004

6

Adrenals

0.132

0.017

10

0.151

0.006

6

Kidneys

5.61

0.16

10

6.69***

0.15

6

Thymus

0.72

0.09

10

0.78

0.07

6

Brain

5.73

0.37

10

5.58

0.22

6

Spleen

1.62

0.09

10

1.60

0.04

6

Heart

3.08

0.08

10

3.53*

0.15

6

Liver

31.8

0.6

10

35.9**

1.2

6

Statistics: Two sample t-test (two-sided): * p<0.05; ** p<0.01; *** p< 0.001

 

Table 23: Summary of selected macroscopic changes

Changes

Incidence of Gross Lesions (numeric)

Males

Females

0 mg/kg

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

0 mg/kg

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Lungs:

 Spotted surface

0

1

0

0

2

1

0

0

0

0

 Discoloured

0

0

0

1

2

0

1

0

0

4

 Evidence of inflammation

0

1

2

0

0

1

0

4

1

0

 Focal foamy appearance

0

0

0

0

0

0

0

0

1

0

Thoracic cavity:

 Hydrothorax

0

0

0

2

1

0

1

0

0

2

 Haemothorax

0

0

1

0

0

0

0

0

0

2

 Filled with whitish liquid

0

4

4

2

0

0

1

6

3

0

 Turbid liquid

0

0

0

0

0

1

0

0

0

0

 Evidence of inflammation

1

1

1

0

0

0

0

0

0

0

 Thickening of the mediastinum

0

0

0

0

0

2

1

1

1

0

 Adhesions of thoracic tissues  

1

1

4

3

0

2

2

7

2

0

Adrenals:

 Enlarged

0

0

0

0

2

0

0

0

0

10

Testes:

 Bi-lateral small

0

0

0

0

3

 

 

Table 24: Summary of selected macroscopic changes – Recovery period (males only)

Changes

Incidence of Gross Lesions (numeric)

Males

0 mg/kg

5000 mg/kg

Lungs:

 Evidence of inflammation

1

0

Thoracic cavity:

 Hydrothorax

2

0

 Adhesion of lung-lobes and thoracic wall

2

0

Testes:

 Soft tissue

0

1

 Bi-lateral small

0

2

Table 25: Summary of selected microscopic changes

Changes

Incidence of Lesions (numeric)

Males

Females

0 mg/kg

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

0 mg/kg

40 mg/kg

200 mg/kg

1000 mg/kg

5000 mg/kg

Adrenals:

(15)

(15)

(15)

(15)

(14)

(15)

(15)

(13)

(15)

(15)

Increased ratio cortex/medulla

0

0

0

0

9***

0

0

1

0

12***

Focal increased cortical vacuolation

0

0

0

1

0

0

0

0

0

0

Diffuse increased cortical vacuolation

0

2

0

0

0

1

0

0

0

0

Focal medullary mononuclear-cell infiltrate

1

0

0

0

0

0

0

0

0

0

Kidneys:

(15)

(15)

(15)

(15)

(15)

(14)

(15)

(15)

(15)

(15)

Cortical proximal tubular brown pigment accumulation:

 

Very slight

0

0

0

0

7**

0

0

0

0

6*

slight

0

0

0

0

3

0

0

0

0

2

Total incidence for score expanded finding

0

0

0

0

10***

0

0

0

0

8**

Basophilic tubules:

 

Very slight

6

9

7

5

2

0

3

0

5*

0

Slight

0

0

4

9***

2

0

0

0

0

0

Total incidence for score expanded finding

6

9

11

14**

4

0

3

0

5*

0

Uni-lateral hydronephrosis

0

0

0

0

0

0

1

1

0

0

Focal mononuclear-cell infiltrate

0

1

0

0

0

0

1

1

0

0

Medullary mineralisation

1

1

0

1

0

0

3

1

1

0

Corticomedullary mineralisation

0

0

0

0

0

9

7

8

9

0***

Cortical mineralisation

0

0

2

0

0

0

0

1

0

0

Liver:

(15)

(15)

(15)

(15)

(15)

(15)

(15)

(15)

(15)

(15)

Centrilobular hepatocellular hypertrophy:

 

Slight

1

0

0

9**

12***

0

0

1

2

12***

Total incidence for score expanded finding

1

0

0

9**

12***

0

0

1

2

12***

Decreased glycogen content

1

0

0

0

12***

1

0

1

0

0

Increased glycogen content

0

3

3

0

1

0

0

0

0

0

Aggregates of res cells and necrotic hepatocytes:

 

Very slight

7

5

4

0**

3

4

4

6

4

5

Slight

3

3

5

0

0

3

2

1

2

0

Total incidence for score expanded finding

10

8

9

0***

3*

7

6

7

6

5

Periportal mononuclear-cell infiltrate

1

0

1

0

1

2

1

3

1

0

Centrilobular mononuclear-cell infiltrate

0

1

0

0

0

2

0

0

1

0

Focal hepatocellular necrosis

0

0

0

0

1

0

0

0

0

0

Hepatocellular vacuolation

2

0

0

0

1

4

0

0

0

0

Focal subcapsular mineralisation

0

0

1

0

0

0

0

0

0

0

Lungs:

(15)

(15)

(15)

(15)

(14)

(15)

(15)

(15)

(15)

(15)

Fibrotic activation

1

0

0

0

1

1

0

0

0

0

Pleuritis

1

1

2

3

1

3

1

1

2

0

Focal pleural thickening

0

1

2

3

0

3

1

6

1

0

Focal bronchiolar epithelial hyperplasia

0

0

0

0

1

0

0

0

0

0

Focal increased septal cellularity

0

0

0

2

0

2

2

3

0

1

Mineralisation of arterial wall

10

3*

6

3

7

9

6

0***

1**

4

Focal bronchiolar cellular debris

0

0

1

0

0

0

0

0

0

0

Focal pneumonitis

0

0

3

0

0

1

0

3

0

0

Brown pigment accumulation

1

0

1

0

1

2

1

2

0

2

Nerve - Peripheral:

(14)

(15)

(14)

(15)

(15)

(15)

(15)

(15)

(15)

(14)

Blue axonal swelling

4

5

9

7

11*

5

6

11

6

7

Spleen:

(15)

(15)

(15)

(15)

(14)

(13)

(15)

(10)

(15)

(15)

Brown pigment accumulation

 

Very slight

10

13

11

14

1**

0

0

1

1

0

Slight

0

0

4

1

11***

13

13

8

12

6***

Moderate

0

0

0

0

0

0

2

1

2

9***

Total incidence for score expanded finding

10

13

15*

15*

12

13

15

10

15

15

Extra medullary haematopoiesis

0

0

0

0

1

0

0

0

0

0

White-pulp depletion

0

0

0

0

1

0

0

0

0

0

Thymus:

(15)

(14)

(14)

(14)

(13)

(14)

(15)

(14)

(14)

(15)

Involution:

 

Very slight

3

3

5

5

5

4

7

1

4

6

Slight

0

0

0

0

4*

0

0

0

0

4

Moderate

0

0

0

0

0

1

0

1

0

0

Total incidence for score expanded finding

3

3

5

5

9*

5

7

2

4

10

Focal haemorrhage

0

1

0

1

0

0

0

0

1

3

Starry sky appearance

0

1

0

1

0

0

0

0

1

3

Thoracic cavitya:

(1)

(3)

(6)

(4)

(1)

(6)

(2)

(7)

(4)

(2)

In mediastinum inflammatory cell infiltrate

1

1

5

3

1

4

1

6

3

2

Distended lymph vessels

1

3

2

1

0

4

1

6

3

0

Testes:

(15)

(15)

(15)

(14)

(14)

 

Tubular atrophy:

 

Very slight

0

1

1

1

1

Slight

0

1

0

0

0

Severe

0

0

0

0

2

Very severe

0

0

0

0

1

Total incidence for score expanded finding

0

2

1

1

4*

Epididymides:

(15)

 

 

 

(14)

Focal interstitial mononuclear-cell infiltrate

5

0*

Prostate:

(15)

 

 

 

(14)

 

 

 

 

 

 

Focal interstitial mononuclear-cell infiltrate

5

0*

Figures in brackets represent the number of animals examined microscopically

a. Microscopic verification of gross observations only, no statistical analysis performed

Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01

 

Table 26: Summary of selected microscopic changes – Recovery period (males only)

Changes

Incidence of Lesions (numeric)

Males

0 mg/kg

5000 mg/kg

Adrenals:

(10)

(6)

Diffuse increased cortical vacuolation

6

1

Kidneys:

(10)

(6)

Proximal tubular brown pigment accumulation

7

6

Basophilic tubules

4

0

Medullary mineralisation

1

0

Liver:

(10)

(6)

Aggregates of res cells and necrotic hepatocytes

4

3

Periportal mononuclear-cell infiltrate

0

1

Centrilobular mononuclear-cell infiltrate

0

1

Lungs:

(10)

(6)

Focal pleuritis

1

0

Focal pleural thickening

2

0

Focal increased septal cellularity

1

2

Focal accumulation of alveolar macrophages

0

2

Single granuloma

1

0

Mineralisation of arterial wall

7

1

Increased perivascular lymphoid aggregates

0

2

Nerve-Peripheral:

(10)

(6)

No abnormality detected

10

6

Spleen:

(10)

(6)

Brown pigment accumulation:

 

Slight

7

3

Moderate

3

3

Total incidence for score expanded finding

10

6

Extra medullary haematopoiesis

1

2

Testes:

(9)

(6)

Tubular atrophy:

 

Very slight

0

1

Slight

1

3

Very severe

0

2

Total incidence for score expanded finding

1

6**

Tubular mineralisation

1

4

Thymus:

(9)

(6)

No abnormality detected

9

6

Figures in brackets represent the number of animals examined microscopically

Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01

Table 27: Summary of selected microscopic changes – Neuro-histology

Changes

Incidence of Lesions (numeric)

Males

Females

0 mg/kg

1000 mg/kg

5000 mg/kg

0 mg/kg

1000 mg/kg

5000 mg/kg

Nerve-Peripheral:

(5)

(5)

(5)

(5)

(5)

(6)

Axonal distention:

 

 

 

 

 

 

Very slight

0

0

0

0

0

3

Slight

0

0

5**

0

0

0

Total incidence for score expanded finding

0

0

5**

0

0

3

Minimal intra-axonal myelin deposits

5

3

4

3

4

3

Slight intra-axonal myelin deposits

0

2

0

1

1

2

Moderate intra-axonal myelin deposits

0

0

1

1

0

1

Figures in brackets represent the number of animals examined microscopically

Significance of difference in a pairwise (Fisher’s) test between each treatment and control incidence: * p<0.05; ** p<0.01

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:

This data is being read across from the source study that tested technical hexane based on analogue read across.

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-hexane
EC Number:
203-777-6
EC Name:
N-hexane
Cas Number:
110-54-3
Molecular formula:
C6H14
IUPAC Name:
hexane
Details on test material:
- Analytical purity:
Technical grade
- Composition of test material, percentage of components:
n-hexane - 58%; benzene 0.060% weight (0.045% vol.)
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.)

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

open allclose all
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)

Target system / organ toxicity

open allclose all
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

Any other information on results incl. tables

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<