Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2011 - 23 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, United Kingdom
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 194-257 g; Females: 161-206 g
- Housing: Polypropylene cages with stainless steel mesh lids and softwood flake bedding (except for urine collection: metabolic cages)
- Diet: Ground diet, ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Monthly (dietary admixtures stable for at least 6 weeks at -18 °C)
- Mixing appropriate amounts with (Type of food): Basal laboratory diet
- Storage temperature of food: -18 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of selected dietary admixtures were analysed for homogeneity and concentration prior to use by gas chromatography using an external standard technique.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuously in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
200 other: mg/kg diet (15.3 mg/kg bw)
Dose / conc.:
2 000 other: mg/kg diet (154.9 mg/kg bw)
Remarks:
154.9 mg/kg bw/day
Dose / conc.:
6 000 other: mg/kg diet (464 mg/kg day)
Remarks:
464.1 mg/kg bw/day
Dose / conc.:
20 000 other: mg/kg diet (1504.6 mg/kg bw)
Remarks:
1504.6 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: 14-day range finding study
- Rationale for animal assignment: Random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked: overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes (signs of functional/behavioural toxicity)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 7 and at end of study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Hb, RBC, Hct, MCH, MCV, MCHC, WBC, Neut, Lymph, Mono, Eos, Bas, PLT, CT, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 7 and at end of study
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, ASAT, ALAT, AP, creatinine, total cholesterol, total bilirubin, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: Week 7 and at end of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, specific gravity, ketones, bilirubin, pH, protein, glucose, urobilinogen, blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12 only
- Dose groups that were examined: All dose groups
- Battery of functions tested: motor activity, forelimb/hindlimb grip strength, sensory reactivity to auditory, visual and proprioceptive stimuli

OESTROUS CYCLE: Yes
- Time schedule: during weeks 6&7 and weeks 12&13
- Method: daily vaginal smear was stained with giemsa stain, stage of oestrus was examined microscopically
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Examinations: full external and internal examination

ORGAN WEIGHTS: Yes
- Tissues measured: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid/parathyroid, thymus, uterus and cervix

HISTOPATHOLOGY: Yes
- Dose groups that were examined: control and high dose group, except for kidneys, liver and adrenals (all groups)
- Tissues examined: Adrenals, Oesophagus, Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Cervix, Salivary glands (submaxillary), Coagulating gland, Sciatic nerve, Colon, Seminal vesicles, Duodenum, Skin (hind limb), Epididymides, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes, Jejunum, Thymus, Kidneys, Thyroid/parathyroid, Liver, Tongue, Lungs (with bronchi), Trachea, Lymph nodes (cervical and mesenteric), Urinary bladder, Mammary glands, Uterus, Muscle (skeletal), Vagina
- Staining: haematoxylin & eosin, Mallory's Heidenhain (for male kidney)
Other examinations:
After necropsy:
- Thyroid hormone assessment: blood samples collected for analysis of thyroid hormone, in case pituitary-thyroid axis effects identified
- Sperm assessment: 1. weighing of testis and epididymides (excl. connective tissue), 2. analysis of homogenisation resistant spermatids in testis (DNA specific fluorescent staining), 3. determination of number of motile, progressively motile and non-motile sperm, 4. morphometric analysis (eosin staining), 5. analysis of homogenisation resistant spermatids in cauda epidydimis (eosin staining)
Statistics:
For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected throughout the study period. One female treated with 6000 ppm showed generalised fur loss from Day 40 onwards. Observations of this nature are commonly observed in group housed animals and in the absence of a similar effect in high dose animals this observation is considered not to be indicative of test item toxicity.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 1 in "Any other information on results"
A reduction in overall body weight gain was detected in animals of either sex treated with 20000 ppm. Statistically significant reductions in body weight gains (P<0.05 or P<0.01) were evident in males treated with 20000 ppm during Weeks 1, 4, 10 and 12 and in females during Weeks 1 and 6. This effect also extended to males treated with 6000 ppm during Weeks 1, 4 and 12 and in males treated with 2000 ppm during Weeks 4 and 12. No toxicologically significant effects were detected in animals of either sex treated with 200 ppm. Females treated with 200 ppm showed a statistically significant increase in body weight gain (P<0.05) during Week 3 and females from all treatment groups showed a statistically significant increase in body weight gain (P<0.01) during Week 11. An increase in this parameter cannot be regarded as an adverse effect of treatment and as such, the intergroup differences were considered to be of no toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 20000 ppm showed a reduction in food consumption throughout the study period. No such effects were detected in animals of either sex treated with 200, 2000 or 6000 ppm. Reduction in food consumption at this high dose can be due to the palatability of the test substance and is considered to be of no toxicological importance.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency was adversely affected at 20000 ppm however this was confined to weeks when body weight gain was statistically reduced. No such effects were detected in animals of either sex treated with 200, 2000 or 6000 ppm. Reduction in food consumption at this high dose can be due to the palatability of the test substance and is considered to be of no toxicological importance.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspections of the water consumption did not result in any treatment related effects throughout the study.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment related ocular effects detected during opthalmoscopic examinations.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in haematological parameters measured. During Week 13 assessments females treated with 20000 and 6000 ppm showed statistically significant increases in haemoglobin (P<0.05) and hematocrit (P<0.05) values. Females from all treatment groups also showed a statistically significant increase in clotting time (P<0.05). In the absence of a true dose related response or any associated changes, these findings were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- During Week 7 and 13 assessments males treated with 20000 ppm showed statistically significant reductions in chloride concentration (P<0.05 or P<0.01) and aspartate aminotransferase levels (P<0.05 or P<0.01) and an increase in cholesterol levels (P<0.05 or P<0.01). The effect on chloride concentration (P<0.05) was also evident at 6000 ppm males during Week 7 assessments and on aspartate aminotransferase levels (P<0.01) at 6000 ppm males during Week 13 assessments. Week 13 evaluations also showed reductions in sodium concentration (P<0.05), alanine aminotransferase (P<0.01) and bile acid levels (P<0.05) in males treated with 20000 ppm and alanine aminotransferase levels (P<0.01) in males treated with 6000 ppm. The increase in chloride and sodium levels were only 1-1.5%. Althought statistically significant, these effects are not biological relevant. The reductions in bile acids and the enzymes levels of aspartate aminotransferase and alanine aminotransferase and increases in cholesterol in males treated with either 20000 or 6000 ppm are most likely linked to the liver weight findings and the histopathological effects seen in this organ and can be considered of an adaptive nature.
- No toxicologically significant effects were detected in males treated with 2000 or 200 ppm or in females from all treatment groups.
- Females treated with 20000 ppm at Week 7 assessments showed a statistically significant reduction in aspartate aminotransferase levels (P<0.01). This effect was not recorded during Week 13 assessments, therefore, it is considered fortuitous and of no toxicological importance. Females treated with 20000 or 6000 ppm during Week 13 evaluations showed a statistically significant increase in calcium concentration (P<0.05). In the absence of any histopathological correlates this effect is not considered toxicologically important.
- See table 2 and 3 in "Any other information on results" for the results of the different parameters described above.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related effects were detected in the urinalytical values of all treated animals.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant changes in the behavioural parameters measured. Open-field assessments showed an isolated incident of pilo erection and tiptoe gait in one 6000 ppm male during Week 12. In the absence of similar effects detected during the daily clinical observations or in animals of either sex treated at 20000 ppm these observations were considered not to be indicative of systemic toxicity.
There were no toxicologically significant changes in functional performance parameters measured. Males treated with 20000 ppm showed a statistically significant increase in the overall mobility (P<0.05) whilst females from this treatment group showed statistically significant reductions in the final 20% of activity (P<0.01) and overall activity (P<0.01). In the absence of any supporting clinical observations to suggest a neurotoxic effect, these findings were considered to be of no toxicological significance. Females from all treatment groups showed statistically significant reductions in overall mobility (P<0.05 or P<0.01). In the absence of a dose-related response or any supporting clinical observations to suggest a neurotoxic effect, these findings were considered to be of no toxicological significance.
There were no treatment related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and were of no toxicological importance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- All treated males showed a statistically significant increase in kidney (P<0.05 or P<0.01) weight, both absolute and relative to terminal body weights. This is a result of the accumulation of the alpha 2μ-microglobulin, which is unique to the mature male rat and has no relevance for other species including man. Males treated with 20000 ppm also showed a statistically significant increase in adrenal (P<0.01) and relative liver (P<0.01) weight. These effects are considered to be adaptive rather then representing a serious adverse effect. No toxicologically significant effects were detected in females from any treated group.
- All treated males showed statistically significant reductions in pituitary (P<0.05) and prostate (P<0.05) weight, both absolute and relative to terminal body weights whilst females treated with 6000 or 200 ppm showed a statistically significant reduction in liver (P<0.05 or P<0.01) weight, both absolute and relative to terminal body weights. In the absence of a true dose related response or any histopathological correlates these findings were considered not to be of toxicological significance. The statistically significant reduction detected in males treated with 20000 ppm in absolute liver weight was considered of no toxicological importance due to the significantly reduced size of high dose animals when compared to the control group.
- See table 4 in "Any other information on results" for the results of the relative organs weight for the different organs described above.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Three males treated with 20000 ppm had pale kidneys at necropsy. No such effects were detected in females treated with 20000 ppm or animals of either sex treated with 6000, 2000 or 200 ppm. One male treated with 2000 ppm had flaccid and small testes at necropsy. In the absence of any supportive histopathological correlations to suggest a systemic toxic effect of the test item, these findings were considered to be of no toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Kidneys: Hyaline droplets nephropathy was found in all treated males. The alpha 2μ-globulin nature of this finding was confirmed by additional Mallory’s Heidenhain staining performed on male kidneys. The nephropathy consisted of increased incidence and mean severity of renal tubular degeneration/ regeneration in all males treated with 20000, 6000 or 2000 ppm and in most males treated with 200 ppm, along with elevated mean severity degrees of tubular hyaline droplets. These observed effects are due to the accumulation of the alpha 2μ-microglobulin, which is unique to the mature male rat. Therefore, this finding has no relevance for other species including man.
- Liver: Centrilobular to midzonal hepatocellular hypertrophy was recorded at minimal severity degrees in males treated with 20000, 6000 or 2000 ppm. Elevated incidence and severity of mostly diffuse vacuolation was found in males from all treatment groups, this vacuolation did not exceed slight severity degrees. As the histopathological changes of hepatocellular hypetrophy and vacuolation did not exceed minimal or slight severity limits respectively, these effect are considered to be adaptive rather then representing a serious adverse effect.
- Adrenals: Increased incidence in vacuolation of the zona fasciculata was recorded in males from all treatment groups. This finding may be regarded as an adaptive effect occurring as a stress response to the physiological changes observed.
- All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
Other effects:
no effects observed
Description (incidence and severity):
Oestrus Cycle Assessments: There were no treatment related effects on treated female oestrus cycles or proportion of females with anomalous oestrus cycles when compared to the control group.
Sperm Analysis: There were no treatment related effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 504.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Group Mean Weekly Body Weight Values (Mean ± SD)

 

Dose (ppm)

Day 1

Day 8

Day 22

Day 36

Day 50

Day 64

Day 78

Day 91

Male

0

232.2±18.0

300.2±26.3

379.9±36.3

446.5±48.1

473.1±51.3

525.9±66.5

561.2±75.5

572.2±76.4

 

200

225.2±18.0

293.7±25.0

383.0±38.9

456.3±44.8

482.7±48.0

536.8±55.1

572.0±59.1

579.4±63.5

 

2000

216.2±10.8

281.1±16.2

359.1±32.0

417.6±45.7

447.8±49.7

491.3±59.8

522.3±60.5

524.9±62.5

 

6000

216.7±11.6

273.8±14.3

351.7±23.2

410.3±28.7

436.7±34.3

487.6±40.1

520.7±39.8

527.2±44.0

 

20000

219.5±14.4

262.0±16.6

333.9±24.5

387.6±29.5

407.3±33.0

450.8±39.5

475.4±40.1

480.2±41.9

Female

0

178.6±14.8

204.0±17.9

240.2±18.0

266.1±22.6

272.3±21.4

295.1±28.5

306.6±22.8

305.2±21.7

 

200

183.8±16.9

215.5±20.3

259.2±30.8

289.1±38.5

294.2±39.8

308.8±35.9

331.4±41.4

329.2±40.8

 

2000

181.8±9.4

209.3±8.5

248.1±8.7

273.5±8.4

278.8±7.7

300.4±10.2

316.8±11.9

312.3±12.6

 

6000

179.8±11.2

203.5±13.4

239.8±17.2

266.3±22.2

272.7±22.5

293.3±27.4

308.4±27.9

304.5±28.0

 

20000

180.6±9.0

194.9±10.5

224.9±17.8

245.8±18.8

244.0±16.4

265.2±18.9

274.4±20.2

269.8±20.7

* = P<0.05; ** = P<0.01

 

Table 2: Group Mean Blood Chemical Values Week 7 (Mean ± SD)

 

Dose (ppm)

Sodium (mmol/l)

Chloride (mmol/l)

ASAT (IU/l)

ALAT (IU/l)

Cholesterol (mg/dl)

Bile acid (µmol/l)

Male

0

150.5±1.7

101.8±1.6

70.6±11.0

51.8±6.5

67.7±7.4

10.32±5.73

 

200

147.9±2.8

100.4±1.3

69.0±4.6

53.1±10.4

69.9±11.6

8.32±4.64

 

2000

149.8±2.5

100.9±2.1

71.1±9.5

47.9±6.1

69.0±11.2

7.28±4.19

 

6000

148.6±2.9

100.5±1.5*

67.0±5.6

48.7±8.3

74.3±12.0

6.73±2.30

 

20000

149.8±1.7

99.8±1.4**

62.2±8.2*

47.4±7.7

82.2±8.2**

6.90±5.14

Female

0

148.9±1.8

101.9±1.3

80.3±18.3

49.7±11.9

80.3±20.7

13.87±12.18

 

200

147.6±2.5

102.1±1.7

72.2±7.5

50.1±8.0

80.5±14.7

7.42±3.47

 

2000

147.9±2.0

101.6±1.6

73.2±9.9

51.4±10.4

81.6±17.2

10.31±7.44

 

6000

148.4±1.7

102.0±1.9

73.7±8.7

47.8±7.7

80.9±15.2

8.69±4.87

 

20000

148.9±2.0

102.8±2.0

64.0±5.9**

44.7±9.1

84.9±19.3

12.60±13.05

* = P<0.05; ** = P<0.01

 

Table 3: Group Mean Blood Chemical Values Week 13 (Mean ± SD)

 

Dose (ppm)

Sodium (mmol/l)

Chloride (mmol/l)

ASAT (IU/l)

ALAT (IU/l)

Cholesterol (mg/dl)

Bile acid (µmol/l)

Male

0

151.5±2.4

103.2±2.0

67.7±8.8

52.9±6.5

77.0±14.5

12.72±7.34

 

200

149.9±2.6

102.9±1.6

64.0±7.7

49.3±7.6

83.0±10.7

15.18±8.43

 

2000

150.4±1.7

103.1±1.2

63.3±6.2

49.2±6.6

76.2±13.6

17.64±10.03

 

6000

150.3±1.6

102.3±2.2

59.9±5.2**

43.4±6.1**

83.6±20.3

17.06±10.40

 

20000

149.2±1.1*

101.6±1.0**

51.6±4.1**

33.9±3.8**

91.6±10.6*

6.30±3.07*

Female

0

148.2±2.1

103.3±1.3

66.2±16.9

48.9±12.7

87.3±15.3

24.56±23.23

 

200

149.1±1.5

103.5±1.4

70.5±10.7

52.6±8.8

81.7±11.9

15.47±9.83

 

2000

148.5±2.0

102.9±1.4

72.5±30.3

49.9±13.8

83.8±18.3

24.88±15.20

 

6000

149.7±2.0

102.9±1.6

61.3±6.3

49.9±8.0

88.7±19.3

23.51±15.01

 

20000

148.7±1.3

102.5±1.6

59.8±9.6

42.3±10.4

91.7±16.6

51.10±48.12

* = P<0.05; ** = P<0.01

 

Table 4: Group Mean Relative (% of Body Weight) Organ Weights (Mean ± SD)

 

Dose (ppm)

Adrenals

Kidney

Liver

Pituitary

Prostate

Male

0

0.012±0.002

0.596±0.032

3.105±0.242

0.002±0.000

0.154±0.034

 

200

0.012±0.001

0.648±0.025*

3.096±0.211

0.002±0.000*

0.128±0.034*

 

2000

0.012±0.002

0.713±0.052**

2.899±0.168

0.002±0.000*

0.134±0.028*

 

6000

0.014±0.003

0.700±0.061**

3.125±0.263

0.002±0.000*

0.144±0.028*

 

20000

0.017±0.004*

0.772±0068**

3.637±0.182**

0.002±0.000*

0.142±0.039*

Female

0

0.028±0.005

0.639±0.067

3.356±0.233

0.005±0.001

 

 

200

0.025±0.004

0.649±0.048

3.069±0.283**

0.005±0.001

 

 

2000

0.027±0.003

0.653±0.036

3.208±0.197

0.006±0.001

 

 

6000

0.026±0.004

0.674±0.042

3.161±0.138*

0.005±0.002

 

 

20000

0.030±0.005

0.683±0.051

3.423±0.181

0.005±0.001

 

* = P<0.05; ** = P<0.01

Applicant's summary and conclusion

Conclusions:
In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. The NOAEL could be established as the highest dose tested, 20000 ppm or 1500 mg/kg bw/day, under the conditions of this test.
Executive summary:

Introduction and method

In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. The dose levels were 0, 200, 2000, 6000, and 20000 ppm, corresponding to 0, 15.3, 154.9, 464.1, and 1504.6 mg/kg bw/day, respectively. Clinical signs, functional observations, body weight change, dietary intake, and water consumption were monitored during the study. During week 7 and at the end of the study, haematology, blood chemistry and urinalysis were evaluated. Furthermore, oestrus cycle assessment was performed on female animals between week 6 and 7 and week 12 and 13. Opthalmoscopic examination was performed at the start and end of the study in control and high-dose animals. At the end of the study, all animals were subjected to gross necropsy examination and histopathological examination of tissues was performed on animals from control and high-dose. Furthermore, sperm assessment was performed on males at necropsy.

Results

Clinical signs: Reduced overall body weight was observed in male and female animals in the highest dose group, correlated with reduced food consumption and adverse effects on food efficiency, indicating decreased food palatability rather than an adverse effect of the test substance.

Haematology: No effects were found.

Blood biochemistry: Blood chemical investigations showed reductions in chloride and sodium concentration of 1 -5%, which is considered minimal. The minimal decrease in ASAT and ALAT levels is not toxicological relevant. The reduction and increase in bile acids in males and females, respectively, is not considered toxicologically relevant either. The increases in cholesterol in males treated with either 20000 or 6000 ppm are most likely linked to the liver weight findings and the histopathological effects seen in this organ and can be considered of an adaptive nature.

Organ weights and histopathological findings: Relative liver weight is increased in males only up to 17% with slight adaptive and non-adverse centrilobular to midzonal hepatocellular hypertrophy in males only. Increase in adrenal weights, both absolute and relative to terminal body weights detected in males treated with 20000 ppm were correlated with histopathological findings of the adrenal gland. Microscopic examinations revealed increased incidence in vacuolation of the zona fasciculata in all treated males. This finding may be regarded as an adaptive effect occurring as a stress response to the physiological changes observed. Macroscopic examinations also revealed effects on the kidney such as an increase in kidney weights and hyaline droplets nephropathy in all treated males. The hyaline droplets can be directly linked to accumulation of the alpha-2-microglobulin, which is unique to the mature male rat, therefore, this finding is adverse for the male rat but has no relevance for other species including man. No other treatment-related effects were observed.

Conclusion: Under the conditions of the test no toxicologically significant effects were observed both in males and females. Therefore the NOAEL is at the highest dose tested, 20000 ppm or 1500 mg/kg bw/day.