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Administrative data

Description of key information

Subchronic toxicity (OECD 408, GLP) oral:

NOAEL (rat): 300 and 100 mg/kg bw/day in males and females, respectively

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 May - 05 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 138 to 176 g (males), 118 to 147 g (females)
- Housing: on arrival and following randomization, 5 animals of the same sex and same dosing group in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. During motor activity measurements, animals did not have access to food for a maximum of 2 h.
- Water: municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: analysis were performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 46 - 62
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 May 2018 To: 29 August 2018
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 h after adding vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (1.125) and test item (1.1 at 25 °C). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the test facility to select the suitable vehicle and to establish a suitable formulation procedure. Polyethylene glycol 400 (Merck, Darmstadt, Germany) was identified as suitable vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week 1, 6 and 13 for homogeneity. For homogeneity analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%. In addition, stability was tested in samples collected in week 1. Stability of the test item in the vehicle was determined over 5 h at room temperature under normal laboratory light conditions (lowest and highest concentration). Duplicate sets of each sample (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ± 10% of the concentration determined by the initial analysis of each formulation. For concentration analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory.Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Analysis showed that concentrations analyzed were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).
Duration of treatment / exposure:
91 (males) or 92 (females) days
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on the results of the range finding prenatal developmental study (Test Facility Study No. 20148926) and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry:
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on day 1 of treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning on day 1 of treatment until day of necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: day - 5 and then weekly (before dosing), starting on day 1

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: No

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: No

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pre-treatment and at the end of the dosing period in Week 13
- Dose groups that were examined: during pre-treatment in all animals, and at the end of the dosing in vehicle and 1000 mg/kg bw/day dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 in the "Any other information and methods incl. tables" section were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 in the "Any other information and methods incl. tables" section were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13
- Dose groups that were examined: 5 animals per dose group, all dose groups
- Battery of functions tested: grip strength / motor activity / other: hearing ability

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 in the "Any other information and methods incl. tables" section.)

HISTOPATHOLOGY: Yes (see table 3 in the "Any other information and methods incl. tables" section.)
Other examinations:
Estrous Cycle Determination
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
- Time schedule for examinations: week 11 (Day 71) up to and including week 14 (Day 92)
- Dose groups that were examined: females of all dose groups

Sperm analysis
For all surviving males, the following assessments were performed:
1. From all males, sperm samples were taken from the proximal part of the vas deferens (right) at necropsy:
- Sperm motility was assessed from all samples.
- Sperm smears for morphological evaluation were fixed from all samples. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all males of the control and high-dose group.
2. One testis and one epididymis (left) from all males were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for males of the control and high dose group.

Thyroid homone analysis:
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 in the "Any other information and methods incl. tables" section were examined.





Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Variables for inferential analysis: body Weight and body weight gain, food consumption, organ weights, organ weight relative to body weight
Parametric/non parametric tests used: Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F test when Levene’s test was not significant or the Kruskal-Wallis test when group variances were not homogeneous. When the overall F test or Kruskal-Wallis test was significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with 2 groups (the designated control group and 1 other group) were compared using a t-test if Levene’s test was not significant or Wilcoxon Rank-Sum test if it was.

The following pairwise comparisons were made: Low dose group (2) vs. Vehicle dose group (1), Mid dose group (3) vs. Vehicle dose group (1), High dose group (4) vs. Vehicle dose group (1)

Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.



Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100, 300 and 1000 mg/kg bw/day: salivation and ploughing was noted in all animals. For salivation a dose-related trend in severity (up to severe on several occasions in high-dose rats) and time of onset (from day 8 in low-dose rats and from day 1 or 2 at the higher dose levels) was observed. The salivation was considered to be a physiological response and/or behavioural sign of discomfort in reaction to the bolus administration of the test item rather than a sign of systemic toxicity considering its severity (mostly slight or moderate) and the time of occurrence (i.e. within half an hour after dosing).

All other clinical signs noted occurred incidentally and represented normal background findings. At the incidence observed, these findings were considered not to be treatment-related. (Please refer to table 4 in the "Any other information on results incl. tables" section.)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1000 mg/kg bw/day: two females died unscheduled. One female was found dead in the morning of study day 58. Prior to death, this animal showed no clinical signs of conditional decline and body weight development was normal. Possibly, its death could be related to the gavage procedure, however, this could not be confirmed by macroscopic or microscopic findings, due to cannibalism. The death was considered likely to be procedure-related rather than test-item related. Another female died during blood sampling on the scheduled day of necropsy. This death was considered to be related to the blood sampling procedure under anesthesia and was therefore regarded to be unrelated to treatment with the test item.

300 mg/kg bw/day: one females was euthanized for humane reasons on day 54 of the study, based on deep breathing and weight loss (9% between days 50-54). At macroscopic examination, red foci (multifocal) were noted in the glandular stomach. Microscopic examination showed marked mixed cell inflammation in the bronchus and moderate neutrophilic infiltration in the lumen of the larynx. These microscopic findings and the sudden onset of the moribundity suggested that the conditional decline was related to the gavage procedure rather than being test-item related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: males gained slightly less weight than controls in most weeks of the study (reaching statistical significance in weeks 4 and 9 only). The slight non statistically significant differences in body weights gradually increased up to a mean value of 8 % lower for the high dose animals at the end of the treatment period (non-adverse, due to slight nature of the effect). (Please refer to table 5 in the "Any other information on results incl. tables" section.) Body weights and body weight gain of females showed no treatment-related changes.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: males consumed slightly less food (about 6-9%) than controls on several time periods during the study (weeks 1-2 and the second half of the 13-week treatment period) (non-adverse, due to slight nature of the effect). (Please refer to table 6 in the "Any other information on results incl. tables" section.) Food consumption of treated females was similar to that of controls over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Vehicle, 100, 300 and 1000 mg/kg bw/day: cornea opacity, cornea edema and cornea, opacity multifocal, pinpoint ocurred at similar incidences in all dose groups or only in single animals and were thus considered incidental, non-treatment-related and non-adverse. (Please refer to table 7 in the "Any other information on results incl. tables" section.) Ophthalmic examination of control and high-dose rats in the last week of the treatment period showed no test-item related ocular changes. The incidences of the findings did not indicate a relation with treatment and both the nature and incidence of the findings occurred within the normal range for rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day:
lower number of red blood cells in males (-10%) and females (-13%) (statistically significant),
lower mean corpuscular haemoglobin concentration (MCHC) in males (-9%) and females (-5%) (statistically significant),
lower red blood cell distribution width (RDW) in males (-7%)(statistically significant),
higher mean corpuscular haemoglobin (MCH) in females (+7%) (statistically significant),
higher mean corpuscular volume (MCV) in males and females (both +13%)(statistically significant), lower haemoglobin concentration in males (-9%) and females (-6%) (not statistically significant),
higher number of reticulocytes in males (+37%) and females (+30%) (not statistically significant)

All changes were regarded as non-adverse because of the minor magnitude of the change.

100 and 300 mg/kg bw/day: decreased prothrombin time in males (statistically significant). This finding was considered non-treatment-related and non-adverse due to the lack of a dose-related response.

100 mg/kg bw/day: higher MCHC in females (statistically significant). This isolated finding was considered non-treatment-related and non adverse due to the lack of a dose-related response.

(Please refer to table 8 in the "Any other information on results incl. tables" section.)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day:
higher alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in males (+51% and +39%, respectively) (statistically significant),
higher alkaline phosphatase (ALP) in males (+54%) and females (+46%) (statistically significant),
higher bile acids in males (2.1-fold) (statistically significant). A higher mean bile acids concentration was also noted in females, but the difference from controls (1.9 fold) was not statistically significant and could be explained by the high concentration in a single animal,
lower cholesterol in males (-32%) and females (-28%) (statistically significant),
lower fasting glucose in males (-22%), (statistically significant),
higher creatinine in males (+7%)(statistically significant),
higher potassium in females (+10%).(statistically significant)
lower calcium in females (-3%) (statistically significant)

300 and 1000 mg/kg bw/day:
higher chloride in males (+2%) and (+3%) at the mid an high dose, respectively (statistically significant),
higher potassium in males (statistically significant, non-treatment-related, missing dose-related response)

100 and 300 mg/kg bw/day: higher sodium levels in males and females (statistically significant, non-treatment-related, missing dose-related response)

100 mg/kg bw/day: higher chloride in females (statistically significant, non-treatment-related, missing dose-related response)

Decreases noted in cholesterol (both sexes) and fasting glucose (males only) may also be related to the effect of
the test item on the liver. Slightly higher creatinine, chloride and potassium, and slightly lower calcium, mostly at 1000 mg/kg/day were not associated with adverse anatomic pathology alterations and therefore regarded as non-adverse.

(Please refer to table 9 in the "Any other information on results incl. tables" section.)
Urinalysis findings:
not examined
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):
1000 mg/kg bw/day: higher liver weights (absolute and relative to body weight) were noted in females (statistically significant, treatment-related, adverse), slight increase in relative kidney weights in both sexes (statistically significant, but not considered treatment-related,result of variations in terminal body weights), lower mean uterus weights (absolute and relative to body weight) in females (about -40%, not statistically significant) were considered to reflect normal physiological variation (non-treatment-related)

300 and 1000 mg/kg bw/day: increased heart weights in in mid dose males and high dose females (statistically significant, but not considered treatment-related, result of variations in terminal body weights). In the absence of a dose-related trend, the lower relative heart weight in males was regarded a chance finding.

(Please refer to table 10, 11 and 12 in the "Any other information on results incl. tables" section.)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic findings were within the range of background gross observations encountered in rats of this age and strain and were therefore considered non-treatment-related. (Please refer to table 13 in the "Any other information on results incl. tables" section.)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: centrilobular hypertrophy at minimal to mild degree and periportal hepatocellular vacuolation at minimal degree in the liver (males), increased severity of pigment accumulation in the spleen (both sexes) and increased incidence and severity of diffuse vacuolation in the zona glomerulosa of the adrenal gland (both sexes) (treatment-related, non-adverse). Non-adversity was based on the nature of the finding, the low severity and the absence of any additional degenerative or proliferative changes.

300 and 1000 mg/kg bw/day: in mid dose females and high dose males and females adverse microscopic changes were observed in the liver and consisted of an increased incidence and severity (up to mild degree) of centrilobular mononuclear cell infiltrates accompanied by single cell necrosis as well as increased liver weighs (by about 20%) (treatment-related, adverse).

100 and 300 mg/kg bw/day: centrilobular mononuclear cell infiltration with single cell necrosis was observed in males of the low and mid dose group and in females of the low dose group (treatment-related, non-adverse). Non-adversity was based on the low severity/incidence of the lesions in these dose groups.

All other microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test substance-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations. (Please refer to table 14 in the "Any other information on results incl. tables" section.)
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Neurobehavioural examination - functional tests (effects observed non-treatment-related):
1000 mg/kg bw/day: one male and one female showed low values for total movements and ambulations (below the normal ranges). Habituation profiles of these animals were normal and their lower motor activity was not associated with corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Moreover, all other high-dose animals showed normal motor activity. Therefore, the lower activity recorded for these two animals was not attributed to treatment.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In addition, forelimb and hind limb grip strength showed no treatment-related changes. (Please refer to table 15 and 16 in the "Any other information on results incl. tables" section.)

Estrous cycle determination (effects observed non-treatment-related):
1000 mg/kg bw/day: one female had irregular cycles of, subsequently, 6 and 2 days. Since irregular cycles were noted in only one female and adjacent cycles were normal, this finding was considered non-treatment-related.

Sperm analysis (effects observed non-treatment-related):
1000 mg/kg bw/day: epididymal sperm count was 35% higher compared to the control mean. Since the difference was not statistically significant and was atrributed to low counts in two control males, this finding was not considered treatment-related. (Please refer to table 17 in the "Any other information on results incl. tables" section.)

Thyroid homone analysis:
1000 mg/kg bw/day: T4 levels were lower compared to the control levels (-14% and -22% in males and females, respectively) Since statistical significance was not achieved and T4 levels in almost all high dose animals remained in the concurrent control range, these intergroup differences were considered non-treatment-related. (Please refer to table 9 in the "Any other information on results incl. tables" section.)
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effect observed at this dose level
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect observed at this dose level
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 4: Summary of clinical observations

Dose groups (mg/kg bw/day)

Male

 

Female

Observations

From Day 1 to 93

Vehicle

100

 

300

1000

 

Vehicle

100

 

300

1000

 

eyeball, enlarged

 

 

 

 

 

 

 

 

number of animals affected

1

0

0

0

0

0

0

0

number of times recorded

2

0

0

0

0

0

0

0

% of affected animals

10

0

0

0

0

0

0

0

first to last seen

79 - 86

-

-

-

-

-

-

-

skin, lesion

 

 

 

 

 

 

 

 

number of animals affected

2

2

2

1

0

0

0

1

number of times recorded

4

3

10

5

0

0

0

2

% of affected animals

20

20

20

10

0

0

0

10

first to last seen

9 - 44

9 - 16

9 - 51

9 - 37

-

-

-

23 - 30

breathing, deep

 

 

 

 

 

 

 

 

number of animals affected

0

0

0

0

0

0

1

0

number of times recorded

0

0

0

0

0

0

1

0

% of affected animals

0

0

0

0

0

0

10

0

first to last seen

-

-

-

-

-

-

54 - 54

-

salivation

 

 

 

 

 

 

 

 

number of animals affected

1

10

10

10

2

10

10

10

number of times recorded

1

839

900

826

2

848

872

879

% of affected animals

10

100

100

100

20

100

100

100

first to last seen

62 - 62

8 - 91

2 - 91

1 - 91

54 - 62

7 - 92

1 - 92

1 - 92

skin, scab

 

 

 

 

 

 

 

 

number of animals affected

0

1

0

0

0

0

0

0

number of times recorded

0

2

0

0

0

0

0

0

% of affected animals

0

10

0

0

0

0

0

0

first to last seen

-

6 - 7

-

-

-

-

-

-

ploughing

 

 

 

 

 

 

 

 

number of animals affected

0

10

10

10

0

10

10

10

number of times recorded

0

839

840

763

0

845

811

815

% of affected animals

0

100

100

100

0

100

100

100

first to last seen

-

8 - 91

8 - 91

8 - 91

-

8 - 92

8 - 92

8 - 92

 

Table 5: Summary of body weight gains (g)

Males

 

Days - Change

 

Dose group (mg/kg bw/day)

 

-5-1

1-8

8-15

15-22

22-29

29-36

36-43

 

Vehicle

Mean

34.30

45.30

43.10

32.80

27.70

20.30

13.50

 

 

SD

3.47

4.19

5.93

5.25

4.11

3.27

4.77

 

 

N

10

10

10

10

10

10

10

 

100

Mean

35.00

46.70

45.80

36.80

23.40

21.20

11.50

 

 

SD

2.62

3.77

5.67

4.54

5.42

4.32

2.80

 

 

N

10

10

10

10

10

10

10

 

300

Mean

36.20

48.90

42.40

36.70

26.50

21.30

17.50

 

 

SD

3.12

4.38

5.78

5.89

2.72

4.22

5.02

 

 

N

10

10

10

10

10

10

10

 

1000

Mean

32.20

41.50

42.67

29.89

20.33b

16.22

14.33

 

 

SD

6.51

6.31

3.91

3.10

4.69

5.29

7.84

 

 

N

10

10

9

9

9

9

9

 

Males

 

Days - Change

Dose group

 

43-50

50-57

57-64

64-71

71-78

78-85

85-91

 

Vehicle

Mean

11.90

10.00

9.20

12.80

9.90

6.50

4.40

 

 

SD

3.57

5.58

4.71

4.85

3.41

5.23

3.63

 

 

N

10

10

10

10

10

10

10

 

100

Mean

11.60

10.00

11.00

10.30

8.10

5.10

10.20

 

 

SD

5.27

3.56

2.26

4.35

2.60

3.87

8.77

 

 

N

10

10

10

10

10

10

10

 

300

Mean

11.60

13.00

11.00

10.00

10.20

6.90

5.20

 

 

SD

3.66

4.88

4.45

3.43

3.05

3.03

3.26

 

 

N

10

10

10

10

10

10

10

 

1000

Mean

10.11

9.56

4.44a

8.22

7.78

4.11

2.78

 

 

SD

5.25

5.92

3.54

5.36

3.67

3.62

5.70

 

 

N

9

9

9

9

9

9

9

 

Females

 

Days - Change

Dose group

 

-5-1

1-8

8-15

15-22

22-29

29-36

36-43

 

Vehicle

Mean

19.20

21.00

20.40

14.00

10.50

8.60

4.70

 

 

SD

3.58

4.92

4.62

3.65

4.84

4.30

3.86

 

 

N

10

10

10

10

10

10

10

 

100

Mean

18.40

23.20

18.60

15.10

11.20

11.30

2.80

 

 

SD

2.41

3.74

3.27

5.40

4.18

3.02

5.83

 

 

N

10

10

10

10

10

10

10

 

300

Mean

15.80

23.20

17.10

14.20

8.30

8.90

5.50

 

 

SD

2.70

5.98

4.89

3.08

3.40

5.67

5.21

 

 

N

10

10

10

10

10

10

10

 

1000

Mean

19.70

23.00

18.60

14.80

7.90

10.10

6.40

 

 

SD

3.74

4.62

3.44

4.83

2.88

1.91

2.37

 

 

N

10

10

10

10

10

10

10

 

Females

 

Days - Change

Dose group

 

43-50

50-54

50-57

57-64

64-71

71-78

78-85

85-91

Vehicle

Mean

4.10

--

5.10

3.60

5.50

4.10

3.50

3.10

 

SD

5.38

--

3.03

3.37

3.72

3.90

4.72

3.25

 

N

10

--

10

10

10

10

10

10

100

Mean

6.20

--

5.40

4.40

4.40

4.00

1.40

3.40

 

SD

7.63

--

5.02

4.60

4.12

6.51

3.86

3.63

 

N

10

--

10

10

10

10

10

10

300

Mean

4.40

-22 .00z

6.44

4.67

6.33

2.00

3.44

2.22

 

SD

7.52

--

5.77

5.34

2.78

4.30

4.48

3.93

 

N

10

1

9

9

9

9

9

9

1000

Mean

5.30

--

4.70

1.89

4.89

2.11

3.00

-0 .67

 

SD

5.31

--

3.47

4.40

3.14

5.93

2.74

4.42

 

N

10

--

10

9

9

9

9

9

Significantly different from control group 1 value :a=p≤0.05,b=p≤0.01 (Dunnett)

Group excluded from statistical analysis (N<3): z

 

Table 6 Summary of food consumption (g/animal/day)

Males

 

Days (From – To)

Dose group (mg/kg bw/day)

 

1-8

8-15

15-22

22-29

29-36

36-43

43-50

Vehicle

Mean

21.85y

24.45y

25.25y

25.30y

25.45y

24.60y

24.40y

 

SD

0.07

0.21

0.21

0.28

0.21

0.57

0.42

 

N

2

2

2

2

2

2

2

100

Mean

21.15

23.70

25.00

25.40

24.65

23.35

23.10

 

SD

0.49

0.14

0.14

0.14

0.07

0.49

1.41

 

N

2

2

2

2

2

2

2

 

% Diff G1

-3 .20

-3 .07

-0 .99

0.40

-3 .14

-5 .08

-5 .33

300

Mean

21.30

23.25

24.65

25.10

24.95

24.40

24.25

 

SD

1.27

1.06

0.92

0.71

1.34

1.98

1.91

 

N

2

2

2

2

2

2

2

 

% Diff G1

-2 .52

-4 .91

-2 .38

-0 .79

-1 .96

-0 .81

-0 .61

1000

Mean

20.10

22.70

24.45

24.65

24.45

23.75

22.70

 

SD

--

--

0.49

1.06

0.35

0.21

0.00

 

N

1

1

2

2

2

2

2

 

% Diff G1

-8 .01

-7 .16

-3 .17

-2 .57

-3 .93

-3 .46

-6 .97

Males

 

Days (From – To)

 

 

 

 

 

 

Dose group

 

50-57

57-64

64-71

71-78

78-85

85-91

 

Vehicle

Mean

23.80y

23.40y

23.75y

23.50y

22.70y

23.15y

 

 

SD

0.85

0.85

0.49

0.99

1.27

0.78

 

 

N

2

2

2

2

2

2

 

100

Mean

23.65

22.40

22.70

22.55

21.80

22.45

 

 

SD

0.35

0.28

0.28

0.07

0.14

0.49

 

 

N

2

2

2

2

2

2

 

 

 

-0 .63

-4 .27

-4 .42

-4 .04

-3 .96

-3 .02

 

300

Mean

23.70

22.90

23.50

23.10

22.75

22.95

 

 

SD

2.26

2.55

2.26

2.26

1.63

2.33

 

 

N

2

2

2

2

2

2

 

 

 

-0 .42

-2 .14

-1 .05

-1 .70

0.22

-0 .86

 

1000

Mean

23.15

22.05

22.20

22.10

21.45

22.55

 

 

SD

0.35

0.21

0.28

0.14

0.35

1.20

 

 

N

2

2

2

2

2

2

 

 

 

-2 .73

-5 .77

-6 .53

-5 .96

-5 .51

-2 .59

 

Females

 

Days (From – To)

Dose group

 

1-8

8-15

15-22

22-29

29-36

36-43

43-50

Vehicle

Mean

15.60y

16.30y

16.35y

16.45y

16.20y

15.80y

16.60y

 

SD

0.42

0.57

0.21

0.07

0.14

0.14

0.14

 

N

2

2

2

2

2

2

2

100

Mean

15.40

16.05

16.30

16.55

16.50

16.25

16.75

 

SD

0.14

0.35

0.28

0.07

0.57

0.92

0.78

 

N

2

2

2

2

2

2

2

 

 

-1 .28

-1 .53

-0 .31

0.61

1.85

2.85

0.90

300

Mean

15.35

16.05

16.40

16.60

16.60

16.50

17.15

 

SD

0.07

0.21

0.14

0.14

0.28

0.14

0.07

 

N

2

2

2

2

2

2

2

 

 

-1 .60

-1 .53

0.31

0.91

2.47

4.43

3.31

1000

Mean

15.55

16.25

16.55

16.70

17.00

17.05

16.50

 

SD

0.35

0.35

0.21

1.13

0.28

0.35

1.13

 

N

2

2

2

2

2

2

2

 

 

-0 .32

-0 .31

1.22

1.52

4.94

7.91

-0 .60

Females

 

Days (From – To)

Dose group

 

50-57

57-64

64-71

71-78

78-85

85-91

 

Vehicle

Mean

16.40y

15.75y

16.20y

16.25y

15.85y

16.10y

 

 

SD

0.28

0.35

0.28

0.49

0.78

0.57

 

 

N

2

2

2

2

2

2

 

100

Mean

16.20

16.00

16.15

16.70

15.55

16.15

 

 

SD

0.71

0.99

1.06

0.85

0.78

0.78

 

 

N

2

2

2

2

2

2

 

 

 

-1 .22

1.59

-0 .31

2.77

-1 .89

0.31

 

300

Mean

16.55

16.50

16.50

16.65

16.15

16.70

 

 

SD

0.78

0.00

0.57

0.21

0.21

0.57

 

 

N

2

2

2

2

2

2

 

 

 

0.91

4.76

1.85

2.46

1.89

3.73

 

1000

Mean

16.65

15.35

16.10

15.95

15.70

16.25

 

 

SD

0.35

1.34

0.00

0.07

0.57

0.78

 

 

N

2

2

2

2

2

2

 

 

 

1.52

-2 .54

-0 .62

-1 .85

-0 .95

0.93

 

All Groups excluded from statistical analysis (N<3 in Control): y

 

Table 7: Summary of ophthalmoscopy findings

 

Males

Females

Findings

Vehicle

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Vehicle

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Cornea, opacity

6

1

 

3

1

1

1

1

Cornea edema

2

2

2

1

1

1

1

 

Cornea, opacity multifocal, pinpoint

 

 

2

1

 

 

 

 

 

Table 8: Summary of haematology and coagulation values

End Of Treatment

Dose groups (mg/kg bw/day)

Control

100

300

1000

Males

 

WBC

mean

6.6

7.1

6.1

6.2

10E9/L

st.dev

1.7

1.2

0.9

0.9

 

n

10

10

10

9

Neutrophils

mean

0.9

1.2

1.0

1.1

10E9/L

st.dev

0.4

0.2

0.1

0.4

 

n

10

10

10

9

Lymphocytes

mean

5.5

5.7

4.9

4.9

10E9/L

st.dev

1.4

1.1

0.9

0.9

 

n

10

10

10

9

Monocytes

mean

0.1

0.1

0.1

0.2

10E9/L

st.dev

0.0

0.1

0.0

0.1

 

n

10

10

10

9

Eosinophils

mean

0.1

0.1

0.1

0.1

10E9/L

st.dev

0.0

0.0

0.0

0.0

 

n

10

10

10

9

Basophils

mean

0.0

0.0

0.0

0.0

10E9/L

st.dev

0.0

0.0

0.0

0.0

 

n

10

10

10

9

Red blood cells

mean

9.41

9.23

9.38

8.45 **

10E12/L

st.dev

0.27

0.28

0.31

0.47

 

n

10

10

10

9

Reticulocytes

mean

178.8

199.5

172.2

245.0

10E9/L

st.dev

21.9

19.5

19.3

64.3

 

n

10

10

10

9

RDW

mean

12.3

12.9

12.2

11.4 *

%

st.dev

0.5

0.9

0.6

0.7

 

n

10

10

10

9

Haemoglobin

mean

10.2

10.1

10.0

9.3 **

mmol/L

st.dev

0.3

0.3

0.3

0.4

 

n

10

10

10

9

Haematocrit

mean

0.510

0.499

0.496

0.514

L/L

st.dev

0.015

0.010

0.008

0.025

 

n

10

10

10

9

MCV

mean

54.2

54.1

52.9

61.0 **

fL

st.dev

1.7

1.2

1.2

3.7

 

n

10

10

10

9

MCH

mean

1.08

1.09

1.07

1.10

fmol

st.dev

0.04

0.04

0.02

0.03

 

n

10

10

10

9

MCHC

mean

19.95

20.18

20.17

18.14 **

mmol/L

st.dev

0.25

0.43

0.32

0.61

 

n

10

10

10

9

Platelets

mean

684

740

717

675

10E9/L

st.dev

109

43

68

73

 

n

10

10

10

9

PT

 

mean

 

17.8

 

17.0 **

 

17.2 *

 

18.0

s

st.dev

0.5

0.4

0.5

0.3

 

n

9

8

8

6

APTT

mean

20.8

20.1

20.8

20.1

s

st.dev

1.6

2.1

1.6

0.9

 

n

10

9

10

9

Females

 

 

 

 

 

WBC

 

mean

 

3.9

 

3.8

 

3.9

 

4.6

10E9/L

st.dev

0.9

1.5

0.9

1.3

 

n

9

10

9

8

Neutrophils

mean

0.4

0.7

0.6

0.6

10E9/L

st.dev

0.2

0.3

0.2

0.2

 

n

9

10

9

8

Lymphocytes

mean

3.3

2.9

3.1

3.8

10E9/L

st.dev

0.8

1.5

0.7

1.1

 

n

9

10

9

8

Monocytes

mean

0.1

0.1

0.1

0.1

10E9/L

st.dev

0.0

0.0

0.1

0.1

 

n

9

10

9

8

Eosinophils

mean

0.1

0.1

0.1

0.1

10E9/L

st.dev

0.0

0.0

0.0

0.0

 

n

9

10

9

8

Basophils

mean

0.0

0.0

0.0

0.0

10E9/L

st.dev

0.0

0.0

0.0

0.0

 

n

9

10

9

8

Red blood cells

mean

8.29

8.14

8.35

7.21 **

10E12/L

st.dev

0.59

0.56

0.11

0.50

 

n

10

10

9

8

Reticulocytes

mean

213.7

205.4

218.7

277.8

10E9/L

st.dev

35.2

22.2

33.9

60.0

 

n

10

10

9

8

RDW

mean

11.0

11.0

11.1

11.0

%

st.dev

0.3

0.6

0.5

0.5

 

n

10

10

9

8

Haemoglobin

mean

9.3

9.4

9.5

8.7

mmol/L

st.dev

0.7

0.7

0.2

0.7

 

n

10

10

9

8

Haematocrit

mean

0.461

0.451

0.462

0.454

L/L

st.dev

0.035

0.033

0.012

0.032

 

n

10

10

9

8

MCV

mean

55.7

55.5

55.4

63.0 **

fL

st.dev

1.2

1.3

1.0

2.2

 

n

10

10

9

8

MCH

mean

1.13

1.15

1.14

1.21 **

fmol

st.dev

0.03

0.04

0.02

0.02

 

n

10

10

9

8

MCHC

mean

20.25

20.72 *

20.66

19.16 **

mmol/L

st.dev

0.34

0.30

0.35

0.54

 

n

10

10

9

8

Platelets

mean

779

750

864

744

10E9/L

st.dev

119

91

138

93

 

n

10

10

9

8

 

PT

mean

17.4

17.2

18.7

18.5

s

st.dev

1.2

0.6

2.4

2.2

 

n

10

10

9

8

APTT

mean

20.7

20.7

21.0

19.6

s

st.dev

1.6

1.1

1.8

1.8

 

n

10

10

9

8

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 9: Summary of clinical chemistry values

End Of Treatment

Dose groups (mg/kg bw/day)

Control

100

300

1000

Males

ALAT

mean

39.6

44.2

45.1

59.9 **

U/L

st.dev

5.0

7.0

5.9

20.1

 

n

10

10

10

9

ASAT

mean

69.1

77.2

81.3

96.1 **

U/L

st.dev

9.6

9.8

9.9

19.9

 

n

10

10

10

9

ALP

mean

102

98

98

157 **

U/L

st.dev

22

20

19

47

 

n

10

10

10

9

Total protein

mean

65.8

66.6

66.2

64.4

g/L

st.dev

1.3

3.3

1.7

1.9

 

n

10

10

10

9

Albumin

mean

33.3

33.7

33.6

33.8

g/L

st.dev

0.6

1.3

0.5

1.1

 

n

10

10

10

9

Total bilirubin

mean

2.6

2.6

2.8

2.7

umol/L

st.dev

0.2

0.3

0.3

0.2

 

n

10

10

10

9

Urea

mean

7.0

7.4

7.5

7.5

mmol/L

st.dev

0.6

0.9

0.9

0.3

 

n

10

10

10

9

Creatinine

mean

34.5

34.5

34.5

36.9 **

umol/L

st.dev

1.1

1.5

1.6

1.8

 

n

10

10

10

9

Glucose

mean

10.44

10.41

10.05

8.14 **

mmol/L

st.dev

1.70

1.56

1.30

1.13

 

n

10

10

10

9

Cholesterol

mean

1.95

1.86

1.78

1.33 **

mmol/L

st.dev

0.22

0.19

0.28

0.33

 

n

10

10

10

9

Bile Acids

mean

25.2

25.6

17.3

53.4 **

umol/L

st.dev

13.4

19.3

8.9

24.3

 

n

10

10

10

9

Sodium

mean

141.8

143.3 **

143.6 **

142.7

mmol/L

st.dev

1.0

1.1

0.9

1.2

 

n

10

10

10

9

Potassium

mean

3.74

3.83

4.02 *

3.82

mmol/L

st.dev

0.13

0.23

0.13

0.34

 

n

10

10

10

9

Chloride

mean

102

103

104 **

105 **

mmol/L

st.dev

1

1

1

2

 

n

10

10

10

9

Calcium

mean

2.63

2.66

2.68

2.63

mmol/L

st.dev

0.03

0.05

0.05

0.06

 

n

10

10

10

9

 

Inorg.Phos

 

mean

 

1.69

 

1.63

 

1.75

 

1.78

mmol/L

st.dev

0.21

0.13

0.09

0.16

 

n

10

10

10

9

Total T4

mean

3.61

3.97

4.04

3.09

ug/dL

st.dev

0.59

0.63

1.04

0.39

 

n

10

10

10

9

females

ALAT

 

mean

 

36.6

 

38.7

 

37.3

 

41.6

U/L

st.dev

7.9

11.2

9.4

9.1

 

n

10

10

9

9

ASAT

mean

73.5

83.8

72.8

71.9

U/L

st.dev

8.8

27.1

5.1

8.3

 

n

10

10

9

9

ALP

mean

41

53

39

60 *

U/L

st.dev

13

13

7

26

 

n

10

10

9

9

Total protein

mean

68.4

66.7

69.6

65.9

g/L

st.dev

2.8

1.8

3.3

2.2

 

n

10

10

9

9

Albumin

mean

36.7

35.9

37.8

36.2

g/L

st.dev

1.5

1.3

1.5

1.1

 

n

10

10

9

9

Total bilirubin

mean

3.0

3.0

3.0

3.2

umol/L

st.dev

0.4

0.3

0.3

0.4

 

n

10

10

9

9

Urea

mean

7.6

7.4

7.7

8.1

mmol/L

st.dev

0.6

0.6

1.2

1.1

 

n

10

10

9

9

Creatinine

mean

40.6

39.2

38.5

39.0

umol/L

st.dev

2.0

1.7

3.3

1.6

 

n

10

10

9

9

Glucose

mean

7.12

6.74

6.74

7.42

mmol/L

st.dev

1.15

0.63

1.13

1.28

 

n

10

10

9

9

Cholesterol

mean

1.95

1.72

1.70

1.40 *

mmol/L

st.dev

0.49

0.48

0.44

0.27

 

n

10

10

9

9

Bile Acids

mean

19.7

20.2

19.1

36.6

umol/L

st.dev

8.3

12.2

12.5

42.9

 

n

10

10

9

9

Sodium

mean

141.3

143.0 **

144.2 **

142.1

mmol/L

st.dev

1.0

0.7

0.8

1.1

 

n

10

10

9

9

Potassium

mean

3.31

3.47

3.33

3.63 *

mmol/L

st.dev

0.30

0.20

0.10

0.25

 

n

10

10

9

9

Chloride

mean

105

106 **

107 **

106 *

mmol/L

st.dev

1

1

1

1

 

n

10

10

9

9

Calcium

mean

2.69

2.64

2.69

2.61 **

mmol/L

st.dev

0.07

0.05

0.04

0.06

 

n

10

10

9

9

Inorg.Phos

mean

1.46

1.52

1.59

1.56

mmol/L

st.dev

0.22

0.09

0.19

0.14

 

n

10

10

8

9

Total T4

mean

2.39

2.54

2.38

1.86

ug/dL

st.dev

0.79

0.69

0.85

0.52

 

n

10

10

9

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 10: Summary of absolute organ weights

Males

Group

(mg/kg bw/day)

 

Body weight

Brain

Epididymis

Gland Adrenal

Gland

Prostate

Gland

Sem Ves

Gland

Thyroid

 

 

g

g

g

g

g

g

g

control

Mean

375.1

2.0836

1.1901

0.05781

0.8821

1.2902

0.01566

 

SD

36.4

0.1148

0.1098

0.00720

0.2420

0.1764

0.00231

 

N

10

10

10

10

10

10

10

100

Mean

374.7

2.0428

1.2244

0.05831

0.8008

1.4507

0.01603

 

SD

36.8

0.0851

0.0970

0.00938

0.2282

0.2786

0.00411

 

N

10

10

10

10

10

10

10

 

%Diff G1

-0 .1

-1 .9581

2.8821

0.86490

-9 .2166

12.4399

2.36271

300

Mean

387.8

2.0560

1.2114

0.05979

0.8479

1.2558

0.01675

 

SD

33.6

0.0735

0.1281

0.00682

0.2522

0.2168

0.00255

 

N

10

10

10

10

10

10

10

 

%Diff G1

3.4

-1 .3246

1.7898

3.42501

-3 .8771

-2 .6663

6.96041

1000

Mean

344.7

2.0511

1.1616

0.05176

0.6903

1.1749

0.01590

 

SD

27.4

0.0681

0.0580

0.00805

0.1408

0.2073

0.00287

 

N

9

9

9

9

9

9

9

 

%Diff G1

-8 .1

-1 .5593

-2 .3985

-10 .47301

-21 .7398

-8 .9375

1.53257

Males

 

 

Heart

Kidney

Liver

Spleen

Testis

Thymus

 

control

Mean

g

g

g

g

g

g

 

 

SD

1.0080

2.4660

9.3318

0.5655

3.6530

0.2906

 

 

N

0.1234

0.3757

1.5440

0.1083

0.3178

0.0637

 

100

Mean

10

10

10

10

10

10

 

 

SD

0.9990

2.6050

9.4615

0.5563

3.7483

0.2642

 

 

N

0.0947

0.2145

1.0497

0.0484

0.3845

0.0722

 

 

%Diff G1

10

10

10

10

10

10

 

300

Mean

-0 .8929

5.6367

1.3899

-1 .6269

2.6088

-9 .0847

 

 

SD

0.9664

2.5880

9.6277

0.5626

3.7919

0.2858

 

 

N

0.0628

0.2555

0.8865

0.0876

0.3279

0.0575

 

 

%Diff G1

10

10

10

10

10

10

 

1000

Mean

-4 .1270

4.9473

3.1709

-0 .5128

3.8024

-1 .6518

 

 

SD

0.9701

2.5402

9.2477

0.5166

3.7129

0.2332

 

 

N

0.0927

0.1934

0.7626

0.0630

0.2191

0.0423

 

 

%Diff G1

9

9

9

9

9

9

 

 

 

-3 .7588

3.0098

-0 .9016

-8 .6551

1.6394

-19 .7446

 

Females

Group

(mg/kg bw/day)

 

Body weight

Brain

Gland Adrenal

Gland

Thyroid

Heart

Kidney

Liver

control

Mean

g

g

g

g

g

g

g

 

SD

223.8

1.9236

0.06453

0.01339

0.6761

1.5852

5.7994

 

N

12.8

0.0888

0.01075

0.00190

0.0379

0.1127

0.7506

100

Mean

10

10

10

10

10

10

10

 

SD

223.5

1.8803

0.06921

0.01389

0.6916

1.6120

5.6842

 

N

15.5

0.0542

0.00910

0.00195

0.0600

0.1250

0.5325

 

%Diff G1

10

10

10

10

10

10

10

300

Mean

-0 .1

-2 .2510

7.25244

3.73413

2.2926

1.6906

-1 .9864

 

SD

213.4

1.8793

0.06820

0.01299

0.6957

1.5870

5.8367

 

N

18.3

0.1129

0.00810

0.00247

0.0607

0.1268

0.8101

 

%Diff G1

9

9

9

9

9

9

9

1000

Mean

-4 .6

-2 .3012

5.68728

-2 .99560

2.8940

0.1136

0.6426

 

SD

218.1

1.9086

0.05995

0.01453

0.7194

1.7080

6.8550b

 

N

22.6

0.0454

0.01010

0.00292

0.0815

0.1649

0.6071

 

%Diff G1

8

8

8

7

8

8

8

Females

 

 

Ovary

Spleen

Thymus

Uterus

 

 

 

control

Mean

g

g

g

g

 

 

 

 

SD

0.1350

0.3937

0.2846

0.7898

 

 

 

 

N

0.0186

0.0425

0.0475

0.4280

 

 

 

100

Mean

10

10

10

10

 

 

 

 

SD

0.1513

0.3997

0.2863

0.8129

 

 

 

 

N

0.0191

0.0294

0.0306

0.3222

 

 

 

 

%Diff G1

10

10

10

10

 

 

 

300

Mean

12.0741

1.5240

0.5973

2.9248

 

 

 

 

SD

0.1328

0.4026

0.2352a

0.7933

 

 

 

 

N

0.0199

0.0545

0.0213

0.3912

 

 

 

 

%Diff G1

9

9

9

9

 

 

 

1000

Mean

-1 .6461

2.2493

-17 .3499

0.4474

 

 

 

 

SD

0.1365

0.4288

0.2893

0.4728

 

 

 

 

N

0.0251

0.0471

0.0619

0.1671

 

 

 

 

%Diff G1

8

8

8

8

 

 

 

 

 

1.1111

8.9027

1.6339

-40 .1431

 

 

 

Significantly different from control group 1 value : a=p≤0.05,b=p≤0.01 (Dunn)

 

Table 11: Summary of organ weights relative to body weight

Males

Group

(mg/kg bw/day)

 

Brain

Epididymis

Gland Adrenal

Gland

Prostate

Gland

Sem Ves

Gland

Thyroid

Heart

 

 

%

%

%

%

%

%

%

control

Mean

0.55840

0.31813

0.0155

0.23473

0.34545

0.0042

0.26838

 

SD

0.03954

0.02148

0.0019

0.05825

0.04674

0.0005

0.01434

 

N

10

10

10

10

10

10

10

100

Mean

0.54873

0.32851

0.0156

0.21599

0.38493

0.0043

0.26699

 

SD

0.04436

0.03037

0.0020

0.06566

0.05075

0.0009

0.01329

 

N

10

10

10

10

10

10

10

 

%Diff G1

-1 .73141

3.26065

0.6025

-7 .98467

11.42982

1.9021

-0 .51709

300

Mean

0.53272

0.31351

0.0154

0.21703

0.32337

0.0043

0.24992a

 

SD

0.03520

0.03331

0.0015

0.05592

0.04923

0.0006

0.01376

 

N

10

10

10

10

10

10

10

 

%Diff G1

-4 .59967

-1 .45401

-0 .2193

-7 .54035

-6 .39119

3.6075

-6 .87522

1000

Mean

0.59775

0.33879

0.0151

0.19946

0.34277

0.0046

0.28179

 

SD

0.04170

0.03066

0.0025

0.03098

0.06930

0.0008

0.01982

 

N

9

9

9

9

9

9

9

 

%Diff G1

7.04767

6.49182

-2 .5076

-15 .02842

-0 .77639

10.8140

4.99732

Males

 

 

Kidney

Liver

Spleen

Testis

Thymus

 

 

control

Mean

%

%

%

%

%

%

%

 

 

SD

0.65756

2.47895

0.15014

0.97666

0.07756

 

 

 

N

0.07344

0.23420

0.01989

0.06736

0.01581

 

 

100

Mean

10

10

10

10

10

 

 

 

SD

0.69842

2.52911

0.14915

1.00536

0.07043

 

 

 

N

0.05872

0.18531

0.01297

0.11174

0.01783

 

 

 

%Diff G1

10

10

10

10

10

 

 

300

Mean

6.21360

2.02360

-0 .66122

2.93860

-9 .18779

 

 

 

SD

0.66741

2.48456

0.14471

0.98160

0.07360

 

 

 

N

0.03279

0.13234

0.01562

0.08770

0.01235

 

 

 

%Diff G1

10

10

10

10

10

 

 

1000

Mean

1.49844

0.22631

-3 .61257

0.50572

-5 .10703

 

 

 

SD

0.73750a

2.68451

0.15068

1.08346

0.06791

 

 

 

N

0.02286

0.11401

0.02146

0.10781

0.01348

 

 

 

%Diff G1

9

9

9

9

9

 

 

 

 

12.15681

8.29233

0.36042

10.93501

-12 .44638

 

 

Females

Group

(mg/kg bw/day)

 

Brain

Gland Adrenal

Gland

Thyroid

Heart

Kidney

Liver

Ovary

control

Mean

%

%

%

%

%

%

%

 

 

0.86159

0.0289

0.0060

0.30240

0.70923

2.59247

0.06058

 

SD

0.05597

0.0048

0.0009

0.01375

0.05009

0.32206

0.00943

 

N

10

10

10

10

10

10

10

100

Mean

0.84435

0.0310

0.0062

0.30964

0.72227

2.54130

0.06786

 

SD

0.05406

0.0036

0.0009

0.02016

0.04813

0.11461

0.00910

 

N

10

10

10

10

10

10

10

 

%Diff G1

-2 .00053

7.3580

4.0112

2.39290

1.83943

-1 .97381

12.03147

300

Mean

0.88446

0.0320

0.0061

0.32681a

0.74485

2.72978

0.06200

 

SD

0.06995

0.0030

0.0012

0.02617

0.03862

0.24753

0.00539

 

N

9

9

9

9

9

9

9

 

%Diff G1

2.65433

10.8381

2.1268

8.07136

5.02358

5.29637

2.35777

1000

Mean

0.88217

0.0275

0.0066

0.32993a

0.78421b

3.15242 b

0.06242

 

SD

0.08159

0.0037

0.0014

0.02071

0.04232

0.21928

0.00805

 

N

8

8

7

8

8

8

8

 

%Diff G1

2.38831

-4 .8216

9.3446

9.10242

10.57337

21.59899

3.04074

Females

 

 

Spleen

Thymus

Uterus

 

 

 

 

control

Mean

%

%

%

 

 

 

 

 

 

0.17605

0.12699

0.35102

 

 

 

 

 

SD

0.01824

0.01865

0.18264

 

 

 

 

 

N

10

10

10

 

 

 

 

100

Mean

0.17904

0.12851

0.36804

 

 

 

 

 

SD

0.01031

0.01480

0.15613

 

 

 

 

 

N

10

10

10

 

 

 

 

 

%Diff G1

1.70044

1.19996

4.84611

 

 

 

 

300

Mean

0.18829

0.11036

0.36826

 

 

 

 

 

SD

0.01537

0.00751

0.17956

 

 

 

 

 

N

9

9

9

 

 

 

 

 

%Diff G1

6.95674

-13 .09221

4.90872

 

 

 

 

1000

Mean

0.19804

0.13413

0.21903

 

 

 

 

 

SD

0.02761

0.03454

0.08351

 

 

 

 

 

N

8

8

8

 

 

 

 

 

%Diff G1

12.49191

5.62264

-37 .60137

 

 

 

 

Significantly different from control group 1 value :a=p≤0.05,b=p≤0.01 (Dunn)

 

Table 12: Mean percent liver weight differences from control groups

 

Males

Females

Dose Level (Mg/Kg bw/day):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

Absolute

1

3

-1

-2

1

18**

Relative To Body Weight

2

0

8

-2

5

22**

**: P<0.01

 

 

 

 

 

 

 

Table 13: Summary of gross pathology findings

 

Males

Female

Dose group (mg/kg bw/day)

Vehicle

100

300

1000

Vehicle

100

300

1000

Number of animals

10

10

10

9

10

10

9

8

Adipose Tissue

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Bone, Femur

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Bone, Sternum

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Bone Marrow, Femur

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Bone Marrow, Sternum

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Brain

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Cervix

 

 

 

 

 

 

 

 

Submitted

-

-

-

-

10

10

9

8

No Visible Lesions

-

-

-

-

10

10

9

8

Epididymis

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

No Visible Lesions

10

10

10

9

-

-

-

-

Esophagus

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

10

10

10

9

10

10

9

8

Eye

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

No Visible Lesions

9

10

10

9

10

10

9

8

Enlargement

1

0

0

0

0

0

0

0

 

Galt

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Adrenal

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Clitoral

 

 

 

 

 

 

 

 

 

Submitted

No Visible Lesions Focus

GLAND, COAGULATING

-

-

-

-

10

10

9

8

 

No Visible Lesions

-

-

-

-

8

10

9

8

 

Focus

-

-

-

-

2

0

1

0

 

Gland; Coagulating

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

 

No Visible Lesions

10

10

10

8

-

-

-

-

 

Abnormal Appearance

0

0

0

1

-

-

-

-

 

Gland, Harderian

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Lacrimal

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Mammary

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Parathyroid

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Pituitary

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Preputial

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

 

No Visible Lesions

10

10

10

8

-

-

-

-

 

Small

0

0

0

1

-

-

-

-

 

Gland, Prostate

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

 

No Visible Lesions

10

10

10

9

-

-

-

-

 

Gland, Salivary, Mandibular

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Salivary, Sublingual

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Gland, Seminal Vesicle

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

 

No Visible Lesions

10

10

10

9

-

-

-

-

 

Gland, Thyroid

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Heart

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Kidney

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

9

10

9

10

10

9

8

 

Dilatation

0

1

0

0

0

0

0

0

 

Large Intestine, Cecum

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Large Intestine, Colon

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Large Intestine, Rectum

 

 

 

 

 

 

 

 

 

Submitted

10

10

0

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Larynx

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Liver

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

9

10

9

9

10

10

8

8

 

Hernia

1

0

0

0

0

0

0

0

 

Discoloration

0

0

1

0

0

0

1

0

 

Abnormal Consistency

0

0

1

0

0

0

0

0

 

Small

0

0

0

0

0

0

1

0

 

Lung

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Lymph Node Iliac

 

 

 

 

 

 

 

 

 

Submitted

-

-

-

-

1

0

0

0

 

No Visible Lesions

-

-

-

-

-

-

-

-

 

Lymph Node, Mandibular

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

9

9

9

7

 

Discoloration

0

0

0

0

1

1

0

1

 

Lymph Node, Mesenteric

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Muscle, Diaphgram

 

 

 

 

 

 

 

 

 

Submitted

1

0

0

0

-

-

-

-

 

No Visible Lesions

1

-

-

-

-

-

-

-

 

Muscle, Skeletal

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Nasopharynx

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Nerve, Optic

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Nerve, Sciatic

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Ovary

 

 

 

 

 

 

 

 

 

Submitted

-

-

-

-

10

10

9

8

 

No Visible Lesions

-

-

-

-

10

10

9

8

 

Pancreas

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Skin

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Small Intestine,

 

 

 

 

 

 

 

 

 

Duodenum

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Small Intestine, Ileum

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Small Intestine,

 

 

 

 

 

 

 

 

 

Jejunum

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Spinal Cord

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Spleen

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Stomach

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

9

10

9

9

9

7

9

6

 

Focus

1

0

1

0

0

1

0

1

 

Irregular Surface

0

0

0

0

1

2

0

1

 

Testis

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

-

-

-

-

 

No Visible Lesions

10

10

10

9

-

-

-

-

 

Thymus

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

9

9

10

8

9

8

 

Focus

0

0

1

0

0

1

0

0

 

Discoloration

0

0

0

0

0

1

0

0

 

Tongue

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Trachea

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Urinary Bladder

 

 

 

 

 

 

 

 

 

Submitted

10

10

10

9

10

10

9

8

 

No Visible Lesions

10

10

10

9

10

10

9

8

 

Uterus

 

 

 

 

 

 

 

 

 

Submitted

-

-

-

-

10

10

9

8

 

No Visible Lesions

-

-

-

-

5

4

4

7

 

Fluid Accumulation

-

-

-

-

5

6

5

1

 

Vagina

 

 

 

 

 

 

 

 

 

Submitted

-

-

-

-

10

10

9

8

 

No Visible Lesions

-

-

-

-

10

10

9

8

 

 

Table 14: Summary test item-related microscopic findings – scheduled euthanasia animals

 

Males

Females

Dose level (mg/kg):

 

Vehicle

100

300

1000

Vehicle

100

300

1000

LIVER a

10

10

10

9

10

10

9

8

Centrilobular infiltrate, mononuclear

 

 

 

 

 

 

 

 

Minimal

-

2

4

3

-

1

3

2

Mild

-

-

2

4

-

-

2

5

Centrilobular single cell necrosis

 

 

 

 

 

 

 

 

Minimal

-

-

2

2

-

1

1

3

Mild

-

-

-

3

-

-

1

2

Hypertrophy, centrilobular

 

 

 

 

 

 

 

 

Minimal

-

-

-

3

-

-

-

-

Mild

-

-

-

3

-

-

-

-

Vacuolation, periportal

 

 

 

 

 

 

 

 

Minimal

-

-

-

4

-

-

-

1

SPLEEN a

10

10

10

9

10

10

9

8

Pigment

 

 

 

 

 

 

 

 

Minimal

7

7

8

-

3

2

5

1

Mild

3

1

2

4

7

8

4

3

Moderate

-

-

-

5

-

-

-

4

ADRENAL GLAND a

10

10

10

9

10

10

9

8

Vacuolation, zona glomerulosa

 

 

 

 

 

 

 

 

Minimal

1

2

1

5

-

-

-

2

Mild

-

-

-

3

-

-

-

3

a = Number of tissues examined from each group.

 

Table 15: Summary of functional tests

Males

Dose

(mg/kg bw/day)

group 1

control

group 2

100

group 3

300

group 4

1000

At Week 13

Hearing

 

median

 

0

 

0

 

0

 

0

Score 0/1

n

5

5

5

5

Pupil L

median

0

0

0

0

Score 0/1

n

5

5

5

5

Pupil R

median

0

0

0

0

Score 0/1

n

5

5

5

5

Static R

median

0

0

0

0

Score 0/1

n

5

5

5

5

Grip Fore

mean

1411

1387

1420

1281

Gram

st.dev

166

165

252

129

 

n

5

5

5

5

Grip Hind

mean

634

688

706

593

Gram

st.dev

124

72

83

40

 

n

5

5

5

5

Females

Dose

(mg/kg bw/day)

group 1

control

group 2

100

group 3

300

group 4

1000

At Week 13

Hearing

 

median

 

0

 

0

 

0

 

0

Score 0/1

n

5

5

5

5

Pupil L

median

0

0

0

0

Score 0/1

n

5

5

5

5

Pupil R

median

0

0

0

0

Score 0/1

n

5

5

5

5

Static R

median

0

0

0

0

Score 0/1

n

5

5

5

5

Grip Fore

mean

1101

1193

1060

1248

Gram

st.dev

72

141

148

79

 

n

5

5

5

5

Grip Hind

mean

569

502

550

483

Gram

st.dev

78

112

60

55

 

n

5

5

5

5

 

Table 16: Motor activity test summary

Males

Dose

(mg/kg bw/day)

group 1

control

group 2

100

group 3

300

group 4

1000

Total Movements

mean1

2411

2421

2897

2710

 

st.dev

616

828

868

1227

 

n

5

5

5

5

Ambulations

mean1

435

424

494

613

 

st.dev

235

221

169

353

 

n

5

5

5

5

Females

Dose

(mg/kg bw/day)

group 1

control

group 2

100

group 3

300

group 4

1000

Total Movements

mean1

3630

3216

3656

3024

 

st.dev

908

829

684

1157

 

n

5

5

5

5

Ambulations

mean1

950

705

956

840

 

st.dev

219

183

125

352

 

n

5

5

5

5

 

Table 17: Summary of sperm analysis

Dose Group

(mg/kg bw/day)

 

Vehicel

100

300

1000

Necropsy

Nr of cells exam

 

mean

 

270

 

284

 

240

 

331

 

st.dev

58

98

35

113

 

n

10

10

10

9

Motile sperm

mean

76

78

72

81

%

st.dev

11

5

14

6

 

n

10

10

10

9

Progressiv.sperm

mean

10

15

16

15

%

st.dev

7

5

7

9

 

n

10

10

10

9

Spermcount epi

mean

454.4

---

---

500.9

10e6/Gram

st.dev

107.1

---

---

135.8

 

n

8

0

0

9

Normal morph

mean

163

---

---

170

No of cells

st.dev

14

---

---

10

 

n

10

0

0

9

Detached head

mean

1

---

---

1

No of cells

st.dev

1

---

---

2

 

n

10

0

0

9

Abnormal head

mean

0

---

---

0

No of cells

st.dev

0

---

---

0

 

n

10

0

0

9

Coiled tail

mean

25

---

---

24

No of cells

st.dev

9

---

---

11

 

n

10

0

0

9

Other tail

mean

10

---

---

4

No of cells

st.dev

8

---

---

3

 

n

10

0

0

9

Abnormal neck

mean

1

---

---

0

No of cells

st.dev

1

---

---

0

 

n

10

0

0

9

Combined

mean

0

---

---

0

No of cells

st.dev

0

---

---

0

 

n

10

0

0

9

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- Repeated dose toxicity: oral

Repeated dose toxicity following the oral route was tested in a GLP-compliant 90-Day gavagestudy inWistar rats (Crl: WI(Han)) according to OECD 408 (Charles River Laboratories 2019). In this study, 10 rats per sex and dose level were administered the test substance, dissolved in polyethylene glycol 400 at 100, 300 and 1000 mg/kg bw/day. Control animals received polyethylene glycol 400, only. Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity (weeks 1, 6 and 13). Stability of the test item in the vehicle was assessed in week 1. Analysis showed that analysed concentrations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).

Three unscheduled deaths (one mid-dose female and two high-dose females) were observed during the study period, but none was considered treatment-related. Unscheduled deaths were most likely related to the oral gavage or blood sampling procedure. Salivation and ploughing was noted in all treated animals. For salivation a dose-related trend in severity (up to severe on several occasions in high-dose rats) and time of onset (from day 8 in low-dose rats and from day 1 or 2 at the higher dose levels) was observed. The salivation was considered to be a physiological response and/or behavioural sign of discomfort in reaction to the bolus administration of the test item rather than a sign of systemic toxicity considering its severity (mostly slight or moderate) and the time of occurrence (i.e. within half an hour after dosing). All other clinical signs noted occurred incidentally and represented normal background findings and were thus not considered treatment-related. Males at 1000 mg/kg/day showed slight and therefore non-adverse, reduced body weight gain and food consumption. There were no treatment-related adverse effects observed on body weight or food consumption in females.

 

Adverse microscopic changes were observed in the liver at 300 mg/kg/day (females only) and 1000 mg/kg/day (both sexes) and consisted of an increased incidence and severity (up to mild degree) of centrilobular mononuclear cell infiltrates accompanied by single cell necrosis as well as increased liver weighs (by about 20%) in females treated at 1000 mg/kg only.

 

Non-adverse, treatment-related centrilobular mononuclear cell infiltration with single cell necrosis was observed in males at 100 and 300 mg/kg/day and in females at 100 mg/kg/day. Non-adversity was based on the low severity/incidence of the lesions in these dose groups.

 

Other non-adverse microscopic changes at the highest dose level were centrilobular hypertrophy and hepatocellular vacuolation in the liver (males), increased severity of pigment in the spleen (both sexes), and increased incidence and severity of diffuse vacuolation in the zona glomerulosa of the adrenal gland (both sexes). Due to the low severity and the absence of any additional degenerative or proliferative changes, these findings were not considered to be adverse.

 

Treatment-related higher liver weights (absolute and relative to body weight) were noted in females and were considered adverse, due to histopathology correlates.

Other organ weight findings at high dose, slight increase in relative kidney weights in both sexes and lower mean uterus weights (absolute and relative to body weight, not statistically significant) in females were considered to reflect normal physiological variation and were thus not considered treatment-related. In addition, increased heart weights in in mid dose males and high dose females were also not considered treatment related, due to the absence of a dose-related trend and regarded a chance finding.

 

Clinical chemistry showed changes in several liver-related parameters at 1000 mg/kg/day, mostly in males, namely: increases in alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP) and bile acids. Decreases noted in cholesterol (both sexes) and fasting glucose (males only) may also be related to the effect of the test item on the liver. The other clinical chemistry findings (slightly higher creatinine, chloride and potassium, and slightly lower calcium, mostly at 1000 mg/kg/day) were not associated with adverse anatomic pathology alterations and therefore regarded as non-adverse.

Treatment-related changes in red blood cell parameters were noted at 1000 mg/kg/day, generally in both sexes, and consisted of decreases in the number of red blood cells, haemoglobin, MCHC and RDW, and increases in MCV, MCH and reticulocytes. These changeswere regarded as non-adversebecause of the minor magnitude of the change.

All opthalmology findings occurred at similar incidences in all dose groups or only in single animals and were thus considered incidental, non-treatment-related and non-adverse.

There were no indications of possible reproductive toxicity as evidenced by the absence of treatment-related changes in the length and regularity of the estrous cycle, sperm parameters (motility, concentration, morphology), and weight and morphology of male and female reproductive organs.

 

Thyroid hormone analysis revealed lower T4 levels in the high dose group compared to the control levels (-14% and -22% in males and females, respectively). Since statistical significance was not achieved and T4 levels in almost all high dose animals remained in the concurrent control range, these intergroup differences were considered non-treatment-related.

 

Neurobehavioural examination revealed low values for total movements and ambulations in one male and one female (below the normal ranges). However, habituation profiles of these animals were normal and their lower motor activity was not associated with corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Moreover, all other high-dose animals showed normal motor activity. Therefore, the lower activity recorded for these two animals was not attributed to treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In addition, forelimb and hind limb grip strength showed no treatment-related changes.

 

Based on effects observed on the liver at 300 mg/kg bw/day in females and 1000 mg kg bw/day in both sexes, the LOAEL/NOAEL for female rats was determined at 300 mg/kg bw/day and 100 mg/kg bw/day and for male rats at 1000 mg/kg bw/day and 300 mg/kg bw/day.

Justification for classification or non-classification

Adverse effects, in form of liver toxicity were observed in the sub-chronic rat study with oral test substance administration at an dose of 1000 and 300 mg/kg bw/day and above in males and females, respectively. The dose at which adverse effects were observed does exceed the current valide cut-off level for classification in category 2 of 10 < c ≤ 100 mg/kg bw/day (oral route) (Annex I: 3.9.2.9.7.) and thus, the data requirements for classification are not met according to Regulation (EC) No. 1272/2008 (CLP).

The available data on oral repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

 

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