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Description of key information

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) and OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Method: daily oral gavage administration of WS400102 at dose levels of 100, 300 and 1000 mg/kg/day up to 35 days for male and unpaired female rats and ca. 48 days for paired female rats
Changes in haematology, clinical chemistry parameters, slightly increased liver weights (particulary in males), histological minimal changes in kidneys (in females) were observed.
No adverse effects or test material related findings were observed at the low or mid-dose levels. The NOAEL was considered to be 300 mg/kg day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Wistar Crl:WI rats
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: Males: 340 g – 395 g, Females: 230 g - 265 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg bw

According to the results of a stability study, the test material was stable in Propylene glycol in concentration range of 20-200 mg/mL for 24 hours at room temperature (RT, 15-30ºC) and for up to three days when stored refrigerated at 2-8ºC (protected from light) and the homogeneity of the formulations was satisfactory.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of WS400102 formulations for concentration and homogeneity was performed using validated HPLC-UV method (CiToxLAB study code 11/350-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400102 from propylene glycol was in the range from 97 to 105%.
Duration of treatment / exposure:
Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
Satellite females (nulliparous and nonpregnant): 35 days
Frequency of treatment:
daily, 7 days/week
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:
Main groups: 12 animals/sex/dose
Satellite group (20 female rats): 5 animals/dose
Offspring were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on available data and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (7.5.1 Dose Range Finding Study 7 days rat_WS400102), the dose levels selected for the this study were 100, 300 and 1000 mg/kg bw/day.
Positive control:
No
Observations and examinations performed and frequency:
Observations and examinations performed and frequency
see Table 1a-c: Schedule and Dosing scheme ("Any other information on material and methods incl. tables")
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for signs of morbidity and mortality
- Time schedule: daily, general clinical observations
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly
Observations in a standard arena: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule: All adult Main and Satellite animals were weighed on Day 0, afterwards at least weekly and at termination.
Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).

FOOD CONSUMPTION: yes
-Time schedule: at least weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy: Day 35
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: Subgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: bloodcells, coagulation (APIT, PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy: Day 35
- Animals fasted: Yes (overnight)
- How many animals:Subgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: Glucose, T-Bilirubin, Urea, Cholesterol, Creatinine, Phosphorus, Sodium, Potassium, Calcium, Chlorid, Total protein, Albumin, AST, ALT, ALKP, GGT, Bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy
- Metabolism cages used for collection of urine: Yes (ca. 16 h)
- How many animals: Subgroup A (5 main males/group), satellite females (5/group)
- Parameters examined: Leukocytes, Nitrit, pH, protein, Glucose, Urobilinogen, Bilirubin, Ketones, Erythrocytes, Specific gravity, Sediment, Volume, Colour/Appearance.

NEUROBEHAVIOURAL EXAMINATION, Neurotoxicity: yes, FOB
- Time schedule: during the last exposure week (on Day 32)
- Dose groups that were examined: Main animals, 5 males/group, “subgroup A” and satellite females of all dose levels
- Battery of functions tested: sensory activity / grip strength / motor activity /general physical condition and behaviour of animals; a modified Irwin test was performed.


Sacrifice and pathology:
Sacrifice and pathology
GROSS PATHOLOGY: Yes
- Animals: each adult animal, pups (PND4)
- Time schedule for the adults: on Day 35, i.e. one day after the last treatment following overnight period food deprivation, or PND5

HISTOPATHOLOGY: Yes, see Table 1-3 ("Any other information on materials and methods incl. tables")
Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In both sexes in the high dose group (1000mg/kg bw/day)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In both sexes in the high dose group (1000mg/kg bw/day)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In both sexes in the high dose group (1000mg/kg bw/day). Effects sharper in males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Hepatic enlargement in high dose level groups
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings noted in the liver of males at 1000 mg/kg bw/day (High dose). Slightly effects in kidneys of females
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study. There were no clinical signs related to treatment.

BODY WEIGHT AND WEIGHT GAIN
There was no adverse effect of test item on body weight or body weight gain. Body weights were comparable to the control in all treated groups.

HAEMATOLOGY
Both males and females (satellite) at 1000 mg/kg (High dose) had decreased erythrocyte (RBC) count, haemoglobin concentration and haematocrit value. Compared to the control mean, the decrease was in the range of 8-10% in males and 10-12% in females. The differences were statistically significant. See Table 1 "Any other information on results incl. tables".
Females at 1000 mg/kg (High dose) had decreased leucocyte (WBC) counts. The difference attained statistical significance (p<0.01).

CLINICAL CHEMISTRY
Compared to the control mean, alkaline phosphatase activity (ALKP) was slightly elevated in males at 1000 mg/kg bw/day (High dose) and the mean value attained statistical significance.
Females at 1000 mg/kg bw/day (High dose) had elevated albumin (Alb.) concentration and consequently higher total protein (Tot.Prot) concentration and increased albumin to globulin (A/G) ration. The differences were statistically significant.
Increased calcium, sodium and chloride concentrations were measured in females at 1000 mg/kg bw/day.
See Table 2 "Any other information on results incl. tables".

URINALYSIS
There was no effect of treatment noted during urinalysis.
Urine sediment analysis showed similar results in all experimental groups including control.

NEUROBEHAVIOUR
There were no treatment related effects.There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.There was no effect of treatment noted during the assessment of foot splay or grip strength.

ORGAN WEIGHTS
Compared to controls, higher weights of liver were recorded for males at 1000 mg/kg bw/day (High dose). Liver of the High dose Satellite females were also slightly higher. The values for males were approximately 23-26% higher, than control and the differences attained statistical significance (p<0.01). In females, the increase was approximately of 16-19% and was statistically significant for body weight relative mean value.
See Table 3 and 4 "Any other information on results incl. tables".

GROSS PATHOLOGY
Test material-related macroscopic findings were observed in the liver at a dose level of 1000 mg/kg bw/day. The hepatic enlargement was recorded in 4 of 12 High Dose males.
Other occasional observations were considered to be incidental or within the physiological range.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test material related microscopic findings were noted in the liver of males at 1000 mg/kg bw/day (High dose). Minimal to mild diffuse hepatocellular vacuolation of all lobes was seen in 6 of 9 males. Minimal to mild centrilobular hypertrophy was present in 4 of 9 High Dose males. The liver changes corresponded with organ weight changes and with macroscopic observations (enlargement).
In the Satellite females, minimal bilateral vacuolation and presence of cytoplasmic eosinophilic granules in the cortical tubules of kidneys were observed in 2/5 High dose animals.
See Table 5 "Any other information on results incl. tables".
Other changes were regarded as incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 - <= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: slight changes in haematology, clinical chemistry, kidney (females only), liver (males only) in high dose animals
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Mean values of erythrocyte (RBC) count, haemoglobin concentration and haematocrit value in males and females

Groups/Dose (mg/kg/day)

Control

100

300

1000

 

Males

 

RBC

(M/uL)

8.34

8.14

7.81*

7.68*

DN

Difference (%)

-2

-6

-8

 

Haemoglobin

(g/dL)

14.5

14.4

13.6*

13.0**

DN

Difference (%)

-1

-6

-10

 

Haematocrit

(%)

42.4

42.6

40.1*

38.8**

DN

Difference (%)

0.5

-5

-8

 

Females (satellite)

 

RBC

(M/uL)

7.81

7.54

7.48

7.02*

DN

Difference (%)

-3

-4

-10

 

Haemoglobin

(g/dL)

15.3

14.9

14.7

13.5**

DN

Difference (%)

-3

-4

-12

 

Haematocrit

(%)

43.2

41.5

41.3

38.1**

DN

Difference (%)

-4

-4

-12

 

 * = p<0.05.   ** = p<0.01 DN = Duncan's Multiple Range Test; % differences vs. control

Table 2: Mean values of selected clinical chemistry parameters in males and females

Groups/Dose (mg/kg/day)

Control

100

300

1000

 

Males

 

ALKP

(U/L)

90.4

97.4

90.8

127.2**

DN

Difference (%)

8

0

41

 

Females (satellite)

 

Albumin

 (g/L)

35.9

37.0

39.0

42.9**

DN

Difference (%)

3

9

19

 

Incidence

1/5

3/5

5/5

 

Total protein

 (g/L)

63.8

65.0

66.6

71.4**

DN

Difference (%)

2

4

12

 

A/G

1.30

1.34

1.40

1.54**

DN

Difference (%)

3

8

18

 

Calcium

(mmol/L)

2.52

2.58

2.64*

2.70**

DN

Difference (%)

3

5

7

 

Sodium (mmol/L)

144.2

145.2

147.1*

148.7**

DN

Difference (%)

1

2

3

 

Chloride (mmol/L)

99.9

101.1

102.5

104.6**

DN

Difference (%)

1

3

5

 

 * = p<0.05.   ** = p<0.01 DN = Duncan's Multiple Range Test;

% differences vs. control ,Incidence: incidence of elevated values

Table 3: Mean liver weights of males and satellite groups of females

/Dose (mg/kg/day)

Control

100

300

1000

 

Males

 

Liver weight absolute (g)

14.5

14.3

15.4

17.9**

DN

Difference (%)

- 2

6

23

 

body weight relative

 (%)

3.07

3.08

3.19

3.88**

DN

Difference (%)

0

4

26

 

brain weight relative

(%)

663

651

705

822**

DN

Difference (%)

- 2

6

24

 

Females (Satellite)

 

Liver weight absolute (g)

7.5

7.5

8.5

8.7

NS

Difference (%)

0

13

16

 

body weight relative

 (%)

2.81

2.85

3.04

3.35*

DN

Difference (%)

1

8

19

 

brain weight relative

(%)

358

378

402

417

NS

Difference (%)

6

12

16

 

*= p<0.05   ** = p<0.01 DN = Duncan's Multiple Range Test; NS = not significant

% differences vs. control

Table 4: Mean kidney weights of satellite females

Groups/Dose (mg/kg/day)

Control

100

300

1000

 

Kidneys weight absolute (g)

1.78

1.82

2.08

1.92

NS

Difference (%)

3

17

8

 

body weight relative

 (%)

0.66

0.70

0.75*

0.74*

DN

Difference (%)

5

13

11

 

brain weight relative

(%)

85

92

99

92

NS

Difference (%)

9

17

9

 

*= p<0.05. DN = Duncan's Multiple Range Test; NS = not significant ,  % differences vs. control

Table 5: Incidence of the liver lesions in males

Groups/Dose (mg/kg/day)

Control

100

300

1000

No. of examined

5

0

0

9

Without alteration

5/5

-

-

3/9

Vacuolation: total

0/5

-

-

6/9

minimal

0

-

-

2

mild

0

-

-

4

Hypertrophy: total

0/5

-

-

4/9

minimal

0

-

-

2

mild

0

-

-

2

Conclusions:
For the repeated dose toxicity endpoint the NOAEL of WS400102 administered at oral route to Wistar rats for 35 consecutive days is considered to be the mid-dose level of 300 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The subacute key study is fully adequate for assessment of repeat dose oral toxicity over 35 days, as it is of high quality and reliability. It comprises the OECD Guidelines for Testing of Chemicals No. 422 and No. 407.
System:
other: slight changes in haematology, clinical chemistry, kidney (females only), liver (males only) in high dose animals
Organ:
kidney
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

Justification for classification or non-classification

A NOAEL of 300 mg/kg bw/day after 35 days does not necessitate any classification regarding repeated exposure according to European classification rules [ REGULATION (EC) 1272/2008].