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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:
20 May- 23 Sept 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid
EC Number:
940-877-5
Cas Number:
2241455-89-8
Molecular formula:
C9H12N2O4
IUPAC Name:
2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo Holland
- Females nulliparous and non-pregnant: not reported
- Age at study initiation: 27 - 29 days
- Weight at study initiation: 68.9 - 103.3 g (males), 68.8 - 105.1 g (females)
- Housing: up to 5 rats of one sex per cage, in clear polysulfone solid bottomed cages, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analysis of water, diet and bedding material are kept on file at ERBC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 May 2021 To: 23 September 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulation was prepared weekly according to stability data obtained from ERBC Study No. A4260, stirred for at least 15 minutes before dosing and maintained under magnetic stirring during administration.

VEHICLE
- Justification for use and choice of vehicle: corn oil, no justification provided
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in ERBC Study no. A4260 in the range from 20 to 200 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
Preparations of the test item were prepared as suspensions in corn oil. Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots in different positions. For the intermediate levels, only concentration was assessed by taking two different analytical aliquots. Each analytical aliquot was analysed separately.Concentration was evaluated as the mean of the single determinations and homogeneity as the coefficient of variation of the sextuplicate set.
In ERBC Study no. A4260, a 28 hour and 8 day stability at room temperature were verifed in the range from 20 to 200 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defned period of storage, are still acceptable (85%-115% for concentration and CV < 10% for homogeneity).
The proposed preparation procedure for the test item was checked in the range from 20 to 200 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4260 to confrm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the preparations prepared on Day 1 and Week 13 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
13 weeks + 4 weeks recovery period (for 5 males and 5 females of the control and high dose group, respectively)
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
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: Dose levels were selected in consultation with the Sponsor, based on the results obtained in a previous oral toxicity study in rats (OECD 422) and preliminary non-GLP study (ERBC Study No. E0590), in which no adverse effects were noted at all dose levels (100, 300 and 1000 mg/kg bw/day).
- Rationale for animal assignment: The rats were allocated to the 4 groups by computerised stratifed randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for clinical biochemistry: At the end of recovery period, erroneously the animals were not fasted overnight, but they were under conditions of food deprivation in the morning, before blood collection.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study. Observations were performed at the same time interval each day (2 - 2.5 hours post-dose).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week during the study from the start of treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
- The weight of food consumed by each cage of rats was recorded at weekly intervals starting from treatment. The group mean daily intake per rat was calculated.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the commencement of treatment and during Week 13 of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide.
- Dose groups that were examined: All (prior to treatment), control and high dose group (Week 13).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No (but under conditions of food deprivation)
- How many animals: All
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy and at the end of the recovery period.
- Animals fasted: No (but under conditions of food deprivation)
- How many animals: All
- Parameters checked in table [No.1] were examined.

PLASMA/SERUM HORMONES: Yes (Thyroid hormone determination (T3, T4 and TSH))
- Time of blood sample collection: Prior to necropsy
- How many animals: Main phase animals
- Parameters checked: Thyroid hormones T3, T4 and TSH

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12 of treatment and once during Week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength was performed. Motor activity was assessed once during Week 12 of treatment and once during Week 4 of recovery by an automated activity recording.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
OESTRUS CYCLE: Yes
Time schedule: At the end of the study, just prior to necropsy, vaginal smears were taken from all surviving female animals, and the oestrous cycle phase recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 2)
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the signifcance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verifed by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modifed t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological fnding was carried out by means of a non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the treatment period, rales were observed in 3 out of 10 males dosed at 1000 mg/kg bw/day. Ocular discharge or decreased activity were also seen in 2 of these males. Ocular discharge was also observed in a single male animal dosed at 100 mg/kg bw/day, while a palpable mass was described in a second male of the same group. No signifcant clinical signs were observed during recovery period. No treatment-related clinical signs were observed in the females during treatment and recovery periods.

No changes of toxicological signifcance were found at the weekly clinical examination, which included an evaluation of neurotoxicity.
Slight decreases in the number of rearing, statistically signifcant, were occasionally observed during the treatment-period in males and/or females dosed at 100, 300 and 1000 mg/kg bw/day, when compared to controls. Due to the occasional occurrence of these change and in the absence of other signs, they were not considered to be of toxicological relevance (non-treatment-related, non-adverse). (Please refer to table 5 in the "any other information on results incl. tables" section)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two cases of unscheduled death occured.
One control male was sacrificed for humane reasons on Day 35, after swallowing the catheter. Piloerection and salivation were observed in this animal the day before humane sacrifice. At post mortem examination, this animal had oedematous consistency of salivary glands and unilateral minimal pelvic dilation in the kidney; microscopically there was a mild diffuse oedema in the surrounding connective tissue of salivary glands. Moderate proteinaceus plug was noted in the urinary bladder with uncertain relationship to the dilated renal pelvis. The cause of illness could be attributed to a misdosing

A high dose female was sacrifced for humane reasons on Day 48. Decreased activity, dyspnoea, hunched posture, rales, staining around perianal region and swollen abdomen were observed prior to sacrifce. At post mortem examination, this animal had distended gastrointestinal tract with abnormal gaseous content (stomach, duodenum, ileum, jejunum, ileum, caecum and colon), reduced size of the thymus and abnormal yellow staining of the skin in the urogenital region. Microscopically there was marked atrophy of the thymus, acinar cell hypertrophy in the pancreas, minimal, focal, mucosal ulceration of the non glandular region of the forestomach associated with inflammatory reaction in the submucosa. (The factors contributory to illness status of this female could not be determined.)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to control animals, no treatment-related changes were noted in mean body weights and body weight gain in both genders, during the treatment and recovery periods of the study. At the end of the treatment period, there were no differences in terminal body weight in treated males and females, when compared to their controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in food consumption in male and female animals, during the treatment and recovery periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were detected in both eyes of all surviving animals, from control and all treated groups, when they were re-examined during Week 13 of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
White blood cells were increased in males dosed at 1000 mg/kg bw/day (58%). Changes involved mainly lymphocytes and monocytes. At the end of the recovery period treated males showed leucocytes values comparable to controls, confirming complete reversibility. The other statistically signifcant differences between control and treated males (haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentration) were within the range of expected biological variation and/or not dose-related, therefore they were considered to be incidental.
There were no changes observed in coagulation paramaters. (Please refer to table 3 in the "any other information on results incl. tables" section)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed. The statistically signifcant differences recorded between control and treated animals (alkaline phosphatase, aspartate aminotransferase, creatinine, calcium, sodium and phosphorus in males, glucose and phosphorus in females) were within the range of expected biological variation and/or not dose-related, therefore they were considered to be unrelated to treatment.
Recovery phase: No treatment-related changes were observed. In males, calcium was still slightly higher than controls. As for the dosing phase, this change was considered to be incidental. The other statistically signifcant differences recorded (alkaline phosphatase and sodium in females) were not recorded at the end of the Dosing phase, therefore they were considered to be unrelated to treatment. (Please refer to table 4 in the "any other information on results incl. tables" section)
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: .Thyroid hormones (T3, T4 and TSH). For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No relevant differences that could be considered treatment-related were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
A slight statistically signifcant increase of mean landing footsplay was recorded in the males treated at 1000 mg/kg bw/day (19%) at the end of treatment period. No changes were observed at measurements performed at the end of recovery. These increases were not associated with other fndings, therefore they were not considered to be adverse. All the other statistically signifcant changes were considered to be incidental.
Motor activity measurements performed at the end of the treatment and recovery periods did not show any signifcant differences between treated animals and controls. (Please refer to table 7 in the "any other information on results incl. tables" section)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Increases in relative (% to body weight) testes and adrenal gland weights were mainly present in high dose males, when compared to control group animals. The increase in testes weights was dose-related, reaching statistical signifcance in males receiving test item at 1000 mg/kg bw/day (+12%). Adrenal gland weight changes were not statistically signifcant. Since no macroscopic or microscopic correlation were noted for the adrenal gland and testes weight changes, these
organ weight variations were considered to be unrelated to treatment. Any other organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.
(Please refer to table 6 in the "any other information on results incl. tables" section)

Recovery sacrifice: At the end of the recovery period, there were no treatment-related changes in the terminal body weight or in the absolute and relative organ weights. In particular, there were no organ weight changes in the testes and adrenal gland. Any organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, there were no treatment-related macroscopic fndings in males and females receiving up to and including 1000 mg/kg bw/day.
Any macroscopic observations, including the subcutaneous masses observed in a 100 mg/kg bw/day male and in a control female, were within the range of expected spontaneous fndings in SD rats of the same age considered incidental and unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related microscopic observationsin males and females receiving up to and at 1000 mg/kg bw/day.
There were no treatment-related microscopic observations in the testes examined with PAS special stain.
There were no treatment-related effects on physiology of the oestrous cycle (oestrous, metestrus, diestrus and proestrus) in control and high dose females.
Any microscopic observations were within the range of expected spontaneous fndings in SD rats of the same age considered incidental and unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Oestrus cyle: No treatment-related anomalies were observed in the oestrous cycle of the treated females at the end of the treatment period, when compared to controls.

Thyroid hormone determination (T3, T4 and TSH): no changes were observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects of toxicological relevance observed.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 3: Selected haematology data (males)

Treatment Group (mg/kg bw/day)

Main study

Recovery phase

Control

100

300

1000

Control

1000

Number

10

10

9

10

4

5

HGB (g/dL)

14.43±

0.533

13.86± *D

0.372

14.09±
0.558

14.77±
0.362

14.00±
0.245

14.08±
0.432

HCT (%)

44.59±

1.730

42.75± *D

0.925

43.97±

1.572

44.80±

0.961

42.75±

0.592

42.62±

1.632

LYM (x10^3/µL)

4.139±

1.1188

4.068±

1.2046

4.732±

0.9578

6.868± +D

1.1948

3.168±
1.0994

4.472±
1.6311

MON (x10^3/µL)

0.181±
0.0905

0.199±
0.0677

0.168±

0.0823

0.304± +D

0.0829

0.178±

0.0846

0.224±

0.0783

BAS (x10^3/µL)

0.023±

0.0166

0.021±
0.0088

0.020±
0.0112

0.043±+D
0.0134

0.0023±

 0.0126

0.026±
0.0114

LUC (x10^3/µL)

0.019±
0.0166

0.020±
0.0141

0.024±
0.0113

0.054±+D
0.0158

0.020±

0.0082

0.068±

0.0554

EOSR (%)

1.40±

0.356

1.19±

0.281

0.94±

0.606

0.77± +C

0.231

1.73±

0.492

1.22±

0.487

LUCR (%)

0.33±
0.195

0.38±
0.132

0.42±

0.156

0.63± +D

0.134

0.45±

0.129

1.12±

1.047

*D Dunnett LSD Test Significant at the 0.05 level

+D Dunnett LSD Test Significant at the 0.01 level

*C Cochran and Cox Test Significant at the 0.05 level

+C Cochran and Cox Test Significant at the 0.01 level

 

HGB: Haemoglobin

HCT: Haematocrit

LYMR/LYM: Lymphocytes (relative or absolute)

MONR/MON: Monocytes (relative or absolute)

EOSR/EOS: Eosinophils (relative or absolute) % or 10^3/µL
BASR/BAS: Basophils (relative or absolute) % or 10^3/µL
LUCR/LUC: Large unstained cells (relative or absolute)

 

Data shown as mean± Standard deviation

 

Table 4: Selected clinical chemistry data

Sex

Treatment Group (mg/kg bw/day)

Main study

Recovery phase

Control

100

300

1000

Control

1000

Number

10

10

9

10

4

5

M

ALP (U/L)

205.78±

42.116

201.75±

26.236

171.18± *D
23.076

165.61± *D
28.486

298.10±
58.376

235.40±
39.130

M

AST (U/L)

116.79±

17.488

96.17± *D

10.224

122.47±

21.731

95.40± *D

11.661

154.38±

37.481

155.14±

40.320

M

GLU (mg/dL)

156.33±

26.113

190.72± +C

14.039

142.73±

39.284

184.03±

36.541

138.18±
29.278

204.68±
62.246

F

GLU (mg/dL)

154.13±

45.932

144.36±

18.219

112.81± *C

20.487

118.29± *C

17.696

196.58±

19.242

198.70±

11.716

M

CREA (mg/dL)

0.379±
0.0431

0.387±
0.0662

0.365±

0.0310

0.329± +C

0.0213

0.313±

0.0222

0.346±

0.0385

M

Ca (mmol/L)

2.440±

0.0389

2.487±
0.0485

2.484±
0.0570

2.564±+D
0.0932

2.435±

 0.0311

2.484± *D
0.0152

M

Na (mmol/L)

140.61±
1.680

141.16±
0.809

142.06± *C
0.747

140.82±
0.673

140.63±

0.310

139.80±

0.919

M

IP (mg/dl)

6.704±

0.6921

8.405± +C

1.3624

7.158±

0.8612

6.313±

0.2748

6.168±

0.7605

6.790±

0.2571

F

IP (mg/dl)

5.167±
0.6691

5.875±
0.9450

6.411± +D

0.6256

6.497± +D

1.1293

6.168±

0.5034

6.412±

0.8027

 

*D Dunnett LSD Test Significant at the 0.05 level

+D Dunnett LSD Test Significant at the 0.01 level

*C Cochran and Cox Test Significant at the 0.05 level

+C Cochran and Cox Test Significant at the 0.01 level

 

M = male, F = female

ALP: Alkaline phosphatase

AST: Aspartate aminotransferase

GLU: Glucose

CREA: Creatinine

Ca: Calcium

Na: Natrium

IP:Inorganic phosphorus

 

Data shown as mean± Standard deviation

 

 

Table 5: Selected open field measurements

Sex

Day

Treatment Group (mg/kg bw/day)

Main study

Control

100

300

1000

Number

15

10

10

15

M

Allocation Day 5

RE

7.3±

1.05

6.0± +D

0.82

5.8± +D
0.92

6.0± +D
1.00

M

Dosing Day 5

RE

7.5±

2.61

6.1±

2.23

6.8±
2.10

6.2±
1.52

M

Dosing Day 13

RE

7.1±

2.37

6.2±

1.93

6.3±
3.09

6.7±
3.02

M

Dosing Day 20

RE

8.4±

1.24

8.4±

1.26

8.7±
1.16

6.9± +D
1.41

M

Dosing Day 27

RE

5.3±

1.68

3.2± +D

1.69

3.3± +D
1.77

4.8± 
0.94

M

Dosing Day 34

RE

3.7±

1.23

4.2± 

0.92

4.5± 
0.85

4.3± 
0.82

M

Dosing Day 41

RE

5.8±

1.53

5.5± 

2.42

4.7± 
1.06

6.0± 
2.78

M

Dosing Day 48

RE

5.9±

2.53

5.0± 

1.89

6.5± 
1.58

5.4± 
0.83

M

Dosing Day 55

RE

8.1±

2.95

7.1± 

5.15

6.1± 
1.52

4.3± #C 
1.67

M

Dosing Day 62

RE

6.1±

1.41

6.3± 

1.34

5.9± 
0.99

6.8± 
1.66

M

Dosing Day 69

RE

4.9±

1.38

5.2± 

1.32

4.8± 
1.32

5.6± 
1.35

M

Dosing Day 76

RE

6.9±

2.11

6.9± 

2.23

5.7± 
1.64

7.5± 
2.50

M

Dosing Day 83

RE

7.4±

1.50

6.7± 

2.91

7.2± 
2.70

7.0± 
1.89

M

Dosing Day 90

RE

6.4±

1.55

7.2± 

2.20

7.1± 
1.79

4.7 *D± 
1.23

F

Allocation Day 6

RE

14.4±

2.50

12.8±

2.49

8.9± +D

2.42

9.2± +D

1.21

F

Dosing Day 6

RE

14.1±

1.79

13.4±

2.12

12.9±

2.47

13.8±

2.04

F

Dosing Day 14

RE

13.2±

1.74

12.4±

2.59

12.1±

1.20

12.9±

2.28

F

Dosing Day 21

RE

14.9±

1.87

13.7±

2.71

15.9±

1.37

13.1± *D

2.02

F

Dosing Day 28

RE

7.8±

1.37

10.3± +D

1.42

9.1±

1.85

6.9±

1.96

F

Dosing Day 35

RE

7.1±

2.36

10.7± *C

4.08

7.5±

1.72

8.7± *C

1.23

F

Dosing Day 42

RE

13.1±

4.65

10.8±

4.18

10.1±

2.85

10.0±

2.04

F

Dosing Day 49

RE

10.2±

3.30

10.1±

3.87

10.5±

2.07

9.6±

2.47

F

Dosing Day 56

RE

10.3±

2.31

10.3±

1.95

9.6±

2.22

9.1±

1.92

F

Dosing Day 63

RE

11.2±

2.37

11.9±

2.13

10.9±

2.92

11.3±

2.43

F

Dosing Day 70

RE

8.7±

2.49

9.5±

2.46

10.4±

1.51

10.0±

2.11

F

Dosing Day 77

RE

8.7±

2.22

8.0±

2.71

10.0±

1.76

9.8±

2.22

F

Dosing Day 84

RE

10.9±

2.00

10.6±

2.01

11.4±

1.84

11.2±

1.31

F

Dosing Day 91

RE

10.5±

2.20

10.7±

2.54

11.1±

1.73

10.9±

1.82

*D Dunnett LSD Test Significant at the 0.05 level

+D Dunnett LSD Test Significant at the 0.01 level

*C Cochran and Cox Test Significant at the 0.05 level

+C Cochran and Cox Test Significant at the 0.01 level

RE: Rearing

Data shown as mean± Standard deviation

 

 

Table 6: Selected Organ weight to terminal body weight (%) in males

Treatment Group (mg/kg bw/day)

Number

Testes

 

Adrenal gland

Control group

10

0.9274±

0.08141

0.0131±

0.00174

100

10

0.9407±

0.04834

0.0131±

0.00111

300

10

1.0133±

0.11447

0.0131±

0.00136

1000

10

1.0397± *D
0.07421

0.0146± *D

0.00124

Recovery phase

Control group

4

0.0111±

0.00034

0.9127±

0.03394

1000

5

0.0112±

0.00168

0.9293±
0.04947

*D =Dunnett LSD Test Significant at the 0.05 level

Data shown as mean± Standard deviation

 

Table 7: Selected landing foot splay data

Sex

Treatment Group (mg/kg bw/day)

Main study

Recovery phase

Control

100

300

1000

Control

1000

Number

14

10

10

15

4

4

M

LAN 1 (cm)

5.64±

1.345

6.45±

1.066

6.95± *D
0.862

6.87± *D
1.316

6.88±
1.109

7.40±
1.517

M

LAM (cm)

5.661±

0.9383

6.725±

0.9534

6.752±

1.2343

6.719± *D

1.2607

7.375±

0.9242

7.450±

0.7786

 

*D Dunnett LSD Test Significant at the 0.05 level

+D Dunnett LSD Test Significant at the 0.01 level

*C Cochran and Cox Test Significant at the 0.05 level

+C Cochran and Cox Test Significant at the 0.01 level

 

LAN1: Landing foot splay 1 (first measurement of distance between ink blots (cm))

LAN2: Landing foot splay 2 (second measurement of distance between ink blots (cm))

LAM: Landing foot splay mean (averaged measurements of distance between ink blots (cm))

Data shown as mean± Standard deviation

 

 

Applicant's summary and conclusion

Conclusions:
No adverse effects were observed at all dose levels investigated following treatment with the test item, when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg bw/day when compared to controls. One case of poor health conditions, which caused a premature sacrifice for humane reasons occurred in one female animal dosed at 1000 mg/kg bw/day. The relation to treatment with the test item of the illness status of this female cannot be established on the basis of macroscopic and microscopic findings. Based on these findings, it can be concluded that the NOAEL for this study is >=1000 mg/kg bw/day.