Registration Dossier

Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 2016 to 03 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 204 to 255 g (males), 152 to 195 g (females)
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) , ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at löeast 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 11 May 2016 (first day of treatment) to 07 September 2016 (final day of necropsy)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly for the first two weeks and then fortnightly thereafter and stored at approximately 4 ºC in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 1.67, 16.67, 50 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days.
Samples of test item formulations were taken on six occasions and analyzed for concentration. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.
Duration of treatment / exposure:
90 d + 28 d recovery
Frequency of treatment:
daily, 7 d/week
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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: based on previous dose range finding study (Envigo Research Limited Study Number QB13VN; Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat)
- Rationale for animal assignment (if not random): randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 28 d
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin color, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- for each cage group at weekly intervals

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: Yes
- Time schedule for examinations: daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all control and treated animals were examined pre-treatment and all non-recovery control and non-recovery high dose animals were examined before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Bile acids , Gamma glutamyl transpeptidase, Triglycerides (Trigs)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (with cervix)

HISTOPATHOLOGY: Yes
samples preserved:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Gross lesions, Heart, Ileum (including Peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (including coagulating gland), Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/Parathyroid, Tongue, Trachea, Urinary bladder, Uterus (with cervix), Vagina

All tissues from non-recovery control and 200 mg/kg bw/day dose group animals and any animals that died during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of possible treatment-related mesenteric lymph node and adrenal changes, examination was subsequently extended to include similarly prepared sections of the mesenteric lymph nodes and adrenals from animals in the low, intermediate and recovery dose groups.

Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Incidences of increased salivation were evident in all animals of both sexes treated with 300 mg/kg bw/day from Day 5 (males) and Day 8 (females) until the termination of treatment. Episodes of noisy respiration were also evident in all animals of both sexes treated with 300 mg/kg bw/day from Day 1 (males) and Day 8 (females) onwards. Incidences of labored respiration, decreased respiratory rate, hunched posture and lethargy were also evident in some males treated with 300 mg/kg bw/day and a decreased respiratory rate was evident in one female from this treatment group. Throughout the treatment-free, twenty-eight day period, four males and four females that were previously given 300 mg/kg bw/day continued to show episodes of noisy respiration. The females that were sacrificed in extremis also showed labored respiration, decreased respiratory rate, pilo-erection and hunched posture and the female that was terminated on Day 27 also had gasping respiration and a distended abdomen. The male that was sacrificed in extremis also showed gasping respiration.
At 100 mg/kg bw/day, increased salivation and noisy respiration were evident throughout the treatment period albeit to a lesser extent than at 300 mg/kg bw/day. One male from this treatment group also showed hunched posture on Day 10 only.
No such effects were detected in males treated with 10 mg/kg bw/day however one female from this treatment group had noisy respiration on Day 26 only.
One control female had generalized fur loss between Days 81 and 91. This was considered to be incidental.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females and one male treated with 300 mg/kg bw/day were sacrificed in extremis on Days 8, 27 and 85. A further female treated with 300 mg/kg bw/day was found dead on Day 82. There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 300 mg/kg bw/day showed lower body weight gain throughout the treatment period (excluding Weeks 3 and 10). Statistical significance (p<0.05-0.01) was achieved during Weeks 1, 2, 4, 5 and 13. Consequently overall body weight gain was lower than controls. Significant improvement in body weight gain was evident in males previous treated with 300 mg/kg bw/day during the twenty-eight day treatment-free period. Statistically significant increases (p<0.05-0.01) when compared to controls were evident in these males throughout the treatment-free period.
No toxicologically significant effects were detected in treated females or in males treated with 100 or 10 mg/kg bw/day.
A statistically significant increase (p<0.05) in body weight gain during Week 10 and a statistically significant reduction (p<0.05) in body weight gain during Week 13 was evident in males treated with 100 and 10 mg/kg bw/day. A true dose related response was not evident and no adverse effect on overall body weight gain was evident in these males. Therefore the intergroup differences were considered not to be of toxicological importance. Females treated with 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during Week 6. No adverse effect on overall body weight gain was evident in these females. Therefore, in isolation, the intergroup difference was considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 300 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. Females from this treatment group showed a reduction in food consumption from Week 10 onwards. Incidences of reduced food conversion efficiency were also evident in these animals during the treatment period and generally followed the fluctuations seen in body weight gain. During the twenty-eight day treatment-free period, recovery in both food consumption and food conversion efficiency were evident in animals of both sexes that were previously given 300 mg/kg bw/day.
No such effects were detected in animals of either sex treated with 100 or 10 mg/kg bw/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Incidences of reduced food conversion efficiency were evident in high dose animals during the treatment period and generally followed the fluctuations seen in body weight gain. During the twenty-eight day treatment-free period, recovery in both food consumption and food conversion efficiency were evident in animals of both sexes that were previously given 300 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not reveal any inter-group differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmoscopic examination of animals of both sexes from the non-recovery control and 300 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 300 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in neutrophils and a statistically significant reduction (p<0.05) in lymphocytes. A statistically significant increase (p<0.05) in neutrophils was also present in males previously treated with 300 mg/kg bw/day at the end of the twenty-eight day treatment-free period. The majority of individual values for both parameters at 300 mg/kg bw/day were outside of historical control ranges and some individual neutrophil values at 100 mg/kg bw/day and in the recovery animals were also outside of the historical control range. All individual lymphocyte values at 100 mg/kg bw/day were within historical control range.
No toxicologically significant effects were detected in treated females or in males treated with 10 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in hemoglobin, hematocrit and mean corpuscular volume. Recovery males that were previously given 300 mg/kg bw/day also showed a statistically significant increase (p<0.01) in hematocrit at the end of the treatment-free period. The majority of individual values for all parameters were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in monocytes. The majority of individual values were within historical control range and in the absence of a similar effect in 300 mg/kg bw/day females or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Following the treatment-free period, males that were previously given 300 mg/kg bw/day showed a statistically significant increase (p<0.05) in erythrocyte count. The majority of individual values were within historical control range and in the absence of a similar effect in 300 mg/kg bw/day males at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 300 and 100 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.01) in bile acids. Males treated with 300 mg/kg bw/day and recovery males that were previously given 300 mg/kg bw/day showed a statistically significant reduction (P<0.05-0.01) in triglycerides. Females treated with 300 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in urea, total protein, albumin, albumin/globulin ratio, creatinine and bilirubin and statistically significant reductions (p<0.01) in cholesterol and chloride concentration. The effect on urea, total protein, albumin and chloride concentration also extended to females treated with 100 mg/kg bw/day with females from the 10 mg/kg bw/day dose group also showing a statistically significant increase in albumin. The majority of individual values for these parameters for both sexes were within historical control ranges and although these intergroup differences may indicate minor perturbations in hepatic metabolism, in the absence of any associated microscopic hepatic changes evident these differences were considered not to represent an adverse effect of treatment.
Animals of both sexes treated with 300 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in alanine aminotransferase and males treated with 300 mg/kg bw/day also showed a statistically significant increase (p<0.01) in aspartate aminotransferase. These intergroup differences may again indicate minor perturbation in hepatic metabolism and the majority of individual values did exceed historical control ranges, however, in the absence of any associated microscopic hepatic changes evident, the intergroup differences were considered not to represent an adverse effect of treatment.
Females from all treatment groups showed a statistically significant increase (p<0.05-0.01) in calcium concentration. Individual values for all treated females were actually below the historical control range and all individual values for control females were also below the historical control range which may have contributed to these differences. Therefore the intergroup differences were considered to reflect biological variation rather than a true effect of treatment.
Following the treatment-free period, males that were previously given 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total protein whilst females that were previously given 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in phosphorus. The majority of individual values were within historical control ranges and in the absence of a similar effect in 300 mg/kg bw/day animals at the end of the treatment period or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
At the end of the treatment period, males treated with 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. The majority of individual values for absolute weights were within the historical control range; however, the majority of individual values for relative weights were outside of the historical control range. No associated microscopic spleen changes were evident in these animals, therefore, the intergroup difference was considered not to be of toxicological significance. Males treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in absolute and relative liver weight and a statistically significant increase (p<0.05) in absolute and relative heart weight. Females treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.05) in heart weight both absolute and relative to terminal body weight. The majority of individual values for all parameters were within historical control ranges and in the absence of similar effects in 300 mg/kg bw/day animals or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected in surviving animals.
The male treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 85 had gaseous distension in the stomach and ileum, pale and enlarged lungs and a reddened glandular region in the stomach. This animal had tracheal ulceration and bronchopneumonia, indicative of test item irritancy through reflux, and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 8 had a dark liver and reddened lungs with clear fluid present. This female had edema and inflammation in the lungs and ulceration of the trachea, also indicative of test item irritancy through reflux, and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was sacrificed in extremis on Day 27 had gaseous distention in the intestines and reddened and enlarged lungs. This female had hemorrhage in the lungs as well as minimal inflammatory change, possibly through technical error and this is considered to be the cause of death.
The female treated with 300 mg/kg bw/day that was found dead on Day 82 had gaseous distension in the duodenum, ileum and jejunum. Histological examination did not reveal any obvious cause of death. Some tissues were notably autolysed.
A number of animals across most dose groups including controls showed reddened lungs. Such findings are common in this type of study and were considered unrelated to treatment with the test item. One non-recovery control male had an enlarged urinary bladder which contained brown fluid. Two non-recovery females treated with 300 mg/kg bw/day and one female treated with 100 mg/kg bw/day had increased pelvic space in one or both kidneys. The left kidney for one of the females treated with 300 mg/kg bw/day was also fluid filled. Following the treatment-free period, one control female had increased pelvic space in the left kidney with this kidney also being fluid-filled, one male that was previously given 300 mg/kg bw/day had small testes and one male that was previously given 300 mg/kg bw/day had a small and dark spleen. In the absence of any associated treatment-related microscopic changes, these findings were considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:
Mesenteric Lymph Node:
Histiocytosis was present in the mesenteric lymph node of all non-recovery animals of both sexes treated with 300 mg/kg bw/day, varying from minimal to moderate in severity. It was also present in all animals of both sexes treated with 100 mg/kg bw/day at minimal or mild severity. No such effects were detected in animals of both sexes treated with 10 mg/kg bw/day. Following the twenty-eight day recovery period, histiocytosis at minimal or mild severity was present in all animals of both sexes that were previously treated with 300 mg/kg bw/day.

The following microscopic abnormalities were evident, however, these were considered to reflect individual variation or an equivocal finding rather than an effect of treatment:

Spleen:
Increased hemosiderin was present in the spleen of one non-recovery control female and two non-recovery females treated with 300 mg/kg bw/day at a mild severity after evaluation and blind reading of all animals. This was considered not to indicate an effect of treatment and reflects individual variation.

Thymus:
Minimal thymic atrophy was present in two non-recovery males treated with 300 mg/kg bw/day. Whilst thymic atrophy is often considered to be related to stress, without other obvious signs in these animals (adrenal cortical hypertrophy for example) or a corresponding reduction in organ weight, it was considered to be of equivocal significance and therefore cannot be attributed to treatment.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
haematology
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
haematology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

*         Significantly different from control group p<0.05

**        Significantly different from control group p<0.01

n        Data not appropriate for statistical analysis

.         not examined

 

 

Group Mean Body Weight Values

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

1

8

15

22

27

29

36

43

50

57

1(M)

Mean

229.7

259.5

288.6

309.5

.

327.7

343.7

359

373.2

383.4

 

S.D.

11.3

15.6

22.2

24.8

.

29.1

30

32.8

35.3

36.8

 

N

20

20

20

20

0

20

20

20

20

20

2(M)

Mean

232.8

264.6

294.9

319.3

.

340.7

359.5

374.9

388.6

399.7

 

S.D.

11.8

18.5

21.9

24.1

.

27.1

32.7

36.7

39.8

42.4

 

N

10

10

10

10

0

10

10

10

10

10

3(M)

Mean

237.9

270.2

296.1

320.7

.

339.3

354.3

369.9

379.2

390.9

 

S.D.

10

14.4

15.3

16.8

.

20.7

23.2

25

28.2

29.5

 

N

10

10

10

10

0

10

10

10

10

10

4(M)

Mean

229.1

248.9

266.7

289.9

.

306.3

315.3

327.5

335.1

343.5

 

S.D.

8.2

10.8

16.5

18.5

.

22.4

25.7

22.7

25.3

29

 

N

20

20

20

20

0

20

20

20

20

20

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

64

71

78

85

91

98

105

112

119

1(M)

Mean

395.5

403.8

408.8

415.8

422.9

419.6

426.1

431.4

430.3

 

S.D.

38.6

39.1

41.3

42.6

44.8

45.7

44.8

43.7

47.1

 

N

20

20

20

20

20

10

10

10

10

2(M)

Mean

414.2

425

434

441.1

446.4

.

.

.

.

 

S.D.

46.3

47.3

49.5

52.5

54.7

.

.

.

.

 

N

10

10

10

10

10

0

0

0

0

3(M)

Mean

406

417.9

426

433.1

436.8

.

.

.

.

 

S.D.

30

28.7

27.8

30.1

28.6

.

.

.

.

 

N

10

10

10

10

10

0

0

0

0

4(M)

Mean

355.3

365.5

369.2

374.6

373.5

385.5

400.2

409.9

415.3

 

S.D.

30.3

33.7

31.3

30.7

32

33.9

32.4

31.3

32.4

 

N

20

20

20

20

19

10

10

10

10

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

1

8

15

22

27

29

36

43

50

57

1(F)

Mean

167.6

183.5

200.4

212.8

.

225.6

232.9

242.7

249.9

253.5

 

S.D.

8.3

9.3

9.4

9.9

.

14.2

14

14.6

15.8

17.8

 

N

20

20

20

20

0

20

20

20

20

20

2(F)

Mean

172.6

190

210.3

221.3

.

232.8

241

250.2

256

263.4

 

S.D.

6.1

11.3

11.6

13.3

.

19.8

20.1

20.6

22.1

22.3

 

N

10

10

10

10

0

10

10

10

10

10

3(F)

Mean

168.8

183.6

203

212.8

.

220.7

228.4

237.7

244.9

248.4

 

S.D.

8.4

11

12.2

15.4

.

19.3

19.9

19.4

21.9

26.3

 

N

10

10

10

10

0

10

10

10

10

10

4(F)

Mean

171.4

186.9

203.2

211.7

184

227.9

236.2

242

249.8

254.8

 

S.D.

10.2

12.1

15.6

17.3

.

16.4

16.9

18.6

19

20.9

 

N

20

20

19

19

1

18

18

18

18

18

  

Group

(sex)

 

Day Numbers Relative to Start Date                                        

64

71

78

85

91

98

105

112

119

1(F)

Mean

256.2

262.6

266.4

265.6

270.6

274.7

273.6

274.5

274.7

 

S.D.

19.4

18.2

18.7

19

17.6

17.7

19.4

20.5

19.4

 

N

20

20

20

20

20

10

10

10

10

2(F)

Mean

266.4

269.3

276

279.7

281.7

.

.

.

.

 

S.D.

21.8

25.5

19.7

22.9

21.5

.

.

.

.

 

N

10

10

10

10

10

0

0

0

0

3(F)

Mean

251.2

258

261.3

259.8

264.4

.

.

.

.

 

S.D.

24.2

23

24.5

27.3

23.6

.

.

.

.

 

N

10

10

10

10

10

0

0

0

0

4(F)

Mean

259.3

265.2

264.2

266.9

267.5

269.5

269.5

273.1

275

 

S.D.

20.5

20.6

18.4

18.9

22.6

30.5

27.9

28.8

29.8

 

N

18

18

18

17

17

8

8

8

8

 

Group Mean Body Weight Gains

Group

(sex)

 

Day Numbers Relative to Start Date                                        

1 – 8

8-15

15-22

22-27

22-29

29-36

36-43

43-50

50-57

1(M)

Mean

29.8

29.1

20.9

.

18.3

16

15.4

14.2

10.3

 

S.D.

6.6

11.3

4

.

5.7

4.3

6

4.8

3.9

 

N

20

20

20

.

20

20

20

20

20

2(M)

Mean

31.8

30.3

24.4

.

21.4

18.8

15.4

13.7

11.1

 

S.D.

7.4

4.8

4.4

.

4.3

6.7

5.1

5

4.1

 

N

10

10

10

.

10

10

10

10

10

3(M)

Mean

32.3

25.9

24.6

.

18.6

15

15.6

9.3

11.7

 

S.D.

5

4.1

5.4

.

5.6

5.6

2.4

5.9

5.4

 

N

10

10

10

.

10

10

10

10

10

4(M)

Mean

19.9**

17.8**

23.3

.

16.4**

9.1*

12.2

7.6

8.4

 

S.D.

7

10.9

8.1

.

15.1

9.7

7.4

11.1

7.5

 

N

20

20

20

.

20

20

20

20

20

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

57-64

64-71

71-78

78-85

85-91

91-98

98-105

105-112

112-119

1(M)

Mean

12.1

8.3

5

7

7.1

3.5

6.5

5.3

1.1

 

S.D.

4.1

3.5

5.3

2.7

4.6

3.1

4.9

3.2

8.9

 

N

20

20

20

20

20

10

10

10

10

2(M)

Mean

14.5

10.8*

9

7.1

5.3*

.

.

.

.

 

S.D.

5.5

3.3

2.5

4.8

4.7

.

.

.

.

 

N

10

10

10

10

10

.

.

.

.

3(M)

Mean

15.1

11.9*

8.1

7.1

3.7*

.

.

.

.

 

S.D.

4.7

3

5.7

7.7

2.8

.

.

.

.

 

N

10

10

10

10

10

.

.

.

.

4(M)

Mean

11.8

10.3*

3.7

5.5

2.1*

7.6*

14.7**

9.7**

5.4*

 

S.D.

6.3

7.6

9.6

11.2

10.4

5

5

3.4

4

 

N

20

20

20

20

19

10

10

10

10

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

1 – 8

8-15

15-22

22-27

22-29

29-36

36-43

43-50

50-57

1(F)

Mean

15.9

16.9

12.5

.

12.8

7.3

9.9

7.2

3.6

 

S.D.

5.9

5.7

4.5

.

7.2

7.4

5.2

4.9

6.1

 

N

20

20

20

.

20

20

20

20

20

2(F)

Mean

17.4

20.3

11

.

11.5

8.2

9.2

5.8

7.4

 

S.D.

6.4

7.8

4.5

.

9.4

6.5

6.2

6.3

4.9

 

N

10

10

10

.

10

10

10

10

10

3(F)

Mean

14.8

19.4

9.8

.

7.9

7.7

9.3

7.2

3.5

 

S.D.

5.2

6

6.9

.

6.2

6

5.9

7.2

6.9

 

N

10

10

10

.

10

10

10

10

10

4(F)

Mean

15.5

15.9

8.5

 4.0

14.9

8.2

5.8*

7.8

5

 

S.D.

4.5

7.2

9.3

.

10.8

5.7

6.2

7.9

9.7

 

N

20

19

19

1

18

18

18

18

18

 

Group

(sex)

 

Day Numbers Relative to Start Date                                        

57-64

64-71

71-78

78-85

85-91

91-98

98-105

105-112

112-119

1(F)

Mean

2.8

6.4

3.8

0.8

5.1

1.8

1.1

0.9

0.2

 

S.D.

6.8

6

5.2

6.2

5.8

4.8

3.6

3.3

4

 

N

20

20

20

20

20

10

10

10

10

2(F)

Mean

3

2.9

6.7

3.7

2

.

.

.

.

 

S.D.

5.7

9.6

10.5

6.3

7.2

.

.

.

.

 

N

10

10

10

10

10

.

.

.

.

3(F)

Mean

2.8

6.8

3.3

1.5

4.6

.

.

.

.

 

S.D.

6.1

5.1

7

4.5

7.4

.

.

.

.

 

N

10

10

10

10

10

.

.

.

.

4(F)

Mean

4.5

5.8

1.0

2.7

0.6

2.9

0

3.6

1.9

 

S.D.

9.6

7.3

9.6

5.3

8.5

6.4

4.4

4.8

2.6

 

N

18

18

18

17

17

8

8

8

8

 

Group Mean Hematological Values

Non-recovery animals:       

 

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

 

g/dl

10^12/l

%

pg

fl

g/dl

10^9/l

10^9/l

1(M)

Mean

15.7

9.151

47.64

17.17

52.06

32.98

7.62

1.098

 

S.D.

0.58

0.394

2.15

0.48

1.09

0.48

1.16

0.375

 

N

10

10

10

10

10

10

10

10

2(M)

Mean

15.71

9.086

47.56

17.33

52.44

32.99

7.66

1.284

 

S.D.

0.31

0.365

0.72

0.97

2.67

0.3

1.45

0.401

 

N

10

10

10

10

10

10

10

10

3(M)

Mean

15.68

9.136

47.87

17.18

52.44

32.77

6.49

1.442*

 

S.D.

0.58

0.441

2.04

0.68

1.81

0.48

0.97

0.269

 

N

10

10

10

10

10

10

10

10

4(M)

Mean

16.42**

9.239

49.86*

17.8

54.01*

32.93

8.17

3.326**

 

S.D.

0.54

0.364

1.83

0.77

2.13

0.29

1.72

1.531

 

N

9

9

9

9

9

9

9

9

       

 

 

Lymph

Mono

Eos

Bas

CT

PLT

APTT

Group

(sex)

 

10^9/l

10^9/l

10^9/l

10^9/l

Seconds

10^9/l

Seconds

1(M)

Mean

6.441

0.006n

0.076

0.000n

9.53

585

16.01

 

S.D.

1.026

0.019

0.065

0

0.46

79.6

1.52

 

N

10

10

10

10

10

10

10

2(M)

Mean

6.302

0.000n

0.074

0.000n

9.65

575

16.29

 

S.D.

1.259

0

0.089

0

0.76

90

1.14

 

N

10

10

10

10

10

10

10

3(M)

Mean

4.983*

0.007n

0.059

0.000n

9.71

579.9

16.67

 

S.D.

0.94

0.022

0.058

0

0.89

75

1.11

 

N

10

10

10

10

10

10

10

4(M)

Mean

4.838*

0.009n

0.09

0.000n

9.91

581.9

16.34

 

S.D.

2.089

0.027

0.075

0

1.05

43.7

1.8

 

N

9

9

9

9

9

9

9

 

 

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

 

g/dl

10^12/l

%

pg

fl

g/dl

10^9/l

10^9/l

1(F)

Mean

14.94

8.267

44.72

18.09

54.1

33.44

4.56

1.088

 

S.D.

0.45

0.262

1.43

0.44

1.19

0.31

1.35

1.371

 

N

10

10

10

10

10

10

10

10

2(F)

Mean

14.83

8.116

44.34

18.3

54.71

33.45

4.49

0.681

 

S.D.

0.56

0.448

1.63

0.47

1.48

0.31

0.77

0.174

 

N

10

10

10

10

10

10

10

10

3(F)

Mean

15.32

8.215

45.85

18.67

55.89

33.4

4.69

1.073

 

S.D.

0.43

0.428

1.28

0.91

2.48

0.24

1.35

0.479

 

N

10

10

10

10

10

10

10

10

4(F)

Mean

15.06

8.368

45.4

18.02

54.27

33.2

4.64

1.046

 

S.D.

0.63

0.454

2.19

0.38

1.06

0.34

1.36

0.513

 

N

9

9

9

9

9

9

9

9

       

 

 

Lymph

Mono

Eos

Bas

CT

PLT

APTT

Group

(sex)

 

10^9/l

10^9/l

10^9/l

10^9/l

Seconds

10^9/l

Seconds

1(F)

Mean

3.419

0.000n

0.057

0.000n

9.06

621.4

14.69

 

S.D.

1.019

0

0.055

0

0.94

49.5

1.66

 

N

10

10

10

10

10

10

10

2(F)

Mean

3.762

0.000n

0.048

0.000n

8.86

617.4

15.24

 

S.D.

0.686

0

0.059

0

0.69

103.6

2.04

 

N

10

10

10

10

10

10

10

3(F)

Mean

3.583

0.014*,n

0.022

0.000n

8.88

598.7

15.79

 

S.D.

1.443

0.024

0.029

0

0.49

47

1.02

 

N

10

10

10

10

10

10

10

4(F)

Mean

3.563

0.000n

0.034

0.000n

8.61

650.3

14.51

 

S.D.

1.014

0

0.051

0

0.34

52.7

1.6

 

N

9

9

9

9

9

9

9

            

Recovery animals:

 

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

 

g/dl

10^12/l

%

pg

fl

g/dl

10^9/l

10^9/l

1(M)

Mean

16.01

9.047

47.84

21.46

52.9

33.45

7.45

1.222

 

S.D.

0.53

0.442

1.51

12.52

2.81

0.44

1.15

0.283

 

N

10

10

10

10

10

10

10

10

4(M)

Mean

16.51

9.410*

49.81**

17.57

53.1

33.14

7.53

1.726*

 

S.D.

0.54

0.318

1.26

1.01

2.51

0.47

0.98

0.575

 

N

10

10

10

10

10

10

10

10

       

 

 

Lymph

Mono

Eos

Bas

CT

PLT

APTT

Group

(sex)

 

10^9/l

10^9/l

10^9/l

10^9/l

Seconds

10^9/l

Seconds

1(M)

Mean

6.168

0.000n

0.059

0.000n

9.92

591.4

15.19

 

S.D.

0.972

0

0.069

0

0.61

101.7

2.02

 

N

10

10

10

10

10

10

10

4(M)

Mean

5.743

0.000n

0.061

0.000n

10.06

612.9

14.61

 

S.D.

0.824

0

0.064

0

0.57

70.3

0.84

 

N

10

10

10

10

10

10

10

       

 

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Group

(sex)

 

g/dl

10^12/l

%

pg

fl

g/dl

10^9/l

10^9/l

1(F)

Mean

15.56

8.466

45.72

18.41

54.02

34.06

4.24

0.678

 

S.D.

0.62

0.383

1.77

0.68

1.68

0.35

0.65

0.151

 

N

10

10

10

10

10

10

10

10

4(F)

Mean

15.7

8.371

46.1

18.81

55.15

34.09

4.68

0.931

 

S.D.

0.47

0.453

1.39

0.96

2.06

0.57

1.2

0.362

 

N

8

8

8

8

8

8

8

8

       

 

 

Lymph

Mono

Eos

Bas

CT

PLT

APTT

Group

(sex)

 

10^9/l

10^9/l

10^9/l

10^9/l

Seconds

10^9/l

Seconds

1(F)

Mean

3.524

0.000n

0.04

0.000n

8.58

584.9

13.88

 

S.D.

0.67

0

0.045

0

0.7

66.1

1.48

 

N

10

10

10

10

10

10

10

4(F)

Mean

3.718

0.000n

0.028

0.000n

8.65

589.5

14.29

 

S.D.

1.061

0

0.04

0

0.41

98.7

1.48

 

N

8

8

8

8

8

8

8

 

Group Mean Blood Chemical Values

Non-recovery animals:       

 

 

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Cl

Ca++

P

Group

(sex)

 

mg/dl

mg/dl

g/dl

g/dl

Ratio

mmol/l

mmol/l

mmol/l

mmol/l

mmol/l

1(M)

Mean

33.5

175.6

6.432

3.62

1.294

146.2

4.75

102.7

1.62

1.39

 

S.D.

6.9

17.6

0.225

0.13

0.097

2.1

0.968

1.9

0.097

0.27

 

N

10

10

10

10

10

10

10

10

10

10

2(M)

Mean

32.2

170.7

6.192

3.44

1.23

146.3

4.704

103.4

1.581

1.28

 

S.D.

6.6

27.6

0.947

0.63

0.128

1.7

0.423

1.3

0.158

0.29

 

N

10

10

10

10

10

10

10

10

10

10

3(M)

Mean

32.2

170.1

6.276

3.51

1.275

145.5

4.627

103.3

1.643

1.28

 

S.D.

5.7

16

0.282

0.2

0.074

2.1

0.315

1.7

0.081

0.15

 

N

10

10

10

10

10

10

10

10

10

10

4(M)

Mean

34.1

166.7

6.161

3.49

1.307

148

5.213

101.9

1.617

1.54

 

S.D.

7

28.7

0.727

0.42

0.088

2.5

1.65

2.2

0.22

0.41

 

N

9

9

9

9

9

9

9

9

9

9

       

 

 

yGT

ASAT

ALAT

AP

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

 

IU/l

IU/l

IU/l

IU/l

mg/dl

mg/dl

mg/dl

mg/dl

µmol/l

1(M)

Mean

0.00n

85.1

63.6

113.4

0.783

272.9

77.4

0.102

10.42

 

S.D.

0

23.3

8.9

26.9

0.057

130.5

9.9

0.041

3.86

 

N

10

10

10

10

10

10

10

10

10

2(M)

Mean

0.00n

74.9

58.9

119.9

0.823

267.9

84.6

0.117

8.18

 

S.D.

0

15.1

14.4

32.8

0.2

173.2

20.2

0.022

2.78

 

N

10

10

10

10

10

10

10

10

10

3(M)

Mean

0.00n

102.4

86.5*

109.3

0.797

165.6

78.3

0.118

7.24*

 

S.D.

0

22.9

25.5

15.4

0.071

43.7

17.2

0.018

4.62

 

N

10

10

10

10

10

10

10

10

10

4(M)

Mean

0.00n

128.9**

89.3*

88.1

0.773

135.7**

64

0.11

4.27**

 

S.D.

0

39.4

27.4

32.9

0.112

72

14

0.045

0.94

 

N

8

9

9

9

9

9

9

9

9

       

 

 

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Cl

Ca++

P

Group

(sex)

 

mg/dl

mg/dl

g/dl

g/dl

Ratio

mmol/l

mmol/l

mmol/l

mmol/l

mmol/l

1(F)

Mean

38.3

149

7.239

4.31

1.48

148

4.953

105.3

1.784

1.58

 

S.D.

3.9

16.3

0.377

0.36

0.185

2.1

1.156

1.8

0.143

0.27

 

N

10

10

10

10

10

10

10

10

10

10

2(F)

Mean

33.7

153

7.457

4.56*

1.578

148

4.41

105.5

1.881*

1.48

 

S.D.

3.5

13.3

0.517

0.31

0.07

2.3

0.289

1.8

0.067

0.23

 

N

10

10

10

10

10

10

10

10

10

10

3(F)

Mean

43.0*

149

7.837**

4.77**

1.556

147.6

4.838

103.6*

1.934**

1.76

 

S.D.

4.8

13.7

0.401

0.24

0.12

2.2

0.459

1.5

0.079

0.45

 

N

10

10

10

10

10

10

10

10

10

10

4(F)

Mean

51.7**

155.9

7.918**

4.92**

1.644*

146

5.576

103.1**

1.942**

1.68

 

S.D.

6.8

17.7

0.54

0.25

0.14

2.6

3.729

1.6

0.09

0.47

 

N

9

9

9

9

9

9

9

9

9

9

 

 

 

yGT

ASAT

ALAT

AP

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

 

IU/l

IU/l

IU/l

IU/l

mg/dl

mg/dl

mg/dl

mg/dl

µmol/l

1(F)

Mean

0.00n

102.1

63.9

66.2

0.799

141

74.4

0.077

11.63

 

S.D.

0

39.9

13.4

43.9

0.074

68.7

9.4

0.044

9.05

 

N

10

10

10

10

10

10

10

10

10

2(F)

Mean

0.10n

86.9

63.2

72.4

0.818

159.4

71.8

0.104

15.38

 

S.D.

0.32

15.6

11.4

16.5

0.046

41.7

13.3

0.02

10.3

 

N

10

10

10

10

10

10

10

10

10

3(F)

Mean

0.00n

101.9

102.8**

55.4

0.826

161.7

69.4

0.098

12.83

 

S.D.

0

50.3

15.9

9.9

0.042

52.5

11.9

0.037

12.52

 

N

10

10

10

10

10

10

10

10

10

4(F)

Mean

0.00n

124

111.1**

50.2

0.862*

127.3

59.4**

0.116*

8.88

 

S.D.

0

64.7

36.4

19.8

0.062

46.5

8.8

0.047

5.03

 

N

9

9

9

9

9

9

9

9

9

 

Recovery animals:

 

 

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Cl

Ca++

P

Group

(sex)

 

mg/dl

mg/dl

g/dl

g/dl

Ratio

mmol/l

mmol/l

mmol/l

mmol/l

mmol/l

1(M)

Mean

46.3

158.7

6.98

3.95

1.297

147.4

5.013

103.8

2.805

1.81

 

S.D.

6.9

19.4

0.16

0.1

0.089

2.1

0.865

2.6

0.262

0.13

 

N

10

10

10

10

10

10

10

10

10

10

4(M)

Mean

43.7

160.4

6.766*

3.89

1.367

145.9

4.744

102.9

2.811

1.79

 

S.D.

4

7.8

0.25

0.18

0.12

1.5

0.347

1.1

0.087

0.21

 

N

10

10

10

10

10

10

10

10

10

10

 

 

 

yGT

ASAT

ALAT

AP

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

 

IU/l

IU/l

IU/l

IU/l

mg/dl

mg/dl

mg/dl

mg/dl

µmol/l

1(M)

Mean

0.00n

86.7

66.2

135

0.789

237.1

94.5

0.116

13.78

 

S.D.

0

21.9

15.3

34.2

0.115

55.8

18.7

0.018

8.3

 

N

10

10

10

10

10

10

10

10

10

4(M)

Mean

0.00n

111.6

84.8

121

0.783

164.5*

87.2

0.104

15.68

 

S.D.

0

47

46.9

20

0.065

67.1

12.8

0.015

10.32

 

N

10

10

10

10

10

10

10

10

10

 

 

 

Urea

Glucose

total Prot.

Albumin

A/G

Na+

K+

Cl

Ca++

P

Group

(sex)

 

mg/dl

mg/dl

g/dl

g/dl

Ratio

mmol/l

mmol/l

mmol/l

mmol/l

mmol/l

1(F)

Mean

51.2

159.6

7.116

4.35

1.568

145.9

4.716

103.5

2.672

1.35

 

S.D.

15.1

15.7

0.576

0.38

0.081

3.5

0.925

2.1

0.102

0.22

 

N

10

10

10

10

10

10

10

10

10

10

4(F)

Mean

57.9

163.8

7.559

4.61

1.565

144.8

4.335

102.9

2.699

1.14*

 

S.D.

9.2

12.8

0.423

0.29

0.066

1.5

0.514

2.2

0.088

0.2

 

N

8

8

8

8

8

8

8

8

8

8

 

 

 

yGT

ASAT

ALAT

AP

Creat

Tri

Chol

Bili

Bile acids

Group

(sex)

 

 

 

 

 

 

 

 

 

 

1(F)

Mean

0.00n

139.5

88.9

50

0.776

175.3

89.8

0.112

16.31

 

S.D.

0

82.4

36.1

11.5

0.191

62.8

14.2

0.027

17.81

 

N

10

10

10

10

10

10

10

10

10

4(F)

Mean

0.00n

100.6

65.9

52.4

0.87

156.4

91.6

0.101

15

 

S.D.

0

28.7

20.1

11

0.121

73.1

10.9

0.016

9.01

 

N

8

8

8

8

8

8

8

8

8

 

Histopathology

Non-recovery animals

 

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg bw/d)

0

10

100

300

0

10

100

300

Mesenteric lymph node

Histiocytosis

 

 

 

 

 

 

 

 

Minimal

0

0

5

0

0

0

3

0

Slight

0

0

5

7

0

0

7

6

Moderate

0

0

0

2

0

0

0

3

Total

0

0

10

9

 0

 0

10 

 9

Plasmacytosis

 

 

 

 

 

 

 

 

Mild

0

0

0

0

0

0

0

1

Spleen

Hematopoiesis

 

 

 

 

 

 

 

 

Mild

1

.

.

0

2

1

4

1

Hemosiderin increased

 

 

 

 

 

 

 

 

mild

0

.

.

0

1

0

0

3

Thymus

Atrophy

 

 

 

 

 

 

 

 

Minimal

0

.

.

2

0

.

.

0

N

10

10

10

9

10

10

10

9

 

Recovery animals:                

 

1M

4M

1F

4F

Dose (mg/kg bw/d)

 

 

 

 

Mesenteric lymph node

Histiocytosis

 

 

 

 

Minimal

0

2

2

1

Mild

0

6

0

6

Moderate

0

2

0

1

Total

0

10

0

8

Ectasia

 

 

 

 

mild

0

1

0

0

Spleen

Hematopoiesis

 

 

 

 

Mild

.

.

0

1

N

10

10

10

8

BADGE-MXDA fertility parameters

 

Summary Incidence of Necropsy Findings– fertility related parameters

 

Males

 

0 (control)

10 mg/kg bw/d

100 mg/kg bw/d

200 mg/kg bw/d

Group:

1

2

3

4

Testes

 

 

 

 

Submitted

20

10

10

19

No Visible Lesions

20

10

10

18

small

0

0

0

1

 

 

Group Mean Organ Weights with Corresponding Relative (% of Bodyweight) Organ Weights– fertility related parameters

Non-recovery animals

 

 

0 (control)

10 mg/kg bw/d

100 mg/kg bw/d

200 mg/kg bw/d

0 (control)

10 mg/kg bw/d

100 mg/kg bw/d

200 mg/kg bw/d

Group:

 

1

2

3

4

1

2

3

4

Sex

 

M

M

M

M

F

F

F

F

Epididymides

Mean (g)

S.D.    

N

1.62830

0.18488      

10

1.67464   

0.15323   

10

1.74595   

0.21200   

10

1.53051                                                                           

0.20489                                                                           

9

-

-

-

-

 

Mean (%)

S.D.    

N

0.380     

0.031     

10

0.380     

0.060     

10

0.401     

0.052     

10

0.418                                                                          

0.064                                                                          

9

-

-

-

-

Testes

Mean (g)

S.D.    

N

3.77051   

0.42495   

10

3.81649   

0.19625   

10

3.93235   

0.29053   

10

3.75139                                                                           

0.37542                                                                           

9

-

-

-

-

 

Mean (%)

S.D.    

N

0.883     

0.098     

10

0.866

0.106

10

0.903

0.076

10

1.021

0.107

9

-

-

-

-

Ovaries

Mean (g)

S.D.    

N

-

-

-

-

0.12624   

0.02200   

9

0.10666   

0.02534   

10

0.11020   

0.02475   

10

0.12373                               

0.02204                               

9

 

Mean (%)

S.D.    

N

-

-

-

-

0.046     

0.008     

9

0.038     

0.008     

10

0.042     

0.009     

10

0.047                              

0.007                              

9

Uterus And Cervix

Mean (g)

S.D.    

N

-

-

-

-

0.99179   

0.37925

10

0.96745

0.39192

10

0.88045   

0.27004   

10

0.83181                               

0.46430

9

 

Mean (%)

S.D.    

N

-

-

-

-

0.371     

0.141     

10

0.344     

0.136     

10

0.332     

0.096     

10

0.316                              

0.175                              

9

 

Recovery animals

 

 

0 (control)

200 mg/kg bw/d

0 (control)

200 mg/kg bw/d

Group:

 

1

4

1

4

Sex

 

M

M

F

F

Epididymides

Mean (g)

S.D.    

N

1.52527 

0.18201 

10

1.50451                                                                                                   

0.24307

10

-

-

 

Mean (%)

S.D.    

N

0.356

0.040

10

0.364

0.065

10

-

-

Testes

Mean (g)

S.D.    

N

3.70899 

0.40210 

10

3.63139

0.53509

10

-

-

 

Mean (%)

S.D.    

N

0.865   

0.072   

10

0.878

0.147

10

-

-

Ovaries

Mean (g)

S.D.    

N

-

-

0.09885 

0.02070 

10

0.08621                                                                                 

0.01204

8

 

Mean (%)

S.D.    

N

-

-

0.036   

0.008   

10

0.032

0.005

8

Uterus And Cervix

Mean (g)

S.D.    

N

-

-

0.88128 

0.21986 

10

0.82868

0.31206

8

 

Mean (%)

S.D.    

N

-

-

0.321   

0.073   

10

0.300

0.113

8

 

 

 

 

 

 

Conclusions:
The oral (gavage) administration of BADGE-MXDA for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 300 mg/kg bw/day resulted in treatment-related effects in animals of both sexes treated with 300 and 100 mg/kg bw/day. These findings included the death of one female treated with 300 mg/kg bw/day, the necessary early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in the hematology parameters measured (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in animals of both sexes treated with 10 mg/kg bw/day, therefore, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.
Executive summary:

The present study was designed to investigate the systemic toxicity of the test item BADGE-MXDA in accordance with OECD Guideline 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 100 and 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Propylene Glycol). Two recovery groups, each of ten males and ten females, were treated with the high dose (300 mg/kg bw/day) or the vehicle alone for up to ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on all non-recovery control and high dose animals during Week 12 of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

Two females and one male treated with 300 mg/kg bw/day were sacrificedin extremison Days 8, 27 and 85. A further female treated with 300 mg/kg bw/day was found dead on Day 82. There were no further unscheduled deaths.

Clinical Observations

Increased salivation and noisy respiration was evident in animals of both sexes treated with 300 mg/kg bw/day and to a lesser extent in animals of both sexes treated with 100 mg/kg bw/day throughout the treatment period. Incidences of labored respiration, decreased respiratory rate, hunched posture and lethargy were also evident in males treated with 300 mg/kg bw/day and a decreased respiratory rate was evident in one female from this treatment group. The females that were sacrificedin extremisalso showed labored respiration, decreased respiratory rate, pilo-erection and hunched posture and one of the females also had gasping respiration and a distended abdomen. The male that was sacrificedin extremisalso showed gasping respiration. No such effects were detected in males treated with 10 mg/kg bw/day, however, one female from this treatment group had noisy respiration on Day 26 only.

Behavioral Assessment

Weekly behavioral assessments revealed instances of noisy respiration in some animals of both sexes treated with 300 mg/kg bw/day, in some females treated with 100 mg/kg bw/day and in one male and one female treated with 10 mg/kg bw/day. One of the males treated with 300 mg/kg bw/day also showed an isolated incidence of a decreased respiratory rate and pilo-erection.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 300 mg/kg bw/day showed lower body weight gain throughout the treatment period, consequently overall body weight gain was lower than controls. Significant improvement in body weight gain was evident in males previously treated with 300 mg/kg bw/day during the twenty-eight day treatment-free period. No such adverse effects were detected in treated females or in males treated with 100 or 10 mg/kg bw/day. 

Food Consumption

Males treated with 300 mg/kg bw/day showed a reduction in food consumption throughout the treatment period. Females from this treatment group showed a reduction in food consumption from Week 10 onwards. Incidences of reduced food conversion efficiency were also evident in these animals during the treatment period. Improvement in food consumption and food conversion efficiency was evident during the twenty-eight day treatment-free period. No such effects were detected in animals of both sexes treated with 100 or 10 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmoscopy

Opthalmoscopic examination of animals of both sexes from the non-recovery control and 300 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related

differences.

Hematology

Males treated with 300 and 100 mg/kg bw/day showed an increase in neutrophils and a reduction in lymphocytes. The effect on neutrophils was also present in males previously treated with 300 mg/kg bw/day at the end of the twenty-eight day treatment-free period. No such effects were detected in treated females or in males treated with 10 mg/kg bw/day.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Necropsy

No toxicologically significant macroscopic abnormalities were detected in surviving animals.

The male treated with 300 mg/kg bw/day that was sacrificedin extremison Day 85 had gaseous distension in the stomach and ileum, pale and enlarged lungs and a reddened glandular region in the stomach. This animal had tracheal ulceration and bronchopneumonia, indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificedin extremison Day 8 had a dark liver and reddened lungs with clear fluid present. This female had edema and inflammation in the lungs and ulceration of the trachea, also indicative of test item irritancy through gastric reflux, and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was sacrificedin extremison Day 27 had gaseous distention in the intestines and reddened and enlarged lungs. This female had hemorrhage in the lungs as well as minimal inflammatory change, possibly through technical error and this is considered to be the cause of death.

The female treated with 300 mg/kg bw/day that was found dead on Day 82 had gaseous distension in the duodenum, ileum and jejunum. Histological examination did not reveal any obvious cause of death. Some tissues were notably autolysed.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured

Histopathology

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Node: Histiocytosis was present in the mesenteric lymph nodes of all non-recovery animals of both sexes treated with 300 mg/kg bw/day, varying from minimal to moderate in severity. It was also present in all animals of both sexes treated with 100 mg/kg bw/day at minimal or mild severity. No such effects were detected in animals of both sexes treated with 10 mg/kg bw/day. Following the twenty-eight day recovery period, histiocytosis at minimal or mild severity was present in all animals of both sexes that were previously treated with 300 mg/kg bw/day.

Histiocytosis in the mesenteric lymph nodes is known to occur in response to the oral administration of some test items and is likely to represent accumulation of material, indicating delayed clearance of an exogenous or endogenous material.

The following microscopic abnormalities were evident, however, these were considered to reflect individual variation or an equivocal finding rather than an effect of treatment:

Spleen: Increased hemosiderin was present in the spleen of one non-recovery control female and two non-recovery females treated with 300 mg/kg bw/day at a mild severity after evaluation and blinded reading of all animals. This was considered not to indicate an effect of treatment and reflects individual variation.

Thymus: Minimal thymic atrophy was present in two non-recovery males treated with 300 mg/kg bw/day. Whilst thymic atrophy is often considered to be related to stress, without other obvious signs in these animals (adrenal cortical hypertrophy for example) or a corresponding reduction in organ weight, it was considered to be of equivocal significance and therefore cannot be attributed to treatment.

Conclusion

The oral (gavage) administration of BADGE-MXDA for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 300 mg/kg bw/day resulted in treatment-related effects in animals of both sexes treated with 300 and 100 mg/kg bw/day. These findings included the death of one female treated with 300 mg/kg bw/day, the necessary early termination of two females and one male treated with 300 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption (300 mg/kg bw/day males only), changes in the hematology parameters measured (300 mg/kg bw/day males only) and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in animals of both sexes treated with 10 mg/kg bw/day, therefore, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2016 and 15 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
San Juan
Remarks:
Sprague-Dawley Crl:CD (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Weight at study initiation: 197 to 276 g
- Fasting period before study: no
- Housing: individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 20 May 2016 (first day of treatment) to 08 June 2016 (final day of necropsy)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services (Envigo Study Number: CQ79LS). Results showed the formulations to be stable for at least twenty-one days. Formulations were prepared on three occasions and stored at approximately +4 °C in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 4.17, 16.7, 58.3/41.67 (Dose Level/Concentration reduced from the 26 May 2016 (Days 7 to 9 of gestation))
- Amount of vehicle (if gavage): 6 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of test item formulations and were analyzed for concentration of BADGE-MXDA at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (Animals were delivered in two batches containing females prior to Day 3 of gestation.)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 3 to Day 19 of gestation
Frequency of treatment:
daily
Duration of test:
18 d (aminals were sacrificed on day 20 of gestation)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
Dose Level reduced to 250 mg/kg bw/d from the 26 May 2016 (Days 7 to 9 of gestation)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in collaboration with the Study Monitor and were based on previous toxicity data (Envigo Research Limited Study Number DP46NV).
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period; during the dosing period, observations were recorded immediately before dosing, up to thirty minutes after dosing and one hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: ovaries and uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- other: placental weight
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Historical control data:
attached as .pdf file
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Noisy respiration and increased salivation were evident in the majority of females at the high dose level throughout the treatment period. In addition, decreased respiratory rate, labored respiration and hunched posture was evident in one surviving female on Day 8, decreased respiratory rate and labored/gasping respiration was evident in one surviving female on Day 10 and diarrhoea was evident in one surviving female on Day 17. One female also had generalized fur loss on days 19 and 20.
All of the females that were sacrificed in extremis showed increased salivation and respiratory pattern changes (noisy respiration, gasping/labored respiration and/or decreased respiratory rate). In addition, the female that was sacrificed in extremis on Day 5 had chromodacryorrhea, hunched posture, pilo-erection and lethargy, the female that was sacrificed in extremis on Day 7 had lethargy, hunched posture, pilo-erection and a distended abdomen, one of the females that was sacrificed in extremis on Day 14 had hunched posture and the other female that was sacrificed in extremis on Day 14 had hunched posture, a distended abdomen and was dehydrated. The female that was sacrificed in extremis on Day 16 also had lethargy and a mass under the left forelimb.
Instances of noisy respiration were evident in seven females treated with 100 mg/kg bw/day on Days 7, 12, 14, 15 or 17 and one female treated with 100 mg/kg bw/day had increased salivation on Day 12 only.
No such effects were detected in females treated with 25 mg/kg bw/day.
One control female had diarrhoea on Day 16 only.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females from the high dose group were sacrificed in extremis on Days 5 and Day 7 of gestation due to excessive body weight losses and adverse clinical signs. Following the reduction of the high dose level to 250 mg/kg bw/day a further three females were sacrificed in extremis on Days 14 and 16 and two females were found dead on Day 18 of gestation.
There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females from the high dose group showed a reduction in body weight gain between Days 3 and 5 of gestation, with the majority of females showing actual body weight losses during this period. Two of these females were subsequently terminated on Days 5 and 7 due to excessive body weight loss. Following the reduction of the high dose level, improvement in body weight gain was evident in the surviving females however further body weight losses were evident in two females that were prematurely terminated and in the two females which were found dead. Body weight gain in the surviving high dose females was comparable to controls between Days 11 and 20. Cumulative body weight gain was statistically significantly reduced (p<0.05-0.01) in surviving females throughout the treatment period and body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (p<0.01) when compared to controls.
An initial reduction in body weight gain was evident in females treated with 100 mg/kg bw/day between Days 3 and 4 of gestation, with actual body weight losses being evident in some females. As a consequence of the initial reduction in body weight gain a slight reduction in cumulative body weight gain (achieving statistical significance between Days 3 and 14 only; p<0.05) was evident in these females and body weight gain when adjusted for gravid uterus weight was also slightly lower than controls. However statistical significance was not achieved for body weight gains or for body weight gain when adjusted for gravid uterus weight and body weight gains from Day 4 onwards were comparable to controls.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 25 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females from the high dose group showed a statistically significant reduction (p<0.01-0.001) in food consumption between Days 3 and 11. Food consumption between Days 11 and 20 was comparable to controls. Females treated with 100 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in food consumption between Days 3 and 5. Recovery in these females was evident thereafter.
No differences as compared to the control group were detected on food consumption in females treated with 25 mg/kg bw/day.
Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in food consumption between Days 11 and 14 however in the absence of a similar effect in the high dose females during this period the intergroup difference was most likely to represent biological variation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All of the females from the high dose group that were sacrificed in extremis and one of the females that was found dead had gaseous distension in the stomach and gastro-intestinal tract. The female that was sacrificed in extremis on Day 7 also had a distended abdomen and the female that was sacrificed in extremis on Day 16 had a mass under the left forelimb that was filled with a pale brown viscous liquid. The remaining female that was found dead had a fluid filled thoracic cavity.
No macroscopic abnormalities were detected in the surviving females of the high dose group or in females treated with 100 or 25 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), or pre- or post-implantation losses at 25, 100 or 350/250 mg/kg bw/day.
Intergroup differences for mean placental weights did not indicate any obvious effects of maternal treatment at 25, 100 or 350/250 mg/kg bw/day.
Statistical analysis of the data did not reveal any significant intergroup differences.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction (p<0.05-0.01) in the number of fetuses/litters showing incomplete ossification of the parietal was evident in all treated groups. Group mean values were within historical control ranges and the observation of one variant at a lower incidence compared with controls is not significant when evaluated in isolation. In the absence of any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This takes account of this finding being seen regularly on this study type amongst control group fetuses.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
There was no obvious effect of maternal treatment on litter data as assessed by live litter size and sex ratio at 25, 100 or 350/250 mg/kg bw/day.
Intergroup differences for mean fetal and litter weights did not indicate any obvious effects of maternal treatment at 25, 100 or 350/250 mg/kg bw/day.
Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 and subsequent detailed visceral and skeletal examination indicated any adverse effect of maternal exposure on fetal development.
A statistically significant reduction (p<0.05-0.01) in the number of fetuses/litters showing incomplete ossification of the parietal was evident in all treated groups. Group mean values were within historical control ranges and the observation of one variant at a lower incidence compared with controls is not significant when evaluated in isolation. In the absence of any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality. This takes account of this finding being seen regularly on this study type amongst control group fetuses.
Statistical analysis of the remaining data did not reveal any significant intergroup differences

Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

results tables are attached as .pdf file below

Conclusions:
The oral (gavage) administration of BADGE-MXDA to pregnant rats during gestation at dose levels of 25, 100 and 350 mg/kg bw/day (reduced to 250 mg/kg bw/day from Days 7 to 9 of gestation), resulted in treatment related effects at 350/250 mg/kg bw/day. The reduction in body weight gain and reduction in food consumption together with adverse clinical signs detected when the females were initially dosed at 350 mg/kg bw/day, resulted in the early sacrifice of two females (Day 5 and Day 7 of gestation). Following the reduction of the high dose level to 250 mg/kg bw/day, improvement was evident in some of the remaining females however a further three females were sacrificed early on Days 14 and 16 of gestation and two females were found dead on Day 18 of gestation. Although an initial reduction in body weight gain was noted for the 100 mg/kg bw/day dose group, full recovery was evident thereafter and only minimal clinical observations were evident. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.
Executive summary:

The study was performed to investigate the effects of BADGE-MXDA on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. The study was designed to comply with OECD Guideline 414 (adopted 22 January 2001).

The test item was administered by gavage to three groups each of twenty-four time-mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels of 25, 100, and 350 mg/kg bw/day. Following the early termination of two females treated with 350 mg/kg bw/day and adverse clinical signs in the remaining high dose females, the high dose level was reduced to 250 mg/kg bw/day from the 26 May 2016 (Days 7 to 9 of gestation). A further group of twenty-four time-mated females was exposed to the vehicle only (Propylene Glycol) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weights, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

…….

Mortality

Two females from the high dose group were sacrificedin extremison Days 5 and Day 7 of gestation due to excessive body weight losses and adverse clinical signs. Following the reduction of the high dose level to 250 mg/kg bw/day a further three females were sacrificed in extremis on Days 14 and 16 and two females were found dead on Day 18 of gestation. There were no further unscheduled deaths.

Clinical Observations

Noisy respiration and increased salivation were evident in the majority of females at the high dose level throughout the treatment period. Isolated incidences of decreased respiratory rate, gasping/labored respiration, hunched posture, diarrhoea or generalized fur loss were also evident in four surviving females from this dose group. In addition to increased salivation and respiratory pattern changes the females that were sacrificedin extremisalso showed incidences of chromodacryorrhea, hunched posture, dehydration, pilo-erection, lethargy, distended abdomen and/or a mass under the left forelimb. 

Instances of noisy respiration were also evident in females treated with 100 mg/kg bw/day and one female treated with 100 mg/kg bw/day had increased salivation on Day 12 only.

No such effects were detected in females treated with 25 mg/kg bw/day.

Body Weight

Females from the high dose group showed a reduction in body weight gain between Days 3 and 5 of gestation, with actual body weight losses being evident in some females. Following the reduction of the high dose level, improvement in body weight gain was evident in the surviving females however further body weight losses were evident in the females that were prematurely terminated on Days 14 and 16 or which were found dead on Day 18. Body weight gain in the surviving high dose females was comparable to controls between Days 11 and 20. Cumulative body weight gain was reduced in these females throughout the treatment period and body weight gain when adjusted for gravid uterus weight was also reduced when compared to controls.

A reduction in body weight gain was evident in females treated with 100 mg/kg bw/day between Days 3 and 4 of gestation, with actual body weight losses being evident in some females. Recovery was however evident thereafter. No such effects were evident in females treated with 25 mg/kg bw/day.

Food Consumption

Females from the high dose group showed a reduction in food consumption between Days 3 and 11. Food consumption between Days 11 and 20 was comparable to controls. Females treated with 100 mg/kg bw/day also showed a reduction in food consumption between Days 3 and 5. Recovery was evident thereafter. No differences as compared to the control group were detected on food consumption in females treated with 25 mg/kg bw/day.

Water Consumption

No differences as compared to the control group were detected on water consumption.


 

Post Mortem Studies

All of the females from the high dose group that were sacrificedin extremisand one of the females that was found dead had gaseous distension in the stomach and gastro-intestinal tract. The female that was sacrificedin extremison Day 7 also had a distended abdomen and the female that was sacrificedin extremison Day 16 had a mass under the left forelimb that was filled with a pale brown viscous liquid. The remaining female that was found dead had a fluid filled thoracic cavity. No treatment related macroscopic abnormalities were detected in the surviving females of the high dose group or in females treated with 100 or 25 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 25, 100 or 350/250 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 25, 100 or 350/250 mg/kg bw/day.

Fetal Examination

External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 350/250 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 25, 100 or 350/250 mg/kg bw/day.

Conclusion

The oral (gavage) administration of BADGE-MXDA to pregnant rats during gestation at dose levels of 25, 100 and 350 mg/kg bw/day (reduced to 250 mg/kg bw/day from Days 7 to 9 of gestation), resulted in treatment related effects at 350/250 mg/kg bw/day. The reduction in body weight gain and reduction in food consumption together with adverse clinical signs detected when the females were initially dosed at 350 mg/kg bw/day, resulted in the early sacrifice of two females (Day 5 and Day 7 of gestation). Following the reduction of the high dose level to 250 mg/kg bw/day, improvement was evident in some of the remaining females however a further three females were sacrificed early on Days 14 and 16 of gestation and two females were found dead on Day 18 of gestation. Although an initial reduction in body weight gain was noted for the 100 mg/kg bw/day dose group, full recovery was evident thereafter and only minimal clinical observations were evident. Consequently, 100 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 250 mg/kg bw/day.

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion