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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Sep 2017 to 30 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Sep 2017 to 30 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion BV, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
other: Wistar Han IGS rats (Crl:WI(Han)), (SPF)
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: (P) females 14 weeks, males 13 weeks
- Weight at study initiation: (P) females 210.0 to 247.2 g, males 316.7 to 371.4 g
- Fasting period before study: no
- Housing: The rats were housed in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced at last once per week with fresh portions and topped up when necessary. Each batch of VRF1 (FG) diet is analyzed by the supplier for nutrients and contaminants.
- Water: ad libitum, tap-water. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility.
- Acclimation period: quarantine 9 Aug 2017 to 18 Aug 2017.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%):at least 45% and not exceeding 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
4 Sep 2017 to 30 Oct 2017
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials in a refrigerator (2-10°C) in aliquots sufficient for one day (one vial/group/day). The vehicle for dosing the controls was similarly stored. During the daily administration all dilutions were continuously stirred on a magnetic stirrer.

DETAILS ON EXPOSURE
A dosing volume of 10 mL/kg body weight/day was used for all groups. The dosing volume was adjusted based on the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volumes were not adjusted after gestation day 14. A fixed volume based on the body weight on gestation day 14 was used for females between gestation day 14 up to the end of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity, stability and content of the test substance in dosing dilutions was conducted using an HPLC-MS method.
HOMOGENEITY: The homogeneity (and content) was assessed in the batch of gavage solutions, prepared for study 21025-02 on 4 September 2017. Three samples of each gavage solution, taken at different locations in the container, and 1 sample of the control solution (0 mg/mL) were analyzed. Each sample was analyzed in duplicate.
For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the diet container). The test substance was considered to be homogeneously distributed in the diet if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
STABILITY: The stability of the test substance in gavage solution was examined in the batch of gavage solutions prepared on 4 September 2017. Samples were analyzed directly after preparation, after storage in the animal room for at least 4 hours, and after storage in a refrigerator (2-10 °C) for at least one week. For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in the solution if p ≥ 0.01 and/or if the relative decrease in the mean concentration after storage was ≤ 10%.
CONTENT: The content of the test substance was determined in two other batches of gavage solutions, prepared on 25 September 2017 (all groups), 9 October (high dose group; after storage for 7 days in a refrigerator) and 16 October 2017 (low- and mid-dose groups). The content of the test substance in gavage was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Duration of treatment / exposure:
Males: 2 weeks pre-mating, during mating and up to the day of sacrifice, in total 30 days of treatment.
Females: 2 weeks pre-mating, during mating, during gestation and lactation until sacrifice (day 14 of lactation).
Frequency of treatment:
Daily
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the sponsor based on the results of a 2-week dose-range finding study with the test item in rats (Triskelion report V21025/01, Oral (gavage) dose-range finding study with Di-(2-Hydroxyethyl) Disulfide in rats, 22 August 2017). In this dose-range finding dose levels of 0, 5, 25 and 75 mg/kg body weight/day were administered to groups of 4 male and 4 female rats. The main effect noted was an increase in liver weights of high-dose females. The weight of the kidneys was statistically significantly increased in low- and high-dose females, but was not noticeably affected in the mid-dose group.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning all abnormalities, signs of ill health or reactions to treatment were recorded and the afternoon, checks for dead or moribund animals were made.
- At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of adult male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 1 in ‘Any other information on methods incl. tables’ were examined.
- The following parameters were calculated: Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 2 in ‘Any other information on methods incl. tables’ were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once, in the week before sacrifice.
- Dose groups that were examined: All groups, 5 males/group and 5 females with a litter/group.
- Battery of functions tested: Functional Observational Battery (FOB) and spontaneous motor activity.

IMMUNOLOGY: No

OTHER: BLOOD SAMPLING FOR HORMONE DETERMINATIONS
- Time schedule for examinations: During scheduled necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: all adult male and female animals and serum was stored in a freezer (at ≤-18°C).
- The blood samples taken from the adult males and from the two pups per litter sacrificed on PND 13 were analyzed for T4 hormone levels. Analysis was performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method based on the manufacturer’s protocol.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Five high-dose females were found dead during the study. On 12 October 2017, all remaining females of the high-dose group were killed intercurrently for animal welfare reasons and because it was anticipated that insufficient pregnant females would remain to allow a meaningful evaluation (see Annex 13; Amendment 2 to Study plan P21025/02). A necropsy was performed on the animals that died or were killed intercurrently. The tissues and organs listed below were preserved for all males and females. Organs were not weighed at interim sacrifice.
Prior to sacrifice, all surviving adult male and female animals were fasted overnight (water was freely available). The animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia and then subjected to macroscopic examination for pathological changes.
At scheduled necropsy, the organs of the adult animals indicated in the Table 3 in ‘Any other information on materials and methods incl. tables’ were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Samples of the tissues and organs of the adult animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde, except for the testes and epididymides which were preserved in Bouin’s fixative.
The reproductive organs of all male and female animals were preserved. The number of implantation sites in the uterus were counted.
The other organs/tissues were preserved of five adult animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected).
In addition to the organs and tissues in the Table 3, all gross lesions were preserved.

HISTOPATHOLOGY: Yes
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which was stained with PAS haematoxylin.
Microscopic examination (by light microscopy) was performed on the preserved organs of the male animals of the control (group 1) and high-dose group (group 4) and on the preserved organs of the female animals of the control (group 1) and mid-dose group (group 3).
The organs and tissues were not examined in females of the terminated high-dose group. In this group only organs showing gross lesions were examined microscopically.
In addition, organs showing gross lesions of animals of all other groups were examined microscopically. Organs marked with an asterisk (the Levator ani plus bulbocavenosus muscle complex, Cowper’s glands and glans penis) were preserved after weighing but not further examined.
Statistics:
The statistical procedures for analysis of data are described in Table 4 in 'Any other information on materials and methods incl. tables'. Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
Clinical signs:
no effects observed
Description (incidence and severity):
Despite the mortality in high-dose females, there were no noticeable clinical signs during the pre-mating period, mating and gestation period or the lactation period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Five females of the high-dose group were found dead during the study. The remaining females in the high-dose group were humanely killed for animal welfare reasons and because it was anticipated that insufficient pregnant females would remain to allow a meaningful evaluation. There was no mortality in females of the other groups or in males of any dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight changes of females were not affected in any group during the premating period, nor in the remaining low- and mid-dose groups during the gestation period or the lactation period.
Body weights and body weight changes of males of the high-dose group were reduced during the post-mating period (Table 1 to 3 in 'Any other information on results incl. tables). The differences with the controls were statistically significant from day 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls (Table 4 to 6 in 'Any other information on results incl. tables'. Food consumption was not affected in females of the low- and mid-dose group during the gestation period or the lactation period.
There were no treatment-related effects on food consumption of male animals during the pre- and post-mating period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological findings can be found in Tables 7 to 10 in 'Any other information on results incl. tables'.
There were no treatment-related effects in red blood cell and coagulation parameters at sacrifice in males of all dose groups and in females of the low- and mid-dose groups.
MCHC was slightly, though statistically significantly reduced in females of the low- and mid-dose groups. No significance was attached to this finding because there was no dose-response relationship and it was not accompanied by significant changes in the main parameters from which this calculated value was derived.
Total and differential white blood cell counts were not affected in males of all dose groups and in females of the remaining low- and mid-dose groups. No haematological data were obtained for the female high-dose group that was terminated intercurrently.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant changes in clinical chemistry variables were noted in high-dose males, namely increases in ASAT and ALAT activity and urea and inorganic phosphate concentrations, and decreases in triglycerides and sodium concentration (Table 11 and 12 in 'Any other information on results incl. tables').
In females of the remaining low- and mid-dose groups there were no statistically significant differences with the controls. No clinical biochemistry data were obtained for the female high-dose group that was terminated intercurrently.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.
Some treatment-related effects were, however observed in male or female animals of the high-dose groups during weekly detailed clinical examinations or during functional observations at the end of the study. These effects included sliding with the ventral parts of the head and neck over the bottom of the open field and salivation. At a few occasions, repetitive movements of mouth and jaws, a hunched posture, piloerection, low activity, large number of urine pools, vocalisation when prodded or nasal haemorrhagic discharge were observed in some animals of the high-dose group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative weight of the kidneys was statistically significantly increased in males of the high-dose group (21%). The mean relative liver weight of high-dose males was about 17% increased but the differences with the controls were not statistically significant (Table 13 to 16 in 'Any other information on results incl. tables').
Relative organ weights were not significantly affected in females of the remaining low- and mid-dose groups.
A few statistically significant differences with the controls were noted in absolute organ weights. Absolute seminal vesicle weights were increased in the mid-dose group and decreased in the high-dose group. The absolute weight of Cowpers gland was decreased in the high-dose group. In females of the mid-dose group, the absolute weight of the thyroids was decreased. Because these findings were not reflected in significant changes in the relative weights of these organs, they are not considered of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of ulceration and yellow/white deposition in the stomach was increased in females of the low- and mid-dose groups. The other macroscopic observations at necropsy revealed no treatment-related abnormalities (including the high-dose females died or were killed intercurrently). The findings were unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination was performed on control- and high-dose males and on control- and mid-dose females.
The incidence of focal necrosis in the stomach was increased in females of the low- and mid-dose groups both compared to concurrent and historical control data.
There were no other treatment-related histopathological changes. A statistically significantly lower incidence of microhaemorrhages was noted in the thymus of high-dose males. This finding is the result of the relatively high incidence of this irrelevant change in the control group rather than related to treatment and is considered a chance finding.
The other histopathological changes observed were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
T4 LEVELS:
No statistically significant effects were observed on T4-hormones in males (Table 17 in 'Any other information on results incl. tables').
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1. Body weight males and females during the study - Day(s) Relative to Start Date.

Sex: Male

 

 

 

Bodywt

day -x (g)

 

[G]

Bodywt

day 0 (g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-3

0

7

14

21

28

0 mg/kg

Mean SD

N

334.29

13.48

12

342.37

14.89

12

351.50

15.41

12

357.64

16.73

12

361.62

15.90

12

371.83

17.41

12

5 mg/kg

Mean

SD

N

334.28

12.93

12

342.63

13.42

12

351.81

15.98

12

360.26

16.38

12

362.18

18.33

12

372.53

20.28

12

20 mg/kg

Mean

SD

N

334.87

14.41

12

342.88

16.56

12

353.29

17.23

12

363.03

17.48

12

366.46

18.30

12

375.33

18.40

12

75 mg/kg

Mean SD

N

334.45

14.63

12

342.63

14.08

12

349.85

14.23

12

353.13

14.69

12

354.18 *

13.72

12

354.71 **

15.17

12

Sex: Female

 

 

 

 

 

 

 

0 mg/kg

Mean SD

N

217.33

7.91

12

224.18

6.20

12

224.54

6.76

12

225.78

6.67

12

 

 

5 mg/kg

Mean

SD

N

217.60

7.85

12

223.11

8.41

12

223.72

6.10

12

225.83

7.06

12

 

 

20 mg/kg

Mean

SD

N

217.39

7.95

12

222.53

7.52

12

223.29

8.52

12

227.41

10.00

12

 

 

75 mg/kg

Mean SD

N

217.75

8.55

12

225.13

11.25

12

222.74

12.68

11

225.50

11.32

11

 

 

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

 

Table 2. Body weight females gestation - Day(s) Relative to Mating (Litter: A).

Sex: Female

 

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

0

7

14

20

0 mg/kg

Mean

227.42

248.33

272.11

332.17

 

SD

6.62

9.30

11.81

15.63

 

N

12

12

12

12

5 mg/kg

Mean

228.77

250.03

271.59

326.74

 

SD

7.27

10.17

11.62

16.41

 

N

12

12

12

12

20 mg/kg

Mean

226.59

247.58

268.65

321.56

 

SD

7.09

10.69

12.34

18.18

 

N

11

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 3. Body weight females lactation - Day(s) Relative to Littering (Litter: A).

Sex: Female

 

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

0

4

7

13

0 mg/kg

Mean

255.99

274.28

277.06

286.32

 

SD

10.14

13.88

14.36

13.78

 

N

12

12

12

12

5 mg/kg

Mean

251.76

266.01

266.68

278.53

 

SD

9.92

11.35

14.21

12.49

 

N

12

12

12

12

20 mg/kg

Mean

252.52

266.53

271.63

282.53

 

SD

13.32

12.74

13.33

11.85

 

N

12

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 4. Food consumption males during the study - Daily Food Cons Per Animal (g)

Sex: Male

Day(s) Relative to Animal start date

0 - 7

7 - 14

21 - 28

0 mg/kg

Mean

20.6

18.8

18.3

 

SD

0.6

1.1

0.4

 

N

3

3

3

5 mg/kg

Mean

20.4

17.5

18.0

 

SD

0.2

0.5

0.1

 

N

3

3

3

20 mg/kg

Mean

20.9

18.8

18.6

 

SD

0.8

0.6

0.7

 

N

3

3

3

75 mg/kg

Mean

19.8

17.8

18.6

 

SD

1.5

0.5

1.8

 

N

3

3

3

0 mg/kg

Mean

13.9

13.0

 

SD

0.4

0.5

 

N

3

3

 

5 mg/kg

Mean

14.4

13.5

 

SD

0.4

0.6

 

N

3

3

 

20 mg/kg

Mean

14.2

13.4

 

SD

0.2

0.1

 

N

3

3

 

75 mg/kg

Mean

12.8 **

13.4

 

SD

0.1

0.8

 

N

3

3

 

Dunnett** = p < 0.01

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 5. Food consumption females gestation - Daily Food Cons Per Animal (g).

Sex: Female

Day(s) Relative to Mating (Litter: A)

0 - 7

7 - 14

14 - 20

0 mg/kg

Mean

15.22

16.81

18.12

 

SD

1.42

1.45

0.88

 

N

12

12

12

5 mg/kg

Mean

15.61

16.48

18.16

 

SD

1.45

2.10

1.42

 

N

12

12

12

20 mg/kg

Mean

14.66

16.07

17.98

 

SD

1.81

1.99

1.68

 

N

11

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 6. Food consumption females lactation - Daily Food Cons Per Animal (g)

Sex: Female

Day(s) Relative to Littering (Litter: A)

0 - 4

4 - 7

7 - 13

0 mg/kg

Mean

28.99

41.42

48.46

 

SD

5.33

5.46

4.37

 

N

12

12

12

5 mg/kg

Mean

27.67

41.36

45.78

 

SD

4.37

6.38

4.30

 

N

12

12

12

20 mg/kg

Mean

26.95

40.46

47.51

 

SD

3.18

5.43

3.85

 

N

12

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 7. Red blood cell and coagulation parameters - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

 

[G]

Hb (mmol/L)

 

[G]

PCV

(L/L)

 

[G]

MCV

(fL)

  

[G1]

MCH

(fmol)

  

[G1]

MCHC

(mmol/L)

  

[G1]

Reticulo cytes

(%)

 [G1]

Thrombo cytes

(10E9/L)

 [G1]

Prothrom Time

(s)

 [G1]

0 mg/kg

Mean SD

N

8.850

0.424

5

9.46

0.34

5

0.4840

0.0196

5

54.71

0.62

5

1.070

0.026

5

19.55

0.42

5

1.988

0.170

5

805.4

100.8

5

19.80

1.05

5

5 mg/kg

Mean SD

N

9.066

0.370

5

9.68

0.24

5

0.4984

0.0150

5

55.00

1.04

5

1.068

0.021

5

19.43

0.26

5

2.160

0.344

5

834.2

111.4

5

20.52

0.67

5

20 mg/kg

Mean SD

N

8.774

0.177

5

9.46

0.44

5

0.4864

0.0193

5

55.43

1.41

5

1.078

0.035

5

19.45

0.39

5

1.958

0.156

5

772.8

78.3

5

20.66

0.99

5

75 mg/kg

Mean SD

N

9.926

1.139

5

10.64

1.28

5

0.5468

0.0780

5

54.95

1.65

5

1.072

0.024

5

19.51

0.56

5

2.042

0.315

5

792.0

136.1

5

20.08

1.05

5

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett

 

Table 8. Red blood cell and coagulation parameters - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

 

[G]

Hb (mmol/L)

 

 [G]

PCV

(L/L)

 

 [G]

MCV

(fL)

 

 [G]

MCH

(fmol)

  

[G]

MCHC

(mmol/L)

 

 [G]

Reticulo cytes

(%)

 [G]

Thrombo cytes

(10E9/L)

 [G]

Prothrom Time

(s)

 [G]

0 mg/kg

Mean SD

N

7.974

0.586

5

9.58

0.59

5

0.4776

0.0263

5

59.99

2.36

5

1.203

0.037

5

20.05

0.22

5

3.488

0.510

5

895.0

216.2

5

21.46

1.11

5

5 mg/kg

Mean SD

N

7.992

0.457

5

9.42

0.32

5

0.4820

0.0110

5

60.44

3.19

5

1.181

0.065

5

19.54 * 0.42

5

3.294

0.182

5

1102.8

85.1

5

21.66

1.47

5

20 mg/kg

Mean SD

N

7.376

0.443

5

9.04

0.38

5

0.4630

0.0182

5

62.90

3.53

5

1.228

0.077

5

19.52 * 0.22

5

3.862

0.583

5

978.6

120.5

5

21.66

0.82

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05

 

Table 9. Total and differential white blood cell counts - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G1]

Eosino Absolute

(10E9/L)

 

[G2]

Baso Absolute

(10E9/L)

 

[G2]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G2]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

5.38

1.68

5

4.13

1.22

5

1.02

0.45

5

0.111

0.095

5

0.025

0.014

5

0.082

0.040

5

77.22

5.45

5

18.42

3.52

5

2.28

2.48

5

0.44

0.18

5

1.46

0.41

5

5 mg/kg

Mean SD

N

6.38

1.18

5

5.05

1.04

5

1.09

0.14

5

0.085

0.025

5

0.014

0.009

5

0.120

0.031

5

78.86

2.33

5

17.32

2.33

5

1.34

0.32

5

0.20

0.10

5

1.90

0.42

5

20 mg/kg

Mean SD

N

6.66

0.96

5

5.19

1.10

5

1.20

0.25

5

0.136

0.104

5

0.022

0.011

5

0.087

0.037

5

77.26

7.62

5

18.42

5.34

5

2.28

2.21

5

0.32

0.16

5

1.36

0.65

5

75 mg/kg

Mean SD N

6.38

0.94

5

5.14

0.88

5

0.99

0.13

5

0.116

0.125

5

0.028

0.029

5

0.089

0.041

5

80.36

4.14

5

15.68

2.64

5

1.88

2.17

5

0.40

0.35

5

1.36

0.50

5

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks [G2] - Ancova/Anova & Dunnett(Log)

 

Table 10. Total and differential white blood cell counts - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G]

Eosino Absolute

(10E9/L)

 

[G]

Baso Absolute

(10E9/L)

 

[G]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G1]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

6.44

3.82

5

3.04

1.79

5

3.11

1.92

5

0.064

0.032

5

0.019

0.019

5

0.184

0.105

5

46.16

4.79

5

48.34

3.09

5

1.16

0.42

5

0.38

0.33

5

3.36

1.34

5

5 mg/kg

Mean SD

N

7.06

2.99

5

3.85

1.45

5

2.83

1.54

5

0.112

0.040

5

0.019

0.011

5

0.223

0.137

5

57.12

8.91

5

37.44

9.06

5

2.12

1.73

5

0.24

0.09

5

2.82

1.46

5

20 mg/kg

Mean SD N

6.30

1.27

5

3.28

0.86

5

2.70

0.81

5

0.065

0.013

5

0.012

0.007

5

0.226

0.090

5

52.12

9.22

5

42.80

10.45

5

1.06

0.29

5

0.20

0.12

5

3.56

1.11

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

 

Table 11. Clinical chemistry - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

 

 

 

[G]

ASAT (U/L)

 

 

[G1]

ALAT (U/L)

 

 

[G1]

GGT (U/L)

 

 

[G2]

Bilirub Total

(umol/L)

 

[G]

Creatin ine

(umol/L)

 

[G]

Bile Acids

(umol/L)

 

[G1]

Total Protein

(g/L)

 

[G]

Albumin (g/L)

 

 

[G]

Albumin/ Globulin

 

 

[G]

Glucose Plasma

(mmol/L)

 

[G]

Cholest erol

(mmol/L)

 

[G1]

Phospho lipids

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G]

Urea (mmol/L)

 

 

[G]

PO4

(mmol/L)

 

 

[G1]

Ca (mmol/L)

 

 

[G1]

Cl (mmol/L)

 

 

[G1]

K

(mmol/L)

 

 

[G]

Na (mmol/L)

 

 

[G1]

0 mg/kg

Mean SD

N

108.6

32.5

5

90.0

7.9

5

47.0

4.5

5

0.00

0.00

5

1.42

0.43

5

42.8

3.3

5

9.64

1.91

5

63.2

2.6

5

33.0

1.4

5

7.602

1.614

5

1.914

0.230

5

1.728

0.102

5

1.138

0.143

5

6.24

0.59

5

2.574

0.283

5

2.818

0.080

5

99.0

1.6

5

5.98

0.48

5

146.2

1.1

5

1.093

0.029

5

5 mg/kg

Mean SD

N

103.0

34.4

5

86.2

7.2

5

40.4

5.7

5

0.00

0.00

5

1.26

0.71

5

40.6

4.7

5

17.06

12.29

5

65.0

2.5

5

34.8

1.1

5

8.816

1.836

5

1.816

0.249

5

1.666

0.156

5

0.956

0.163

5

6.82

1.64

5

2.908

0.299

5

2.912

0.069

5

98.2

0.4

5

6.16

0.80

5

146.4

0.5

5

1.155

0.068

5

20 mg/kg

Mean SD

N

95.6

8.5

5

84.6

2.4

5

46.8

10.4

5

0.00

0.00

5

1.52

0.28

5

40.4

2.3

5

18.88

18.68

5

62.6

3.2

5

33.6

1.5

5

8.992

2.111

5

1.552

0.171

5

1.524

0.129

5

0.864

0.394

5

6.32

0.44

5

2.718

0.286

5

2.846

0.103

5

99.0

1.0

5

5.72

0.43

5

146.8

0.8

5

1.160

0.047

5

75 mg/kg

Mean SD

N

139.6

34.6

5

110.6 * 22.9

5

103.2 ** 33.3

5

0.00

0.00

5

1.38

0.42

5

39.2

1.9

5

28.63

13.15

4

62.6

3.2

5

34.0

1.4

5

8.640

1.449

5

1.424

0.519

5

1.458

0.318

4

0.624 **

0.087

5

8.14 * 1.18

5

3.132 * 0.216

5

2.788

0.096

4

98.2

1.3

5

6.14

0.71

5

143.2 ** 1.3

5

1.193

0.080

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log): * = p < 0.05; ** = p < 0.01

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 12. Clinical chemistry - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

 

 

 

[G]

ASAT (U/L)

 

 

[G1]

ALAT (U/L)

 

 

[G]

GGT (U/L)

 

 

[G2]

Bilirub Total

(umol/L)

 

[G]

Creatin ine

(umol/L)

 

[G]

Bile Acids

(umol/L)

 

[G]

Total Protein

(g/L)

 

[G]

Albumin (g/L)

 

 

[G]

Albumin/ Globulin

 

 

[G]

Glucose Plasma

(mmol/L)

 

[G]

Cholest erol

(mmol/L)

 

[G1]

Phospho lipids

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G1]

Urea (mmol/L)

 

 

[G1]

PO4

(mmol/L)

 

 

[G]

Ca (mmol/L)

 

 

[G1]

Cl (mmol/L)

 

 

[G1]

K

(mmol/L)

 

 

[G1]

Na (mmol/L)

 

 

[G1]

0 mg/kg

Mean SD

N

90.6

21.1

5

140.0

22.9

5

61.2

8.6

5

1.94

4.01

5

1.28

0.26

5

48.4

3.6

5

32.28

21.66

5

63.2

3.5

5

33.0

2.2

5

7.606

1.306

5

2.896

0.598

5

2.906

0.403

5

2.114

1.095

5

9.16

1.57

5

3.174

0.568

5

2.792

0.151

5

95.0

2.3

5

5.20

0.33

5

143.6

2.8

5

1.095

0.081

5

5 mg/kg

Mean SD

N

118.4

52.7

5

120.4

19.9

5

63.0

14.7

5

0.18

0.35

5

1.36

0.15

5

50.0

2.7

5

30.12

15.89

5

64.6

3.2

5

34.6

1.8

5

7.892

1.399

5

2.338

0.506

5

2.582

0.430

5

1.830

0.990

5

9.42

1.61

5

3.040

0.493

5

2.850

0.190

5

96.0

3.4

5

5.06

0.50

5

144.0

2.8

5

1.155

0.063

5

20 mg/kg

Mean SD

N

137.0

44.1

5

123.4

25.1

5

57.8

12.9

5

0.10

0.22

5

1.14

0.38

5

47.4

4.4

5

17.80

13.34

5

61.6

5.2

5

32.4

3.0

5

7.952

0.179

5

2.592

0.472

5

2.778

0.401

5

2.296

0.784

5

9.66

1.29

5

3.078

0.061

5

2.812

0.076

5

98.0

1.0

5

5.28

0.46

5

145.2

1.9

5

1.110

0.060

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 13. Absolute organ weights - Day(s): 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G1]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G1]

Thymus (g)

[G]

Thyroid (g)

[G]

Testes (g)

[G1]

Epididy mides

(g)

 

[G1]

Prostate (g)

[G2]

Seminal vesicles

(g)

 

[G1]

LABC

Muscle (g)

 

[G1]

Cowpers Glands

(g)

 

[G2]

Glans Penis (g)

[G1]

0 mg/kg

Mean SD

N

358.58

16.31

12

2.064

0.018

5

1.028

0.051

5

0.0540

0.0076

5

1.998

0.147

5

8.408

0.327

5

0.5860

0.0394

5

0.0200

0.0073

12

3.563

0.305

12

1.193

0.101

12

0.939

0.162

12

1.193

0.169

12

1.0571

0.1190

12

0.1046

0.0143

12

0.1376

0.0326

12

0.3228

0.0997

5

5 mg/kg

Mean SD

N

358.58

18.26

12

2.098

0.031

5

1.030

0.034

5

0.0548

0.0068

5

2.140

0.100

5

8.282

0.398

5

0.5792

0.0889

5

0.0171

0.0051

12

3.715

0.299

12

1.208

0.130

12

0.918

0.175

12

1.226

0.199

12

1.0701

0.1283

12

0.0953

0.0147

12

0.1344

0.0280

12

0.3028

0.0779

5

20 mg/kg

Mean SD

N

361.33

17.67

12

2.052

0.108

5

1.044

0.072

5

0.0578

0.0063

5

2.144

0.058

5

8.712

0.414

5

0.6410

0.0425

5

0.0190

0.0075

11

3.638

0.192

12

1.209

0.076

12

0.973

0.121

12

1.371 *

0.144

12

1.0884

0.1006

12

0.1048

0.0321

12

0.1236

0.0304

12

0.3246

0.0468

5

75 mg/kg

Mean SD

N

334.93 ** 14.23 12

2.052

0.073

5

0.952

0.052

5

0.0582

0.0048

5

2.256

0.191

5

9.106

1.883

5

0.5178

0.0755

5

0.0153

0.0058

12

3.512

0.148

12

1.136

0.113

12

0.923

0.240

12

1.007 *

0.197

12

0.9757

0.0854

12

0.0878 **

0.0227

12

0.1280

0.0308

12

0.2466

0.0606

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 14. Absolute organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G]

Thymus (g)

[G]

Thyroid (g)

[G2]

Ovaries (g)

[G2]

Uterus (g)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

1.912

0.094

5

0.866

0.084

5

0.0754

0.0038

5

1.516

0.087

5

8.764

0.905

5

0.5480

0.0701

5

0.2218

0.0456

5

0.0186

0.0046

12

0.0884

0.0136

12

0.5245

0.1042

12

5 mg/kg

Mean SD

N

254.78

10.69

12

1.936

0.062

5

0.832

0.058

5

0.0710

0.0120

5

1.558

0.080

5

8.452

0.569

5

0.5558

0.0735

5

0.1796

0.0348

5

0.0155

0.0044

11

0.0921

0.0143

12

0.5579

0.1135

12

20 mg/kg

Mean SD

N

258.29

11.63

12

1.974

0.056

5

0.824

0.047

5

0.0738

0.0124

5

1.624

0.077

5

8.594

0.414

5

0.5850

0.0386

5

0.1914

0.0272

5

0.0141 *

0.0020

12

0.0883

0.0058

12

0.5187

0.0680

12

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log): * = p < 0.05 Litter: A = First litter

 

Table 15. Relative organ weights - Day(s): 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G1]

Spleen rel.wgt

(g/kg body wgt)

[G]

Thymus rel.wgt

(g/kg body wgt)

[G2]

Thyroid rel.wgt

(g/kg body wgt)

[G]

Testes rel.wgt

(g/kg body wgt)

[G1]

Epididy rel.wgt

(g/kg body wgt)

[G2]

Prostate rel.wgt

(g/kg body wgt)

[G2]

Sem ves rel.wgt

(g/kg body wgt)

[G1]

LABC Muscle rel.wgt

(g/kg body wgt)

[G1]

Cowpers Gl. rel.wgt

(g/kg body wgt)

[G2]

Glans Penis rel.wgt

(g/kg body wgt)

[G1]

0 mg/kg

Mean SD

N

358.58

16.31

12

5.803

0.320

5

2.887

0.147

5

0.1514

0.0193

5

5.622

0.563

5

23.63

1.25

5

1.648

0.148

5

0.0557

0.0195

12

9.928

0.571

12

3.325

0.222

12

2.617

0.407

12

3.326

0.440

12

2.9454

0.2731

12

0.2918

0.0382

12

0.3844

0.0941

12

0.910

0.305

5

5 mg/kg

Mean SD

N

358.58

18.26

12

5.918

0.312

5

2.904

0.149

5

0.1544

0.0190

5

6.031

0.282

5

23.34

1.03

5

1.627

0.201

5

0.0475

0.0139

12

10.378

0.909

12

3.380

0.449

12

2.558

0.466

12

3.422

0.554

12

2.9883

0.3578

12

0.2665

0.0433

12

0.3740

0.0707

12

0.855

0.234

5

20 mg/kg

Mean SD

N

361.33

17.67

12

5.560

0.443

5

2.823

0.169

5

0.1559

0.0108

5

5.805

0.300

5

23.55

0.35

5

1.737

0.167

5

0.0520

0.0189

11

10.090

0.683

12

3.349

0.180

12

2.696

0.352

12

3.799

0.399

12

3.0124

0.2457

12

0.2914

0.0882

12

0.3399

0.0680

12

0.881

0.155

5

75 mg/kg

Mean SD

N

334.93 ** 14.23

12

6.214

0.264

5

2.886

0.237

5

0.1764

0.0173

5

6.829 **

0.577

5

27.62

6.18

5

1.571

0.256

5

0.0453

0.0165

12

10.501

0.604

12

3.398

0.387

12

2.769

0.786

12

2.997

0.522

12

2.9155

0.2580

12

0.2626

0.0677

12

0.3840

0.0987

12

0.748

0.195

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 16. Relative organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G]

Spleen rel.wgt

(g/kg body wgt)

[G1]

Thymus rel.wgt

(g/kg body wgt)

[G]

Thyroid rel.wgt

(g/kg body wgt)

[G2]

Ovaries rel.wgt

(g/kg body wgt)

[G]

Uterus rel.wgt

(g/kg body wgt)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

7.382

0.308

5

3.348

0.359

5

0.2914

0.0178

5

5.860

0.425

5

33.83

3.24

5

2.117

0.274

5

0.855

0.161

5

0.0713

0.0201

12

0.3366

0.0510

12

1.997

0.408

12

5 mg/kg

Mean SD

N

254.78

10.69

12

7.699

0.404

5

3.309

0.280

5

0.2820

0.0464

5

6.191

0.296

5

33.57

1.88

5

2.213

0.332

5

0.713

0.135

5

0.0612

0.0174

11

0.3618

0.0563

12

2.188

0.424

12

20 mg/kg

Mean SD N

258.29

11.63

12

7.623

0.326

5

3.178

0.105

5

0.2864

0.0586

5

6.278

0.483

5

33.18

1.60

5

2.265

0.252

5

0.737

0.087

5

0.0544

0.0059

12

0.3428

0.0294

12

2.017

0.318

12

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log) [G2] - Kruskal-Wallis & Dunnett on Ranks Litter: A = First litter

able 17. Hormone determinations (T4)

 

(P) Male

T4

(ng/ml)

 

[G]

0 mg/kg

Mean

308.77

 

SD

116.64

 

N

12

5 mg/kg

Mean

309.56

 

SD

58.01

 

N

12

20 mg/kg

Mean

368.28

 

SD

114.96

 

N

12

75 mg/kg

Mean

314.03

 

SD

142.44

 

N

12

[G] - Ancova/Anova & Dunnett(Log)

Conclusions:
In this GLP compliant OECD 422 study, the No-Observed-Adverse-Effect-Level (NOAEL) was 20 mg/kg bw/day for both sexes, based on mortality in high-dose females and on effects on growth, clinical chemistry and weights of liver and kidney in high-dose males.
Executive summary:

In this GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422 the test substance was administered by oral gavage to four groups of male and female Wistar Han rats. Each group consisted of 12 animals per sex. The test substance was administrated at 0, 5, 20 and 75 mg/kg bw/day dissolved in tap water during a pre-mating period of 2 weeks, during mating, gestation and until day 13 of lactation. Males were exposed for 30 days before sacrifice. The content, homogeneity and stability of the test substance in the vehicle (tap water) was confirmed by analysis. Cage side observations, including checks for dead or moribund animals, were performed twice a day. Near the end of gestation, females were checked twice a day for parturition. Body weights for males and females were recorded weekly. Food consumption was measured per cage and recalculated to food intake per animal per day. In the week before sacrifice, neurobehavioural examination was performed on 5 animals per group per sex using Functional Observational Battery (FOB) and spontaneous motor activity in the arena test. Prior to scheduled sacrifice, blood was withdrawn from the abdominal aorta under CO2/O2 anaesthesia for assessing haematology and clinical chemistry parameters. Furthermore, the blood was analysed for T4 hormone levels in all males. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically.

Mortality was limited to the high dose level females, of which 5 died and the rest were killed humanely for animal welfare reasons. No treatment- related clinical signs were observed during the study. Body weights and body weight changes of males of the high-dose group were reduced during the post-mating period. During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls. There was no treatment-related effects on food consumption of male animals during the pre- and post-mating period. Statistically significant changes in clinical chemistry variables were noted in high-dose males, namely increases in ASAT and ALAT activity and urea and inorganic phosphate concentrations, and decreases in triglycerides and sodium concentration. No statistically significant effects were observed on T4-hormones in males. Haematological and clinical chemistry data were not obtained for the female high-dose group that was terminated intercurrently. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. The relative weight of the kidneys was statistically significantly increased in males of the high-dose group (21%). The mean relative liver weight of high-dose males was about 17% increased but the differences with the controls were not statistically significant. Relative organ weights were not significantly affected in females of the remaining low- and mid-dose groups. The incidence of ulceration and yellow/white deposition in the stomach was increased in females of the low- and mid-dose groups. The other macroscopic observations at necropsy revealed no treatment-related abnormalities. Microscopic evaluation did, however, not reveal any corroborating histopathological changes. The incidence of local macroscopic (ulceration) and microscopic changes (focal necrosis) in the stomach was, however, increased in low- and mid-dose females, both compared to concurrent and historical control data. Although these changes were not clearly elevated in high-dose females nor in males they may represent a local treatment-related effect, because in high-dose females and males the duration of exposure was shorter. Therefore, it cannot be excluded that the prolonged exposure to the test substance induced local reaction in the stomach. Other histopathological findings were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.

Based on mortality in high-dose females and on effects on growth, clinical chemistry and weights of liver and kidney in high-dose males, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic effects was placed at 20 mg/kg body weight/day. Local reactions in the stomach were, however, noted in females of the low- and mid-dose groups.

 

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Sep 2017 to 30 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion BV, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
other: Wistar Han IGS rats (Crl:WI(Han)), (SPF)
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: (P) females 14 weeks, males 13 weeks
- Weight at study initiation: (P) females 210.0 to 247.2 g, males 316.7 to 371.4 g
- Fasting period before study: no
- Housing: The rats were housed in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced at last once per week with fresh portions and topped up when necessary. Each batch of VRF1 (FG) diet is analyzed by the supplier for nutrients and contaminants.
- Water: ad libitum, tap-water. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility.
- Acclimation period: quarantine 9 Aug 2017 to 18 Aug 2017.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%):at least 45% and not exceeding 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
4 Sep 2017 to 30 Oct 2017
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials in a refrigerator (2-10°C) in aliquots sufficient for one day (one vial/group/day). The vehicle for dosing the controls was similarly stored. During the daily administration all dilutions were continuously stirred on a magnetic stirrer.

DETAILS ON EXPOSURE
A dosing volume of 10 mL/kg body weight/day was used for all groups. The dosing volume was adjusted based on the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volumes were not adjusted after gestation day 14. A fixed volume based on the body weight on gestation day 14 was used for females between gestation day 14 up to the end of pregnancy.
Details on mating procedure:
- M/F ratio per cage:1/1 animals in the same dose group.
- Length of cohabitation: Animals were caged together until mating occurred.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity, stability and content of the test substance in dosing dilutions was conducted using an HPLC-MS method.
HOMOGENEITY: The homogeneity (and content) was assessed in the batch of gavage solutions, prepared for study 21025-02 on 4 September 2017. Three samples of each gavage solution, taken at different locations in the container, and 1 sample of the control solution (0 mg/mL) were analyzed. Each sample was analyzed in duplicate.
For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the diet container). The test substance was considered to be homogeneously distributed in the diet if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
STABILITY: The stability of the test substance in gavage solution was examined in the batch of gavage solutions prepared on 4 September 2017. Samples were analyzed directly after preparation, after storage in the animal room for at least 4 hours, and after storage in a refrigerator (2-10 °C) for at least one week. For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in the solution if p ≥ 0.01 and/or if the relative decrease in the mean concentration after storage was ≤ 10%.
CONTENT: The content of the test substance was determined in two other batches of gavage solutions, prepared on 25 September 2017 (all groups), 9 October (high dose group; after storage for 7 days in a refrigerator) and 16 October 2017 (low- and mid-dose groups). The content of the test substance in gavage was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Duration of treatment / exposure:
Males: 2 weeks pre-mating, during mating and up to the day of sacrifice, in total 30 days of treatment.
Females: 2 weeks pre-mating, during mating, during gestation and lactation until sacrifice (day 14 of lactation).
Frequency of treatment:
Daily
Details on study schedule:
After arrival, the animals were quarantined and acclimatized for 9 days. Followed by a 2-week pre-treatment period during which vaginal smears were made in every female rat (including surplus females) for monitoring the estrous cycle. At the end of the pre-treatment period, the animals were allocated to the different groups and the treatment was started at the first day of the pre-mating period. After a 2-week premating period, each female was caged with one male from the same dose group. Upon evidence of copulation, taking 1 to 13 pre-coital days during which treatment continued, the females were caged individually for the birth and rearing of their pups. Males, females that delivered and females that appeared not pregnant were sacrificed. Treatment continued until day 13 of lactation. Pups were sacrificed at day 13 of lactation. Parental female animals were sacrificed at day 14 of lactation (except for the high-dose group, see below). Male animals were sacrificed after the mating period after 30 days of treatment.
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the sponsor based on the results of a 2-week dose-range finding study with the test item in rats (Triskelion report V21025/01, Oral (gavage) dose-range finding study with Di-(2-Hydroxyethyl) Disulfide in rats, 22 August 2017). In this dose-range finding dose levels of 0, 5, 25 and 75 mg/kg body weight/day were administered to groups of 4 male and 4 female rats. The main effect noted was an increase in liver weights of high-dose females. The weight of the kidneys was statistically significantly increased in low- and high-dose females, but was not noticeably affected in the mid-dose group.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning all abnormalities, signs of ill health or reactions to treatment were recorded and the afternoon, checks for dead or moribund animals were made.
- At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Litters were evaluated on the day of birth (i.e. day 0 of lactation). To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of adult male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 1 in ‘Any other information on methods incl. tables’ were examined.
- The following parameters were calculated: Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 2 in ‘Any other information on methods incl. tables’ were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once, in the week before sacrifice.
- Dose groups that were examined: All groups, 5 males/group and 5 females with a litter/group.
- Battery of functions tested: Functional Observational Battery (FOB) and spontaneous motor activity.

IMMUNOLOGY: No

OTHER: BLOOD SAMPLING FOR HORMONE DETERMINATIONS
- Time schedule for examinations: During scheduled necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: all adult male and female animals and serum was stored in a freezer (at ≤-18°C).
- In addition, blood samples were collected and serum was stored in a freezer (at ≤-18°C):
A) from the surplus pups per litter at culling on lactation day 4. Blood samples were collected by decapitation from all surplus pups, and serum was pooled per litter and stored for possible determination of serum T4 levels.
B) from two pups per litter at sacrifice on PND 13. Individual blood samples were collected from the heart whilst under CO2/O2 anaesthesia, from two pups per litter at sacrifice on lactation day 13. Serum was prepared and stored for determination of serum T4 levels. Serum samples were discarded after analysis or after issuing the final report.
- The blood samples taken from the adult males and from the two pups per litter sacrificed on PND 13 were analyzed for T4 hormone levels. Analysis was performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method based on the manufacturer’s protocol.
Oestrous cyclicity (parental animals):
Number of adult females with normal or abnormal estrous cycle and cycle duration
Sperm parameters (parental animals):
Parameters examined of the male reproduction organs in P male parental generations are listed in Table 4 in ‘Any other information on materials and methods incl. tables’.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, if possible, four pups per sex per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups, runts (pups that were 25% less in body weight than the mean body weight of the pups in the control group) and grossly malformed pups were evaluated on day 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on day 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.
The number of culled pups, total number of pups lost (dead, missing, cannibalised) and number and incidence of total litters lost were also recorded.
At lactation day 4, the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD was reported as such, corrected for body weight and for the cube root of body weight.
On PND 13 all surviving male pups were examined for the presence of nipples and/or areolas.
Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
Yes, any grossly malformed pups were sacrificed and examined. The thyroid gland was collected in two selected pups per litter on PND 13 and examined microscopically. On any stillborn pups or pups dying during the study, a necropsy was performed and macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after 30 days of treatment.
- Maternal animals: All surviving animals, after 14 days of lactation.

GROSS PATHOLOGY: Yes
Prior to sacrifice, all surviving adult male and female animals were fasted overnight (water was freely available). The animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia and then subjected to macroscopic examination for pathological changes.
At scheduled necropsy, the organs of the adult animals indicated in the Table 3 in ‘Any other information on materials and methods incl. tables’ were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Samples of the tissues and organs of the adult animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde, except for the testes and epididymides which were preserved in Bouin’s fixative.
The reproductive organs of all male and female animals were preserved. The number of implantation sites in the uterus were counted.
The other organs/tissues were preserved of five adult animals/sex/group (surviving males with the lowest identification numbers in each cage; females with a litter were selected).
In addition to the organs and tissues in the Table 3, all gross lesions were preserved.
Organs were not weighed at interim sacrifice.

HISTOPATHOLOGY: Yes
Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which was stained with PAS haematoxylin.
Microscopic examination (by light microscopy) was performed on the preserved organs of the male animals of the control (group 1) and high-dose group (group 4) and on the preserved organs of the female animals of the control (group 1) and mid-dose group (group 3).
The organs and tissues were not examined in females of the terminated high-dose group. In this group only organs showing gross lesions were examined microscopically.
In addition, organs showing gross lesions of animals of all other groups were examined microscopically. Organs marked with an asterisk (the levator ani plus bulbocavenosus muscle complex, Cowper’s
glands and glans penis) were preserved after weighing but not further examined.
Postmortem examinations (offspring):
At necropsy of the litter on day 13 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques and necropsied. The thyroid gland was collected in two selected pups per litter on PND 13 and examined microscopically.
Statistics:
The statistical procedures for analysis of data are described in Table 4 in 'Any other information on materials and methods incl. tables'. Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
Reproductive indices:
The reproductive indices can be found in Table 5 in 'Any other information on materials and methods incl. tables'.
Offspring viability indices:
The reproductive indices can be found in Table 6 in 'Any other information on materials and methods incl. tables'.
Clinical signs:
no effects observed
Description (incidence and severity):
Despite the mortality in high-dose females, there were no noticeable clinical signs during the pre-mating period, mating and gestation period or the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Five females of the high-dose group were found dead during the study. The remaining females in the high-dose group were humanely killed for animal welfare reasons and because it was anticipated that insufficient pregnant females would remain to allow a meaningful evaluation. There was no mortality in females of the other groups or in males of any dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight changes of females were not affected in any group during the premating period, nor in the remaining low- and mid-dose groups during the gestation period or the lactation period.
Body weights and body weight changes of males of the high-dose group were reduced during the post-mating period (Table 1 to 3 in 'Any other information on results incl. tables). The differences with the controls were statistically significant from day 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls (Table 4 to 6 in 'Any other information on results incl. tables'. Food consumption was not affected in females of the low- and mid-dose group during the gestation period or the lactation period.
There were no treatment-related effects on food consumption of male animals during the pre- and post-mating period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological findings are presented in Tables 7 to 10 in 'Any other information on results incl. tables'.
There were no treatment-related effects in red blood cell and coagulation parameters at sacrifice in males of all dose groups and in females of the low- and mid-dose groups.
MCHC was slightly, though statistically significantly reduced in females of the low- and mid-dose groups. No significance was attached to this finding because there was no dose-response
relationship and it was not accompanied by significant changes in the main parameters from which this calculated value was derived.
Total and differential white blood cell counts were not affected in males of all dose groups and in females of the remaining low- and mid-dose groups. No haematological data were obtained for the female high-dose group that was terminated intercurrently.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry findings are presented in Tables 11 and 12 in 'Any other information on results incl. tables'.
Statistically significant changes in clinical chemistry variables were noted in high-dose males, namely increases in ASAT and ALAT activity and urea and inorganic phosphate concentrations,
and decreases in triglycerides and sodium concentration (Table 3 in 'Any other information on results incl. tables'.
In females of the remaining low- and mid-dose groups there were no statistically significant differences with the controls. No clinical biochemistry data were obtained for the female high-dose group that was terminated intercurrently.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.
Some treatment-related effects were, however observed in male or female animals of the highdose groups during weekly detailed clinical examinations or during functional observations at the end of the study. These effects included sliding with the ventral parts of the head and neck over the bottom of the open field and salivation. At a few occasions, repetitive movements of mouth and jaws, a hunched posture, piloerection, low activity, large number of urine pools, vocalization when prodded or nasal haemorrhagic discharge were observed in some animals of the high-dose group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination was performed on control- and high-dose males and on control- and mid-dose females.
The incidence of focal necrosis in the stomach was increased in females of the low- and mid-dose groups both compared to concurrent and historical control data.
There were no other treatment-related histopathological changes. A statistically significantly lower incidence of microhemorrhages was noted in the thymus of high-dose males. This finding is the result of the relatively high incidence of this irrelevant change in the control group rather than related to treatment and is considered a chance finding.
The other histopathological changes observed were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
T4 LEVELS:
No statistically significant effects were observed on T4-hormones in males (Table 23 in 'Any other information on results incl. tables').
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
During the 2 weeks pre-mating period, the mean length of the longest cycle was statistically significantly increased in high-dose females (Table 17 in 'Any other information on results incl. tables).
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Four high-dose females that were placed with males were not mated within 7 days, and the male rats were replaced by rats of the same group that had successfully mated. As a result the male mating index was reduced and the pre-coïtal time was prolonged in the high-dose group. Because most high-dose females eventually were mated, the female mating index was not clearly affected in the high-dose group. Male and female mating indices and fertility indices were not affected in the low- and mid-dose group.
All females of the control-, low- and mid-dose group delivered live born pups, and there were no females with only stillborn pups. The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) were comparable and accounted 0(0), 0 (0), 2 (3) for the control-, low-, and mid-dose group, respectively.
The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance (Table 18 in ‘Any other information on results incl. tables’). Because in the mid-dose group three pups were stillborn and another pup died after delivery, the number of live pups per litter at PND 0 and PND 4 (pre-culling) became statistically significantly reduced in this group.
In the mid-dose group, the mean number of implantation sites was also low compared to the controls, although still within the range of recent historical control data (Table 19 in ‘Any other information on results incl. tables).
Post implantation loss was not statistically significantly affected in females of the former mid-dose group. Therefore the lower number of live pups per litter at PND 0 was mainly due to a lower number of implantations in mid-dose females.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related signs in pups during the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance. Because in
the mid-dose group three pups were stillborn and another pup died after delivery, the number of live pups per litter became statistically significantly reduced in this group. In the mid-dose
group, the mean number of implantation sites was also low compared to the controls (Table 18 in 'any other information on results incl. tables').
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related differences in mean pup weights between the test groups and the controls on PND 0, 4 and 7, no runts were observed(Table 24 in 'Any other information on results incl. tables').
On lactation day 13, the weight of the male pups of the low-dose group was statistically significantly lower than in controls. This is considered a chance finding because there were neither significant changes in male pup weight at the next higher level nor in total pup weight.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on anogenital distance or anogenital distance corrected for cube root of bodyweight of male and female pups (Table 22 in 'Any other information on results incl. tables').
There were no effects on nipple retention of male pups on PND 13.
After culling on PND 4, no pups were lost. No differences were observed on sex ratio on PND 0 and PND 13 among the groups (Table 18 in 'Any other information on results incl. tables').
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative weights of the thyroid on PND 13 were slightly, though statistically significantly increased in male pups of the former mid-dose group.
Female pup thyroid weight and total pup thyroid weight (both sexes combined) were, however, not significantly affected (Table 21 in 'Any other information on results incl. tables').
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations of pups at necropsy on PND 13 and stillborn pups or pups that died revealed no abnormalities.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic examinations of the thyroid gland of pups killed on PND 13 revealed no treatment-related abnormalities.
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant effects observed on T4-hormones (Table 23 in 'Any other information on results incl. tables').
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1. Parental body weight males and females during the study - Day(s) Relative to Start Date.

Sex: Male

 

 

 

Bodywt

day -x (g)

 

[G]

Bodywt

day 0 (g)

 

[G]

Bodywt

 (g)

 

[C]

Bodywt

 (g)

 

[C]

Bodywt

 (g)

 

[C]

Bodywt

 (g)

 

[C]

-3

0

7

14

21

28

0 mg/kg

Mean SD

N

334.29

13.48

12

342.37

14.89

12

351.50

15.41

12

357.64

16.73

12

361.62

15.90

12

371.83

17.41

12

5 mg/kg

Mean

SD

N

334.28

12.93

12

342.63

13.42

12

351.81

15.98

12

360.26

16.38

12

362.18

18.33

12

372.53

20.28

12

20 mg/kg

Mean

SD

N

334.87

14.41

12

342.88

16.56

12

353.29

17.23

12

363.03

17.48

12

366.46

18.30

12

375.33

18.40

12

75 mg/kg

Mean SD

N

334.45

14.63

12

342.63

14.08

12

349.85

14.23

12

353.13

14.69

12

354.18 *

13.72

12

354.71 **

15.17

12

Sex: Female

 

 

 

 

 

 

 

0 mg/kg

Mean SD

N

217.33

7.91

12

224.18

6.20

12

224.54

6.76

12

225.78

6.67

12

 

 

5 mg/kg

Mean

SD

N

217.60

7.85

12

223.11

8.41

12

223.72

6.10

12

225.83

7.06

12

 

 

20 mg/kg

Mean

SD

N

217.39

7.95

12

222.53

7.52

12

223.29

8.52

12

227.41

10.00

12

 

 

75 mg/kg

Mean SD

N

217.75

8.55

12

225.13

11.25

12

222.74

12.68

11

225.50

11.32

11

 

 

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

 

Table 2. Parental body weight females gestation - Day(s) Relative to Mating (Litter: A).

Sex: Female

 

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

0

7

14

20

0 mg/kg

Mean

227.42

248.33

272.11

332.17

 

SD

6.62

9.30

11.81

15.63

 

N

12

12

12

12

5 mg/kg

Mean

228.77

250.03

271.59

326.74

 

SD

7.27

10.17

11.62

16.41

 

N

12

12

12

12

20 mg/kg

Mean

226.59

247.58

268.65

321.56

 

SD

7.09

10.69

12.34

18.18

 

N

11

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 3. Parental body weight females lactation - Day(s) Relative to Littering (Litter: A).

Sex: Female

 

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

Bodywt

 (g)

 

[G]

0

4

7

13

0 mg/kg

Mean

255.99

274.28

277.06

286.32

 

SD

10.14

13.88

14.36

13.78

 

N

12

12

12

12

5 mg/kg

Mean

251.76

266.01

266.68

278.53

 

SD

9.92

11.35

14.21

12.49

 

N

12

12

12

12

20 mg/kg

Mean

252.52

266.53

271.63

282.53

 

SD

13.32

12.74

13.33

11.85

 

N

12

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 4. Food consumption males during the study - Daily Food Cons Per Animal (g)

Sex: Male

Day(s) Relative to Animal start date

0 - 7

7 - 14

21 - 28

0 mg/kg

Mean

20.6

18.8

18.3

 

SD

0.6

1.1

0.4

 

N

3

3

3

5 mg/kg

Mean

20.4

17.5

18.0

 

SD

0.2

0.5

0.1

 

N

3

3

3

20 mg/kg

Mean

20.9

18.8

18.6

 

SD

0.8

0.6

0.7

 

N

3

3

3

75 mg/kg

Mean

19.8

17.8

18.6

 

SD

1.5

0.5

1.8

 

N

3

3

3

0 mg/kg

Mean

13.9

13.0

 

SD

0.4

0.5

 

N

3

3

 

5 mg/kg

Mean

14.4

13.5

 

SD

0.4

0.6

 

N

3

3

 

20 mg/kg

Mean

14.2

13.4

 

SD

0.2

0.1

 

N

3

3

 

75 mg/kg

Mean

12.8 **

13.4

 

SD

0.1

0.8

 

N

3

3

 

Dunnett** = p < 0.01

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 5. Food consumption females gestation - Daily Food Cons Per Animal (g).

Sex: Female

Day(s) Relative to Mating (Litter: A)

0 - 7

7 - 14

14 - 20

0 mg/kg

Mean

15.22

16.81

18.12

 

SD

1.42

1.45

0.88

 

N

12

12

12

5 mg/kg

Mean

15.61

16.48

18.16

 

SD

1.45

2.10

1.42

 

N

12

12

12

20 mg/kg

Mean

14.66

16.07

17.98

 

SD

1.81

1.99

1.68

 

N

11

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 6. Food consumption females lactation - Daily Food Cons Per Animal (g)

Sex: Female

Day(s) Relative to Littering (Litter: A)

0 - 4

4 - 7

7 - 13

0 mg/kg

Mean

28.99

41.42

48.46

 

SD

5.33

5.46

4.37

 

N

12

12

12

5 mg/kg

Mean

27.67

41.36

45.78

 

SD

4.37

6.38

4.30

 

N

12

12

12

20 mg/kg

Mean

26.95

40.46

47.51

 

SD

3.18

5.43

3.85

 

N

12

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 7. Red blood cell and coagulation parameters - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

 

[G]

Hb (mmol/L)

 

[G]

PCV

(L/L)

 

[G]

MCV

(fL)

  

[G1]

MCH

(fmol)

  

[G1]

MCHC

(mmol/L)

  

[G1]

Reticulo cytes

(%)

 [G1]

Thrombo cytes

(10E9/L)

 [G1]

Prothrom Time

(s)

 [G1]

0 mg/kg

Mean SD

N

8.850

0.424

5

9.46

0.34

5

0.4840

0.0196

5

54.71

0.62

5

1.070

0.026

5

19.55

0.42

5

1.988

0.170

5

805.4

100.8

5

19.80

1.05

5

5 mg/kg

Mean SD

N

9.066

0.370

5

9.68

0.24

5

0.4984

0.0150

5

55.00

1.04

5

1.068

0.021

5

19.43

0.26

5

2.160

0.344

5

834.2

111.4

5

20.52

0.67

5

20 mg/kg

Mean SD

N

8.774

0.177

5

9.46

0.44

5

0.4864

0.0193

5

55.43

1.41

5

1.078

0.035

5

19.45

0.39

5

1.958

0.156

5

772.8

78.3

5

20.66

0.99

5

75 mg/kg

Mean SD

N

9.926

1.139

5

10.64

1.28

5

0.5468

0.0780

5

54.95

1.65

5

1.072

0.024

5

19.51

0.56

5

2.042

0.315

5

792.0

136.1

5

20.08

1.05

5

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett

 

Table 8. Red blood cell and coagulation parameters - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

 

[G]

Hb (mmol/L)

 

 [G]

PCV

(L/L)

 

 [G]

MCV

(fL)

 

 [G]

MCH

(fmol)

  

[G]

MCHC

(mmol/L)

 

 [G]

Reticulo cytes

(%)

 [G]

Thrombo cytes

(10E9/L)

 [G]

Prothrom Time

(s)

 [G]

0 mg/kg

Mean SD

N

7.974

0.586

5

9.58

0.59

5

0.4776

0.0263

5

59.99

2.36

5

1.203

0.037

5

20.05

0.22

5

3.488

0.510

5

895.0

216.2

5

21.46

1.11

5

5 mg/kg

Mean SD

N

7.992

0.457

5

9.42

0.32

5

0.4820

0.0110

5

60.44

3.19

5

1.181

0.065

5

19.54 * 0.42

5

3.294

0.182

5

1102.8

85.1

5

21.66

1.47

5

20 mg/kg

Mean SD

N

7.376

0.443

5

9.04

0.38

5

0.4630

0.0182

5

62.90

3.53

5

1.228

0.077

5

19.52 * 0.22

5

3.862

0.583

5

978.6

120.5

5

21.66

0.82

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05

 

Table 9. Total and differential white blood cell counts - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G1]

Eosino Absolute

(10E9/L)

 

[G2]

Baso Absolute

(10E9/L)

 

[G2]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G2]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

5.38

1.68

5

4.13

1.22

5

1.02

0.45

5

0.111

0.095

5

0.025

0.014

5

0.082

0.040

5

77.22

5.45

5

18.42

3.52

5

2.28

2.48

5

0.44

0.18

5

1.46

0.41

5

5 mg/kg

Mean SD

N

6.38

1.18

5

5.05

1.04

5

1.09

0.14

5

0.085

0.025

5

0.014

0.009

5

0.120

0.031

5

78.86

2.33

5

17.32

2.33

5

1.34

0.32

5

0.20

0.10

5

1.90

0.42

5

20 mg/kg

Mean SD

N

6.66

0.96

5

5.19

1.10

5

1.20

0.25

5

0.136

0.104

5

0.022

0.011

5

0.087

0.037

5

77.26

7.62

5

18.42

5.34

5

2.28

2.21

5

0.32

0.16

5

1.36

0.65

5

75 mg/kg

Mean SD N

6.38

0.94

5

5.14

0.88

5

0.99

0.13

5

0.116

0.125

5

0.028

0.029

5

0.089

0.041

5

80.36

4.14

5

15.68

2.64

5

1.88

2.17

5

0.40

0.35

5

1.36

0.50

5

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks [G2] - Ancova/Anova & Dunnett(Log)

 

Table 10. Total and differential white blood cell counts - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G]

Eosino Absolute

(10E9/L)

 

[G]

Baso Absolute

(10E9/L)

 

[G]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G1]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

6.44

3.82

5

3.04

1.79

5

3.11

1.92

5

0.064

0.032

5

0.019

0.019

5

0.184

0.105

5

46.16

4.79

5

48.34

3.09

5

1.16

0.42

5

0.38

0.33

5

3.36

1.34

5

5 mg/kg

Mean SD

N

7.06

2.99

5

3.85

1.45

5

2.83

1.54

5

0.112

0.040

5

0.019

0.011

5

0.223

0.137

5

57.12

8.91

5

37.44

9.06

5

2.12

1.73

5

0.24

0.09

5

2.82

1.46

5

20 mg/kg

Mean SD N

6.30

1.27

5

3.28

0.86

5

2.70

0.81

5

0.065

0.013

5

0.012

0.007

5

0.226

0.090

5

52.12

9.22

5

42.80

10.45

5

1.06

0.29

5

0.20

0.12

5

3.56

1.11

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

 

Table 11. Clinical chemistry - Day: 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

 

 

 

[G]

ASAT (U/L)

 

 

[G1]

ALAT (U/L)

 

 

[G1]

GGT (U/L)

 

 

[G2]

Bilirub Total

(umol/L)

 

[G]

Creatin ine

(umol/L)

 

[G]

Bile Acids

(umol/L)

 

[G1]

Total Protein

(g/L)

 

[G]

Albumin (g/L)

 

 

[G]

Albumin/ Globulin

 

 

[G]

Glucose Plasma

(mmol/L)

 

[G]

Cholest erol

(mmol/L)

 

[G1]

Phospho lipids

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G]

Urea (mmol/L)

 

 

[G]

PO4

(mmol/L)

 

 

[G1]

Ca (mmol/L)

 

 

[G1]

Cl (mmol/L)

 

 

[G1]

K

(mmol/L)

 

 

[G]

Na (mmol/L)

 

 

[G1]

0 mg/kg

Mean SD

N

108.6

32.5

5

90.0

7.9

5

47.0

4.5

5

0.00

0.00

5

1.42

0.43

5

42.8

3.3

5

9.64

1.91

5

63.2

2.6

5

33.0

1.4

5

7.602

1.614

5

1.914

0.230

5

1.728

0.102

5

1.138

0.143

5

6.24

0.59

5

2.574

0.283

5

2.818

0.080

5

99.0

1.6

5

5.98

0.48

5

146.2

1.1

5

1.093

0.029

5

5 mg/kg

Mean SD

N

103.0

34.4

5

86.2

7.2

5

40.4

5.7

5

0.00

0.00

5

1.26

0.71

5

40.6

4.7

5

17.06

12.29

5

65.0

2.5

5

34.8

1.1

5

8.816

1.836

5

1.816

0.249

5

1.666

0.156

5

0.956

0.163

5

6.82

1.64

5

2.908

0.299

5

2.912

0.069

5

98.2

0.4

5

6.16

0.80

5

146.4

0.5

5

1.155

0.068

5

20 mg/kg

Mean SD

N

95.6

8.5

5

84.6

2.4

5

46.8

10.4

5

0.00

0.00

5

1.52

0.28

5

40.4

2.3

5

18.88

18.68

5

62.6

3.2

5

33.6

1.5

5

8.992

2.111

5

1.552

0.171

5

1.524

0.129

5

0.864

0.394

5

6.32

0.44

5

2.718

0.286

5

2.846

0.103

5

99.0

1.0

5

5.72

0.43

5

146.8

0.8

5

1.160

0.047

5

75 mg/kg

Mean SD

N

139.6

34.6

5

110.6 * 22.9

5

103.2 ** 33.3

5

0.00

0.00

5

1.38

0.42

5

39.2

1.9

5

28.63

13.15

4

62.6

3.2

5

34.0

1.4

5

8.640

1.449

5

1.424

0.519

5

1.458

0.318

4

0.624 **

0.087

5

8.14 * 1.18

5

3.132 * 0.216

5

2.788

0.096

4

98.2

1.3

5

6.14

0.71

5

143.2 ** 1.3

5

1.193

0.080

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log): * = p < 0.05; ** = p < 0.01

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 12. Clinical chemistry - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

 

 

 

[G]

ASAT (U/L)

 

 

[G1]

ALAT (U/L)

 

 

[G]

GGT (U/L)

 

 

[G2]

Bilirub Total

(umol/L)

 

[G]

Creatin ine

(umol/L)

 

[G]

Bile Acids

(umol/L)

 

[G]

Total Protein

(g/L)

 

[G]

Albumin (g/L)

 

 

[G]

Albumin/ Globulin

 

 

[G]

Glucose Plasma

(mmol/L)

 

[G]

Cholest erol

(mmol/L)

 

[G1]

Phospho lipids

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G1]

Urea (mmol/L)

 

 

[G1]

PO4

(mmol/L)

 

 

[G]

Ca (mmol/L)

 

 

[G1]

Cl (mmol/L)

 

 

[G1]

K

(mmol/L)

 

 

[G1]

Na (mmol/L)

 

 

[G1]

0 mg/kg

Mean SD

N

90.6

21.1

5

140.0

22.9

5

61.2

8.6

5

1.94

4.01

5

1.28

0.26

5

48.4

3.6

5

32.28

21.66

5

63.2

3.5

5

33.0

2.2

5

7.606

1.306

5

2.896

0.598

5

2.906

0.403

5

2.114

1.095

5

9.16

1.57

5

3.174

0.568

5

2.792

0.151

5

95.0

2.3

5

5.20

0.33

5

143.6

2.8

5

1.095

0.081

5

5 mg/kg

Mean SD

N

118.4

52.7

5

120.4

19.9

5

63.0

14.7

5

0.18

0.35

5

1.36

0.15

5

50.0

2.7

5

30.12

15.89

5

64.6

3.2

5

34.6

1.8

5

7.892

1.399

5

2.338

0.506

5

2.582

0.430

5

1.830

0.990

5

9.42

1.61

5

3.040

0.493

5

2.850

0.190

5

96.0

3.4

5

5.06

0.50

5

144.0

2.8

5

1.155

0.063

5

20 mg/kg

Mean SD

N

137.0

44.1

5

123.4

25.1

5

57.8

12.9

5

0.10

0.22

5

1.14

0.38

5

47.4

4.4

5

17.80

13.34

5

61.6

5.2

5

32.4

3.0

5

7.952

0.179

5

2.592

0.472

5

2.778

0.401

5

2.296

0.784

5

9.66

1.29

5

3.078

0.061

5

2.812

0.076

5

98.0

1.0

5

5.28

0.46

5

145.2

1.9

5

1.110

0.060

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 13. Absolute organ weights - Day(s): 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G1]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G1]

Thymus (g)

[G]

Thyroid (g)

[G]

Testes (g)

[G1]

Epididy mides

(g)

 

[G1]

Prostate (g)

[G2]

Seminal vesicles

(g)

 

[G1]

LABC

Muscle (g)

 

[G1]

Cowpers Glands

(g)

 

[G2]

Glans Penis (g)

[G1]

0 mg/kg

Mean SD

N

358.58

16.31

12

2.064

0.018

5

1.028

0.051

5

0.0540

0.0076

5

1.998

0.147

5

8.408

0.327

5

0.5860

0.0394

5

0.0200

0.0073

12

3.563

0.305

12

1.193

0.101

12

0.939

0.162

12

1.193

0.169

12

1.0571

0.1190

12

0.1046

0.0143

12

0.1376

0.0326

12

0.3228

0.0997

5

5 mg/kg

Mean SD

N

358.58

18.26

12

2.098

0.031

5

1.030

0.034

5

0.0548

0.0068

5

2.140

0.100

5

8.282

0.398

5

0.5792

0.0889

5

0.0171

0.0051

12

3.715

0.299

12

1.208

0.130

12

0.918

0.175

12

1.226

0.199

12

1.0701

0.1283

12

0.0953

0.0147

12

0.1344

0.0280

12

0.3028

0.0779

5

20 mg/kg

Mean SD

N

361.33

17.67

12

2.052

0.108

5

1.044

0.072

5

0.0578

0.0063

5

2.144

0.058

5

8.712

0.414

5

0.6410

0.0425

5

0.0190

0.0075

11

3.638

0.192

12

1.209

0.076

12

0.973

0.121

12

1.371 *

0.144

12

1.0884

0.1006

12

0.1048

0.0321

12

0.1236

0.0304

12

0.3246

0.0468

5

75 mg/kg

Mean SD

N

334.93 ** 14.23 12

2.052

0.073

5

0.952

0.052

5

0.0582

0.0048

5

2.256

0.191

5

9.106

1.883

5

0.5178

0.0755

5

0.0153

0.0058

12

3.512

0.148

12

1.136

0.113

12

0.923

0.240

12

1.007 *

0.197

12

0.9757

0.0854

12

0.0878 **

0.0227

12

0.1280

0.0308

12

0.2466

0.0606

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 14. Absolute organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G]

Thymus (g)

[G]

Thyroid (g)

[G2]

Ovaries (g)

[G2]

Uterus (g)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

1.912

0.094

5

0.866

0.084

5

0.0754

0.0038

5

1.516

0.087

5

8.764

0.905

5

0.5480

0.0701

5

0.2218

0.0456

5

0.0186

0.0046

12

0.0884

0.0136

12

0.5245

0.1042

12

5 mg/kg

Mean SD

N

254.78

10.69

12

1.936

0.062

5

0.832

0.058

5

0.0710

0.0120

5

1.558

0.080

5

8.452

0.569

5

0.5558

0.0735

5

0.1796

0.0348

5

0.0155

0.0044

11

0.0921

0.0143

12

0.5579

0.1135

12

20 mg/kg

Mean SD

N

258.29

11.63

12

1.974

0.056

5

0.824

0.047

5

0.0738

0.0124

5

1.624

0.077

5

8.594

0.414

5

0.5850

0.0386

5

0.1914

0.0272

5

0.0141 *

0.0020

12

0.0883

0.0058

12

0.5187

0.0680

12

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log): * = p < 0.05 Litter: A = First litter

 

Table 15. Relative organ weights - Day(s): 30 Relative to Start Date

Sex: Male

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G1]

Spleen rel.wgt

(g/kg body wgt)

[G]

Thymus rel.wgt

(g/kg body wgt)

[G2]

Thyroid rel.wgt

(g/kg body wgt)

[G]

Testes rel.wgt

(g/kg body wgt)

[G1]

Epididy rel.wgt

(g/kg body wgt)

[G2]

Prostate rel.wgt

(g/kg body wgt)

[G2]

Sem ves rel.wgt

(g/kg body wgt)

[G1]

LABC Muscle rel.wgt

(g/kg body wgt)

[G1]

Cowpers Gl. rel.wgt

(g/kg body wgt)

[G2]

Glans Penis rel.wgt

(g/kg body wgt)

[G1]

0 mg/kg

Mean SD

N

358.58

16.31

12

5.803

0.320

5

2.887

0.147

5

0.1514

0.0193

5

5.622

0.563

5

23.63

1.25

5

1.648

0.148

5

0.0557

0.0195

12

9.928

0.571

12

3.325

0.222

12

2.617

0.407

12

3.326

0.440

12

2.9454

0.2731

12

0.2918

0.0382

12

0.3844

0.0941

12

0.910

0.305

5

5 mg/kg

Mean SD

N

358.58

18.26

12

5.918

0.312

5

2.904

0.149

5

0.1544

0.0190

5

6.031

0.282

5

23.34

1.03

5

1.627

0.201

5

0.0475

0.0139

12

10.378

0.909

12

3.380

0.449

12

2.558

0.466

12

3.422

0.554

12

2.9883

0.3578

12

0.2665

0.0433

12

0.3740

0.0707

12

0.855

0.234

5

20 mg/kg

Mean SD

N

361.33

17.67

12

5.560

0.443

5

2.823

0.169

5

0.1559

0.0108

5

5.805

0.300

5

23.55

0.35

5

1.737

0.167

5

0.0520

0.0189

11

10.090

0.683

12

3.349

0.180

12

2.696

0.352

12

3.799

0.399

12

3.0124

0.2457

12

0.2914

0.0882

12

0.3399

0.0680

12

0.881

0.155

5

75 mg/kg

Mean SD

N

334.93 ** 14.23

12

6.214

0.264

5

2.886

0.237

5

0.1764

0.0173

5

6.829 **

0.577

5

27.62

6.18

5

1.571

0.256

5

0.0453

0.0165

12

10.501

0.604

12

3.398

0.387

12

2.769

0.786

12

2.997

0.522

12

2.9155

0.2580

12

0.2626

0.0677

12

0.3840

0.0987

12

0.748

0.195

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 16. Relative organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G]

Spleen rel.wgt

(g/kg body wgt)

[G1]

Thymus rel.wgt

(g/kg body wgt)

[G]

Thyroid rel.wgt

(g/kg body wgt)

[G2]

Ovaries rel.wgt

(g/kg body wgt)

[G]

Uterus rel.wgt

(g/kg body wgt)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

7.382

0.308

5

3.348

0.359

5

0.2914

0.0178

5

5.860

0.425

5

33.83

3.24

5

2.117

0.274

5

0.855

0.161

5

0.0713

0.0201

12

0.3366

0.0510

12

1.997

0.408

12

5 mg/kg

Mean SD

N

254.78

10.69

12

7.699

0.404

5

3.309

0.280

5

0.2820

0.0464

5

6.191

0.296

5

33.57

1.88

5

2.213

0.332

5

0.713

0.135

5

0.0612

0.0174

11

0.3618

0.0563

12

2.188

0.424

12

20 mg/kg

Mean SD N

258.29

11.63

12

7.623

0.326

5

3.178

0.105

5

0.2864

0.0586

5

6.278

0.483

5

33.18

1.60

5

2.265

0.252

5

0.737

0.087

5

0.0544

0.0059

12

0.3428

0.0294

12

2.017

0.318

12

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log) [G2] - Kruskal-Wallis & Dunnett on Ranks Litter: A = First litter

Table 17. Oestrus cyclicity

 

 

 

Pre-treatment

 

 

 

 

 

 

Pre-mating

 

 

 

 

 

 

 

 

 

Control

Low dose

Mid dose

High dose

Control

Low dose

Mid dose

High dose

Number of females evaluated

 

n

12

12

12

12

12

12

12

11

Number of acyclic females

 

n

0

0

0

0

0 f

0

0

1

 

 

%

0.0

0.0

0.0

0.0

0.0

0.0

0.0

9.1

Length of the longest cycle

4

n

12

12

11

12

12

12

12

6

 

 

 

5

n

0

0

1

0

0

0

0

4

>5

n

0

0

0

0

0

0

0

0

Mean length of the longest cycle (days)

 

 

 

 

 

mean

4.00

4.00

4.08

4.00

4.00 kw

4.00

4.00

4.40**

 

sd

0.00

0.00

0.29

0.00

0.00

0.00

0.00

0.52

 

n

12

12

12

12

12

12

12

10

Number of animals with prolonged oestrus period

 

 

 

n

0

0

0

0

0 f

0

0

0

 

%

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Number of complete cycles per animal in 14 (pretreatment) or 15 (premating) days

 

 

 

 

 

mean

2.3

2.4

2.4

2.3

2.8 kw

2.8

2.8

2.6

 

sd

0.49

0.51

0.51

0.49

0.39

0.45

0.45

0.52

 

n

12

12

12

12

12

12

12

10

Table 18. Delivery report and litter report

Sex: Female

 

Day(s) Relative to Littering

0 mg/kg

5 mg/kg

20 mg/kg

Pups Delivered (Total) [k]

Mean SD

Sum Sum

 

Sum

11.8

1.6

142

142

100.0

0

0.0

10.9

1.8

131

131

100.0

0

0.0

10.2

1.9

122

119

97.5

3

2.5

Liveborn [f]

Live Birth Index (%)

Stillborn day 0 [f]

Stillborn Index (%)

Live Pups/Litter day 0 [k] 1

Mean

11.8

10.9

9.8 *

 

SD

1.7

1.8

1.7

 

N

12

12

12

 

Sum

141 1

131

118 1

Live Pups/Litter day 4 Pre [k]

Mean

11.7

10.9

9.8 *

 

SD

1.7

1.8

1.7

 

N

12

12

12

 

Sum

140

131

117

Culled pups

Sum

44

36

23

Live Pups/Litter day 4 Post [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Live Pups/Litter day 7 [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Live Pups/Litter day 13 [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Dead, Missing, Cannibalized d0-d4

 

2

0

2

Dead, Missing, Cannibalized d5-d7

 

0

0

0

Dead, Missing, Cannibalized d8-d13

 

0

0

0

No. of litters lost entirely d0-d4 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d5-d7 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d8-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d0-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

Viability Index 0-4 (%)

 

99

100

98

Viability Index 4-13 (%)

 

100

100

100

Live Males on Day 0

Mean

5.3

4.4

4.9

 

SD

1.1

1.4

2.1

 

N

12

12

12

 

Sum

64

53

59

Sex Ratio Day 0 - Males (%)

 

45.4

40.5

50.0

Live Males on Day 13

Mean

4.0

3.6

3.8

 

SD

0.0

0.8

1.2

 

N

12

12

12

 

Sum

48

43

46

Sex Ratio Day 13 - Males

 

50.0

45.3

48.9

Implantation Sites Total

Mean

12.6

11.8

11.2

 

SD

1.6

1.9

2.0

 

N

12

12

12

 

Sum

151

142

134

No. of lost implantations

Sum

9

11

15

Post-Implantation Loss % [k]

Mean

5.7

7.5

10.4

 

SD

8.2

8.8

11.5

1 Pups that were liveborn but died on day 0 are not included

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Live birth index: no. of liveborn pups *100/no. of total pups delivered

Stillborn index: no. of stillborn pups *100/no. of total pups delivered

Viability index 0-4: no. of live pups on day 4 *100/no. of liveborn pups

Viability index 4-13: no. of live pups on day 13 * 100/no. of live pups post cull

Sex ratio day n: no. of live male pups on day n *100/ no. of live pups on day n

Post-implantation loss: no. of implant sites - no. of liveborn *100/no. of implant sites

Table 19. Historical ranges of number of delivered pups per litter and number of implantation sites.

Study

Total pups delivered per litter (mean)

Implantation sites

(mean)

20624

12.8

13.4

20718/02

11.8

13.4

20785/02

11.6

12.1

20810/02

10.3

11.0

20853/02

11.5

13.2

20881/02

10.1

11.3

20890/02

12.0

12.2

20934/02

11.6

12.7

21025/02

11.8

12.6


Table 20. Pup organ weights

 days relative to littering

 

 

 

 

 

 

Mean Pup

TermBW /L (g)

 

[g]

Mean Pup (M)

TermBW /L (g)

 

[g]

Mean Pup (F)

TermBW /L (g)

 

[g]

Mean Pup

Thyroid wgt (g)

 

[g]

Mean Pup (M)

Thyroid wgt (g)

 

[g]

Mean Pup (F)

Thyroid wgt (g)

 

[g]

13

13

13

13

13

13

0 mg/kg

Mean SD

N

33.742

3.187

12

34.075

3.159

12

32.964

3.233

11

0.0042

0.0015

12

0.0040

0.0016

12

0.0045

0.0015

11

5 mg/kg

Mean

SD

N

32.817

2.477

12

33.417

2.608

12

32.217

2.992

12

0.0043

0.0009

12

0.0048

0.0011

10

0.0041

0.0010

12

20 mg/kg

Mean SD

N

34.504

2.504

12

34.908

2.890

12

34.100

2.512

12

0.0048

0.0009

12

0.0056 *

0.0013

11

0.0040

0.0012

12

[g] - Anova & Dunnett: * = p < 0.05

Litter: A = First litter

Table 21. Pup thyroid weights

 days relative to littering

 

 

 

Mean Pup

Thyroid relw (g/kg

body wgt) [g]

Mean Pup (M)

Thyroid relw (g/kg

body wgt) [g]

Mean Pup (F)

Thyroid relw (g/kg

body wgt) [g]

13

13

13

0 mg/kg

Mean

0.1246

0.1188

0.1381

 

SD

0.0452

0.0475

0.0465

 

N

12

12

11

5 mg/kg

Mean

0.1325

0.1428

0.1272

 

SD

0.0231

0.0277

0.0274

 

N

12

10

12

20 mg/kg

Mean

0.1380

0.1606 *

0.1183

 

SD

0.0232

0.0343

0.0329

 

N

12

11

12

[g] - Anova & Dunnett: * = p < 0.05

Litter: A = First litter

 Table 22. Pup anogenital distance

 

 

 

 

 

 

 

Mean Male

Pup BW /L (g)

 

 

[G]

Mean Male

Pup AGD /L (mm)

 

[G]

AGDcorrected

for BW - M (mm/g3)

 

[G]

Mean Female

Pup BW /L (g)

 

 

[G]

Mean Female

Pup AGD /L (mm)

 

[G]

AGDcorrected

for BW - F (mm/g3)

 

[G1]

4

4

4

4

4

4

0 mg/kg

Mean SD

N

11.53

1.49

12

6.202

0.671

12

2.755

0.324

12

10.92

1.37

12

3.682

0.370

12

1.667

0.192

12

5 mg/kg

Mean

SD

N

11.83

1.19

12

6.688

0.485

12

2.943

0.230

12

11.58

1.15

12

3.863

0.405

12

1.713

0.203

12

20 mg/kg

Mean SD

N

12.41

1.55

12

6.521

0.464

12

2.822

0.168

12

11.97

1.37

12

3.665

0.231

12

1.607

0.114

12

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

Litter: A = First litter

L = per Litter

Table 23. Hormone determinations (T4)

 

(P) Male

(F1) Male

(F1) Female

T4

(ng/ml)

 

[G]

T4

(ng/ml)

 

[G]

T4

(ng/ml)

 

[G]

0 mg/kg

Mean

308.77

64.47

66.05

 

SD

116.64

16.41

22.40

 

N

12

12

12

5 mg/kg

Mean

309.56

81.61

80.88

 

SD

58.01

29.82

32.34

 

N

12

12

12

20 mg/kg

Mean

368.28

88.43

87.89

 

SD

114.96

35.35

28.66

 

N

12

12

12

75 mg/kg

Mean

314.03

-

-

 

SD

142.44

-

-

 

N

12

-

-

[G] - Ancova/Anova & Dunnett(Log)

Table 24. Pup body weight per litter - Day(s) Relative to Littering (Litter: A)

 

 

 

Mean Total

Pup BW/L d0 (g)

 

[G]

Mean Total

Pup BW /L (g)

 

[G1]

Mean Total

Pup BW /L (g)

 

[G1]

Mean To

Pup BW (g)

 

[G1]

tal

/L

0

4

7

13

0 mg/kg

Mean

6.20

11.21

18.46

33.93

 

SD

0.59

1.42

2.10

3.17

 

N

12

12

12

12

5 mg/kg

Mean

6.47

11.69

18.69

32.86

 

SD

0.52

1.15

1.71

2.75

 

N

12

12

12

12

20 mg/kg

Mean

6.51

12.18

18.94

34.22

 

SD

0.62

1.40

1.60

2.23

 

N

12

12

12

12

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett {Covariate: Pup bodyweight on day zero}

Litter: A = First litter

L = per Litter

Conclusions:
In this GLP compliant OECD 422 study, the No-Observed-Adverse-Effect-Level (NOAEL) was 20 mg/kg bw/day for both sexes, based on mortality in high-dose females and on effects on growth, clinical chemistry and weights of liver and kidney in high-dose males. Because there were no relevant effects on reproductive performance or on developmental parameters in the mid-dose group, a NOAEL for reproduction could not be determined.
Executive summary:

In the GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422 the test substance was administered by oral gavage to four groups of male and female Wistar rats. Each group contained 12 animals per sex. The test substance was administrated at 0, 5, 20 and 75 mg/kg bw/day dissolved in tap water during a pre-mating period of 2 weeks, during mating, gestation and until day 13 of lactation. Males were exposed for 30 days before sacrifice. The content, homogeneity and stability of the test substance in the vehicle (tap water) was confirmed by analysis. Cage side observations, including checks for dead or moribund animals, were performed twice a day. Near the end of gestation, females were checked twice a day for parturition. Litters were checked once a day. The parent generation were subjected to the following examinations. Body weights for males and females were recorded weekly. Food consumption was measured per cage and recalculated to food intake per animal per day. In the week before sacrifice, neurobehavioural examination was performed on 5 animals per group per sex using Functional Observational Battery (FOB) and spontaneous motor activity in the arena test. Prior to scheduled sacrifice, blood was withdrawn from the abdominal aorta under CO2/O2 anaesthesia for assessing haematology and clinical chemistry parameters. Furthermore, the blood was analysed for T4 hormone levels in all males. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically. Reproductive performance was assessed based on oestrogenic cycle, number of implantation sites, number of pregnant females, number of viable/stillborn litter and the pre-coital time period.

The F1 generations was subjected to the following examinations. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups, runts and grossly malformed pups were evaluated on day 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on day 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group. At lactation day 4, the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD was reported as such, corrected for body weight and for the cube root of body weight. On PND 13 all surviving male pups were examined for the presence of nipples and/or areolas. T4 hormone levels were evaluated in 2 pups per litter. Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Any grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups or pups dying during the study and on the surviving pups on day 13 of lactation. The pups were evaluated externally for gross abnormalities and the thyroid gland was collected in two selected pups per litter on PND 13 and examined microscopically.

For the parent generation, mortality was limited to the high dose level females, of which 5 died and the rest was killed for animal welfare reasons. No clinical signs were observed during the study. Body weights and body weight changes of males of the high-dose group were reduced during the post-mating period. During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls. There was no treatment-related effects on food consumption of male animals during the pre- and post-mating period. Statistically significant changes in clinical chemistry variables were noted in high-dose males, namely increases in ASAT and ALAT activity and urea and inorganic phosphate concentrations, and decreases in triglycerides and sodium concentration. No statistically significant effects were observed on T4-hormones in males. Haematological and clinical chemistry data were not obtained for the female high-dose group that was terminated intercurrently. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. The relative weight of the kidneys was statistically significantly increased in males of the high-dose group (21%). The mean relative liver weight of high-dose males was about 17% increased but the differences with the controls were not statistically significant. Relative organ weights were not significantly affected in females of the remaining low- and mid-dose groups. The incidence of ulceration and yellow/white deposition in the stomach was increased in females of the low- and mid-dose groups. The other macroscopic observations at necropsy revealed no treatment-related abnormalities. Microscopic evaluation did, however, not reveal any corroborating histopathological changes. The incidence of local macroscopic (ulceration) and microscopic changes (focal necrosis) in the stomach was, however, increased in low- and mid-dose females, both compared to concurrent and historical control data. Although these changes were not clearly elevated in high-dose females nor in males they may represent a local treatment-related effect, because in high-dose females and males the duration of exposure was shorter. Therefore it cannot be excluded that the prolonged exposure to the test substance induced local reaction in the stomach. Other histopathological findings were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.

Reproductive performance was not affected in the control-, low- and mid-dose groups. All females of the control-, low- and mid-dose group delivered live born pups, and there were no females with only stillborn pups. Post implantation loss was not statistically significantly affected in females of the former mid-dose group. The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance. In the mid-dose group, the mean number of implantation sites was also low compared to the controls, although still within the range of recent historical control data. During the 2 weeks pre-mating period, the mean length of the longest cycle was statistically significantly increased in high-dose females.

For the F1 generation, there were no treatment-related signs in pups during the lactation period. Mortality was observed, but was not treatment-related. There were no treatment-related differences in mean pup weights, anogenital distance, nipple retention of male pups, differences in T4 levels. Macroscopic and microscopic examinations did not reveal any abnormalities. The absolute and relative weights of the thyroid on PND 13 were slightly, though statistically significantly increased in male pups of the former mid-dose group.

Based on mortality in high-dose females and on effects on growth, clinical chemistry and weights of liver and kidney in high-dose males, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic effects was placed at 20 mg/kg body weight/day. Local reactions in the stomach were, however, noted in females of the low- and mid-dose groups. Because there were no relevant effects on reproductive performance or on developmental parameters, the NOAEL for reproduction could not be determined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion BV, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-dithiobisethanol
EC Number:
217-576-6
EC Name:
2,2'-dithiobisethanol
Cas Number:
1892-29-1
Molecular formula:
C4H10O2S2
IUPAC Name:
2-[(2-hydroxyethyl)disulfanyl]ethan-1-ol

Test animals

Species:
rat
Strain:
other: Wistar Han IGS rats (Crl:WI(Han)), (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: (P) females 14 weeks, males 13 weeks
- Weight at study initiation: (P) females 210.0 to 247.2 g, males 316.7 to 371.4 g
- Fasting period before study: no
- Housing: The rats were housed in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced at last once per week with fresh portions and topped up when necessary. Each batch of VRF1 (FG) diet is analyzed by the supplier for nutrients and contaminants.
- Water: ad libitum, tap-water. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility.
- Acclimation period: quarantine 9 Aug 2017 to 18 Aug 2017.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%):at least 45% and not exceeding 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
4 Sep 2017 to 30 Oct 2017

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials in a refrigerator (2-10°C) in aliquots sufficient for one day (one vial/group/day). The vehicle for dosing the controls was similarly stored. During the daily administration all dilutions were continuously stirred on a magnetic stirrer.

DETAILS ON EXPOSURE
A dosing volume of 10 mL/kg body weight/day was used for all groups. The dosing volume was adjusted based on the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volumes were not adjusted after gestation day 14. A fixed volume based on the body weight on gestation day 14 was used for females between gestation day 14 up to the end of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity, stability and content of the test substance in dosing dilutions was conducted using an HPLC-MS method.
HOMOGENEITY: The homogeneity (and content) was assessed in the batch of gavage solutions, prepared for study 21025-02 on 4 September 2017. Three samples of each gavage solution, taken at different locations in the container, and 1 sample of the control solution (0 mg/mL) were analyzed. Each sample was analyzed in duplicate.
For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the diet container). The test substance was considered to be homogeneously distributed in the diet if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.
STABILITY: The stability of the test substance in gavage solution was examined in the batch of gavage solutions prepared on 4 September 2017. Samples were analyzed directly after preparation, after storage in the animal room for at least 4 hours, and after storage in a refrigerator (2-10 °C) for at least one week. For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in the solution if p ≥ 0.01 and/or if the relative decrease in the mean concentration after storage was ≤ 10%.
CONTENT: The content of the test substance was determined in two other batches of gavage solutions, prepared on 25 September 2017 (all groups), 9 October (high dose group; after storage for 7 days in a refrigerator) and 16 October 2017 (low- and mid-dose groups). The content of the test substance in gavage was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.
Details on mating procedure:
- M/F ratio per cage:1/1 animals in the same dose group.
- Length of cohabitation: Animals were caged together until mating occurred.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Males: 2 weeks pre-mating, during mating and up to the day of sacrifice, in total 30 days of treatment.
Females: 2 weeks pre-mating, during mating, during gestation and lactation until sacrifice (day 14 of lactation).
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the sponsor based on the results of a 2-week dose-range finding study with the test item in rats (Triskelion report V21025/01, Oral (gavage) dose-range finding study with Di-(2-Hydroxyethyl) Disulfide in rats, 22 August 2017). In this dose-range finding dose levels of 0, 5, 25 and 75 mg/kg body weight/day were administered to groups of 4 male and 4 female rats. The main effect noted was an increase in liver weights of high-dose females. The weight of the kidneys was statistically significantly increased in low- and high-dose females, but was not noticeably affected in the mid-dose group.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning all abnormalities, signs of ill health or reactions to treatment were recorded and the afternoon, checks for dead or moribund animals were made.
- At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Litters were evaluated on the day of birth (i.e. day 0 of lactation). To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of adult male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 1 in ‘Any other information on methods incl. tables’ were examined.
- The following parameters were calculated: Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to scheduled sacrifice.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: 5 rats/sex/group from the abdominal aorta.
- Parameters reported in Table 2 in ‘Any other information on methods incl. tables’ were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once, in the week before sacrifice.
- Dose groups that were examined: All groups, 5 males/group and 5 females with a litter/group.
- Battery of functions tested: Functional Observational Battery (FOB) and spontaneous motor activity.

IMMUNOLOGY: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: All surviving animals, after 14 days of lactation.
- Organs examined: At scheduled necropsy, the organs of the adult animals indicated in the Table 3 in ‘Any other information on materials and methods incl. tables’ were weighed (paired organs together) as soon as possible after dissection to avoid drying.

OTHER: BLOOD SAMPLING FOR HORMONE DETERMINATIONS
- Time schedule for examinations: During scheduled necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight.
- How many animals: all adult male and female animals and serum was stored in a freezer (at ≤-18°C).
- In addition, blood samples were collected and serum was stored in a freezer (at ≤-18°C):
A) from the surplus pups per litter at culling on lactation day 4. Blood samples were collected by decapitation from all surplus pups, and serum was pooled per litter and stored for possible determination of serum T4 levels.
B) from two pups per litter at sacrifice on PND 13. Individual blood samples were collected from the heart whilst under CO2/O2 anaesthesia, from two pups per litter at sacrifice on lactation day 13. Serum was prepared and stored for determination of serum T4 levels. Serum samples were discarded after analysis or after issuing the final report.
- The blood samples taken from the adult males and from the two pups per litter sacrificed on PND 13 were analyzed for T4 hormone levels. Analysis was performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method based on the manufacturer’s protocol.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, from 2 pups per litter the thyroid gland was collected on PND 13 and examined microscopically.
- Skeletal examinations: No
- Head examinations: No
Statistics:
The statistical procedures for analysis of data are described in Table 4 in 'Any other information on materials and methods incl. tables'. Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
Indices:
The reproductive indices can be found in Table 5 in 'Any other information on materials and methods incl. tables'.
The offspring viability indices can be found in Table 6 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Despite the mortality in high-dose females, there were no noticeable clinical signs during the pre-mating period, mating and gestation period or the lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Five females of the high-dose group were found dead during the study. The remaining females in the high-dose group were humanely killed for animal welfare reasons and because it was anticipated that insufficient pregnant females would remain to allow a meaningful evaluation. There was no mortality in females of the other groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight changes of females were not affected in any group during the premating period, nor in the remaining low- and mid-dose groups during the gestation period or the lactation period (Table 1 to 3 in 'Any other information on results incl. tables').
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls (Table 4 to 5 in 'Any other information on results incl. tables'). Food consumption was not affected in females of the low- and mid-dose group during the gestation period or the lactation period.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Description (incidence and severity):
Haematological findings are represented in Tables 6 and 7 in 'Any other information on results incl. tables'.
There were no treatment-related effects in red blood cell and coagulation parameters at sacrifice in females of the low- and mid-dose groups.
MCHC was slightly, though statistically significantly reduced in females of the low- and mid-dose groups. No significance was attached to this finding because there was no dose-response
relationship and it was not accompanied by significant changes in the main parameters from which this calculated value was derived.
No haematological data were obtained for the female high-dose group that was terminated intercurrently.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry findings are represented in Table 8 in 'Any other information on results incl. tables'.
In females of the remaining low- and mid-dose groups there were no statistically significant differences with the controls. No clinical biochemistry data were obtained for the female high-dose group that was terminated intercurrently.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.
Some treatment-related effects were, however observed in female animals of the high-dose groups during weekly detailed clinical examinations or during functional observations at the end of the study. These effects included sliding with the ventral parts of the head and neck over the bottom of the open field and salivation. At a few occasions, repetitive movements of mouth and jaws, a hunched posture, piloerection, low activity, large number of urine pools, vocalization when prodded or nasal haemorrhagic discharge were observed in some animals of the high-dose group.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Relative organ weights were not significantly affected in females of the remaining low- and mid-dose groups (Table 10 in 'Any other information on results incl. tables).
Statistically significant differences with the controls were noted in absolute organ weights (Table 9 in 'Any other information on results incl. incl. tables'). In females of the mid-dose group, the absolute weight of the thyroids was deceased. Because these findings were not reflected in significant changes in the relative weights of this organs, they are not considered of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of ulceration and yellow/white deposition in the stomach was increased in females of the low- and mid-dose groups. The other macroscopic observations at necropsy revealed no treatment-related abnormalities (including the high-dose females died or were killed intercurrently). The findings were unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination was performed on control- and mid-dose females.
The incidence of focal necrosis in the stomach was increased in females of the low- and mid-dose groups both compared to concurrent and historical control data.
There were no other treatment-related histopathological changes. The other histopathological changes observed were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Only post-implantation loss was examined. Post implantation loss was not statistically significantly affected in females of the former mid-dose group (Table 11 in 'Any other information on results incl. tables') .
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
All females of the control-, low- and mid-dose group delivered live born pups, and there were no females with only stillborn pups (Table 11 in 'Any other information on results incl. tables'). The number of litters with stillborn pups and the total number of stillborn within these litters (between brackets) were comparable and accounted 0(0), 0 (0), 2 (3) for the control-, low-, and mid-dose group, respectively.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Four high-dose females that were placed with males were not mated within 7 days, and the male rats were replaced by rats of the same group that had successfully mated. As a result the male mating index was reduced and the pre-coïtal time was prolonged in the high-dose group. Because most high-dose females eventually were mated, the female mating index was not clearly affected in the high-dose group. Male and female mating indices and fertility indices were not affected in the low- and mid-dose group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance. Because in the mid-dose group three pups were stillborn and another pup died after delivery, the number of live pups per litter at PND 0 and PND 4 (pre-culling) became statistically significantly reduced in this group (Table 2 in ‘Any other information on results incl. tables’).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related differences in mean pup weights between the test groups and the controls on PND 0, 4 and 7 (Table 12 in 'Any other information on results incl. tables') . On lactation day 13, the weight of the male pups of the low-dose group was statistically significantly lower than in controls. This is considered a chance finding because there were neither significant changes in male pup weight at the next higher level nor in total pup weight.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Because in the mid-dose group three pups were stillborn and another pup died after delivery, the number of live pups per litter became statistically significantly reduced in this group (Table 11 in 'any other information on results incl. tables').
Changes in sex ratio:
no effects observed
Description (incidence and severity):
After culling on PND 4, no pups were lost. No differences were observed on sex ratio on PND 0 and PND 13 among the groups (Table 11 in 'Any other information on results incl. tables').
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance (Table 11 in 'any other information on results incl. tables').
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No effects were observed on postnatal survival (Table 11 in 'Any other information on results incl. tables').
External malformations:
no effects observed
Description (incidence and severity):
Macroscopic observations of pups at necropsy on PND 13 and stillborn pups or pups that died revealed no abnormalities.
There were no effects on nipple retention of male pups on PND 13.
There were no treatment-related effects on anogenital distance or anogenital distance corrected for cube root of bodyweight of male and female pups (Table 15 in 'Any other information on results incl. tables').
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
Microscopic examinations of the thyroid gland of pups killed on PND 13 revealed no treatment-related abnormalities.
The absolute and relative weights of the thyroid on PND 13 were slightly, though statistically significantly increased in male pups of the former mid-dose group (Table 13 in 'Any other information on results incl. tables'). Female pup thyroid weight and total pup thyroid weight (both sexes combined) were, however, not significantly affected (Table 14 in 'Any other information on results incl. tables').
Other effects:
no effects observed
Description (incidence and severity):
CLINICAL OBSERVATIONS
There were no treatment-related clinical signs in pups during the lactation period.

T4 HORMONE LEVELS
There were no statistically significant effects observed on T4-hormones (Table 16 in 'Any other information on results incl. tables').

Effect levels (fetuses)

Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1. Maternal body weight during the study - Day(s) Relative to Start Date.

Sex: Female

Bodywt

day -x

(g)

 

[G]

Bodywt

day 0

(g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-3

0

7

14

0 mg/kg

Mean SD

N

217.33

7.91

12

224.18

6.20

12

224.54

6.76

12

225.78

6.67

12

5 mg/kg

Mean

SD

N

217.60

7.85

12

223.11

8.41

12

223.72

6.10

12

225.83

7.06

12

20 mg/kg

Mean

SD

N

217.39

7.95

12

222.53

7.52

12

223.29

8.52

12

227.41

10.00

12

75 mg/kg

Mean SD

N

217.75

8.55

12

225.13

11.25

12

222.74

12.68

11

225.50

11.32

11

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

 

Table 2. Maternal body weight during gestation - Day(s) Relative to Mating (Litter: A).

Sex: Female

 

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

0

7

14

20

0 mg/kg

Mean

227.42

248.33

272.11

332.17

 

SD

6.62

9.30

11.81

15.63

 

N

12

12

12

12

5 mg/kg

Mean

228.77

250.03

271.59

326.74

 

SD

7.27

10.17

11.62

16.41

 

N

12

12

12

12

20 mg/kg

Mean

226.59

247.58

268.65

321.56

 

SD

7.09

10.69

12.34

18.18

 

N

11

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 3: Maternal body weight during lactation - Day(s) Relative to Littering (Litter: A).

Sex: Female

 

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

Bodywt

 

(g)

 

[G]

0

4

7

13

0 mg/kg

Mean

255.99

274.28

277.06

286.32

 

SD

10.14

13.88

14.36

13.78

 

N

12

12

12

12

5 mg/kg

Mean

251.76

266.01

266.68

278.53

 

SD

9.92

11.35

14.21

12.49

 

N

12

12

12

12

20 mg/kg

Mean

252.52

266.53

271.63

282.53

 

SD

13.32

12.74

13.33

11.85

 

N

12

12

12

12

[G] - Ancova/Anova & Dunnett

Litter: A = First litter

 

Table 4: Maternal food consumption gestation - Daily Food Cons Per Animal (g).

Sex: Female

Day(s) Relative to Mating (Litter: A)

0 - 7

7 - 14

14 - 20

0 mg/kg

Mean

15.22

16.81

18.12

 

SD

1.42

1.45

0.88

 

N

12

12

12

5 mg/kg

Mean

15.61

16.48

18.16

 

SD

1.45

2.10

1.42

 

N

12

12

12

20 mg/kg

Mean

14.66

16.07

17.98

 

SD

1.81

1.99

1.68

 

N

11

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

 

Table 5. Maternal food consumption lactation - Daily Food Cons Per Animal (g)

Sex: Female

Day(s) Relative to Littering (Litter: A)

0 - 4

4 - 7

7 - 13

0 mg/kg

Mean

28.99

41.42

48.46

 

SD

5.33

5.46

4.37

 

N

12

12

12

5 mg/kg

Mean

27.67

41.36

45.78

 

SD

4.37

6.38

4.30

 

N

12

12

12

20 mg/kg

Mean

26.95

40.46

47.51

 

SD

3.18

5.43

3.85

 

N

12

12

12

Dunnett

Litter: A = First litter

N=Number of cages

Consumption was measured per cage over the periods shown and expressed as g/animal/day

  

Table 6. Maternal red blood cell and coagulation parameters - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

RBC (10E12/L)

 

[G]

Hb (mmol/L)

 

 

[G]

PCV

(L/L)

 

 

[G]

MCV

(fL)

 

 

[G]

MCH

(fmol)

 

 

[G]

MCHC

(mmol/L)

 

 

[G]

Reticulo cytes

(%)

 

[G]

Thrombo cytes

(10E9/L)

 

[G]

Prothrom Time

(s)

 

[G]

0 mg/kg

Mean SD

N

7.974

0.586

5

9.58

0.59

5

0.4776

0.0263

5

59.99

2.36

5

1.203

0.037

5

20.05

0.22

5

3.488

0.510

5

895.0

216.2

5

21.46

1.11

5

5 mg/kg

Mean SD

N

7.992

0.457

5

9.42

0.32

5

0.4820

0.0110

5

60.44

3.19

5

1.181

0.065

5

19.54 * 0.42

5

3.294

0.182

5

1102.8

85.1

5

21.66

1.47

5

20 mg/kg

Mean SD

N

7.376

0.443

5

9.04

0.38

5

0.4630

0.0182

5

62.90

3.53

5

1.228

0.077

5

19.52 * 0.22

5

3.862

0.583

5

978.6

120.5

5

21.66

0.82

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05

 

Table 7. Maternal total and differential white blood cell counts - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

 

[G]

Neutro Absolute

(10E9/L)

 

[G]

Eosino Absolute

(10E9/L)

 

[G]

Baso Absolute

(10E9/L)

 

[G]

Mono Absolute

(10E9/L)

 

[G]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G1]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean SD

N

6.44

3.82

5

3.04

1.79

5

3.11

1.92

5

0.064

0.032

5

0.019

0.019

5

0.184

0.105

5

46.16

4.79

5

48.34

3.09

5

1.16

0.42

5

0.38

0.33

5

3.36

1.34

5

5 mg/kg

Mean SD

N

7.06

2.99

5

3.85

1.45

5

2.83

1.54

5

0.112

0.040

5

0.019

0.011

5

0.223

0.137

5

57.12

8.91

5

37.44

9.06

5

2.12

1.73

5

0.24

0.09

5

2.82

1.46

5

20 mg/kg

Mean SD N

6.30

1.27

5

3.28

0.86

5

2.70

0.81

5

0.065

0.013

5

0.012

0.007

5

0.226

0.090

5

52.12

9.22

5

42.80

10.45

5

1.06

0.29

5

0.20

0.12

5

3.56

1.11

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

 

Table 8. Maternal clinical chemistry - Day: 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ALP (U/L)

 

 

 

[G]

ASAT (U/L)

 

 

[G1]

ALAT (U/L)

 

 

[G]

GGT (U/L)

 

 

[G2]

Bilirub Total

(umol/L)

 

[G]

Creatin ine

(umol/L)

 

[G]

Bile Acids

(umol/L)

 

[G]

Total Protein

(g/L)

 

[G]

Albumin (g/L)

 

 

[G]

Albumin/ Globulin

 

 

[G]

Glucose Plasma

(mmol/L)

 

[G]

Cholest erol

(mmol/L)

 

[G1]

Phospho lipids

(mmol/L)

 

[G1]

Triglyc erides

(mmol/L)

 

[G1]

Urea (mmol/L)

 

 

[G1]

PO4

(mmol/L)

 

 

[G]

Ca (mmol/L)

 

 

[G1]

Cl (mmol/L)

 

 

[G1]

K

(mmol/L)

 

 

[G1]

Na (mmol/L)

 

 

[G1]

0 mg/kg

Mean SD

N

90.6

21.1

5

140.0

22.9

5

61.2

8.6

5

1.94

4.01

5

1.28

0.26

5

48.4

3.6

5

32.28

21.66

5

63.2

3.5

5

33.0

2.2

5

7.606

1.306

5

2.896

0.598

5

2.906

0.403

5

2.114

1.095

5

9.16

1.57

5

3.174

0.568

5

2.792

0.151

5

95.0

2.3

5

5.20

0.33

5

143.6

2.8

5

1.095

0.081

5

5 mg/kg

Mean SD

N

118.4

52.7

5

120.4

19.9

5

63.0

14.7

5

0.18

0.35

5

1.36

0.15

5

50.0

2.7

5

30.12

15.89

5

64.6

3.2

5

34.6

1.8

5

7.892

1.399

5

2.338

0.506

5

2.582

0.430

5

1.830

0.990

5

9.42

1.61

5

3.040

0.493

5

2.850

0.190

5

96.0

3.4

5

5.06

0.50

5

144.0

2.8

5

1.155

0.063

5

20 mg/kg

Mean SD

N

137.0

44.1

5

123.4

25.1

5

57.8

12.9

5

0.10

0.22

5

1.14

0.38

5

47.4

4.4

5

17.80

13.34

5

61.6

5.2

5

32.4

3.0

5

7.952

0.179

5

2.592

0.472

5

2.778

0.401

5

2.296

0.784

5

9.66

1.29

5

3.078

0.061

5

2.812

0.076

5

98.0

1.0

5

5.28

0.46

5

145.2

1.9

5

1.110

0.060

5

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 9. Maternal absolute organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain (g)

[G]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G]

Thymus (g)

[G]

Thyroid (g)

[G2]

Ovaries (g)

[G2]

Uterus (g)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

1.912

0.094

5

0.866

0.084

5

0.0754

0.0038

5

1.516

0.087

5

8.764

0.905

5

0.5480

0.0701

5

0.2218

0.0456

5

0.0186

0.0046

12

0.0884

0.0136

12

0.5245

0.1042

12

5 mg/kg

Mean SD

N

254.78

10.69

12

1.936

0.062

5

0.832

0.058

5

0.0710

0.0120

5

1.558

0.080

5

8.452

0.569

5

0.5558

0.0735

5

0.1796

0.0348

5

0.0155

0.0044

11

0.0921

0.0143

12

0.5579

0.1135

12

20 mg/kg

Mean SD

N

258.29

11.63

12

1.974

0.056

5

0.824

0.047

5

0.0738

0.0124

5

1.624

0.077

5

8.594

0.414

5

0.5850

0.0386

5

0.1914

0.0272

5

0.0141 *

0.0020

12

0.0883

0.0058

12

0.5187

0.0680

12

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log): * = p < 0.05 Litter: A = First litter

 

Table 10. Maternal relative organ weights - Day(s): 14 Relative to Littering (Litter: A)

Sex: Female

 

 

 

 

 

 

 

 

 

 

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G]

Spleen rel.wgt

(g/kg body wgt)

[G1]

Thymus rel.wgt

(g/kg body wgt)

[G]

Thyroid rel.wgt

(g/kg body wgt)

[G2]

Ovaries rel.wgt

(g/kg body wgt)

[G]

Uterus rel.wgt

(g/kg body wgt)

[G]

0 mg/kg

Mean SD

N

262.91

12.79

12

7.382

0.308

5

3.348

0.359

5

0.2914

0.0178

5

5.860

0.425

5

33.83

3.24

5

2.117

0.274

5

0.855

0.161

5

0.0713

0.0201

12

0.3366

0.0510

12

1.997

0.408

12

5 mg/kg

Mean SD

N

254.78

10.69

12

7.699

0.404

5

3.309

0.280

5

0.2820

0.0464

5

6.191

0.296

5

33.57

1.88

5

2.213

0.332

5

0.713

0.135

5

0.0612

0.0174

11

0.3618

0.0563

12

2.188

0.424

12

20 mg/kg

Mean SD N

258.29

11.63

12

7.623

0.326

5

3.178

0.105

5

0.2864

0.0586

5

6.278

0.483

5

33.18

1.60

5

2.265

0.252

5

0.737

0.087

5

0.0544

0.0059

12

0.3428

0.0294

12

2.017

0.318

12

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log) [G2] - Kruskal-Wallis & Dunnett on Ranks Litter: A = First litter

 

Table 11. Delivery report and litter report

Sex: Female

 

Day(s) Relative to Littering

0 mg/kg

5 mg/kg

20 mg/kg

Pups Delivered (Total) [k]

Mean SD

Sum Sum

 

Sum

11.8

1.6

142

142

100.0

0

0.0

10.9

1.8

131

131

100.0

0

0.0

10.2

1.9

122

119

97.5

3

2.5

Liveborn [f]

Live Birth Index (%)

Stillborn day 0 [f]

Stillborn Index (%)

Live Pups/Litter day 0 [k] 1

Mean

11.8

10.9

9.8 *

 

SD

1.7

1.8

1.7

 

N

12

12

12

 

Sum

141 1

131

118 1

Live Pups/Litter day 4 Pre [k]

Mean

11.7

10.9

9.8 *

 

SD

1.7

1.8

1.7

 

N

12

12

12

 

Sum

140

131

117

Culled pups

Sum

44

36

23

Live Pups/Litter day 4 Post [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Live Pups/Litter day 7 [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Live Pups/Litter day 13 [k]

Mean

8.0

7.9

7.8

 

SD

0.0

0.3

0.4

 

N

12

12

12

 

Sum

96

95

94

Dead, Missing, Cannibalized d0-d4

 

2

0

2

Dead, Missing, Cannibalized d5-d7

 

0

0

0

Dead, Missing, Cannibalized d8-d13

 

0

0

0

No. of litters lost entirely d0-d4 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d5-d7 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d8-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

No. of litters lost entirely d0-d13 [f]

N+ve

0

0

0

 

%

0.0

0.0

0.0

Viability Index 0-4 (%)

 

99

100

98

Viability Index 4-13 (%)

 

100

100

100

Live Males on Day 0

Mean

5.3

4.4

4.9

 

SD

1.1

1.4

2.1

 

N

12

12

12

 

Sum

64

53

59

Sex Ratio Day 0 - Males (%)

 

45.4

40.5

50.0

Live Males on Day 13

Mean

4.0

3.6

3.8

 

SD

0.0

0.8

1.2

 

N

12

12

12

 

Sum

48

43

46

Sex Ratio Day 13 - Males

 

50.0

45.3

48.9

Implantation Sites Total

Mean

12.6

11.8

11.2

 

SD

1.6

1.9

2.0

 

N

12

12

12

 

Sum

151

142

134

No. of lost implantations

Sum

9

11

15

Post-Implantation Loss % [k]

Mean

5.7

7.5

10.4

 

SD

8.2

8.8

11.5

1 Pups that were liveborn but died on day 0 are not included

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Live birth index: no. of liveborn pups *100/no. of total pups delivered

Stillborn index: no. of stillborn pups *100/no. of total pups delivered

Viability index 0-4: no. of live pups on day 4 *100/no. of liveborn pups

Viability index 4-13: no. of live pups on day 13 * 100/no. of live pups post cull

Sex ratio day n: no. of live male pups on day n *100/ no. of live pups on day n

Post-implantation loss: no. of implant sites - no. of liveborn *100/no. of implant sites

 

Table 12. Pup body weight per litter - Day(s) Relative to Littering (Litter: A)

 

 

 

Mean Total

Pup BW/L d0 (g)

 

[G]

Mean Total

Pup BW /L (g)

 

[G1]

Mean Total

Pup BW /L (g)

 

[G1]

Mean To

Pup BW (g)

 

[G1]

tal

/L

0

4

7

13

0 mg/kg

Mean

6.20

11.21

18.46

33.93

 

SD

0.59

1.42

2.10

3.17

 

N

12

12

12

12

5 mg/kg

Mean

6.47

11.69

18.69

32.86

 

SD

0.52

1.15

1.71

2.75

 

N

12

12

12

12

20 mg/kg

Mean

6.51

12.18

18.94

34.22

 

SD

0.62

1.40

1.60

2.23

 

N

12

12

12

12

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett {Covariate: Pup bodyweight on day zero}

Litter: A = First litter

L = per Litter

Table 13. Pup organ weights

 

 

 

 

 

 

 

Mean Pup

TermBW /L (g)

 

[g]

Mean Pup (M)

TermBW /L (g)

 

[g]

Mean Pup (F)

TermBW /L (g)

 

[g]

Mean Pup

Thyroid wgt (g)

 

[g]

Mean Pup (M)

Thyroid wgt (g)

 

[g]

Mean Pup (F)

Thyroid wgt (g)

 

[g]

13

13

13

13

13

13

0 mg/kg

Mean SD

N

33.742

3.187

12

34.075

3.159

12

32.964

3.233

11

0.0042

0.0015

12

0.0040

0.0016

12

0.0045

0.0015

11

5 mg/kg

Mean

SD

N

32.817

2.477

12

33.417

2.608

12

32.217

2.992

12

0.0043

0.0009

12

0.0048

0.0011

10

0.0041

0.0010

12

20 mg/kg

Mean SD

N

34.504

2.504

12

34.908

2.890

12

34.100

2.512

12

0.0048

0.0009

12

0.0056 *

0.0013

11

0.0040

0.0012

12

[g] - Anova & Dunnett: * = p < 0.05

Litter: A = First litter

Table 14. Pup thyroid weights

 

 

 

 

Mean Pup

Thyroid relw (g/kg

body wgt) [g]

Mean Pup (M)

Thyroid relw (g/kg

body wgt) [g]

Mean Pup (F)

Thyroid relw (g/kg

body wgt) [g]

13

13

13

0 mg/kg

Mean

0.1246

0.1188

0.1381

 

SD

0.0452

0.0475

0.0465

 

N

12

12

11

5 mg/kg

Mean

0.1325

0.1428

0.1272

 

SD

0.0231

0.0277

0.0274

 

N

12

10

12

20 mg/kg

Mean

0.1380

0.1606 *

0.1183

 

SD

0.0232

0.0343

0.0329

 

N

12

11

12

[g] - Anova & Dunnett: * = p < 0.05

Litter: A = First litter

 Table 15. Pup anogenital distance

 

 

 

 

 

 

 

Mean Male

Pup BW /L (g)

 

 

[G]

Mean Male

Pup AGD /L (mm)

 

[G]

AGDcorrected

for BW - M (mm/g3)

 

[G]

Mean Female

Pup BW /L (g)

 

 

[G]

Mean Female

Pup AGD /L (mm)

 

[G]

AGDcorrected

for BW - F (mm/g3)

 

[G1]

4

4

4

4

4

4

0 mg/kg

Mean SD

N

11.53

1.49

12

6.202

0.671

12

2.755

0.324

12

10.92

1.37

12

3.682

0.370

12

1.667

0.192

12

5 mg/kg

Mean

SD

N

11.83

1.19

12

6.688

0.485

12

2.943

0.230

12

11.58

1.15

12

3.863

0.405

12

1.713

0.203

12

20 mg/kg

Mean SD

N

12.41

1.55

12

6.521

0.464

12

2.822

0.168

12

11.97

1.37

12

3.665

0.231

12

1.607

0.114

12

[G] - Ancova/Anova & Dunnett

[G1] - Ancova/Anova & Dunnett(Log)

Litter: A = First litter

L = per Litter

Table 16. Hormone determinations (T4) pups

 

(F1) Male

(F1) Female

T4

(ng/ml)

 

[G]

T4

(ng/ml)

 

[G]

0 mg/kg

Mean

64.47

66.05

 

SD

16.41

22.40

 

N

12

12

5 mg/kg

Mean

81.61

80.88

 

SD

29.82

32.34

 

N

12

12

20 mg/kg

Mean

88.43

87.89

 

SD

35.35

28.66

 

N

12

12

[G] - Ancova/Anova & Dunnett(Log)

Applicant's summary and conclusion

Conclusions:
In this GLP compliant OECD 422 study, the No-Observed-Adverse-Effect-Level (NOAEL) was 20 mg/kg bw/day for maternal toxicity, based on mortality in high-dose females. Because there were no relevant effects on reproductive performance or on developmental parameters in the mid-dose group, the NOAEL for developmental toxicity could not be determined.
Executive summary:

In the GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422 the test substance was administered by oral gavage to four groups of male and female Wistar rats. Each group contained 12 animals per sex. The test substance was administrated at 0, 5, 20 and 75 mg/kg bw/day dissolved in tap water during a pre-mating period of 2 weeks, during mating, gestation and until day 13 of lactation. The content, homogeneity and stability of the test substance in the vehicle (tap water) was confirmed by analysis. Cage side observations, including checks for dead or moribund animals, were performed twice a day. Near the end of gestation, females were checked twice a day for parturition. Litters were checked once a day. The parent generation were subjected to the following examinations. Body weights were recorded weekly. Food consumption was measured per cage and recalculated to food intake per animal per day. In the week before sacrifice, neurobehavioural examination was performed on 5 animals per group per sex using Functional Observational Battery (FOB) and spontaneous motor activity in the arena test. Prior to scheduled sacrifice, blood was withdrawn from the abdominal aorta under CO2/O2 anaesthesia for assessing haematology and clinical chemistry parameters. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically. Reproductive performance was assessed based on estrogenic cycle, number of implantation sites, number of pregnant females, number of viable/stillborn litter and the pre-coital time period. The F1 generations was subjected to the following examinations. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups, runts and grossly malformed pups were evaluated on day 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on day 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group. At lactation day 4, the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD was reported as such, corrected for body weight and for the cube root of body weight. On PND 13 all surviving male pups were examined for the presence of nipples and/or areolas. T4 hormone levels were evaluated in 2 pups per litter. Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Any grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups or pups dying during the study and on the surviving pups on day 13 of lactation. The pups were evaluated externally for gross abnormalities and the thyroid gland was collected in two selected pups per litter on PND 13 and examined microscopically.

For the parent generation, mortality was limited to the high dose level females, of which 5 died and the rest was killed for animal welfare reasons. No clinical signs were observed during the study. Female body weights were not affected during the study. During the first week of the premating period, food consumption of high-dose females was slightly, though statistically significantly lower than in controls. No statistically significant changes in clinical chemistry were found for the females. Haematological and clinical chemistry data were not obtained for the female high-dose group that was terminated intercurrently. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. Relative organ weights were not significantly affected in females of the remaining low- and mid-dose groups. The incidence of ulceration and yellow/white deposition in the stomach was increased in females of the low- and mid-dose groups. The other macroscopic observations at necropsy revealed no treatment-related abnormalities. Microscopic evaluation did, however, not reveal any corroborating histopathological changes. The incidence of local macroscopic (ulceration) and microscopic changes (focal necrosis) in the stomach was, however, increased in low- and mid-dose females, both compared to concurrent and historical control data. Although these changes were not clearly elevated in high-dose females nor in males they may represent a local treatment-related effect, because in high-dose females and males the duration of exposure was shorter. Therefore it cannot be excluded that the prolonged exposure to the test substance induced local reaction in the stomach. Other histopathological findings were about equally distributed between the treatment groups and controls or occurred in one or a few animals only. They are common findings in rats of this strain and age and are not considered to be related to treatment.

Reproductive performance was not affected in the control-, low- and mid-dose groups. All females of the control-, low- and mid-dose group delivered live born pups, and there were no females with only stillborn pups. Post implantation loss was not statistically significantly affected in females of the former mid-dose group. The total number of pups delivered per litter (live and stillborn) was reduced in the former mid-dose group but the difference with the controls did not attain statistical significance. In the mid-dose group, the mean number of implantation sites was also low compared to the controls, although still within the range of recent historical control data. During the 2 weeks pre-mating period, the mean length of the longest cycle was statistically significantly increased in high-dose females.

For the F1 generation, there were no treatment-related signs in pups during the lactation period. Mortality was observed, but was not treatment-related. There were no treatment-related differences in mean pup weights, anogenital distance, nipple retention of male pups or differences in T4 levels. Macroscopic and microscopic examinations did not reveal any abnormalities. The absolute and relative weights of the thyroid on PND 13 were slightly, though statistically significantly increased in male pups of the former mid-dose group.

Based on mortality in high-dose females the No-Observed-Adverse-Effect-Level (NOAEL) for maternal systemic effects was placed at 20 mg/kg body weight/day. Local reactions in the stomach were, however, noted in females of the low- and mid-dose groups. Because there were no relevant effects on reproductive performance or on developmental parameters, the NOAEL for developmental toxicity could not be determined.