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REACH

Biphenyl-4,4'-diol

EC number: 202-200-5 | CAS number: 92-88-6

Life Cycle description
Uses advised against

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:: extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-reference

Reason / purpose for cross-reference:: other: The simplified OECD 422 was conducted as the preliminary experiment to the OECD 443 (for the selection of the dose levels.

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

FROM 12 OCTOBER 2020 to 14 JUNE 2021.

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

other: This simplified OECD 422 study was conducted as the preliminary experiment to the OECD 443 (for the selection of the dose levels).

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: the test material was administered to male and females rats to evaluate reproductive performance and to define the dose levels for a subsequent extended one-generation reproductive toxicity study (OECD 443) that was achieved in 2022.

- Short description of test conditions: the test material was administered orally (by gavage) once daily by the oral route (gavage) at dose levels of 200 and 250 mg/kg/day to groups of 10 male and 10 female Wistar rats. A control group of 10 rats/sex received the same volume (5 mL/kg) of the control item (0.5% [w/v] CMC 300-600 centipoises in purified water).
Males were dosed for 30 days, i.e., from 14 days before mating, throughout mating and up to the day before necropsy.
Females were dosed from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 for females that delivered.

- Parameters analysed / observed:
The following observations and examinations were performed: mortality/morbidity, clinical signs, body weights, food consumption, macroscopy at termination and organ weights.
The following reproduction/developmental parameters were also determined: mating and fertility indices, precoital interval time, number of implantation sites, gestation duration, parturition, maternal care, sex ratio and postnatal pup development through to weaning (mortality/morbidity, clinical signs, body weights, sex and macroscopy).

GLP compliance:

Limit test:

Justification for study design:

- Basis for study design:
The study was a simplified reproduction/developmental toxicity screening test ; the objectives of this study were to evaluate the potential of the test material to affect male and female rat reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development and to define the dose levels for a subsequent extended one-generation reproductive toxicity study (OECD 443). This present study can be considered as a preliminary study for the extended one-generation reproductive toxicity study that was carried out subsequently.

- Basis for dose level selection:
The dose levels were selected based on a Prenatal Developmental Study in the Wistar (Han) Rat performed at Charles River in Lyon, France (CRL Study No. AB21564) in which dose levels of 25, 75 and 250 mg/kg/day of test material were administered daily by the oral route (gavage) to 22 pregnant females per group from implantation (GD6) to the day before necropsy (GD20). The findings consisted of transiently reduced body weight gain at 75 mg/kg/day and persistently reduced body weight gain and food consumption at 250 mg/kg/day. These changes impacted mean terminal body weight in the high dose group and were therefore considered as adverse. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 75 mg/kg/day. There was no embryo-fetal toxicity so the NOAEL for prenatal toxicity was set at 250 mg/kg/day.
A Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test (OECD 422 study) was also conducted in the Sprague-Dawley Rat (Crl:CD (SD) IGS (the report was provided by the Sponsor). Twelve male and female rats received the test material, at dose levels of 8, 40 and 200 mg/kg/day for 42 days (corresponding to post-natal Day 4 for females). Findings were limited to white turbid urine noted at 200 mg/kg/day for both sexes. There was no test material-related effect on fertility or pre- and early post-natal development.
The doses of 200 and 250 mg/kg/day were tested in this dose-range finding study to select dose levels for a subsequent extended one-generation reproductive study (OECD 443).

Species:

rat

Strain:

other: CRL:WI(Han) Wistar.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation:
Males: 305.2 g to 365.2 g.
Females: 196.5 g to 259.9 g.
- Age at the initiation of dosing: 10 weeks old.

IN-LIFE DATES: From: 12 October 2020 To: 23 December 2020.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% [w/v] CMC 300-600 centipoises in purified water/Suspension.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulation preparation was carried out at least weekly. Formulation concentrations were 40 and 50 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified.
- Concentration in vehicle: 0 mg/mL, 40 mg/mL and 50 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg/day.
- Lot/batch no. (if required): VWR/18E174115 [22 Nov 2022])
- Purity: 0.5% [w/v] CMC 300-600 centipoises in purified water.

Analytical verification of doses or concentrations:

Duration of treatment / exposure:

- Males were dosed for 30 days, i.e., from 14 days before mating, throughout mating and up to the day before necropsy.
- Females were dosed from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 for females that delivered.
- Apparently unmated females were dose at least 23 days after the last day of the mating period.

Remark: on Day 15 for Male No. 5 (0 mg/kg/day) and on Gestation Day 21 for Female No. 54 (250 mg/kg/day), the animals did not receive the entire dose and the actual volume received was not known. These isolated events were judged without impact on the outcome of the study.

Frequency of treatment:

Once daily.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (control).

Dose / conc.:

200 mg/kg bw/day

Remarks:

Group 2.

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 3.

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Animals identification:
Group 1: males 1 to 10 - females 11 to 20.
Group 2: males 21 to 30, females 31 to 40.
Group 3: Males 41 to 50, females 51 to 60.

Parental animals: Observations and examinations:

Mortality/morbidity, clinical signs, body weights and food consumption observations were performed.
- Clinical signs were checked before and at least once after dosing and once daily on nondosing days; a full clinical examination was performed on each weighing day.
- Body weight was measured weekly for males and non pregnant females.
- Body weight was measured weekly during pre-mating and mating then GD0, GD3, GD6, GD9, GD12, GD15, GD18, GD20, LD1, LD4, LD7, LD14, LD17 and LD21 for pregnant females.
- Food consumption was measured weekly for males.
- Food consumption was measured weekly during pre-mating and then GD0 to GD3, GD3 to GD6, GD6 to GD9, GD9 to GD12, GD12 to GD15, GD15 to GD18, GD18 to GD20, LD1 to LD4, LD4 to LD7, LD7 to LD14, LD14 to LD17 and LD17 to LD21 for females.

Litter observations:

Sex ratio and postnatal pup development through to weaning (mortality/morbidity, clinical signs, body weights, sex) were determined.

Postmortem examinations (parental animals):

Macroscopy at termination and organ weights observations were performed.

Postmortem examinations (offspring):

Macroscopic examinations were performed.

Reproductive indices:

Mating and fertility indices, precoital interval time, number of implantation sites, gestation duration, parturition and maternal care were determined.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related clinical sign in either group for either sex. Isolated clinical signs noted across the groups including hairloss, scabs, sores, palor, thinness, bent tail and maloccluded teeth were considered incidental or related to the pregnancy status of the females.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females at 250 mg/kg/day were euthanized due to loss of the entire litter (Nos. 54 and 57 on LD3 and LD2, respectively). There were no clinicals signs nor macroscopic findings for the females. There was no unscheduled death at 200 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
There was lower mean body weight gain at 200 and 250 mg/kg/day from Day 1 to Day 8 or to Day 15 (250 mg/kg/day) when compared with the control group followed by some recovery but mean body weight gain remained slightly lower over the whole dosing period (Day 1 to Day 29) at 250 mg/kg/day (-10%).
See also Table 1 in the section "Any other information on results incl. tables" below.

Females:
There was lower mean body weight gain at 250 mg/kg/day throughout the premating period (-26%) and at 200 mg/kg/day throughout the lactation period (-30%). In the absence of any dose-relationship and/or any impact on mean terminal body weight, the differences were considered not toxicologically significant.
See also Tables 2, 3 and 4 in the section "Any other information on results incl. tables" below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
There was lower mean food consumption at 250 mg/kg/day during the premating period (Day 1 to Day 15) when compared with the control group (-8%).
See also Table 5 in the section "Any other information on results incl. tables" below.

Females:
There was slightly lower mean food consumption at 250 mg/kg/day during the premating period (-8%) and a non dose-related lower mean food consumption at 200 and 250 mg/kg/day when compared with the control group during the lactation period (-12% and -10%, respectively). In the absence of any dose relationship and/or any associated effects on mean terminal body weight, the differences were considered not toxicologically significant.
See also Tables 6, 7, 8 and 9 in the section "Any other information on results incl. tables" below.

There were no differences in mean food consumption during the gestation period in either group when compared with the control group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- Mating performance and fertility:
There was no test material-related effect on mating performance or fertility. All pairs of rats mated and all mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle) with the exception of Female No. 20 in the control group (7 days). All mated females became pregnant with the exception of 1 female at 200 mg/kg/day (No. 40). In the absence of similar finding in the 250 mg/kg/day group, this isolated case was considered incidental.

- Parturition:
There were 10/10, 9/9 and 10/10 pregnant females that successfully completed delivery in the control, 200 and 250 mg/kg/day groups, respectively.
The mean duration of gestation was comparable in all groups (approximately 22 days).

- Pre-Birth Loss:
The mean number of implantation sites was lower in the 200 mg/kg/day group (12.5) compared with the control group (14.5). However, in the absence of a similar finding in the 250 mg/kg/day group, this was considered incidental.
The percentage of pre-birth loss in the treated groups (3.3% and 4.4% at 200 and 250 mg/kg/day, respectively) was lower than that of the concurrent control group (5.3%).

- Pup Viability and Litter Sizes:
There was no test material-related effect on the mean live litter size in either group.
As a consequence of an incidental difference in the mean number of implantations (see Pre-Birth Loss above), the mean number of delivered pups was incidentally lower in the 200 mg/kg/day group (12.0) compared with the control and 250 mg/kg/day groups (13.8 and 14.2, respectively).
One female in each of the 200 and 250 mg/kg/day groups had 1 stillborn pup (Nos. 33 and 58). These isolated findings were incidental.
The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was lower at 250 mg/kg/day (76.6%) when compared with the control group (100%) and the Historical Control Data range (96.2% to 100%). This was due to 2 females that lost their entire litter (see Section Mortality) and 1 female (No. 58) with 6 missed, dead or cannibalized pups between PND0 and PND4. An effect of the test material on the pup viability cannot be excluded at 250 mg/kg/day.

- Sex ratio:
There was no test material-related effect on the sex ratio in any group.

See also Tables 11 and 12 in the section "Any other information on results incl. tables" below.

Key result

Dose descriptor:

other: -

Effect level:

>= 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Lower body weight gain and slight effect on liver weight.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no pup observations that suggested any association with maternal treatment for the females with litter alive until weaning.
Isolated findings were noted among treated and control groups including thinness, weakness, partial or complete absence of the tail, incomplete hair growth and haematoma and were therefore considered incidental.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of live offspring on Day 21 compared to the number of live offspring on Day 4 was comparable in all groups (100%). No mortality was observed on the period PND 4-21.

Description (incidence and severity):

There was a lower mean pup body weight at 250 mg/kg/day when compared with the control group (-7.4%/-9.2%) and the Historical Control Data (HCD) mean (-10.0%/-11.9%) for both sexes (males/females) on PND1.
This finding was considered adverse at 250 mg/kg/day in view of the magnitude of the maximum change (≥ 10% versus HCD mean) and because the difference was consistent over time for both sexes (-5.0%/-12.3% for males and -7.2/-13.7% for females when compared with the HCD mean/control group on PND21).
There was a lower mean body weight at 200 mg/kg/day when compared with the control group on PND1 and PND21 but the mean values were comparable with that of the HCD mean so
this difference was considered due to the incidentally high mean values in the control group.

See also Tables 13 and 14 in the section "Any other information on results incl. tables" below.

Key result

Dose descriptor:

other: -

Generation:

Effect level:

>= 250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: -

1) P0 - Body Weight

Table 1: P0 - Mean body weight change of males

Sex: Male

Day(s) Relative to Start Date

mg/kg/day

200

mg/kg/day

250

mg/kg/day

-6 -1

Mean

%Diff

34.50 R¹

5.13

31.62

8.59

-8.35

40.84

42.78

18.38

1-8

Mean

%Diff

20.54 I²

3.95

14.10 ww⁴

5.66

-31.35

10.92 www⁶

4.52

-46.84

8-15

Mean

%Diff

21.61 I²

5.05

22.74

7.41

5.23

19.48

4.54

-9.86

15-22

Mean

%Diff

6.55 I,a³

4.55

11.85 d⁵

3.77

80.92

6.94

6.19

5.95

22-29

Mean

%Diff

12.97 I²

5.79

18.26 w⁷

3.83

40.79

18.47 w⁷

5.88

42.41

1-29

Mean

%Diff

61.67 I²

10.46

66.95

10.89

8.56

55.81

11.46

-9.50

SD: standard deviation.

N: Number of values included in the analysis.

% Diff: % difference from control group.

¹ R - Automatic Transformation: Rank.

² I - Automatic Transformation: Identity (No Transformation).

³ I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.

⁴ ww - Test: Williams 2 Sided p < 0.01.

⁵ d - Test: Dunnett 2 Sided p < 0.05.

⁶ www - Test: Williams 2 Sided p < 0.001.

⁷ w - Test: Williams 2 Sided p < 0.05.

Table 2: P0 - Mean pre-mating body weight change of females

Sex: Female

Day(s) Relative to Start Date

mg/kg/day

200

mg/kg/day

250

mg/kg/day

-6 -1

Mean

%Diff

14.93 I¹

4.04

10.17

7.86

-31.88

16.03

6.82

7.37

1-8

Mean

%Diff

8.06 I¹

2.90

7.62

4.54

-5.46

3.77 w²

4.20

-53.23

8-15

Mean

%Diff

10.40 I¹

4.03

11.72

4.34

12.69

9.85

5.42

-5.29

1-15

Mean

%Diff

18.46 I¹

3.56

19.34

6.12

4.77

13.62

6.40

-26.22

¹ I - Automatic Transformation: Identity (No Transformation).

² w - Test: Williams 2 Sided p < 0.05.

Table 3: P0 - Mean gestation body weight change

Sex: Female

Day(s) Relative to Mating

mg/kg/day

200

mg/kg/day

250

mg/kg/day

0-3

Mean

%Diff

12.58 I¹

4.38

9.39

4.43

-25.38

10.52

4.59

-16.38

3-6

Mean

%Diff

9.61 I¹

3.64

10.13

3.92

5.36

5.87

5.61

-38.92

6-9

Mean

%Diff

9.61 I¹

3.15

11.66

4.86

21.36

12.20

4.24

26.95

9-12

Mean

%Diff

13.64 I¹

2.63

12.94

3.24

-5.15

14.11

2.15

3.45

12-15

Mean

%Diff

16.06 I¹

4.72

14.43

4.76

-10.18

14.35

3.64

-10.65

15-18

Mean

%Diff

33.32 I¹

5.66

34.91

11.80

4.78

35.96

6.60

7.92

18-20

Mean

%Diff

28.09 I¹

6.47

24.29

8.75

-13.54

28.87

5.37

2.78

0-20

Mean

%Diff

122.91 I¹

19.58

117.74

25.45

-4.21

121.88

15.50

-0.84

¹ I - Automatic Transformation: Identity (No Transformation).

Table 4: P0 - Mean lactation body weight change

Sex: Female

Day(s) Relative to littering

mg/kg/day

200

mg/kg/day

250

mg/kg/day

1-4

Mean

%Diff

17.49 I¹

6.49

13.69

10.65

-21.74

16.23

6.73

-7.23

4-7

Mean

%Diff

12.19 I¹

5.65

8.35

8.41

-31.50

8.58

9.43

-29.66

7-14

Mean

%Diff

18.62 I¹

7.67

18.60

6.43

-0.11

15.89

8.96

-14.68

14-17

Mean

%Diff

-1.18 R²

6.18

-7.16

2.63

506.99

-1.59

9.90

34.53

17-21

Mean

%Diff

-12.60 I¹

6.10

-9.18

9.81

-27.18

-4.10

12.08

-67.46

1-21

Mean

%Diff

34.52 L³

15.77

24.30

11.91

-29.61

35.00

18.07

1.39

¹ I - Automatic Transformation: Identity (No Transformation).

² R - Automatic Transformation: Rank.

³ L - Automatic Transformation: Log.

2) P0 - Food consumption

Table 5: P0 - Mean pre-mating food consumption of males

Sex: Male

Day(s) Relative to start date

mg/kg/day

200

mg/kg/day

250

mg/kg/day

1-8

Mean

%Diff

21.30 R¹

4.23

22.85

3.93

7.26

21.76

0.45

2.15

8-15

Mean

%Diff

26.33 R¹

1.54

25.39

2.80

-3.57

21.95 SSS²

0.29

-16.66

1-15

Mean

%Diff

23.82 R¹

2.86

24.12

2.98

1.27

21.85 SSS²

0.32

-8.25

¹ R - Automatic Transformation: Rank.

² SSS - Test: Shirley 2 Sided p < 0.001.

Table 6: P0 - Mean pre-mating food consumption of females

Sex: Female

Day(s) Relative to start date

mg/kg/day

200

mg/kg/day

250

mg/kg/day

1-8

Mean

%Diff

18.45 R¹

0.02

17.56 SSS²

0.00

-4.81

16.85 SSS²

0.52

-8.70

8-15

Mean

%Diff

18.24 R¹

0.11

17.74 S³

0.52

-2.76

16.98 SSS²

0.17

-6.92

1-15

Mean

%Diff

18.35 R¹

0.07

17.65 SSS²

0.26

-3.79

16.91 SSS²

0.18

-7.81

¹ R - Automatic Transformation: Rank.

² SSS - Test: Shirley 2 Sided p < 0.001.

³ S - Test: Shirley 2 Sided p < 0.05.

Table 7: P0 - Mean gestation food consumption

Sex: Female

Day(s) Relative to Mating

mg/kg/day

200

mg/kg/day

250

mg/kg/day

0-3

Mean

%Diff

19.94 I¹

2.42

19.90

2.27

-0.16

20.98

2.18

5.25

3-6

Mean

%Diff

21.77 R²

2.72

23.36

1.01

7.31

21.22

3.18

-2.53

6-9

Mean

%Diff

22.42 I¹

2.80

24.46

1.61

9.08

22.80

2.08

1.68

9-12

Mean

%Diff

22.69 I¹

2.76

25.39 w³

1.12

11.92

24.78 w³

2.17

9.24

12-15

Mean

%Diff

24.11 I¹

2.36

25.98

1.11

7.77

25.27

2.29

4.80

15-18

Mean

%Diff

25.19 I¹

2.81

27.60

2.18

9.54

26.36

2.39

4.64

18-20

Mean

%Diff

26.65 I¹

3.41

27.50

2.82

3.19

25.87

2.24

-2.93

0-20

Mean

%Diff

23.08 R²

2.53

24.75

0.80

7.24

23.80

2.06

3.10

¹ I - Automatic Transformation: Identity (No Transformation).

² R - Automatic Transformation: Rank.

³ w - Test: Williams 2 Sided p < 0.05.

Table 8: P0 - Mean lactation food consumption

Sex: Female

Day(s) Relative to littering

mg/kg/day

200

mg/kg/day

250

mg/kg/day

1-4

Mean

%Diff

37.60 R¹

6.41

34.96

7.43

-7.03

32.45

7.13

-13.70

4-7

Mean

%Diff

44.27 R¹

5.09

40.17

9.31

-9.26

40.19 S²

3.05

-9.21

7-14

Mean

%Diff

58.43 R¹

5.92

51.25

12.13

-12.30

52.96 S²

3.82

-9.37

14-17

Mean

%Diff

66.77 R¹

6.83

54.99 S²

14.04

-17.64

58.76 S²

5.39

-11.99

17-21

Mean

%Diff

70.44 R¹

6.14

61.89

15.91

-12.13

65.87

5.27

-6.49

1-21

Mean

%Diff

56.83 R¹

5.51

49.83 S²

11.78

-12.32

51.42 S²

3.31

-9.53

¹ R - Automatic Transformation: Rank.

² S - Test: Shirley 2 Sided p < 0.05.

Table 9: P0 - Mean lactation food consumption

Sex: Female

Day(s) Relative to littering

mg/kg/day

200

mg/kg/day

250

mg/kg/day

1-4

Mean

%Diff

37.60 R¹

6.41

34.96

7.43

-7.03

32.45

7.13

-13.70

4-7

Mean

%Diff

44.27 R¹

5.09

40.17

9.31

-9.26

40.19 S²

3.05

-9.21

7-14

Mean

%Diff

58.43 R¹

5.92

51.25

12.13

-12.30

52.96 S²

3.82

-9.37

14-17

Mean

%Diff

66.77 R¹

6.83

54.99 S²

14.04

-17.64

58.76 S²

5.39

-11.99

17-21

Mean

%Diff

70.44 R¹

6.14

61.89

15.91

-12.13

65.87

5.27

-6.49

1-21

Mean

%Diff

56.83 R¹

5.51

49.83 S²

11.78

-12.32

51.42 S²

3.31

-9.53

¹ R - Automatic Transformation: Rank.

² S - Test: Shirley 2 Sided p < 0.05.

3) P0 - Organ weights

Table 10: P0 –mean body weight / Organ weight

	Necropsy BW g	Kidneys ABS g	Kidneys Rel. Bod. %	Liver ABS g	Liver Rel. Bod. %
Males - Day 31 relative to start date
Group 1 0 mg/kg/day	405.66	2.91651	0.71959	17.81095	4.38437
Group 2 200 mg/kg/day	402.50	2.98348	0.74007	19.91446*	4.93584**
Group 3 250 mg/kg/day	381.12*	2.91491	0.76471*	19.54374*	5.12725**
Females - Day 21 relative to litter date
Group 1 0 mg/kg/day	299.03	2.10478	0.70387	14.80001	4.94527
Group 2 200 mg/kg/day	295.62	2.29860	0.77872*	16.44453*	5.55527**
Group 3 250 mg/kg/day	297.28	2.20881	0.74390*	17.53260**	5.89670**

BW: body weight

ABS: Absolute

Rel.Bod : Relative body weight

* 5% significance level.

** 1% significance level.

4) P0 - Mating performance and fertility / parturition:

Table 11: P0 - summary of mating performance and fertility

GROUP	1	2	3
DOSING	0 mg/kg/day	200 mg/kg/day	250 mg/kg/day
NUMBER OF FEMALES:
Paired	10	10	10
Inseminated	10	10	10
Total litter death	0	0	2
Not pregnant	0	1	0
Mistimed pregnancy	0	1	0
Pregnant	10	9	10
Females with live pups ⁽¹⁾	10	9	10
PRE - COITAL INTERVAL - DAYS
MEAN	3.4	2.3	3.0
S.D.	1.6	0.9	1.1
N	10	10	10
COPULATION INDEX (%)	100	100	100
FERTILITY INDEX (%)	100	90	100
GESTATION INDEX (%)	100	100	100

⁽¹⁾ Including Female No. 39 (mistimed pregnancy).

5) Duration of gestation / litter data

Table 12: Summary of delivery and litter data

Sex: Female Day(s) Relative to littering		0 mg/kg/day	200 mg/kg/day	250 mg/kg/day
Females Completing Delivery [CHSQFS]	N+ve	10	8	10
with Liveborn Pups [CHSQFS]	N+ve	10	8	10
with Stillborn Pups [CHSQFS]	N+ve	0	1	1
with all Stillborn Pups [CHSQFS]	N+ve	0	0	0
with all Dead PND 21 [CHSQFS]	N+ve	0	0	2
Gestation Length (Days) [GEN AN]	Mean SD N	22.6 R¹ 0.5 10	22.3 0.5 8	22.1 S² 0.3 10
Number of Implantation Sites [GEN AN]	Mean SD N Sum	14.5 R¹ 3.1 10 145 R¹	12.5 5.3 8 100	14.9 2.5 10 149
Pre-Birth Loss (%) [GEN AN]	Mean SD N	5.29 R¹ 6.74 10	3.28 7.10 8	4.40 5.02 10
Pups Delivered/Litter [GEN AN]	Mean SD N Sum	13.8 R¹ 3.3 10 138 R¹	12.0 5.1 8 96	14.2 2.2 10 142
Live Pups PND 0 [GEN AN]	Mean SD N Sum	13.8 R¹ 3.3 10 138 R¹	11.9 5.0 8 95	14.1 2.1 10 141
Live Pups PND 1 [GEN AN]	Mean SD N Sum	13.8 R¹ 3.3 10 138 R¹	11.9 5.0 8 95	13.6 2.4 10 136
Live Pups Precull [GEN AN]	Mean SD N Sum	13.8 R¹ 3.3 10 138 R¹	11.8 5.3 8 94	13.5 3.0 8 108
Live Pups Postcull [GEN AN]	Mean SD N Sum	7.8 R¹ 0.6 10 78 R¹	7.0 2.4 8 56	8.0 0.0 8 64
Live Pups PND 7 [GEN AN]	Mean SD N Sum	7.8 R¹ 0.6 10 78 R¹	7.0 2.4 8 56	8.0 0.0 8 64
Live Pups PND 14 [GEN AN]	Mean SD N Sum	7.8 R¹ 0.6 10 78 R¹	7.0 2.4 8 56	8.0 0.0 8 64
Live Pups PND 21 [GEN AN]	Mean SD N Sum	7.8 R¹ 0.6 10 78 R¹	7.0 2.4 8 56	8.0 0.0 8 64
Dead, Miss., Cannib. PND 0 [CHSQFS]	Sum	0	1	1
Dead, Miss., Cannib. PND 1-4 [CHSQFS]	Sum	0	1	33
Dead, Miss., Cannib. PND 5-21 [CHSQFS]	Sum	0	0	0
Dead, Miss., Cannib. PND 0-21 [CHSQFS]	Sum	0	2	34
Live Birth Index (%)	%	100.0	99.0	99.3
Viability Index (PND 0-4) (%)	%	100.0	98.9	76.6
Weaning Index (PND 4-21) (%)	%	100.0	100.0	100.0
Sex Ratio PND 1 - % Males [CHSQFS]	Mean	52.2	51.6	41.6
Sex Ratio PND 21 - % Males [CHSQFS]	Mean	48.3	48.2	50.0

[CHSQFS] - Chi-Squared & Fisher's Exact.

[GEN AN] - Generalised Anova/Ancova Test.

1 R - Automatic Transformation: Rank.

Table 13: Mean Pup Body Weights in Treated Groups Versus Historical Control Data Mean or Control Group

	HCD	Control	Group 2	Group 3
	HCD	0 mkd	200 mkd	250 mkd
	Pup Wt (g)	Pup Wt (g)	Pup Wt (g)	Pup Wt (g)	HCD Mean Versus Group 2/Group 3 (%)	Control Versus Group 2/Group 3 (%)
Males D1	7.0	6.8	6.8	6.3	-2.9/-10.0	0.0/-7.4
Males D21	54.2	58.7	55.0	51.5	+1.5/-5.0	-6.3/-12.3
Females D1	6.7	6.5	6.6	5.9	-1.5/-11.9	+1.5/-9.2
Females D21	52.5	56.4	50.9	48.7	-3.0/-7.2	-9.8/-13.7

mkd : mg/kg/day.

Wt: Weight.

D: Day.

HCD: Historical Control data.

Table 14: Mean pup body weight (grams)

Value

0 mg/kg/day

200 mg/kg/day

250 mg/kg/day

Mean Pup BW - Males d1 [GEN AN]

Mean

%Diff

6.82 L¹

6.76

-0.76

6.25

-8.25

Mean Pup BW - Males d4 [GEN AN]

Mean

%Diff

10.27 I²

9.82

-4.40

9.16

-10.83

Mean Pup BW - Males d7 [GEN AN]

Mean

%Diff

16.62 I²

17.43

4.89

15.52

-6.58

Mean Pup BW - Males d14 [GEN AN]

Mean

%Diff

35.45 R³

33.59

-5.26

31.77 SS⁵

-10.39

Mean Pup BW - Males d21 [GEN AN]

Mean

%Diff

58.68 I²

54.98 w⁴

-6.31

51.46 www⁶

-12.29

Mean Pup BW - Males d4 Postculling [GEN AN]

Mean

%Diff

10.24 I²

9.88

-3.52

9.11

-11.07

Mean Pup BW - Females d1 [GEN AN]

Mean

%Diff

6.50 I²

6.62

1.91

5.93

-8.66

Mean Pup BW - Females d4 [GEN AN]

Mean

%Diff

9.81 I²

10.13

3.35

8.68

-11.53

Mean Pup BW - Females d7 [GEN AN]

Mean

%Diff

16.01 I²

16.47

2.88

14.67

-8.39

Mean Pup BW - Females d14 [GEN AN]

Mean

%Diff

34.45 R³

31.56

-8.40

30.46 SS⁵

-11.59

Mean Pup BW - Females d21 [GEN AN]

Mean

%Diff

56.44 R³

50.86 S⁷

-9.88

48.69 SS⁵

-13.72

Mean Pup BW - Females d4 Postculling [GEN AN]

Mean

%Diff

9.81 I²

10.15

3.50

8.61

-12.23

Mean Pup Body Weight d1 [GEN AN]

Mean

%Diff

6.66 I²

6.66

0.01

6.07

-8.85

Mean Pup Body Weight d4 [GEN AN]

Mean

%Diff

10.04 I²

9.95

-0.84

8.89

-11.39

Mean Pup Body Weight d7 [GEN AN]

Mean

%Diff

16.31 I²

16.65

2.06

15.10

-7.45

Mean Pup Body Weight d14 [GEN AN]

Mean

%Diff

34.94 R³

31.86

-8.82

31.11 SS⁵

-10.95

Mean Pup Body Weight d21 [GEN AN]

Mean

%Diff

57.54 R³

51.87 S⁷

-9.85

50.08 SS⁵

-12.97

Mean Pup BW d4 Postculling [GEN AN]

Mean

%Diff

10.02 I²

10.00

-0.16

8.86

-11.62

%Diff: % difference with the control group.

[GEN AN] - Generalised Anova/Ancova Test.

¹ L - Automatic Transformation: Log.

² I - Automatic Transformation: Identity (No Transformation)].

³ R - Automatic Transformation: Rank.

⁴ w - Test: Williams 2 Sided p < 0.05.

⁵SS - Test: Shirley 2 Sided p < 0.01.

⁶ www - Test: Williams 2 Sided p < 0.001.

⁷ S - Test: Shirley 2 Sided p < 0.05.

Conclusions:

- Parental Toxicity:
Parental findings were limited to a dose-related slightly but statistically significantly lower mean body weight gain and food consumption during the first week of the premating period for both sexes in both treated groups without any impact on subsequent absolute mean body weight through to termination. A slight increase in absolute and relative to body weight increase in liver weight for both sexes was also noted.

- Reproductive Toxicity:
There was no sign of toxicity on reproduction in either group.

- Developmental Toxicity:
Developmental findings consisted of early post-natal pup mortality at 250 mg/kg/day including 2 females with total litter death and 1 female with 7 dead pups compared with no pup mortality in the control group. In addition, there was lower mean pup body weight on PND1 at 250 mg/kg/day and lower mean pup body weight gain from PND1 to PND21 in both groups such that there was a dose-related lower mean pup body weight at 200 and 250 mg/kg/day on PND21. This difference was considered adverse at 250 mg/kg/day in view of the magnitude and the persistence of the change.

- Doses for the Extended One-Generation Toxicity Test (EOGRT):
Based on these findings, the dose of 250 mg/kg/day was considered to be above the maximum tolerated dose for pup viability and the dose of 200 mg/kg/day was associated with only minor parental and pup body weight changes. A high dose between 200 and 250 mg/kg/day would be recommended for the extended one-generation reproductive toxicity study (OECD 443).

Executive summary:

A non GLP reproduction/developmental screening toxicity study was performed to evaluate the potential of the test material to affect male and female rat reproductive performance and early postnatal development and to define the dose levels for a subsequent extended one-generation reproductive toxicity study (OECD 443).

The test material was administered once daily by the oral route (gavage) at dose levels of 200 and 250 mg/kg/day to groups of 10 male and 10 female Wistar rats. A control group of 10 rats/sex received the same volume (5 mL/kg) of the control item (0.5% [w/v] CMC 300-600 centipoises in purified water).

Males were dosed for 30 days, i.e., from 14 days before mating, throughout mating and up to the day before necropsy. Females were dosed from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 for females that delivered.

The following observations and examinations were performed: mortality/morbidity, clinical signs, body weights, food consumption, macroscopy at termination and organ weights.

The following reproduction/developmental parameters were also determined: mating and fertility indices, precoital interval time, number of implantation sites, gestation duration, parturition, maternal care, sex ratio and postnatal pup development through to weaning (mortality/morbidity, clinical signs, body weights, sex and macroscopy).

Parental Toxicity: parental findings were limited to dose-related slightly but statistically significantly lower mean body weight gain and food consumption during the first week of the premating period for both sexes, in both groups, without any impact on subsequent absolute mean body weight through to termination. Slightly higher absolute and relative to body weight mean liver weights were noted for both sexes.

Reproductive Toxicity: there was no sign of toxicity on reproduction in either group.

Developmental Toxicity: developmental findings consisted of early post-natal pup mortality at 250 mg/kg/day including 2 females with total litter death and 1 female with 7 dead pups compared with no pup mortality in the control group. In addition, there was a lower mean pup body weight on PND1 at 250 mg/kg/day and a lower mean pup body weight gain from PND1 to PND21 in both groups such that there was a dose-related lower terminal mean pup body weight at 200 and 250 mg/kg/day on PND21. The effect at 250 mg/kg/day was considered adverse in view of the magnitude and persistence of the change.

Doses for the Extended One-Generation Toxicity Test (EOGRT): based on these findings, the dose of 250 mg/kg/day was considered to be above the maximum tolerated dose for pup development and the dose of 200 mg/kg/day was associated with only minor parental and pup body weight changes. A high dose between 200 and 250 mg/kg/day should be selected for the extended one-generation reproductive toxicity study (OECD 443) in order to elicit tolerable test material-related effects on both the parents and offspring.

Data source

Reference

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

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:: 25 July 2018.
Deviations:: no
Remarks:: None of the technical deviations that occurred during the experimental phase have impacted the results of the study.

GLP compliance:: yes (incl. QA statement)
Limit test:: no
Justification for study design:: SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: see details in the section “Study design” below.
- No extension of cohort 1B was implemented as the information available on potential reproduction toxicity and the results obtained with cohort 1A did not justify it.
- No F2 generation was generated as the results did not justify it.
- Cohorts 2A and 2B were included to evaluate developmental neurotoxicity.
- Cohort 3 was included to evaluate developmental immunotoxicity.
- The oral route of exposure was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Test material

Test material information

Test material form:: solid
Remarks:: White crystalline solid.

Test animals

Species:: rat
Strain:: Wistar
Remarks:: CRL: WI(Han)
Details on species / strain selection:: The rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation:
For the negative control and test material groups: (P) 6 weeks old; (F1) 3 weeks old (PND21).
For the F1 nulliparous and non-pregnant females as positive control of the TDAR (SRBC) Assay: 7 weeks old.
For the F1 nulliparous and non-pregnant females as positive control of the TDAR (KLH) Assay: 8 weeks old.
- Weight: the range of average weight of (P) animals at the initiation of dosing was 145.9-148.1 g for males and 135.7 – 138.7 g for females. The range of average weight of (F1) animals at the initiation of dosing was 59.5-67.8 g for males and 57.4 – 65.8 g for females.
- Fasting period before study: not specified.
- Housing: single or group housed. See also Table 3 in section "Any other information on materials and methods incl. tables".
- Diet: Reference No. A04C-10 (pellets), ad libitum, except during designated procedures. It is considered that there were no known contaminants in the feed that interfered with the objectives of the study.
- Water: filtered (0.2 μm) mains drinking water, ad libitum, except during designated procedures. It is considered that there were no known contaminants in the water that interfered with the outcome of the study.
- Acclimation period: 7 days before the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C.
- Humidity (%):≥ 35%.
Both temperature and relative humidity were within the targets throughout the study.
- Air changes (per hr): 10 or more air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during any designated procedures). Dimmed lighting appeared 15 minutes before the lights were switched on and disappeared 15 minutes after the lights were switched off.

OTHER: Animal Enrichment
For psychological/environmental enrichment, animals were provided with items such as a wooden gnaw block and shredded paper, except when interrupted by study procedures/activities.

IN-LIFE DATES: From: 02 February 2021 To: 19 August 2021.

Administration / exposure

Route of administration:: oral: gavage
Vehicle:: CMC (carboxymethyl cellulose)
Remarks:: The control was 0.5% (w/v) CMC (Batch No. 18E174115) 300-600 centipoises in purified water.
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared according to Test Facility operating standard procedures on a weekly basis and divided into aliquots to be dispensed on each dosing occasion.
Storage conditions: temperature set to maintain +4°C
Any residual volumes from each dosing occasion were discarded.

VEHICLE:
The vehicle was Carboxymethylcellulose (CMC).
- Justification for use and choice of vehicle (if other than water): not specified.
- Concentration in vehicle: the concentration of test material in the vehicle were the following:
* Group 1 (negative control CMC): 0 mg/mL.
* Group 2 (test material 25 mg/kg/day): 5 mg/mL.
* Group 3 (test material 75 mg/kg/day): 15 mg/mL.
* Group 4 (test material 225 mg/kg/day): 45 mg/mL.
* Group 5 (positive control for immunotoxicity, cyclophosphamide 10 mg/kg/day): 0 mg test material/mL (2 mg cyclophosphamide/mL).
- Amount of vehicle (if gavage): the amount of dose formulation administered to the animals was 5 mL/kg/day.
- Lot/batch no. :
Negative control CMC: Batch N. 18E174115. - Purity: 0.5% (w/v) CMC 300-600 centipoises in purified water.
Positive control for TDAR assays: see below in the corresponding section.
Details on mating procedure:: - M/F ratio per cage: after 10 weeks of dosing animals were paired on the basis of 1 male and 1 female from the same group.
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: vaginal plug or presence of sperm in vaginal smear, referred to as gestation day 0 (GD0).
- Mated females were separated from the males once mating had been confirmed and smearing
ceased or when the appearance of the female suggested pregnancy from an undetected
mating.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: - Sample collection and analysis: dose formulation samples were collected for analysis as indicated in Table 1 in the section “Any other information on materials and methods incl. tables”. All samples to be analyzed were transferred to the analytical laboratory at the Test Facility. Samples were stored and analyzed within the stability period as defined in the validation study.
- Analytical method: analyses were performed by UPLC-UV using a validated analytical procedure.
- Concentration analysis: formulations were analyzed for the assessment of achieved concentration of test material in formulations and verification of the absence of test material in the control formulation.
- Homogeneity analysis: the formulations of the test material at concentrations of 1 and 200 mg/mL in the vehicle are homogeneous (based on the analytical results of a previous study).
- Stability analysis: the Sponsor provided data that demonstrate that the test material formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: in a concentration range of 1-200 mg/mL for 11 days at room temperature and +4°C or
26 days at -20°C (based on the analytical results of a previous study).
Duration of treatment / exposure:: The treatment with the test material (or control item) was administered by gavage using a syringe with attached plastic gavage cannula and for the following animals/durations:
- (P) Males: 10 weeks before mating, throughout the mating period and up to the day before necropsy.
- (P) Females: 10 weeks before mating, throughout the mating period, during gestation (the first day of gestation is designated as GD0) and through weaning of their F1 offspring, at least 21 days after parturition, up to and including the day before scheduled necropsy (the day of birth is designated as LD0).
- Apparently unmated females for at least 23 days after the last day of the mating period.
- (F1) Males and Females: during lactation (up to PND21), pups were not dosed directly but could potentially be exposed to the test material in utero, via maternal milk or from exposure to maternal urine/feces. After weaning (PND21), F1 animals were dosed up to and including the day before scheduled necropsy. The F1 surplus and Cohort 2B animals were not dosed.
Frequency of treatment:: Daily.
Details on study schedule:: Additional details on study schedule:

> F0 GENERATION

- F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. Consequently F0-males were euthanised after at least 12 weeks of exposure.
- F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. Consequently F0 Females were euthanised after at least 16 weeks of exposure.

Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily, at the beginning and end.

> F1 GENERATION

- The standardization of litters was performed: 8 pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or anogenital distance (AGD), was not done. Whenever the number of male or female pups prevented having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) was acceptable. No pups of either sex were eliminated when the litter size was below the culling target (8 pups/litter).
- On PND21, pups are selected randomly, and the selected F1 pups are randomly assigned to one of three cohorts of animals, as follows:
Cohort 1 (1A and 1B) = Reproductive/developmental toxicity testing
Cohort 2 (2A and 2B) = Developmental neurotoxicity testing
Cohort 3 = Developmental immunotoxicity testing

- F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99.
- F1 animals from Cohort 1B (KLH – TDAR assay) were sacrificed between PND92 and PND100.
- F1 animals from Cohort 2A were sacrificed after completion of the neurobehavioral tests between PND76 and PND90.
- F1 animals from Cohort 2B were not dosed directly and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis.
- F1 animals from Cohort 3 (SRBC - TDAR assay) were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls.
- Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/kg bw/day
Remarks:: Group 1 (negative control with vehicle alone).

Doses / concentrations 2

Dose / conc.:: 25 mg/kg bw/day
Remarks:: Group 2.

Doses / concentrations 3

Dose / conc.:: 75 mg/kg bw/day
Remarks:: Group 3.

Doses / concentrations 4

Dose / conc.:: 225 mg/kg bw/day
Remarks:: Group 4.

Doses / concentrations 5

Dose / conc.:: 0 mg/kg bw/day
Remarks:: Group 5 (positive control with administration of cyclophosphamide for the TDAR assays).

No. of animals per sex per dose:: - F0: 25 Males and 25 Females per dose.
- F1 cohorts 1A and 1B: 20 Males and 20 Females per dose (including the G5 group for the positive control).
- F1 cohorts 2A, 2B and 3: 10 Males and 10 Females per dose (including the G5 group for the positive control).
See also Tables 2 and 3 in the section “Any other information on materials and methods incl. tables”.
- F1 cohort 2B and F1 surplus animals were not dosed.
- Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

See also Table 2 in section "Any other information on materials and methods incl. tables".
Control animals:: yes
Details on study design:: - Dose selection rationale: dose levels were based on a simplified OECD 421 study in the Wistar rat (Charles River Study No. 20269996). In this study, 10 rat/sex were dosed at 0, 200 and 250 mg/kg/day by oral administration (gavage) from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy for females or from 14 days before mating, throughout mating and up to the day before necropsy (28 days) for males.
Parental toxicity was limited to a dose-related slightly but statistically significantly lower mean body weight gain and food consumption during the first week of the premating period for both sexes in both treated groups without any impact on subsequent absolute mean body weight through to termination.
Test material-related preweaning developmental effects consisted of early postnatal pup mortality at 250 mg/kg/day including 2 females with total litter death and 1 female with 7 dead pups compared with no pup mortality in the control group. In addition, there was lower mean pup body weight at 250 mg/kg/day when compared with the control group (-7.4% and -9.2% for males and females, respectively) and with the historical control data range for both sexes on PND1. Mean pup body weight gain was then lower during the preweaning phase such that there was a dose-related lower mean pup body weight at 200 and 250 mg/kg/day on PND21 when compared with the control group (-6.3%/-9.8% and -12.3%/-13.7% for males/females, respectively).
Based on these findings, the dose of 250 mg/kg/day was considered to be above the maximum tolerated dose for pup viability and the dose of 200 mg/kg/day was associated with only minor parental and pup body weight changes.
- In addition, in the 90-day repeated dose toxicity study conducted in rats at 30, 125 and 500 mg/kg/day, microscopic kidney lesions were seen in most females and in a few males at 500 mg/kg bw/day. The NOAEL was established at 125 mg/kg bw/day.

For this extended 1-generation reproductive toxicity study, a high dose of 225 mg/kg/day was therefore selected to elicit a clear effect on pup body weight but limit the risk of pup mortality in order to ensure sufficient numbers for allocation to the different F1 cohorts. The low dose of 25 and intermediate dose of 75 mg/kg/day were selected to have a ratio of 3 between each dose level to demonstrate a dose-relationship for any potential test material-related findings.

- Animal assignment: at random.

- Fasting period before blood sampling for clinical biochemistry:
F0 and F1-cohort 1A animals were fasted overnight (about 16 hours) before blood sampling for clinical pathology evaluation and for thyroid hormone analysis.
F1 PND4 pups and surplus animals were not fasted before blood sampling for thyroid hormone analysis.
Positive control:: A positive control was used for the T-cell Dependent Antibody Response (TDAR) assays.
- Identification: cyclophosphamide monohydrate.
- Batch No.: MKCJ4697.
- Preparation: each day of dosing, dose formulation of 2.0 mg/mL cyclophosphamide was freshly prepared by dissolving the positive control item in sterile saline solution (0.9% NaCl batch No. 2105125.). No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the positive control item. Dosing formulations of cyclophosphamide were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Dosing formulations were kept at ambient temperature until dosing and were dosed within 6 hours after adding the vehicle to the positive control item.
The dosing formulations were gently inverted until dosing. Any residual volumes were discarded.

Examinations

Parental animals: Observations and examinations:: F0 GENERATION

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage side observations were performed once daily on non-dosing days. During the dosing period, cage side observations were performed before and at least once after dosing. Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

ARENA OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were removed from their cages and observed for specific clinical signs in a standard arena and a detailed clinical observation was performed at the same time as arena observations i.e. once before the first administration of the test material and at least weekly thereafter (except during pairing or delivery).

BODY WEIGHT: Yes.
All females were weighed as follows:
- At least weekly during pre-mating and mating periods (only pre-mating data were reported).
- During the gestation period on GD0, 4, 7, 11, 14, 17 and 20.
- During the lactation period on LD1, 4, 7, 10, 14, 17 and 21.
All males were weighed at least weekly.
Non-pregnant females were also weighed weekly.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes.
Food consumption of each female was recorded for the following periods:
- Weekly during the pre-mating period starting from Day 1.
- During gestation: GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20.
- During lactation: LD1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17, 17 to 21.
Food consumption was recorded for each male weekly during the premating period starting from Day 1.
Food consumption during mating was not recorded for practical reasons.

WATER CONSUMPTION: No.

OTHER:

- Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily (except on days of receipt and study termination where frequency was at least once daily). Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

- Estrous cycle: see below, section "Oestrous cyclicity (parental animals)"

- Mating:
The day of mating was confirmed by the presence of sperm in the vaginal smear or a vaginal plug and were recorded and taken as GD0. Mated females were separated from the males once mating had been confirmed and smearing ceased or when the appearance of the female suggested pregnancy from an undetected mating.

- Pregnancy and Parturition:
Observations for pregnancy and parturition were made daily.
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation and/or body weight measurement could be used to aid in confirmation of pregnancy. Females were allowed to litter normally. LD0 for the dam and PND0 for the offspring was defined as the day when signs of parturition were observed (i.e., blood traces in the cage or pup observed) and used for recording of delivery. Cage debris of pregnant females were examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.

- Hematology:
Blood samples were analyzed for the parameters listed in Table 5 in section “Any other information on materials and methods incl. tables”. In addition, a blood smear was prepared for each animal on each occasion and analyzed when necessary.

- Coagulation:
Blood samples were analyzed for Activated partial thromboplastin time and Prothrombin time.

- Clinical chemistry:
Blood samples were analyzed for the parameters listed in Table 6 in section “Any other information on materials and methods incl. tables”.

- Urinalysis:
Urine samples were processed and analyzed for the parameters specified in Table 7 in section “Any other information on materials and methods incl. tables”.

- Thyroid hormone analysis:
Blood samples were taken from 10 animals/sex/group on the day of scheduled necropsy. Samples were analyzed for Thyroxine (T4, µg/dL) and Thyroid-Stimulating Hormone (TSH, µIU/mL) in accordance with Standard Operating Procedure and using the Immulite 1000®.
Oestrous cyclicity (parental animals):: F0 generation:
Vaginal smears were taken daily to determine the stage of estrous for each female from 14 days prior to mating and until the identification of mating or separation from the male.
On the day of terminal necropsy, or for moribund animals, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):: - Sperm analysis was performed for all surviving males of the F0 generation and all surviving males of the F1 Cohort 1A.
- Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility.
- Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The number of sperm with each type of abnormality was recorded.
Litter observations:: 1) F1 GENERATION UNTIL WEANING

- Number and weight of pups on PND1, 4, 7, 14 and 21.
- General health/mortality and moribundity at least once daily (at the beginning of the working day) along with the mortality check of the dam. Pups were not removed from the cage during observation, unless necessary for confirmation of possible findings.
- Sex of pups on PND1 and 4.
- Anogenital Distance (AGD), measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Presence of areola/nipples examined on PND13 for all remaining males in each litter.
- Thyroid hormone analysis: Blood samples were taken from two F1 pups per litter (1 male and 1 female if possible) on PND4 for analysis of T4 content in accordance with Standard Operating Procedure and using the Immulite 1000®.

2) F1 GENERATION AFTER WEANING

The in-life procedures, observations, and measurements listed below were performed for all F1 animals from weaning (PND21) onwards, except for the animals from Cohort Surplus and Cohort 2B as these animals were necropsied on/before PND24.

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage side observations were performed at least once daily on non-dosing days. During the dosing period, cage side observations were performed before and at least once after dosing. Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

ARENA OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: animals were removed from their cages and observed for specific clinical signs in a standard arena and a detailed clinical observation was performed at the same time as arena observations i.e. from Week 1 and at least weekly thereafter (except during pairing or delivery).

BODY WEIGHT: Yes.
- Time schedule for examinations: animals were weighed at least weekly from weaning onwards. This started on a specific date on which all pups were at least at PND21.

FOOD CONSUMPTION: Yes.
- Food consumption was quantitatively measured at least weekly from Day 1 and on the same day prior to necropsy as all cohorts were necropsied over several days.

WATER CONSUMPTION: No.

OTHER:

- Mortality/Moribundity Checks:
Throughout the study, animals were observed for general health/mortality and moribundity at least twice daily, at the beginning and end of the working day (except on days of receipt and study termination where frequency was at least once daily). Animals were not removed from their cages during observation, unless necessary for identification or confirmation of possible findings.

- Sexual development:
> Balanopreputial skinfold cleavage was monitored daily for all males from PND38. Examinations continued until balanopreputial skinfold cleavage was detected or up to the day of necropsy. Body weight was recorded on the day of acquisition of balanopreputial skinfold cleavage.
> Vaginal opening was monitored daily for all females from PND28 onwards. Vaginal opening was monitored by visual inspection of the vaginal area and body weight was recorded on the day of acquisition of vaginal patency.
> Daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and until the first estrous was determined, in order to determine the time interval between these 2 events.
Daily vaginal lavage was also performed at least 2 weeks before necropsy. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous for each female.
If a vaginal thread was observed, it was recorded daily until no longer present.
> Sperm analysis was performed for all surviving males of the F1 Cohort 1A. Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility. - Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The number of sperm with each type of abnormality was recorded.

- Hematology:
Blood samples collected from F1 animals - cohort 1A were analyzed for the parameters listed in Table 5 in section “Any other information on materials and methods incl. tables”. In addition, a blood smear was prepared for each animal on each occasion and analyzed when necessary.

- Clinical chemistry:
Blood samples from F1 animals - cohort 1A were analyzed for the parameters listed in Table 6 in section “Any other information on materials and methods incl. tables”.

- Urinalysis:
Urine samples from F1 animals - cohort 1A were processed and analyzed for the parameters specified in Table 7 in section “Any other information on materials and methods incl. tables”.

- Thyroid hormone analysis:
Blood samples were taken from 10 animals /sex/group for F1 Cohort 1A animals on the day of scheduled necropsy and from 10 animals /sex/group for F1 Cohort surplus animals on PND21. Samples were analyzed for Thyroxine (T4, µg/dL) and Thyroid-Stimulating Hormone (TSH, µIU/mL) in accordance with Standard Operating Procedure using the Immulite 1000®. Blood samples were collected between 7.00 and 10.30 a.m. by aorta puncture under anaesthesia using isoflurane.

- DEVELOPMENTAL NEUROTOXICITY

1) The neurobehavioral tests are conducted in the cohort 2A: :
> Auditory Startle Reflex (Acoustic Startle) – Habituation:
An auditory reflex test was performed once between PND23-25 in a chamber equipped with a pressure sensor platform. The auditory reflex was monitored by an analysis system (StartleMonitor™ supplied by Kinder Scientific LLC, Poway, USA). The test sessions of auditory startle reflex consisted of a 5-minute acclimation period with a 65 ± 5-dB broadband background white noise.
> Functional Observation Battery (FOB)
Detailed clinical observations were performed once between PND63-75 (10 animals/sex/group) by Functional Observation Battery (FOB) in group-housed animals. FOB parameters were evaluated per Test Facility standard operating procedures (SOPs) and according to Table 4 in section “Any other information on materials and methods incl. tables”.
In a first step, the animal underwent neurobehavioral evaluation in the home cage with ranking of posture, palpebral closure, stereotypies, convulsions and tremors. In a second step, the ease of removal of the animal from the home cage and its reactivity to handling during removal and transfer to the open field was scored. In a third step, the animal was placed in a black open field and observed for 2 minutes in freely moving conditions. Arousal, vocalizations, respiration, gait, stereotypies, convulsions and tremors were ranked. The number of rearing occasions was counted.
At the end of the 2-minute observation period, defecation (aspect of feces) was ranked and the animals underwent a series of additional observations and tests (from the least to the most invasive).
1. The observer handled the animals and ranked fur appearance, lacrimation, salivation, exophthalmos, palpebral closure and erected fur (piloerection).
2. The animal was tested for various reflexes such as response to a touch of the body with a rod, startle response (using a clicker), tail pinch response and pupil response.
3. Body temperature was measured using a rectal probe.
4. The animal was ranked for body tone, forelimb grip strength and air righting reflex (from a height of 30 cm). Forelimb grip strength was measured with a metallic grid connected to a dynamometer. The measure was performed 3 times and the mean value was calculated and reported.
> Locomotor activity (Motor Monitor)
A locomotor activity test was performed once between PND63-75 in an open field in order to assess reactivity to a novel environment and motor activity. Activity was monitored by a photobeam analysis system (Motor Monitor supplied by Kinder Scientific LLC, Poway, USA) for 1 hour. The arena was divided by beams in width and length. The time and the number of beams interrupted by the animal were recorded. Motor activity was divided into 2 types of activity: ambulatory activity (large animal movements like walking) and fine movements (including grooming and head movements).

2) Neuro-histopathology and morphometric analysis in the cohorts 2A and 2B
See section "Postmortem examinations (Offsprings).

- DEVELOPMENTAL IMMUNOTOXICITY

a) Humoral Immunization with KLH (TDAR) - Cohort 1B and Positive Control Animals (cohort 1B, group 5)
Ten F1-animals of Cohort 1B per group and per sex (groups 1 to 4) and all positive control animals (group 5) associated with this cohort were immunized once at 11-12 weeks of age (10 weeks of age for positive control animals), via intravenous injection into the tail vein with 1.0 mL of 300 μg of KLH. This immunization was performed 2 to 4 hours after treatment of the positive control animals with cyclophosphamide.
For positive control animals, cyclophosphamide was administered at 7.5 mg/kg via intraperitoneal injection once daily on 5 consecutive days.
Blood samples were collected (from the jugular vein or other site if deemed necessary) before pre-immunization and on Day 5 post immunization and stored at -20°C until analysis.
Serum levels of KLH-specific IgM antibodies were performed at the Test Site using a validated method documented in the study report.
Positive animals were then euthanized without macroscopic examination.

b) Immunization with SRBC (TDAR) – Cohort 3 and Positive Control Animals
All F1-animals from Cohort 3 and all positive control animals associated with this cohort were immunized once, 5 days before scheduled necropsy (i.e., once between PND48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 0.5 mL of the 4 x 108 SRBC/mL in sterile PBS formulation.
This immunization was performed 2 to 4 hours after dosing of the positive control animals with cyclophosphamide.
The animals were restrained during the injection procedure (without sedation). On the day of SRBC injection, clinical sign observations were performed after this injection for animals of Cohort 3 and the positive control animals.
For positive control animals, cyclophosphamide was administered at 10 mg/kg via intraperitoneal injection, once daily on 5 consecutive days prior to necropsy (i.e., starting at approx. 7 weeks of age).
Blood samples were collected (from the jugular vein or other site if deemed necessary) from all animals of all groups (1 to 5), before pre-immunization and on Day 5 post immunization and stored at ≤-75°C until analysis.
Analysis of samples for the Rat Anti-SRBC IgM was performed at the Test Site using the commercially available kit Rat Anti-SRBC IgM ELISA kit/SRBCM-2 (from Life Diagnostics), based on a validated ELISA method of analysis (U/mL).This kit is marketed for research purpose and not for diagnostics.

c) Splenic Lymphocyte Subpopulation Analysis – F1 Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One male or 1 female per litter were selected representing a maximum number of litter. Half of the spleen sample was transferred to a sterile tube containing RPMI medium supplemented with 20% foetal bovine serum. Samples were analyzed at the Test Facility.
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: all surviving animals were euthanized after successful mating, after a minimum of 12 weeks of dosing.
- Maternal animals: all surviving animals which delivered were euthanized on LD22-24. For the females which failed to deliver, the necropsy was scheduled approximately on GD26 for those with evidence of mating and approximately 26 days after the last day of the mating period for those without evidence of mating.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including external surface, all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass.
Special attention was paid to the organs of the reproductive system.
Any abnormalities observed were recorded and preserved in an appropriate fixative. The number of former implantation sites was recorded for all paired females. The uterus of all adult females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea was counted. Any foetuses were examined externally where possible and discarded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 8 in section “Any other information on materials and methods incl. tables” below were weighed and prepared for microscopic examination.
Histopathological examinations were performed as follows:
- For all macroscopic lesions from all dose group animals.
- For all organs/tissues from all adult animals of Groups 1 (control) and 4 (high dose).
- Reproductive organs for males that failed to sire and females which failed to deliver.
Histology slides were prepared for the organs/tissues as indicated in Table 8.
Slide staining: haematoxylin and eosin.
Target organs were identified in Group 4 animals and included kidneys, heart, stomach, thymus, vagina, ovaries, uterus and cervix and were examined from all animals of Groups 2 and 3, each sex being considered separately.
The tissues identified for processing and microscopic examination are mentioned in Table 8.
Postmortem examinations (offspring):: Terminal procedures are summarized in Table 10 in section “Any other information on materials and methods incl. tables”.

GROSS EXAMINATION OF DEAD PUPS:

Pups (extra pups on PND4 or any moribund pups) were sacrificed by intraperitoneal injection of sodium pentobarbitone and were submitted to a macroscopic examination of the thoracic cavities.
Pups (including any found dead or sacrificed moribund) were necropsied. For any pups found dead or killed moribund, the stomach was examined for the presence of milk and defects or cause of death was evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. There were no external abnormalities collected.

1) F1 COHORT SURPLUS ANIMALS

- The F1 Cohort surplus animals were sacrificed on PND21.
- Terminal body weight was recorded.
- All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded and preserved.
- HISTOPATHOLOGY / ORGAN WEIGHTS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 9 in section “Any other information on materials and methods incl. tables”.

2) F1 GENERATION FROM WEANING ONWARDS

2) a) F1- Cohort 1A animals

- The terminal body weight was recorded.
- Necropsy of Cohort 1A was conducted on PND89 to PND99.
- All animals were submitted to necropsy procedures including an examination of external surface. all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass.
- Special attention was paid to the organs of the reproductive system.
- Any abnormalities observed were recorded and preserved in an appropriate fixative.
- HISTOPATHOLOGY / ORGAN WEIGTHS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 11 in section “Any other information on materials and methods incl. tables”.
Histopathological examination was performed for all organs/tissues from all F1 Cohort 1A animals found dead or killed moribund during the study.
In the first instance, histopathological examination was performed for the control and high dose groups (Groups 1 and 4), unless otherwise indicated (see below). Examination was extended to kidneys for animals in the intermediate dose groups as it was identified as target organs.
From the 10 first animals/sex of all groups, bone marrow (sternum) was evaluated histopathologically, the lymph nodes were weighed and evaluated histopathologically.
From all females of Groups 1 and 4 (20 animals/group), ovarian follicle counts, quantitative evaluation of primordial and small growing follicles, as well as corpora lutea, was performed.
From 10 selected animals/sex/group, after determination of spleen weight, half of the spleen was used for splenic lymphocyte subpopulation analysis and the other half of the spleen was used for histopathology evaluation. From the remaining 10 animals/sex/group, the total spleen was used for histopathology. Where possible, 1 male or 1 female per litter and all litters represented by at least 1 pup were selected.
For the testicles of all males, detailed qualitative examination was performed taking into account the tubular stages of the spermatogenic cycle. In addition an additional slide was prepared and examined, stained with PAS (in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen).
For all females, haematoxylin and eosin stained step sections of ovaries and corpora lutea were prepared for quantitative evaluation of follicles.

2) b) F1- Cohort 1B animals

- The terminal body weight was recorded
- Necropsy of Cohort 1B was conducted on PND92 to PND100.
- All animals were submitted to necropsy procedures including an examination of external surface, all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass. Special attention was paid to the organs of the reproductive system.
- Any abnormalities observed were recorded and preserved in an appropriate fixative.
- HISTOPATHOLOGY / ORGAN WEIGHTS: The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 11 in section “Any other information on materials and methods incl. tables”.
The reproductive organs of all Cohort 1B animals were processed to block stage. There was no further processing and histopathological examination required based on the results of Cohort 1A.

2) c) F1- Cohort 2A and 2B

- The terminal body weight was recorded
- Necropsy for Cohort 2A was conducted on PND76-90 and for Cohort 2B on PND21-22.
- Animals from Cohorts 2A and 2B were first anaesthetized using isoflurane and were subsequently sacrificed by whole body perfusion using 0.9% NaCl, followed by formalin. The animals were not deprived of food overnight before necropsy.
- All animals were subjected to a limited examination, with special attention paid to the reproductive organs.
- The organs identified for weighing and the representative samples of the tissues for collecting are mentioned in Table 12 in section “Any other information on materials and methods incl. tables”.
- HISTOPATHOLOGY / ORGAN WEIGHTS: after sacrifice of Cohort 2A and 2B animals, the cranium was removed, exposing the brain. The skull, including the brain, was placed in 10% buffered formalin for at least 7 days. The fixed brains were weighed. Subsequently, the brain was fixed in 10% buffered formalin together with selected peripheral nervous system tissues. Sections of the brains were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.
For morphometric analysis, 3 sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained.
- Morphometric (quantitative) analyses of central nervous system tissues were performed for Cohort 2A and 2B animals of Groups 1 and 4 in a first instance. Intermediate dose groups (Groups 2 and 3) were not further analysed. Analyses included measurements from neocortical, hippocampal and cerebellar areas selected.
- Specific histopathological examination of the brain and nervous system tissues was performed. Tissues are indicated in Table 12 in section “Any other information on materials and methods incl. tables” for animals in the control and high dose groups (Groups 1 and 4). Histopathological examination was not extended to animals in the intermediate dose groups.

2) d) F1- Cohort 3 and positive control animals

- Necropsy of Cohort 3 animals was conducted on PND53-59. - Positive control animals were euthanized on the same date(s) at approximately 8 weeks of age.
- These animals were not deprived of food overnight before necropsy.
- All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. In case of macroscopic abnormalities, gross lesions were preserved in the most appropriate fixative together with the identification marks.
Statistics:: Data from concurrent controls and historical data from control animals were used to assess effects.
The mean and standard deviation were calculated for all parameters where feasible, using the litter as the basic sample unit.
Body Weight Gains: Calculated between each scheduled interval and against the first body weight of each phase.
Food Consumption: Calculated between each scheduled interval and against the first body weight of each phase.
Additional body weight or food consumption intervals were also presented such as time points corresponding to the last body weight or food consumption recorded for each cohort to elucidate study results.
Data were processed, where appropriate, to give mean values and standard deviations using the litter as the basic sample unit.
See more details in paragraph 11 of section “Any other information on materials and methods incl. tables”.
Reproductive indices:: The following reproductive indices were calculated as follows:
- Pre-coital interval (in days) = Sum of days until successful insemination /Number of inseminated females
- Male and female copulation index (in %) = 100 x Number of inseminated females / Number of paired females
- Male and female fertility index (in %) = 100 x Number of pregnant females / Number of inseminated females
- Irregularity index = Mean standard deviation of length of the estrous cycle / square root (length of the estrous cycle)
- Days in estrus = 100 x Number of estrus days / Number of smears
Offspring viability indices:: The following reproductive indices were calculated as follows:
- Pre-birth loss (in %) = 100 x (Number of implantations – Number of offspring born) / Number of implantations
- Live birth index (in %) = 100 x Number of pups born alive / Number of pups born
- Viability index (in %) = 100 x Number of pups alive on PND4 / Number of pups alive at birth
- Lactation index (in %) = 100 x Number of pups alive on PND21 / Number of pups alive on PND4 (after culling)
- Sex ratio (proportion of male pups in %) = 100 x Number of males / Number of pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: There was no test material-related clinical sign in any group for either sex.
Piloerection, red vaginal discharge and noisy breathing were noted for a few females amongst the treated and control groups and considered related to the littering since they were observed between LD1 and LD3.
One female in the control group (No. 36) had a mass at the abdomen noted from LD7.
Isolated clinical signs were noted amongst treated and control groups for males and/or females including bent tail, broken tail, hypersalivation, scabs, sores, chromodacryorrhea, erythema, and localised or extensive hairloss and were considered incidental and not test material-related.

Arena observations: Clinical signs noted during the arena observations were limited to an exaggerated startle response for 1 male (No. 67) and 1 female (No. 88) at 25 mg/kg/day, firm body for 2 males at 25 mg/kg/day (Nos. 71 and 75) and flacid body for 1 male at 225 mg/kg/day (No. 155). These isolated findings were considered incidental and not test material-related.
Mortality:: no mortality observed
Description (incidence):: There was no unscheduled death in any group.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: There was slightly but statistically significantly lower mean body weight gain from Day 1 to Day 84 for males at 225 mg/kg/day when compared with the control group (-7%). This minor difference was mainly due to lower mean body weight gain towards the end of the dosing period (-41% between Days 58 and 84) but was considered not toxicologically significant in the absence of any effect on mean food consumption and any impact on mean terminal body weight.
There was non dose-related slightly lower mean body weight gain at 75 and 225 mg/kg/day for females during the lactation period (-30% and -27%, respectively) compared with the control group associated with slight reduction in mean food consumption at 225 mg/kg/day only (see Section 8.2.5) considered not toxicologically significant in the absence of dose-related trend or any differences on mean terminal body weight.
There was no test material-related effect on mean body weight gain in any female group during the pre-mating or gestation periods or for males at 25 and 75 mg/kg/day.
See also Tables 16, 17, 19 and 20 in section Any other information on results incl. tables.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: There was sightly but statistically significantly lower mean food consumption at 225 mg/kg/day during the lactation period (-8%) when compared with the control group. This minor difference when compared with the control group was considered not toxicologically significant in the absence of any effect on mean terminal body weight. There was no test material-related effect on mean food consumption in any male group during the pre-mating period (Day 1 to Day 71) or for females during the pre-mating and gestation periods in any group and during the lactation period at 25 and 75 mg/kg/day.
See also Tables 18 and 21 in section Any other information on results incl. tables.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: Hematology and coagulation:
There was no test material-related effect on the hematology and coagulation parameters that was considered adverse in any group for either sex.
There were higher mean neutrophil counts and lower mean lymphocyte counts for females given 225 mg/kg/day compared with control. This finding was considered non adverse in the absence of any associated clinical pathology or histopathology findings or any similar finding for the F1 generation.
Minor statistically significant differences arising between control and treated groups, such as lower monocyte count for males at 75 or 225 mg/kg/day, higher mean corpuscular hemoglobin concentration at 75 and 225 mg/kg/day, and higher mean neutrophil count and lower mean monocyte count at 225 mg/kg/day for females compared with the control group were considered not toxicologically relevant as they were of low magnitude, not dose-related and/or observed for a single gender.
See also Tables 22 and 23 in section "Any other information on results incl. tables".
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Administration of the test material to rats was associated with changes in clinical chemistry parameters at 225 mg/kg/day. These changes are presented in table 24 in section Any other information on results incl. tables.
At 225 mg/kg/day, there were slightly higher sodium concentrations for both sexes and slightly higher phosphorus, urea, and creatinine concentrations for females only compared with the control group.
At 225 mg/kg/day, tryglycerides and Alkaline Phosphatase (ALP) concentrations were slightly higher for both sexes and Alanine Transaminase (ALAT) and Aminotransferase (ASAT) concentrations were slightly higher for females only compared with the control group. Although the ALP and ASAT (for males) and ASAT and ALAT (for females) mean values were disproportionally influenced by 2 atypical animals with exceptionally high concentrations (Male No. 152 and Female No. 192) this correlated with slightly higher mean liver weight for both sexes when compared with the control group.
Minor statistically significant differences arising between the treated and control groups were noted including slightly lower albumin and protein concentrations at 225 mg/kg/day, higher cholesterol at 225 mg/kg/day and glucose concentrations at 75 and 225 mg/kg/day for females, lower cholesterol at 225 mg/kg/day and lower glucose concentration at 75 and 225 mg/kg/day for males compared with the control group. These changes were considered not toxicologically relevant as they were of low magnitude or noted in 1 gender only.
There were no test material-related effects amongst the serum clinical chemistry parameters at 25 and 75 mg/kg/day for either sex.
See also Tables 25 and 26 in section "Any other information on results incl. tables".
Endocrine findings:: effects observed, non-treatment-related
Description (incidence and severity):: There was no test material-related effect on the mean T4 and TSH serum level that was considered toxicologically relevant in any group for either sex.
There was a statistically significantly lower mean T4 serum level for females at 225 mg/kg/day compared with the control group (x0.64) and a higher mean TSH serum level at 225 mg/kg/day for females compared with the control group (x4.88). The difference in TSH level did not reach any statistical significance in any group for adults.
These changes were not toxicologically relevant in the absence of any gender trend, any test item related effect on the thyroid weight, anogenital distance, nipple areola, reproduction and development, any histology examination of the thyroid glands, or similar findings for pups and considered related to the high variability of this parameter. As under the conditions of this study no adverse effect was observed that could be linked to the lower T4 and higher TSH values, this was not taken into account to determine the No Observed Adverse Effect Levels (NOAELs).
There was no differences in the mean TSH serum levels at 25 and 75 mg/kg/day for females
or in any group for males when compared with the control group.
See also Table 27 in section "Any other information on results incl. tables"
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: There were no test material-related effect on urine analysis parameters in any group for either sex.
Behaviour (functional findings):: not examined
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):: Test material-related microscopic findings were present in the kidney, heart, stomach, ovary, vagina, thymus and are summarized in Table 30 in section “Any other information on results incl. tables”.

In the kidney, bilateral tubular mineralization at the corticomedullary junction (minimal or mild) and multifocal cortical tubular dilatation (minimal or mild) were observed for some females at 75 and 225 mg/kg/day. These renal findings were considered test material-related. Of note, the tubular dilatation noted in Male No. 160 (225 mg/kg/day) was unilateral focal and was therefore not comparable to test material-related bilateral renal tubular dilatation present in females.

In the heart, aortic or myocardial mineralization (minimal or mild) was observed for some females at 75 and 225 mg/kg/day. This cardiac finding was considered test material-related.

In the stomach, mineralization of the glandular part (minimal or mild) was observed for some females at 75 and 225 mg/kg/day. It was associated with necrosis of the glandular part (minimal or mild) that grossly correlated with dark foci at necropsy for 2 females dosed at 225 mg/kg/day. These gastric findings were considered test material-related.

In the reproductive tract, diffuse epithelial mucification (minimal to moderate, characterized by the presence of prominent mucus vacuoles in the cytoplasm of superficial cells from the vaginal epithelium) in the vagina was observed at higher incidence and severity at 225 mg/kg/day compared with the control group. Ovarian atrophy (minimal) was present at 225 mg/kg/day and correlated with a lower ovary weight. These findings in the female reproductive tract suggested a delay in the post-gestation recovery of estrous cycling and were considered test material-related.

In the thymus, atrophy (minimal) and increased cortical apoptosis (mild) were noted in several females dosed at 225 mg/kg/day and were considered test material-related. Atrophy correlated with the lower thymus weight noted in some females dosed at 225 mg/kg/day.

In the thyroid gland of some females dosed at 225 mg/kg/day, a follicular cell adenoma was present in Female No. 195, and minimal bilateral diffuse follicular cell hypertrophy was observed in Female No. 184. These sporadic findings in the thyroid gland were considered incidental.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings: neoplastic:: not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:: no effects observed
Description (incidence and severity):: There was no test item material-related effect on mean estrous cycle length and regularity in any group.
See Table 31 in section "Any other information on results incl. tables".
Reproductive function: sperm measures:: no effects observed
Description (incidence and severity):: There was no test material-related effect on sperm count, motility or morphology in any group.
See also Tables 1 to 4 in the file "443-BPD-Attach-F0-Sperm analysis" in the attachments.
Reproductive performance:: no effects observed
Description (incidence and severity):: - Pre-coital time
The majority of mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore comparable in all groups (i.e., 2.8, 2.7, 3.0 and 2.7 days in the control, 25, 75 and 225 mg/kg/day groups, respectively).

- Mating index
There was no test material-related effect on mating performance in any group. After 2 weeks of mating, all pairs of animals mated in all groups.

- Fertility index
There was no test material-related effect on fertility. All mated females became pregnant, with the exception of 2 (Nos. 26 and 35) and 1 (No. 200) females in each of the control and 225 mg/kg/day groups, respectively. The isolated case in the high dose group was not correlated with any microscopic finding of the reproductive organs and was therefore considered incidental.

- Number of implantations sites
There was no test material-related effect on the mean number of implantation sites. Mean values in the treated groups (13.3 in all groups) were comparable with the control mean value (13.8) and above the HCD mean (12.1).

- There was no test item-related effect on the gestation index and duration of gestation in any group.
There were 23, 25, 25 and 24 pregnant females in the control, 25, 75 and 225 mg/kg/day groups, respectively, all of which successfully completed delivery of liveborn pups with the exception of 1 female (No. 176) given 225 mg/kg/day with a total litter resorption. This isolated case was considered not test item-related in the absence of any differences on the mean percentage pre-birth loss between the 225 mg/kg/day and control groups.
The mean duration of gestation was approximately 22 (22.1-22.4) days in each group.

See also Tables 32 and 33 in section “Any other information on results incl. tables”.

Details on results (P0)

For the F0 generation, there were no test item-related findings that were considered toxicologically significant on mean body weight gain, food consumption, clinical condition (including arena observation) or on the hematology, coagulation and urinalysis parameters for either sex in any group.

Higher creatinine and urea concentrations, minor ionic inbalance for females, and higher hepatic enzyme and trygliceride concentrations for both sexes were noted at 225 mg/kg/day compared with the control group and correlated with the pathology findings noted for the kidneys and liver (see below).

The differences in T4 and TSH serum levels were considered not toxicologically relevant in the absence of any gender trend, any test item-related effect on the thyroid weight, reproduction and development, any histology findings in the thyroid glands and any similar findings in the F1 generation (see below Results: F1 generation"). They are considered related to the high variability of this parameter.

At necropsy, there were test item-related findings for F0 females dosed at 225 mg/kg/day in the kidney (minimal to mild tubular dilatation and mineralization, correlating with higher kidney weight), heart (minimal-to-mild myocardial or aortic mineralization), stomach (minimal-to-mild mineralization and/or necrosis of the glandular part, grossly correlating with dark foci), thymus (minimal atrophy and mild increased cortical apoptosis, correlating with decreased organ weight). Mineralization in the kidney, heart and stomach was considered adverse, while other findings were considered secondary to test item-related systemic effects (ovary and thymus), or adaptative changes (liver and adrenal gland). The pathology findings noted on the kidneys were correlated with higher creatinine and urea concentrations and minor ionic inbalance. The higher mean liver weight at 225 mg/kg/day for both sexes correlated with slightly higher mean tryglyceride and hepatic enzyme concentrations when compared with the control group. Ovarian atrophy, lower ovaries weight compared with the control group, and vaginal mucification which suggested a delay in the post-gestation recovery of estrous cyclicity, were noted at necropsy for F0 females at 225 mg/kg/day and were considered probably secondary to test item-related systemic effects on the kidneys, stomach and heart. These findings were not correlated with any test item-related effect on fertility or estrous cycle data and no test item-related effect on the mean count of primordial/small growing follicles (Cohort 1A) and was therefore considered not adverse. In addition, there was no similar finding in the F1 generation.

There was no other test item-related effect on other reproductive parameters of the F0 generation including mating index, fertility index, estrous cyclicity, gestation index, gestation duration, pre-coital time, sperm quality, and pre-birth loss in any group.

Effect levels (P0)

: Key result
Dose descriptor:: NOAEL
Effect level:: >= 75 mg/kg bw/day
Based on:: test mat.
Sex:: female
Basis for effect level:: other:
Remarks on result:: other: No remark.

Results: F1 generation

General toxicity (F1)

Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: 1) F1 pups (before weaning)
No clinical signs occurred amongst pups that were considered to be test material-related.
Incidental clinical signs consisted of occasional cyanotic/cold pups, weakness, thinness, incomplete hair growth, hair loss, sores, haematoma, lacrimation, partial or complete absence of an ear and scabs. In view of their nature and/or sporadic occurrence, including in the control, these findings were considered of no toxicological relevance.

2) F1-post weaning - Cohorts 1A, 1B, 2A and 3
There was no test material-related clinical sign in any group for either sex.
Isolated clinical signs were noted for males and/or females including bent tail, chromodacryorrhea, hypersalivation, lacrimation, localized hair loss, piloerection, scabs, swelling, incomplete hair growth, abscess, limping, malocclusion, red stained fur, scars and wheezing; these signs were considered incidental or related to the dosing procedure.
During arena observation, clinical signs noted were isolated and limited to minor differences in the tactile reflex, alertness and body tone observed sporadically at 75 and 225 mg/kg/day and considered incidental in the absence of any effect on the neurobehavioral tests.
Mortality / viability:: no mortality observed
Description (incidence and severity):: There was no test item-related effect on the mortality of pups in any group at birth until the end of the study.
- At birth: see "Life birth index" and "Lactation index" in the section "Others" below.
- F1-before weaning: see "Viability index" and "Lactation index" in the section "Others" below.
- F1-post weaning - Cohorts 1A, 1B, 2A and 3:
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: 1) F1 pups (before weaning)
There was slightly lower mean pup body weight gain at 225 mg/kg/day, leading to statistically significantly lower mean pup body weight compared with the concurrent control and Historical Control Data mean on PND21 (-8% and -4% for both sexes). However, the mean values (52.3 g and 50.6 g for males and females, respectively) remained within the historical control data range (49.7 to 57.6 for males and 48.8 to 56.0 for females), so this difference was considered not toxicologically significant.
There was no test material-related effect on mean pup body weight at 25 and 75 mg/kg/day.
See also Table 34 in section “Any other information on results incl. tables”.

2) F1-post weaning - Cohorts 1A, 1B, 2A and 3
For males, there was a test material-related slightly lower mean body weight gain after weaning throughout the dosing period for males (Day 1 to Day 70) at 225 mg/kg/day compared with the control group (-10%) leading to a lower mean terminal body weight (-11%) on Day 70 compared with the control group. This difference was considered not adverse in view of the low magnitude of the change and in the absence of any impact on the development such as sexual maturation.
For females, there was lower mean body weight gain during the first week post-weaning at 225 mg/kg/day considered not toxicologically relevant as it was followed by a recovery such that the mean terminal body weight was comparable with that of the control group. In addition there was no test material-related effect on the sexual maturation that was considered toxicologically significant.
There was no test material-related effect on mean body weight gain for females in any group or males at 25 and 75 mg/kg/day.
See also Tables 35 and 36 in section “Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: F1-post weaning - Cohorts 1A, 1B, 2A and 3:
Consistent with the slightly lower mean body weight gain observed, there was a slightly lower mean food consumption for males at 225 mg/kg/day compared with the control group (-8%) during the dosing period (Day 1 to Day 70). This difference was considered not adverse in view of the low magnitude of the change.
For females, slightly lower mean food consumption was noted at 225 mg/kg/day during the first 2 weeks post-weaning compared with the control group, considered not toxicologically significant in the absence of any difference on the mean terminal body weight.
There was no test material-related effect on mean food consumption for females in any group or males at 25 and 75 mg/kg/day.
See also Tables 37 and 38 in section “Any other information on results incl. tables”.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: F1-post weaning - Cohort 1A:
Administration of the test material to rats was associated with changes in hematology parameters at 225 mg/kg/day.
At 225 mg/kg/day, there were slightly higher mean corpuscular volume, mean corpuscular hemoglobin and reticulocyte count and lower white blood cells count including lower lymphocyte, monocyte and basophile counts for males, compared with the control group.
For females, there were slightly lower hematrocrit and hemoglobin concentration, slightly lower red blood cells count and slightly higher mean corpuscular hemoglobin. The difference correlated with spleen minimally increased extramedullary hematopoiesis at higher incidence at 225 mg/kg/day for males and females compared with the control groups, considered not adverse in view of the low magnitude of the change.
There were no changes in hematological parameters at 25 or 75 mg/kg/day for either sex.
See also Table 39 in section “Any other information on results incl. tables”.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: F1-post weaning - Cohort 1A:
There were no test material-related changes in serum clinical chemistry parameters in any group for either sex that were considered toxicologically relevant.
There was statistically significantly lower mean cholesterol serum concentration at 225 mg/kg/day for males (x0.48). In the absence of correlation with any microscopic finding on the liver or any increase in liver weight, this was considered not adverse.
Minor statistically significant differences arising between control and treated animals were noted including slightly higher sodium and alkaline phosphatase concentrations for both sexes at 225 mg/kg/day and a slightly lower mean globulin concentration in males at 225 mg/kg/day when compared with the control group. These changes were considered not toxicologically relevant as they were of low magnitude and/or observed in one sex only.
See also Tables 40 and 41 in section “Any other information on results incl. tables”.
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: There was no test material-related effect on urine analysis parameters in any group for either sex. The variations noted in the pH were part of the background for this strain of rat and therefore considered not toxicologically relevant.
Sexual maturation:: effects observed, non-treatment-related
Description (incidence and severity):: F1-post weaning - Cohort 1A, 1B, 2A and 3:
There was no difference on sexual maturation that was considered toxicologically significant in any group for either sex.
> The time to attain sexual maturation (vaginal opening) was slightly longer for females at 225 mg/kg/day (37.1 days) compared with the control group (34.4 days) but this was likely related to the lower mean body weight at 225 mg/kg/day compared with the control group since mean body weight at sexual maturation was comparable (112 g) with that of the control group (114 g). In addition, the time to attain sexual maturation was within the Historical Control Data mean ± 2 Sd (28.0 to 40.4 days) so the difference was considered not toxicologically significant.
There were no differences in sexual maturation at 25 and 75 mg/kg/day for females, compared with the control group.
> There were no differences in sexual maturation in any group for males. The day of preputial cleavage was 51.6, 51.1, 51.6 and 52.0 for the control group and at 25, 75 and 225 mg/kg, respectively.

See also Table 42 in section “Any other information on results incl. tables”.
Anogenital distance (AGD):: no effects observed
Description (incidence and severity):: There was no test material-related effect on the anogenital distance in any group.
See also Table 43 in section “Any other information on results incl. tables”.
Nipple retention in male pups:: no effects observed
Description (incidence and severity):: Male pups did not present any areolae/nipples in any group.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: 1) F1-post weaning - Cohort 1A, 1B and surplus:

When compared with controls, male and female groups dosed at 225 mg/kg/day had statistically significantly higher mean kidney weights in Cohort 1A and 1B when compared with the control groups; these effects were considered test material-related. In males, higher kidney weights correlated with gross enlargement and granular appearance.
There were no test material-related effect on the mean organ weights for the surplus animals in any group for either sex.
There were other isolated organ weight values that were statistically different from their respective control. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and unrelated to administration of the test material.
See also Tables 44 and 45 in section “Any other information on results incl. tables”.

2) F1 - Cohorts 2A and 2B
- Brain dimension: There were no test material-related differences in the histomorphometric measurements of the brain. The differences were considered to be incidental and part of the biological individual variability.

- Brain Weight: There were no test material-related brain weight changes in males and females from Cohorts 2A and 2B.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: F1-animals

1) Cohort 1A:
In the kidney, granular appearance and/or enlargement noted for 2 males dosed at 225 mg/kg/day were histologically correlated with tubular basophilia, tubular dilatation and inflammatory cell infiltration. Granular appearance noted in one control male did not have any histopathologic correlate. Granular appearance and enlargement were considered test material-related in males dosed at 225 mg/kg/day.
See also Table 46 in section “Any other information on results incl. tables”.

2) Cohort 1B:
Enlarged kidneys (unilateral or bilateral) were noted for males and females dosed at 225 mg/kg/day with higher incidence compared with the control group.
See also Table 47 in section “Any other information on results incl. tables”.

3) Cohorts 1A, 1B, 2A, 2B, 3 and surplus animals:
Other gross findings in Cohorts 1A, 1B, 2A, 2B, 3 and for surplus animals were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings:: effects observed, treatment-related
Description (incidence and severity):: F1-animals

1) Cohort 1A
In the kidney, bilateral multifocal tubular basophilia (minimal to moderate) was observed at higher incidence and severity at 225 mg/kg/day for males and females compared with the control groups. Basophilic tubules were characterized by a thickened basement membrane and were associated with tubular dilatation (minimal to moderate) and chronic inflammatory cell infiltration (minimal to mild). These renal findings were considered test material-related.
In the spleen, minimally increased extramedullary hematopoiesis was observed at higher incidence at 225 mg/kg/day for males and females when compared with the control groups, and was considered test material-related.
Following quantitative microscopic evaluation of ovaries, there were no statistically significant differences in the mean numbers of primordial/small growing follicles and corpora lutea.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
See also Table 48 in section “Any other information on results incl. tables”.

2) Cohort 2A and 2B
- No test material-related microscopic findings were noted in the nervous system following detailed examination of the subregions of the brain (Cohorts 2A and 2B) and spinal cord, dorsal root ganglia and ventral root fibers, skeletal muscle, sciatic and tibial nerves, eye and optic nerves (Cohort 2A). The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered not test material-related.
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: Other results on the F1 generation (DEVELOPMENTAL TOXICITY DATA):

1) GESTATION INDEX AND DURATION
See section above "Results: P0 (first parental generation) - Reproductive performance".

2) PRE-BIRTH LOSS
There was no test item-related effect on the pre-birth data in any group.
The mean percentage of pre-birth loss in treated groups (5.9%, 7.7% and 7.9% at 25, 75 and 225 mg/kg/day, respectively) was lower compared with the concurrent control group (8.3%) and the HCD mean (9.9%).
The mean numbers of pups delivered in the treated groups (12.5, 12.3 and 12.2 at 25, 75 and 225 mg/kg/day, respectively) were comparable with that of the concurrent control group (12.7) and above the historical control data mean (11.0).

3) LIFE BIRTH INDEX
There was no test item-related effect on the number of live offspring at birth in any group.
There were 4, 3, 2 and 5 stillborn pups in the control, 25, 75 and 225 mg/kg/day groups, respectively. The live birth index was consequently comparable in all groups (99.0%, 99.4% and 98.2% at 25, 75 and 225 mg/kg/day, respectively) compared with the control group (98.6%).

4) VIABILITY INDEX (F1 pups)
There was no test item-related effect in any group on the number of live offspring on PND4 (before culling) compared with the number of offspring on PND0 (viability index).
There were 4, 4, 5 and 10 dead, missing or cannibalized pups from 1, 3, 5 and 3 litters in the control, 25, 75 and 225 mg/kg/day groups, respectively, between PND0 and PND4. The viability index was consequently slightly lower at 225 mg/kg/day (96.4%) compared with the control group (98.6%) but remained within the HCD range (96.2% to 100%).

5) LACTATION INDEX (F1 pups)
There was no test item-related effect in any group on the number of live offspring at weaning compared with the number of offspring on PND4, post culling (lactation index).
There was only on dead pup at 75 mg/kg/day (from Female No. 126). This isolated finding in the intermediate dose group was incidental.

6) SEX RATIO (F1 pups)
There was no test item-related effect on the sex ratio in any group.

See also Tables 32 and 33 in section “Any other information on results incl. tables”.

7) THYROID HORMMONE ANALYSIS

1.a) F1 pups - before weaning - on PND 4:
There was no test material-related effect that was considered toxicologically significant on mean T4 serum levels for F1 PND4 pups in any group.
There were statistically significantly higher T4 serum levels at 25 and 225 mg/kg/day (x1.30 - x1.37) compared with the control group considered not toxicologically significant in the absence of any dose relationship or similar finding for PND21 pups and given that all values from the 225 mg/kg/day group remained within the control range with the exception of 2 pups.

1.b) F1 pups - before weaning - on PND 21
There was no test material-related effect that was considered toxicologically significant on mean T4 and TSH serum levels for F1-PND21 pups in any group.
There were slightly higher TSH serum levels in all group for both sexes (x1.39 to x1.79) compared with the control group (statistically significant for males only), considered not toxicologically significant in the absence of any changes in T4 serum levels, in the absence of any dose-relationship and in view of the low magnitude of the changes.
See also Table 60 in section “Any other information on results incl. tables”.

1.c) F1 pups - post weaning - Cohorts 1A
There was statistically significantly lower mean T4 serum level for males at 225 mg/kg/day when compared with the control group (x0.68).
There was no difference on mean T4 serum level for males at 25 and 75 mg/kg/day or for females in any group and no difference on the mean TSH serum level in any group for either sex that were considered toxicologically significant.
See also Table 61 in section “Any other information on results incl. tables”.

8) ESTROUS CYCLE DATA - Cohort 1A

There was no test material-related effect on mean estrous cycle length and regularity in any group. The mean duration from vaginal opening to first estrous was comparable in all groups.
See also Table 62 in section “Any other information on results incl. tables”.

9) SPERM ANALYIS - Cohort 1A

There was no test material-related effect on mean sperm counts, sperm morphology or motility, including the percentage of progressively motile sperm, in any group.
See also Tables 1 to 4 in the file "443-BPD-Attach-F1-Sperm analysis" in the attachments.

Developmental neurotoxicity (F1)

Behaviour (functional findings):: effects observed, non-treatment-related
Description (incidence and severity):: F1-animals

1) Auditory Startle Reflex – Cohort 2A
There was no test material-related effect on the auditory startle reflex test in any group for either sex. The percentage of habituation to repeated acoustic stimuli was comparable between treated and control groups.
See also Tables 49 and 50 in section “Any other information on results incl. tables”.

2) Functional Observation Battery – Cohort 2A
There was no test material-related effect on the FOB parameters in any group for either sex.
See also Tables 51 and 52 in section “Any other information on results incl. tables”.

3) Motor Activity – Cohort 2A
There was no test material-related effect on motor activity (ambulatory or fine movements) in any group for either sex.
During the first 10 minutes of the test, there were no average differences in ambulatory or fine movements in treated groups compared with the control groups for either sex. Thereafter, there was a greater habituation at 225 mg/kg/day for both sexes for ambulatory and fine movements through to the end of the test compared with the control groups and the Historical Control Data range. However, all mean percentage values remained within the mean control value ± Sd. In addition, there were no test material-related findings in the FOB test, including the arena observations, no test material-related brain weight changes and no test material-related microscopic findings of the brain (including histomorphometric measurements). These differences were therefore considered related to the high inter-individual responses in the motor activity test and not test material-related.
See also Table 53 in section “Any other information on results incl. tables”.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:: no effects observed
Description (incidence and severity):: 1) Anti-KLH IgM Analysis (TDAR) – Cohort 1B
There was no test material-related effects for F1-Cohort B animals on the primary anti-KLH IgM response after immunization with KLH in any group for either sex.
See also Tables 54 and 55 in section “Any other information on results incl. tables”.

2) Anti-SRBC IgM Analysis (TDAR) - Cohort 3
There was no test material-related effects for F1-Cohort 3 animals on the primary anti-SRBC IgM response after immunization with SRBC in any group for either sex.
See also Tables 56 and 57 in section “Any other information on results incl. tables”.

3) Splenic Lymphocyte Subpopulation Analysis - Cohort 1A
There was no test material-related effect on the development of splenic subpopulation (assessed by flow cytometry) in any group for either sex.
See also Tables 58 and 59 in section “Any other information on results incl. tables”.

Details on results (F1)

There were no test item-related effects that were considered toxicologically significant on the F1 litter parameters, pup weights, preweaning developmental landmarks (anogenital distance and areolae/nipple retention), neonatal and weanling serum thyroid hormone levels, organ weights or test item-related macroscopic findings for F1 pups that were found dead, culled on PND4, or examined at scheduled necropsy on PND21, i.e., there was no evidence of F1 postnatal toxicity.

For the F1 generation, there was no adverse test item-related effect on mean body weight gain, mean food consumption, clinical parameters, or sexual maturation.

There was no test item-related immunotoxicity (Cohorts 1A and 3), as evidenced by lymphocyte subpopulation analysis and KLH-IgM or SRBC-IgM responses in a TDAR assay.

Lastly, there was no test item-related neurotoxicity (Cohorts 2A and 2B), as indicated by auditory startle responsiveness at weaning or FOB and motor activity at adulthood, or brain morphometry, brain weight and neurohistopathology.

At necropsy, non adverse test item-related effects were observed in the kidney (tubular basophilia and dilatation, chronic inflammatory cell infiltration in both sexes, correlated with gross enlargement and/or granular appearance, and with higher kidney weight) and spleen (increased extramedullary hematopoiesis) of F1 males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of degenerative/necrotic renal changes and any clinical pathology correlate. There was no other test item-related pathology finding.

Effect levels (F1)

: Key result
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: >= 225 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other:
Remarks on result:: other: No remark.

Overall reproductive toxicity

: Key result
Reproductive effects observed:: no
Lowest effective dose / conc.:: 225 mg/kg bw/day
Treatment related:: no

Any other information on results incl. tables

1) Formulations analysis

All analyzed samples for formulations prepared at nominal concentrations of 5, 15 and 45 mg/mL of test material in vehicle (0.5% (w/v) CMC 300-600 centipoises in purified water), taken from each preparation, including the vehicle, on the first day of treatment and on Week 11 and Week 26 of treatment were within the acceptance criteria (± 15%). The deviations for the nominal concentrations ranged from -4.0% to 1.2%. Furthermore, the test material prepared as a suspension in the vehicle was homogenous as the RSD (Relative standard Deviation) on the 6 aliquots (top, middle, bottom) was ≤2.3%.

Finally, no test material was present in the vehicle sample.

Table 13: Formulations analysis – Day 1 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Sampling	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
05-Feb-2021	08-Feb-2021	0	M	0.000	0.000	≤ 2 % LOQ	Yes
				0.000
		5	T	4.914	4.895	-2.1 %	Yes 0.7% RSD
				4.876
			M	4.975	4.960	-0.8 %
				4.946
			B	4.927	4.930	-1.4 %
				4.934
		15	M	15.06	15.02	0.1 %	Yes
				14.98
		45	T	44.53	43.65	-3.0 %	Yes 2.3% RSD
				42.76
			M	45.27	45.55	1.2 %
				45.83
			B	44.65	44.70	-0.7 %
				44.74

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15% and RSD < 10%.

T: Top, M: Middle and B: Bottom of the formulation.

Table 14: Formulations analysis – Week 11 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
15-Apr-2021	19-Apr-2021	0	0.000	0.000	≤ 2 % LOQ	Yes
			0.000
		5	4.833	4.884	-2.3 %	Yes
			4.934
		15	14.67	14.75	-1.7 %	Yes
			14.83
		45	42.54	43.94	-2.3 %	Yes
			45.34

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15%.

Table 15: Formulations analysis – Week 26 results

Date of Formulation	Date of Analysis	Nominal Concentration (mg/mL)	Experimental Concentration (mg/mL)	Mean Concentration (mg/mL)	Deviation from Nominal	Complies (a)
06-Aug-2021	11-Aug-2021	0	0.000	0.000	≤ 2 % LOQ	Yes
			0.000
		5	4.898	4.802	-4.0 %	Yes
			4.706
		15	14.74	14.76	-1.6 %	Yes
			14.78
		45	44.25	44.44	-1.3 %	Yes
			44.62

LOQ (Lower calibration standard): 5 μg/mL.

(a): Deviation from the nominal value complies when within the acceptable limits of ± 15%.

2) F0 - Body weights, body weight changes and food consumption

Table 16: Mean body weight (g) for males (F0) at Day 84

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
84	390.30 I¹	390.81	379.48	373.96

¹ I - Automatic Transformation: Identity (No Transformation).

Table 17: Mean body weight change (g) for males (F0) for the period days 1-84

Period	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-84	243.44 I¹	244.96	232.95	225.85 w²

¹ I - Automatic Transformation: Identity (No Transformation).

² w - Test: Williams 2 Sided p < 0.05.

Table 18: Mean pre-mating food consumption of males (F0) for the period days 1-71

Period	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	23.22 R¹	22.98	23.21	23.53

¹ R - Automatic Transformation: Rank.

Table 19: Mean body weight (g) for females (F0) at end of pre-mating (D71), end of gestation (GD20), end of lactation (LD21)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
D71	242.44 I¹	237.20	239.52	242.69
GD20	355.70 L²	349.58	351.60	351.11
LD21	288.06 I¹	282.86	286.05	279.56

¹ I - Automatic Transformation: Identity (No Transformation).

²L - Automatic Transformation: Log.

Table 20: Mean body weight change (g) for females (F0) for the periods Days 1-71 (pre-mating), GD0-GD20 (gestation) and LD1-LD21 (lactation)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	103.74 I¹	101.50	101.43	103.98
GD0-GD20	112.08 R²	107.14	107.80	108.72
LD1-LD21	21.90 R, k3	24.37	15.28	15.93

¹ I - Automatic Transformation: Identity (No Transformation).

² R - Automatic Transformation: Rank.

³ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

Table 21: Mean food consumption (g) for females (F0) for the periods Days 1-71 (pre-mating), GD0-GD20 (gestation) and LD1-LD21 (lactation)

Day	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Days 1-71	17.55 R¹	17.18	17.12	17.41
GD0-GD20	22.47 I²	21.81	22.02	23.49
LD1-LD21	53.77 R¹	53.40	53.14	49.44 SS³

¹ R - Automatic Transformation: Rank.

² I - Automatic Transformation: Identity (No Transformation).

³ SS - Test: Shirley 2 Sided p < 0.01.

3) F0 - Hematology

Table 22: Group mean hematology results – F0 males - Monocytes counts

Day 85 relative to start date:

Group	M.Abs. Giga/L	M (%)
1	0.233	3.80
2	0.191	3.56
3	0.159*	2.60*
4	0.167*	3.27*

M: Monocytes.

* 5% significance level.

Table 23: Group mean hematology results – F0 females – extract of results

Day 22 relative to litter date:

Group	MCHC g/L	N.Abs. Giga/L	N(%)	L.Abs. Giga/L	L(%)	M.Abs. Giga/L	M (%)
1	320.8	2.565	42.37	2.883	50.26	0.303	4.99
2	322.7	2.411	41.59	2.739	52.61	0.226	3.97
3	329.8*	2.069	36.29	3.027	57.16	0.218	4.26
4	327.4*	4.805**	70.48**	1.340**	23.61**	0.187	2.96*

MCHC: mean corpuscular hemoglobin concentration.

N: Neutrophil.

L: Lymphocyte.

M: Monocytes.

* 5% significance level.

** 1% significance level.

4) F0 - Clinical chemistry

Table 24: Toxicologically Relevant Changes in Serum Clinical Chemistry Parameters in the 225 mg/kg/day Group – Fold changes relative to control group

	Males	Females
Na (mmol/L)	X1.01	X1.01
Triglycerides (mmol/mL)	X1.46	X1.70
ALP (IU/L)	X2.10	X1.48
ASAT (IU/L)	-	X1.76
ALAT (IU/L)	-	X2.15
Chol (mmol/L)	-	X 1.76
Cl (mmol/L)	-	X0.95
P (mmol/L)	-	X 1.39
Urea (mmol/L)	-	X2.19
Creat (mcmol/L)	-	X1.54

-: Absence of change.

Numerical values indicate fold changes of treated group value relative to control group mean value.

Table 25: F0 males – Group mean serum clinical parameters – extract of results

Day 85 relative to start date

Group	Na mmol/L	Gluc mmol/L	Chol mmol/L	Trigs mmol/L	T.Bili µmol/L	ALP BC IU/L	ASAT IU/L
1	142.8	8.597	1.506	1.095	3.01	67.6	98.2
2	144.4	7.896	1.416	1.183	2.81	71.6	84.6
3	143.4	7.081**	1.173**	1.253	2.62**	72.5	90.1
4	144.9*	7.118**	0.639**	1.598*	2.58**	142.2**	93.3*

Na: sodium – Gluc: glucose - Chol: cholesterol - Trigs: Triglycerides - T.Bili: Total Bilirubin.

ALP BC: Alkaline phosphatase - ASAT: Aspartate aminotransferase.

* 5% significance level.

** 1% significance level.

Table 26: F0 females – Group mean serum clinical parameters – extract of results

Day 22 relative to litter date

Group	Na mmol/L	Cl mmol/L	P mmol/L	Gluc mmol/L	Urea mmol/L	Creat µmol/L	Chol mmol/L
1	140.1	99.2	2.979	5.773	9.080	47.8	2.056
2	140.5	100.3	2.839	6.078	9.300	48.0	2.055
3	139.6	99.6	2.786	6.617*	10.440	50.1	2.299
4	142.2**	94.4*	4.131**	7.839**	19.880**	73.4**	3.623**
Group	Trigs mmol/L	Prot g/L	Alb g/L	A.G ratio	ALP BC IU/L	ASAT IU/L	ALAT IU/L
1	2.901	60.83	34.16	1.30	82.9	101.7	27.2
2	1.925	61.67	34.12	1.24	113.6	92.7	25.3
3	2.795	62.87	34.54	1.22	90.5	95.3	25.6
4	4.940	54.53*	29.28**	1.17*	122.5*	178.8	58.6

Na: sodium – Cl: Chloride – P: Phosphorus - Gluc: glucose – Urea : urea nitrogen – Creat: creatinine – Chol: cholesterol - Trigs: Triglycerides - Prot: Total protein – Alb : Albumin – A.G ratio : Albumin/Globulin ratio - ALP BC: Alkaline phosphatase - ASAT: Aspartate aminotransferase – ALAT: Alanine aminotransferase.

* 5% significance level.

** 1% significance level.

5) Thyroid Hormone analysis

Table 27: F0 - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F0-Males Mean	T4 µg/dL	5.63	5.66	5.80	5.07
F0-Males Mean	TSH µIU/mL	0.092	0.108	0.159	0.143
F0-Females Mean	T4 µg/dL	4.66	4.66	3.67	2.97 *
F0-Females Mean	TSH µIU/mL	0.464	0.289	0.501	2.265

*p ≤ 0.05.

6) F0 - Gross pathological findings

Table 28: Summary of gross pathological findings - F0 animals

	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg bw/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	25	25	25	25	25	25	25	25
Liver (Number Examined) Enlargement	(25) 0	(25) 0	(25) 0	(25) 0	(25) 1	(25) 0	(25) 2	(25) 5
Stomach (Number Examined) Dark (depressed) focus, single/several/many, glandular mucosa	(25) 0	(25) 0	(25) 1	(25) 2	(25) 3	(25) 4	(25) 6	(25) 6

7) F0 – Organ weights

Table 29: Summary of Organ Weight Data – Scheduled Euthanasia (F0-animals)

	Male			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	25	25	25	25	25	23
Liver (Number Weighed)^a	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	-7	-3	+6	0	+2	+14
% of body weight	-7	0	+12	+3	+3	+18
Gland, adrenal (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	+1	-3	+11	+7	+2	+22
% of body weight	+1	0	+18	+9	+3	+27
Kidney (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	-2	0	0	-1	-1	+9
% of body weight	-2	+3	+7	+1	0	+14
Thymus (Number Weighed)	(25)	(25)	(25)	(25)	(24)	(23)
Absolute value	+4	-8	-8	+2	-5	-22
% of body weight	+4	-5	-2	+5	-4	-20
Ovary (Number Weighed)	NA	NA	NA	(25)	(24)	(23)
Absolute value				-5	-8	-13
% of body weight				-2	-7	-9

^a All values expressed as percent difference of control group means.

NA: Not Applicable.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group – P ≤ 0.05.

8) F0 – Histopathological findings: non-neoplastic

Table 30: Summary of microscopic findings – scheduled euthanasia (F0)^a Numbers in parentheses represent the number of animals with the finding.

	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	25	25	25	25	25	25	25	25
Kidney (Number Examined)	25	0	0	25	23	25	25	23
Mineralization, tubular, corticomedullary	(0)^a	(0)	(0)	(0)	(0)	(0)	(1)	(8)
Minimal	0	0	0	0	0	0	1	4
Mild	0	0	0	0	0	0	0	4
Dilatation, tubular	(0)	(0)	(0)	(1)	(0)	(0)	(1)	(10)
Minimal	0	0	0	1	0	0	1	6
Mild	0	0	0	0	0	0	0	4
Heart (Number Examined)	25	0	0	25	23	25	25	23
Mineralization, aortic	(0)	(0)	(0)	(0)	(0)	(0)	(1)	(4)
Minimal	0	0	0	0	0	0	1	2
Mild	0	0	0	0	0	0	0	2
Mineralization, myocardial	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(2)
Minimal	0	0	0	0	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Stomach (Number Examined)	25	0	1	25	23	25	25	23
Mineralization, glandular part	(0)	(0)	(0)	(0)	(0)	(0)	(2)	(4)
Minimal	0	0	0	0	0	0	2	3
Mild	0	0	0	0	0	0	0	1
Necrosis, glandular part	(0)	(0)	(0)	(0)	(0)	(0)	(0)	(2)
Minimal	0	0	0	0	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Ovary (Number Examined)	NA	NA	NA	NA	23	25	25	23
Atrophy					(0)	(0)	(0)	(5)
Minimal					0	0	0	5
Vagina (Number Examined)					23	25	25	23
Mucification, epithelial					(2)	(4)	(2)	(12)
Minimal					1	3	1	3
Mild					1	1	1	7
Moderate					0	0	0	2
Thymus (Number Examined)	25	0	0	25	23	25	25	23
Atrophy	(0)			(0)	(0)	(0)	(0)	(3)
Minimal	0			0	0	0	0	3
Apoptosis, increased, cortical	(0)			(0)	(0)	(0)	(0)	(4)
Mild	0	0	0	0	0	0	0	4

NA: Not Applicable.

Table 31: F0 - mean estrous cycle data – Pre-mating period

Parameter	Cycle length (days)	Irregularity index	Percentage of estrus days	Percentage of females acyclic or with acyclic period
Group 1: 0 mg/kg/day MEAN	4.1	0.1	24.0	20
SD	0.2	0.2	4.0
N	20	20	20
Group 2: 25 mg/kg/day MEAN	4.0	0.2	26.7	16
SD	0.2	0.2	4.7
N	21	21	21
Group 3: 75 mg/kg/day MEAN	4.0	0.1	27.2*	8
SD	0.2	0.2	4.0
N	23	23	23
Group 4: 225 mg/kg/day MEAN	4.0	0.1	27.9**	16
SD	0.2	0.2	4.0
N	21	21	21

* p ≤ 0.05.

** p ≤ 0.01.

9) F0 – Reproduction performance

Table 32: F0 Summary of Mating Performance and Fertility

GROUP	1	2	3	4
Dose Level	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Number of females :
Paired	25	25	25	25
Inseminated	25	25	25	25
Not pregnant	2	0	0	1
Total Litter Resorption	0	0	0	1
Mistimed Pregnancy	0	1	0	0
Pregnant	23	25	25	24
Females with live pups	23	25	25	23
Pre Coital interval :
MEAN	2.8	2.7	3.0	2.7
S.D.	1.3	1.1	1.2	0.9
N	25	24	25	25
COPULATION INDEX (%)	100	100	100	100
FERTILITY INDEX (%)	92	100*	100*	96

*p ≤ 0.05.

Table 33: F0 Summary of Delivery and litter data

Sex: Female		0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
Females Completing Delivery [CHSQFS]	N+ve	23	25	25	23
with Liveborn Pups [CHSQFS]	N+ve	23	25	25	23
with Stillborn Pups [CHSQFS]	N+ve	1	1	1	3
with all Stillborn Pups [CHSQFS]	N+ve	0	0	0	0
with all Dead PND 21 [CHSQFS]	N+ve	0	0	0	0
Gestation Length (Days) [GEN AN]	Mean	22.2 R¹	22.4	22.3	22.1
Number of Implantation Sites [GEN AN]	Mean	13.8 R¹	13.3	13.3	13.3
Pre-Birth Loss (%) [GEN AN]	Mean	8.33 R¹	5.93	7.73	7.94
Pups Delivered/Litter [GEN AN]	Mean	12.7 R¹	12.5	12.3	12.2
Live Pups PND 0 [GEN AN]	Mean	12.5 R¹	12.4	12.2	12.0
Live Pups PND 1 [GEN AN]	Mean	12.3 R¹	12.3	12.2	11.5
Live Pups Precull [GEN AN]	Mean	12.3 R¹	12.2	12.0	11.5
Live Pups Postcull [GEN AN]	Mean	7.9 R¹	7.9	7.9	7.6
Live Pups PND 7 [GEN AN]	Mean	7.9 R¹	7.9	7.9	7.6
Live Pups PND 14 [GEN AN]	Mean	7.9 R¹	7.9	7.9	7.6
Live Pups PND 21 [GEN AN]	Mean	7.9 R¹	7.9	7.9	7.6
Dead, Miss., Cannib. PND 0 [CHSQFS]	Sum	4	3	2	5
Dead, Miss., Cannib. PND 1-4 [CHSQFS]	Sum	4	4	5	10
Dead, Miss., Cannib. PND 5-21 [CHSQFS]	Sum	0	0	1	0
Dead, Miss., Cannib. PND 0-21 [CHSQFS]	Sum	8	7	8	15
Live Birth Index (%)	-	98.6	99.0	99.4	98.2
Viability Index (PND 0-4) (%)	-	98.6	98.7	98.4	96.4
Weaning Index (PND 4-21) (%)	-	100.0	100.0	99.5	100.0
Sex Ratio PND 1 - % Males [CHSQFS]	Mean	51.7	47.7	48.2	48.2
Sex Ratio PND 21 - % Males [CHSQFS]	Mean	48.0	49.1	48.5	51.3

[CHSQFS] - Chi-Squared & Fisher's Exact.

[GEN AN] - Generalised Anova/Ancova Test.

¹ R - Automatic Transformation: Rank.

N+ve : number of positive.

10) F1- Body weight and weight changes

Table 34: Mean F1 Pup Body Weights at 225 mg/kg/day vs. Historical Control Data (HCD) Mean or Control Group

	HCD	Control	Group 4
	HCD	0 mg/kg/day	225 mg/kg/day
	Pup Wt (g)	Pup Wt (g)	Pup Wt (g)	Group 4 vs. HCD/Control Mean (%)
Males D1	7.0	6.8	6.7	-4/-1
Males D21	54.2	56.9	52.3	-4/-8
Females D1	6.7	6.5	6.3	-6/-3
Females D21	52.6	55.1	50.6	-4/-8

Wt: Weight.

D: Day.

HCD : Historical Control data.

Table 35: Mean body weight (g) – F1 post weaning Males cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1	67.80 L¹	67.79	67.20	59.46 www⁵
8	111.29 I²	109.99	107.44 w⁴	88.90 www⁵
15	161.31 R³	160.76	157.84	136.62 SSS⁶
22	205.09 I²	203.66	201.97	182.98 www⁵
28	236.57 I²	234.23	238.36	218.42 w⁴
29	247.22 R³	248.30	244.96	224.63 SSS⁶
36	284.80 R³	285.96	282.87	264.43 SSS⁶
43	311.24 R³	313.26	308.72	289.62 SSS⁶
50	333.36 R³	336.58	334.13	308.57 SSS⁶
56	350.30 I²	345.79	346.92	333.20
57	349.54 I²	357.64	348.42	317.31 www⁵
64	366.10 I²	370.84	362.08	328.98 www⁵
70	377.34 I²	380.17	372.87	337.39 www⁵

¹ L - Automatic Transformation: Log.

² I - Automatic Transformation: Identity (No Transformation).

³ R - Automatic Transformation: Rank.

⁴ w - Test: Williams 2 Sided p < 0.05.

⁵ www - Test: Williams 2 Sided p < 0.001.

⁶ SSS - Test: Shirley 2 Sided p < 0.001.

Table 36: Mean body weight (g) – F1 Females post weaning cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1	65.84 L¹	64.18	63.92 w⁶	57.36 www⁷
5	-	-	-	57.70
8	101.57 R²	99.79	99.59	81.69 SSS⁸
15	134.18 I³	131.67	134.06	118.07 www⁷
22	157.39 I,a⁴	151.72 d⁵	156.33	145.71 www⁷
28	172.75 I³	167.84	166.42	158.64 w⁶
29	177.82 I³	171.58	176.52	164.39 www⁷
36	196.60 I³	189.89 w⁶	192.83 w⁶	181.23 www⁷
43	206.93 I³	200.16	204.28	192.79 www⁷
50	214.30 I³	211.18	214.44	204.83 ww⁹
56	229.40 I³	218.40	223.84	210.55
57	225.20 I³	222.43	225.50	214.32 ww⁹
64	232.07 I³	227.86	230.91	221.38 ww⁹
70	234.05 I³	230.18	235.26	225.48

¹ L - Automatic Transformation: Log.

² R - Automatic Transformation: Rank.

³ I - Automatic Transformation: Identity (No Transformation).

⁴ I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.

⁵ d - Test: Dunnett 2 Sided p < 0.05.

⁶ w - Test: Williams 2 Sided p < 0.05.

⁷ www - Test: Williams 2 Sided p < 0.001.

⁸ SSS - Test: Shirley 2 Sided p < 0.001.

⁹ww - Test: Williams 2 Sided p < 0.01.

11) F1- Food consumption

Table 37: Mean food consumption (g/animal/day) – F1 post weaning Males cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1 -> 8	15.32 R,kk ¹	16.09 ddd³	15.51	11.80 SSS⁴
8 -> 15	20.57 R²	20.62	20.56	17.24 SSS⁴
15 -> 22	22.75 R²	22.82	22.94	22.02 SSS⁴
22 -> 28	23.08 R²	23.13	23.20	22.34 S⁵
22 -> 29	24.45 R,k⁶	24.44	24.28	23.79
1 -> 29	20.78 R²	21.09	20.81	18.79 SSS⁴
29 -> 36	26.02 R,k⁶	26.58 d⁷	26.35 d⁷	25.50 S⁵
36 -> 43	25.24 R,kk¹	26.08 dd⁸	25.54	24.57 SS⁹
43 -> 50	25.57 R²	25.71	25.71	24.25 SSS⁴
50 -> 57	25.34 R²	25.67	25.57	23.75 SSS⁴
1 -> 57	23.09 I²	23.50	23.40	21.45 SSS⁴
57 -> 64	24.60 R,kk¹	25.43 d⁷	25.46 d⁷	23.42 SSS⁴
1 -> 64	23.26 R²	23.72	23.63	21.67 SSS⁴
64 -> 70	24.75 R²	24.12	24.82	23.01 SSS⁴
1 -> 70	23.79 R²	23.72	23.92	21.82 SSS⁴

¹ R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01.

² R - Automatic Transformation: Rank.

³ ddd - Test: Dunnett Non-Parametric 2 Sided p < 0.001.

⁴ SSS - Test: Shirley 2 Sided p < 0.001.

⁵S - Test: Shirley 2 Sided p < 0.05.

⁶ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

⁷ d - Test: Dunnett Non-Parametric 2 Sided p < 0.05.

⁸ dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01.

⁹ SS - Test: Shirley 2 Sided p < 0.01.

Table 38: Mean food consumption (g/animal/day) – F1 post weaning Females cohorts 1A, 1B, 2A and 3

Day(s) Relative to Start Date	0 mg/kg/day	25 mg/kg/day	75 mg/kg/day	225 mg/kg/day
1 -> 8	13.22 R,k ¹	12.88 d⁴	13.20	9.85 SSS⁷
8 -> 15	15.58 R,k¹	15.64	15.89 dd⁵	14.18 SSS⁷
15 -> 22	16.89 R,kk²	16.52dd⁵	17.38 S⁶	16.33 S⁶
22 -> 28	16.10 R³	15.95	15.29	15.67
22 -> 29	17.11 R³	16.97	17.19	16.94
1 -> 29	15.68 R,kk²	15.42	15.94	14.55 SSS⁷
29 -> 36	18.22 R³	18.05	18.48	17.76 SS⁸
36 -> 43	17.93 R³	17.99	18.08	17.63 S⁶
43 -> 50	18.09 R,kk²	19.89 dd⁵	18.42	18.06
50 -> 57	18.21 R³	18.30	18.93	18.28
1 -> 57	16.84 R³	16.92	17.11	16.21 SSS⁷
57 -> 64	18.35 R³	18.77	19.11 S⁶	18.82 SS⁸
1 -> 64	17.00 R³	17.10	17.34	16.50 SSS⁷
64 -> 70	18.18 R,kkk⁹	17.41 ddd¹⁰	18.65	17.96
1 -> 70	17.27 R,kk²	17.09	17.64 d⁴	16.52 SSS⁷

¹ R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05.

² R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01.

³ R - Automatic Transformation: Rank.

⁴ d - Test: Dunnett Non-Parametric 2 Sided p < 0.05.

⁵ dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01.

⁶S - Test: Shirley 2 Sided p < 0.05.

⁷ SSS - Test: Shirley 2 Sided p < 0.001.

⁸ SS - Test: Shirley 2 Sided p < 0.01.

⁹ R,kkk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.001.

¹⁰ ddd - Test: Dunnett Non-Parametric 2 Sided p < 0.001.

12) F1- Haematological findings

Table 39: F1 post weaning - cohort 1A - Toxicological Relevant Changes in Haematology between 225 mg/kg/day and Control Groups

	Males	Females
MCV (fL)	X1.03	-
MCH (pg)	X1.04	X1.04
Reti. (Giga/L)	X1.15	-
Reti. (%)	X1.17	-
WBC (Giga/L)	X0.76	-
Lymphocytes (Giga/L)	X0.73	-
Monocytes (Giga/L)	X0.57	-
Basophils (Giga/L)	X0.38	-
RBC (T/L)	-	X0.93
Hb (g/L)	-	X0.97
HCT (L/L)	-	X0.95

-: Absence of change.

Numerical values indicate fold changes of treated group value relative to control group mean value.

MCV: Mean corpuscular volume.

MCH: Mean corpuscular haemoglobin.

Reti. : Reticulocytes.

WBC: White blood cells.

RBC: Red blood cells.

Hb: Hemoglobin.

HCT: Hematocrit.

12) F1 - Clinical biochemistry findings

Table 40: F1 males post weaning - cohort 1A - Group Mean Serum Clinical Chemistry Parameters – extract of results

Group	Dose level mg/kg/day	Na mmol/L	Chol. mmol/L	Glob. g/L	ALP BC IU/L
1	0	141.9	1.456	28.94	110.2
2	25	142.5	1.611	28.23	97.1
3	75	141.6	1.336	29.39	101.9
4	225	142.8*	0.712**	27.08**	135.7*

Na: sodium.

Chol: cholesterol.

Glob: globulin.

ALP BC: Alkaline phosphatase.

*p ≤ 0.05.

**p ≤ 0.01.

Table 41: F1 females post weaning - cohort 1A - Group Mean Serum Clinical Chemistry Parameters – extract of results

Group	Dose level mg/kg/day	Na mmol/L	ALP BC IU/L
1	0	142.4	58.8
2	25	142.2	60.3
3	75	143.0	57.4
4	225	143.6**	79.5**

Na: sodium.

ALP BC: Alkaline phosphatase.

**p ≤ 0.01.

13) F1 – Sexual maturation

Table 42: F1 females - cohorts 1A, 1B, 2A and 3 – Mean sexual maturation data – extract of results

0 mg/kg/day

25 mg/kg/day

75 mg/kg/day

225

mg/kg/day

Day of vaginal opening (Days)

34.4 R¹

34.0

34.5

37.1 SSS²

Body weight (g)

113.71 R¹

113.44

116.65

111.82

¹ R - Automatic Transformation: Rank.

² SSS - Test: Shirley 2 Sided p < 0.001.

14) F1- Anogenital distance

Table 43: F1 animals - Mean Pup Anogenital Distance (normalized to the cube root of body weight)

Dose level

Value

Males

Females

0 mg/kg/day

Mean

1.8

0.2

1.0

0.1

25 mg/kg/day

Mean

1.9

0.2

1.0

0.2

75 mg/kg/day

Mean

1.9

0.1

0.9

0.1

225 mg/kg/day

Mean

1.9

0.2

0.9

0.1

15) F1- organ weight findings

Table 44: F1 cohort 1A Summary of Organ Weight Data - Scheduled Euthanasia

	Males			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	20	20	20	19	20	20
Kidney (Number Weighed)^a	(20)	(20)	(20)	(19)	(20)	(20)
Absolute value	+6	+8	+14	+1	+1	+12
% of body weight	+4	+9	+27	+2	+2	+19

^a All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - p ≤ 0.05.

Table 45: F1 cohort 1B Summary of Organ Weight Data - Scheduled Euthanasia

	Males			Females
Group No.	2	3	4	2	3	4
Dose (mg/kg/day)	25	75	225	25	75	225
Number of Animals per Group	20	20	20	20	20	20
Kidney (Number Weighed)^a	(10)	(10)	(10)	(10)	(10)	(10)
Absolute value	0	+3	+11	-3	+2	+18
% of body weight	-1	+4	+24	-1	+2	+23

^a All values expressed as percent difference of control group means.

Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - p ≤ 0.05.

16) F1 gross pathology findings

Table 46: F1 cohort 1A - Summary of Gross Pathology Findings – Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	19	20	20
Kidney (Number Examined)	20	20	20	20	20	19	20	20
Granular appearance	1	0	0	2	0	0	0	0
Enlarged	0	0	0	1	0	0	0	0

Table 47: F1 cohort 1B - Summary of Gross Pathology Findings – Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	20	20	20
Kidney (Number Examined)	0	0	0	6	2	0	3	13
Enlarged (right and/or left)	0	0	0	5	1	0	3	13

17) F1 histopathology findings

Table 48: F1 cohort 1A - Summary of Microscopic Findings - Scheduled Euthanasia

Sex	Males				Females
Group No.	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	25	75	225	0	25	75	225
Number of Animals per Group	20	20	20	20	20	19	20	20
Kidney (Number Examined)	20	20	20	20	20	19	20	20
Basophilia, tubular	(3)^a	(6)	(7)	(20)	(1)	(0)	(1)	(17)
- Minimal	3	6	7	2	1	0	1	8
- Mild	0	0	0	14	0	0	0	9
- Moderate	0	0	0	4	0	0	0	0
Dilatation, tubular	(0)	(0)	(0)	(16)	(0)	(0)	(0)	(8)
- Minimal	0	0	0	11	0	0	0	7
- Mild	0	0	0	4	0	0	0	1
- Moderate	0	0	0	1	0	0	0	0
Inflammatory cell infiltration, chronic	(1)	(0)	(0)	(12)	(0)	(0)	(0)	(3)
- Minimal	1	0	0	10	0	0	0	3
- Mild	0	0	0	2	0	0	0	0
Spleen (Number Examined)	20	20	20	19	20	19	20	20
Extramedullary hematopoiesis, increased	(4)	(3)	(3)	(10)	(2)	(0)	(1)	(6)
- Minimal	4	3	3	10	2	0	1	6

^a Numbers in parentheses represent the number of animals with the finding.

18) F1 – Behaviour (functional findings)

Auditory startle reflex

Table 49: F1 - cohort 2A males - Group Mean Auditory Startle Reflex – Habituation

	Block No. 1	Block No. 2		Block No. 3		Block No. 4		Block No. 5
Group	Mean	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*
Group 1	0.1957	0.1764	-9.9%	0.1830	-6.7%	0.1536	-21.7%	0.1542	-21.0%
Group 2	0.2147	0.1744	-18.1%	0.1741	-17.3%	0.1777	-15.4%	0.1543	-26.6%
Group 3	0.1740	0.1541	-9.3%	0.1426	-16.2%	0.1378	-19.5%	0.1352	-21.0%
Group 4	0.1750	0.1540	-10.3%	0.1415	-18.6%	0.1396	-17.8%	0.1225	-28.1%

* % difference versus Block 1.

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Table 50: F1 - cohort 2A females - Group Mean Auditory Startle Reflex – Habituation

	Block No. 1	Block No. 2		Block No. 3		Block No. 4		Block No. 5
Group	Mean	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*	Mean	% diff. vs B1*
Group 1	0.2177	0.1786	-16.4%	0.1729	-18.9%	0.1749	-19.3%	0.1635	-23.5%
Group 2	0.2184	0.1991	-6.9%	0.1895	-11.7%	0.1848	-13.1%	0.1801	-15.4%
Group 3	0.1847	0.1820	-1.0%	0.1650	-9.3%	0.1620	-10.6%	0.1452	-19.7%
Group 4	0.1714	0.1395	-18.3%	0.1439	-15.0%	0.1395	-16.4%	0.1329	-21.4%

* % difference versus Block 1.

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Functional Observation Battery (FOB)

Table 51: F1 - cohort 2A males – summary of neurobehavioral evaluation – PND 63-75

Group	1	2	3	4
	Mean	Mean	Mean	Mean
Home cage observations
Posture/Body carriage	/	/	/	/
Convulsions	/	/	/	/
Stereotypy	/	/	/	/
Tremors	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Handling
Ease of removal	/	/	/	/
Handling reactivity	/	/	/	/
Op. field observations
Rearing	8.4	6.5	6.8	6.0
Arousal/alertness	/	/	/	/
Gait/Mobility	/	/	/	/
Vocalizations	/	/	/	/
Tremors	/	/	/	/
Respiration	/	/	/	/
Defecation	-0.9	-0.8	-0.6	-1.0
Stereotypy	/	/	/	/
Convulsions	/	/	/	/
Appearance	/	/	/	/
Salivation	/	/	/	/
Lacrimation	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Exophtalmos	/	/	/	/
Erected fur (piloerection)	/	/	/	/
Touch response/tactile reflex	/	/	/	/
Startle response	/	/	/	/
Tail pinch response	-0.2	-0.1	0.1	/
Pupil response	/	/	/	/
Body temperature (°C)	37.0	37.0	36.9	37.2
Body tone	/	/	/	/
Grip strength	427.1	556.0	569.2	540.8
Air righting reflex	/	/	/	/

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

/: Normal observation.

Op. field: open field.

Table 52: F1 - cohort 2A females – summary of neurobehavioral evaluation – PND 63-75

Group	1	2	3	4
	Mean	Mean	Mean	Mean
Home cage observations
Posture/Body carriage	/	/	/	/
Convulsions	/	/	/	/
Stereotypy	/	/	/	/
Tremors	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Handling
Ease of removal	/	/	/	/
Handling reactivity	0.1	/	0.2	0.1
Op. field observations
Rearing	3.8	6.3	8.1	6.0
Arousal/alertness	0.2	0.1	/	/
Gait/Mobility	/	/	/	/
Vocalizations	/	/	/	/
Tremors	0.1	/	/	/
Respiration	/	/	/	/
Defecation	-1.0	-1.0	-1.0	-1.0
Stereotypy	/	/	/	/
Convulsions	/	/	/	/
Appearance	/	/	/	/
Salivation	/	/	/	0.1
Lacrimation	/	/	/	/
Palpebral closure/Ptosis	/	/	/	/
Exophtalmos	/	/	/	/
Erected fur (piloerection)	/	/	/	/
Touch response/tactile reflex	/	/	/	0.1
Startle response	/	/	/	0.1
Tail pinch response	-0.1	/	-0.1	-0.1
Pupil response	/	/	/	/
Body temperature (°C)	37.7	37.8	37.9	38.0
Body tone	/	/	/	/
Grip strength	463.6	512.4	521.7	587.7
Air righting reflex	/	/	/	/

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

/: Normal observation.

Op. field: open field.

Table 53: F1 - cohort 2A – Motor activity - % of Habituation at 225 mg/kg/day versus HCD range

	HCD Range					225 mg/kg/day
Intervals	2	3	4	5	6	2	3	4	5	6
Male Fine Movements	-27 to -42	-41 to -59	-47 to -59	-57 to -73	-62 to -65	-49	-87	-95	-90	-81
Female Fine Movements	-30 to -41	-39 to -55	-35 to -66	-48 to -72	-57 to -77	-60	-81	-85	-87	-94
Male Ambulation	-36 to -64	-45 to -71	-57 to -70	-60 to -83	-65 to -74	-74	-93	-99	-100	-89
Female Ambulation	-39 to -52	-41 to -66	-45 to -78	-56 to -81	-49 to -80	-78	-93	-92	-92	-98

HCD: Historical Control Data.

Bold: % below the HCB range.

19) F1 – Anti-KLH IgM Analysis (TDAR) – Cohort 1B

Table 54: F1 - cohort 1B females – summary of Anti-KLH IgM (adjusted to pre-dose)

Time-point	Group 1		Group 2	Group 3	Group 4	Cyclophos-phamide 10 mg/kg/day	Global p-value	Test
1	Mean	0.0	0.0	0.0	0.0	0.0	1.0000	(KW)
	StD	0.00	0.00	0.00	0.00	0.00
	N	10	10	10	10	10
5	Mean	22.5	24.2	23.8	21.2	0.4 ***	0.0001 ***	(KW) (DR)
	StD	10.62	11.27	11.98	13.10	0.75
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

(L): Logarithmic transformation; (S): Student; (WMS): Wilcoxon-Mann-Whitney; (A): ANOVA; (KW): Kruskall-Wallis; (D): Dunnett; (DR): Dunnett on Ranks.

*, ** or ***: p-value<0.05, 0.01 or 0.001, respectively.

Table 55: F1 - cohort 1B males – summary of Anti-KLH IgM (adjusted to pre-dose)

Time-point	Group 1		Group 2	Group 3	Group 4	Cyclophos-phamide 10 mg/kg/day	Global p-value	Test
1	Mean	0.0	0.0	0.0	0.0	0.0	1.0000	(KW)
	StD	0.00	0.00	0.00	0.00	0.00
	N	10	10	10	10	10
5	Mean	26.3	32.5	19.2	14.6	-0.1***	<0.0001 ***	(KW) (DR)
	StD	18.16	19.86	14.56	12.62	0.31
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

(L): Logarithmic transformation; (S): Student; (WMS): Wilcoxon-Mann-Whitney; (A): ANOVA; (KW): Kruskall-Wallis; (D): Dunnett; (DR): Dunnett on Ranks.

*, ** or ***: p-value<0.05, 0.01 or 0.001, respectively.

20) F1 – Anti-SRBC IgM Analysis (TDAR) – Cohort 3

Table 56: F1 - cohort 3 males – summary of Anti-SRBC IgM values

	Value	Group 1	Group 2	Group 3	Group 4	Group 5
Pre-Immunization a-SRBC IgM U/mL	Mean	339	280	316	339	436
	St Dev	134.6	104.3	193.5	138.2	269.0
	N	10	10	10	10	9
Post-Immunization a-SRBC IgM U/mL	Mean	11080	6823	13145	12263	327 ***
	St Dev	5524.4	2203.8	14968.8	8709.9	178.3
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Group 5: Positive control animals treated with cyclophosphamide.

***: p-value<0.001

Table 57: F1 - cohort 3 females – summary of Anti-SRBC IgM values

	Value	Group 1	Group 2	Group 3	Group 4	Group 5
Pre-Immunization a-SRBC IgM U/mL	Mean	271	283	287	466	285
	St Dev	83.1	117.8	94.3	313.2	132.7
	N	10	10	10	10	10
Post-Immunization a-SRBC IgM U/mL	Mean	13158	11710	9973	14166	436 ***
	St Dev	8712.2	8519.1	4587.9	8296.1	96.6
	N	10	10	10	10	10

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

Group 5: Positive control animals treated with cyclophosphamide.

***: p-value<0.001

21) F1 – Splenic Lymphocyte Subpopulation Analysis - Cohort 1A

Table 58: F1 – Cohort 1A Splenic Lymphocyte Subpopulation Summary - mean values

Dose level	Group 1		Group 2		Group 3		Group 4
Gender	M	F	M	F	M	F	M	F
All leucocytes	82.30	87.17	86.16	84.60	85.79	86.77	86.00	85.11
Viability	88.23	88.34	88.54	88.07	87.05	88.47	87.85	90.23
All T-lymphocytes	21.80	25.94	24.01	23.20	23.54	25.76	21.95	26.01
T-helper lymphocytes	12.62	15.21	13.99	13.14	13.36	14.79	12.58	14.34
T-cytotoxic	8.68	10.06	9.37	9.38	9.63	10.32	8.79	11.00
B-lymphocytes	36.29	30.24	35.15	32.15	33.09	30.04	32.69	32.20
Natural killer cells	3.10	3.65	3.53	3.75	3.19	3.59	3.53	3.06
NKT-lymphocytes	2.10	2.39	2.44	2.42	2.21	2.43	2.42	2.46

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

M: males - F/ Females.

Table 59: F1 – Cohort 1A Immunophenotyping Fold Changes to Control Animals (Group 1)

Group	Group 1		Group 2		Group 3		Group 4
Gender	M	F	M	F	M	F	M	F
All leucocytes	1.00	1.00	1.05	0.97	1.04	1.00	1.04	0.98
Viability	1.00	1.00	1.00	1.00	0.99	1.00	1.00	1.02
All T-lymphocytes	1.00	1.00	1.10	0.89	1.08	0.99	1.01	1.00
T-helper lymphocytes	1.00	1.00	1.11	0.86	1.06	0.97	1.00	0.94
T-cytotoxic	1.00	1.00	1.08	0.93	1.11	1.03	1.01	1.09
B-lymphocytes	1.00	1.00	0.97	1.06	0.91	0.99	0.90	1.06
Natural killer cells	1.00	1.00	1.14	1.03	1.03	0.98	1.14	0.84
NKT-lymphocytes	1.00	1.00	1.16	1.01	1.05	1.02	1.15	1.03

Group 1: 0 mg/kg/day – Group 2: 25 mg/kg/day - Group 3: 75 mg/kg/day - Group 4: 225 mg/kg/day.

M: males - F/ Females.

22) F1 – Thyroid hormone analysis

Table 60: F1 – pups before weaning - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F1-PND4 Pups Mean	T4 µg/dL	1.11	1.52**	1.09	1.44*
F1-PND21 Male pups Mean	T4 µg/dL	4.52	4.47	4.68	4.52
F1-PND21 Male pups Mean	TSH µIU/mL	0.043	0.072*	0.063**	0.077**
F1-PND21 Female pups Mean	T4 µg/dL	4.62	4.18	4.67	4.93
F1-PND21 Female pups Mean	TSH µIU/mL	0.051	0.081	0.054	0.071

*p ≤ 0.05.

**p ≤ 0.01.

Table 61: F1 cohort 1A – post weaning - Summary of T4 and TSH values

Animals	Hormone	Group 1 0 mg/kg/day	Group 2 25 mg/kg/day	Group 3 75 mg/kg/day	Group 4 225 mg/kg/day
F1-cohort 1A males Mean	T4 µg/dL	7.00	6.50	6.95	4.79***
F1-cohort 1A males Mean	TSH µIU/mL	0.071	0.098	0.100	0.112
F1-cohort 1A females Mean	T4 µg/dL	4.01	3.57	4.19	4.70
F1-cohort 1A females Mean	TSH µIU/mL	0.075	0.054	0.053	0.098

***p ≤ 0.001.

21) F1 – Cohort 1A – Estrous cycle data

Table 62: F1 cohort 1A – Mean time between the day of vaginal opening and the first estrus

Dose level	Value	Time between the day of vaginal opening and the first estrous (days)
Group 1: 0 mg/kg/day	MEAN	3.2
	SD	1.8
	N	20
Group 2: 25 mg/kg/day	MEAN	2.9
	SD	2.1
	N	19
Group 3: 75 mg/kg/day	MEAN	2.7
	SD	1.7
	N	20
Group 4: 225 mg/kg/day	MEAN	2.6
	SD	1.8
	N	20

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following No Observed Adverse Effect Levels (NOAELs) were established for the substance:

- General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach at 225 mg/kg/day for females).

- Reproductive Toxicity (F0): At least 225 mg/kg/day.

- General Toxicity (F1): At least 225 mg/kg/day.

- Developmental Toxicity (F1): At least 225 mg/kg/day.

Executive summary:

This GLP Study following the OECD guideline 443 was carried out to provide an evaluation of the pre and postnatal effects of the test material, on development as well as a thorough evaluation of systemic toxicity in adult male and pregnant and lactating female Wistar Han rats and their offspring.

Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of the test material on the integrity and performance of the adult male and female reproductive systems.

Specifically, but not exclusively, the following parameters were considered: gonadal function, estrous cyclicity, epididymal sperm maturation, mating behaviour, fertility, pregnancy, parturition, and lactation. Developmental neurotoxicity and developmental immunotoxicity assessments were also performed to characterize potential effects on those systems.

STUDY DESIGN

The test material was administered by daily oral gavage at dose levels of 25, 75 and 225 mg/kg/day to groups of 25 males and 25 females Wistar rats (F0 generation). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group of 25 rats/sex received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). The inseminated females were allowed to litter, and development of the offspring was observed up to weaning on Postnatal Day (PND) 21.

The following parameters and endpoints were evaluated in all F0 animals: mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic findings, organ weights and histology findings. F0-males were euthanised after at least 12 weeks of exposure and F0 females after at least 16 weeks of dosing.

The following parameters and endpoints were evaluated in all F1 offspring until weaning (PND21): mortality, clinical signs, body weights, number of pups, pup sex ratio and anogenital distance and areola/nipple retention for male pups.

From weaning, the selected F1-pups were allocated to 5 cohorts (1A, 1B, 2A, 2B and 3) of 20 males and 20 females for the first 2 cohorts, and 10 males and 10 females for the last 3 cohorts. All F1-animals, except Cohort 2B and F1 surplus animals, were dosed by daily oral gavage (once a day) from PND21 up to the day before necropsy at the same dose levels of 25, 75 and 225 mg/kg/day. A fourth group received the same dose volume (5 mL/kg/day) of the control material (0.5% CMC in purified water). Selected F1 animals (with the exception of Cohort 2B) were observed for mortality, clinical signs (including arena observations), body weights, body weight changes, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups, surplus pups and Cohort 1A), sexual maturation, estrous cycles (Cohort 1A), neurobehavioral tests (Cohort 2A), macroscopic findings, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. Humoral response was also evaluated following SRBC administration for Cohort 3 animals and KLH administration for Cohort 1B animals (TDAR assays). F1 animals from Cohort 2B were not dosed directly and were necropsied on/before PND24 for neuro-histopathology and morphometric analysis.

F1 animals from Cohort 3 were sacrificed between PND54 and PND57 or at approximately 12 weeks of age for positive controls. F1 animals from Cohort 2A were sacrificed after completion of the neurobehavioral tests between PND76 and PND90. F1 animals from Cohort 1A were sacrificed after clinical pathology evaluation between PND89 and PND99. F1 animals from Cohort 1B were sacrificed between PND92 and PND100.

Surplus offspring (10/sex/group) not included in any of the F1 cohorts were selected for thyroid hormone analysis and organ weights on PND21.

RESULTS

All formulations at 5, 15 and 45 mg/mL used on Day 1, and in Week 11 and Week 26 of treatment were in agreement with acceptance criteria. Homogeneity was confirmed from the formulations used on Day 1. No test material was detected in the vehicle samples.

F0 generation:

There was no test material-related death in any group.

There was no test material-related clinical sign in any group.

There was no test material-related effect in any group on mean body weight gain or food consumption for either sex that was considered toxicologically significant.

There was no test material-related effect in any group on estrous cyclicity, mating performance (including copulation and pre-coital interval), fertility (including sperm quality), and gestation length.

Higher creatinine and urea concentrations, minor ionic unbalance for females and higher hepatic enzymes and triglyceride concentrations were noted for both sexes at 225 mg/kg/day compared with the control group and correlated with the pathology findings noted for the kidneys and liver.

There were no test material-related effects on T4 and TSH levels that were toxicologically significant in any group for either sex. Changes in hormone serum levels for F0 females and F1 males at 225 mg/kg/day were noted but considered not toxicologically relevant in the absence of any gender trend, any test item‑related effect on the thyroid weight, anogenital distance, nipple areola, reproduction and development, any histology findings in the thyroid glands, or similar findings for pups. They are considered related to the high variability of this parameter. As under the conditions of this study no adverse effect was observed that could be linked to the lower T4 and higher TSH values, this was not taken into account to determine the No Observed Adverse Effect Levels (NOAELs).

At necropsy, there were test material-related findings for females dosed at 225 mg/kg/day in the kidney (minimal to mild tubular dilatation and mineralization, correlating with higher kidney weight), heart (minimal to mild myocardial or aortic mineralization), stomach (minimal to mild mineralization and/or necrosis of the glandular part, grossly correlating with dark foci), thymus (minimal atrophy and mild increased cortical apoptosis, correlating with lower organ weight when compared with the control group). Mineralization in the kidney, heart and stomach was considered adverse, while other findings were considered secondary to test material-related systemic effects (ovary, adrenal gland and thymus), or adaptative changes (liver). The pathology findings noted on the kidneys were correlated with higher creatinine and urea concentrations and minor ionic unbalance. The higher mean liver weight at 225 mg/kg/day for both sexes correlated with slightly higher mean tryglyceride and hepatic enzyme concentrations when compared with the control group. Ovarian atrophy, lower ovaries weight when compared with the control group and vaginal mucification which suggested a delay in the post-gestation recovery of estrous cycle were noted at necropsy at 225 mg/kg/day and were considered probably secondary to test material-related systemic effects noted in kidneys, stomach and heart. These findings were not correlated with any test material-related effect on fertility or estrous cycle data (F0 and F1 generation) and no test material-related effect on the mean count of primordial/small growing follicles (Cohort 1A) and was therefore considered not adverse. In addition, there was no similar finding in the F1 generation.

F1 generation:

There was no test material-related effect on postnatal survival of the F1 offspring in any group. Mean live litter size was comparable in all groups between birth and weaning.

There were no test material-related effects that were considered toxicologically significant on clinical condition, body weight and anogenital distance of the pups, or any evidence of areolae/nipple retention for the male offspring at any dose level.

There were no test material-related effects on T4 and TSH levels for the F1-pups on PND4 and PND21.

There was no test material-related effect on the mean age at attainment of balanopreputial cleavage and vaginal opening or on the duration from vaginal opening to first estrus in any group.

There was no test material-related clinical sign in any group for either sex.

There was a test material-related slightly lower mean body weight gain and reduced mean food consumption throughout the dosing period for males at 225 mg/kg/day compared with the control group leading to slightly, lower mean terminal body weight compared with the control group considered not adverse due to the low magnitude of the change and in the absence of any developmental effects.

There were no test material-related effects on the hematology, coagulation, serum clinical chemistry, urinary parameters or on the thyroid hormone serum levels that were considered toxicologically significant at any dose for either sex (Cohort 1A).

There were no test material-related effects on estrous cyclicity or on sperm quality in any group (Cohort 1A).

There was no test material-related immunotoxicity (Cohorts 1A and 3), as evidenced by the lymphocyte subpopulation analysis or KLH-IgM and SRBC-IgM responses in the TDAR assay.

There were no test material-related findings amongst the auditory startle test (at weaning), and FOB and motor activity, brain morphometry, brain weight and neurohistopathology indicative of any neurotoxicity (Cohorts 2A and 2B).

At necropsy, non-adverse test material-related effects were observed for the kidney and spleen (slightly higher extramedullary hematopoiesis compared with the control group) of males and females dosed at 225 mg/kg/day. The findings for the kidneys were considered non adverse in the absence of any degenerative/necrotic renal changes and any clinical pathology correlate.

CONCLUSION

General Toxicity (F0): At least 75 mg/kg/day (based on adverse pathology findings including renal tubular dilatation and mineralization correlated with higher kidney weight, myocardial or aortic mineralization in the heart, mineralization and/or necrosis of the glandular stomach, kidneys and heart at 225 mg/kg/day for females).

Reproductive Toxicity (F0): At least 225 mg/kg/day.

General Toxicity (F1): At least 225 mg/kg/day.

Developmental Toxicity (F1): At least 225 mg/kg/day.

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