Registration Dossier

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further this study also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity between 49 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.5 – 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 57 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 23) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19468. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 57 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 51-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

Daily

Details on study schedule:

Study initiation date: 27 May 2020
Experimental starting date: 28 May 2020
Acclimatization: Start: 28 May 2020, End: 01 June 2020
Pre-treatment period: Start : 02 June 2020, End: 15 June 2020
Treatment start: Start: 16 June 2020, End: 12 August 2020
Experiment completion Date: 31 August 2020
Submission of Draft report: 31 August 2020
Study completion: 13 November 2020

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes

Details on study design:

Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.

b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LDs 0 and 4 were recorded.

c. The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.

d. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

e. After standardization, the individual pup body weight was measured on LD13.

f. The number of nipples/areolae in male pups was counted on LD 13.

g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.

h. The litters were observed daily to note the number of alive, dead and cannibalized pups.

i. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for defects and/or cause of death.
For apparently non-pregnant rats, the uteri was stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of the embryos (by staining the implantation sites) thereby, for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.

Statistics:

Parameters such as body weight, body weight change, food consumption, oestrous cycle, gestation length (days), mean litter size, mean viable litter size, ano-genital distance, sex ratio, survival index and pup body weight, laboratory Investigations – haematology, coagulation, clinical chemistry, urinalysis & thyroid hormone profile, organ weights, organ weight ratios (organ to body weight and organ to brain weight), no. of implantations, post implantation loss (%) were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square was found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroscedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.

Reproductive indices:

11.1 Reproductive Performance Data of Parents
a. Male mating index (%)

Number of males with evidence of mating
= ------------------------------------------------------------------- x 100
Number of males cohabited

b. Male fertility index (%)

Number of males siring a litter/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

Number of females mated
= ------------------------------------------ x 100
Number of females cohabited

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of mating

e. Mean number of implantations/group

Total number of implantations
= ---------------------------------------
Total number of pregnant animals

f. Post implantation loss (%)

Number of implantations - Number of live pups
= ------------------------------------------------------------------- x 100
Number of implantations

Offspring viability indices:

11.2 Litter Data
a. Mean litter size per group

Total Number of pups born
= -------------------------------------------------
Total Number of littered animals

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

c. Live birth index (%)

No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)

d. Day 4 survival index (%)

Number of viable pups on lactation Day 4
= -------------------------------------------------------- x 100
Number of viable pups born

e. Sex Ratio/ Percentage of male offspring (%)

No. of male pups born
= -------------------------------- x 100
Total no. of pups born

f. Ano-genital Distance Ratio (mm/g1/3 )

Ano-genital distance
= --------------------------------
Cube root of body weight

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs and mortality observed throughout the treatment period in either sex at all the doses tested, except for an incidental observation of dehydration, piloerection and weakness in a female rat (Rx7557) of high dose group on days 42 and 43. This dam was sacrificed on Day 43 due to total litter loss. The yellowish colour faeces was observed at all the tested doses in both the sexes which recovered by Day 2 of recovery period. This yellowish coloured faecal matter is related to physical nature of the test item.
There were no abnormalities observed in pups

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
The significantly lower weekly absolute weight change during Days 52-59 in recovery group females was considered incidental as the mean body weights were not altered by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was not altered throughout treatment and recovery period when compared to the concurrent vehicle control.
Incidences of significantly decreased food consumption during Days 1-8 in 1000 mg/kg bwt/day recovery dose group males and during Days 8-14 in both 111 and 333 mg/kg bwt/day dosed females and significantly increased food consumption during Days 43-50 in 1000 mg/kg bwt/day recovery dose group males was observed. These isolated statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the hematology parameters including PT and APTT values in both males and females. A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical chemistry parameters were not affected by test item administration. Increased creatinine kinase activity in 1000 mg/kg bwt/day dose males was considered as incidental change as it was not associated with any microscopic changes in muscle tissues examined. Increased creatinine concentration in 1000 mg/kg bwt/day dose females was considered as incidental change as it was not associated microscopic changes in kidneys examined. A few other variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for an incidence of lower distance travelled at interval 1 and lower total distance travelled in recovery males treated at 1000 mg/kg bwt/day.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic changes at any of the dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter Date: Test item had no treatment-related effects on the mean litter size, mean viable litter size and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.
Body Weight of Pups: The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Anogental Distance (AGD): No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex. An incidence of higher ano-genital distance in female pups at 111 mg/kg bwt/day was considered as not related to treatment as this difference was not dose related and was minimal (̴ 3%).
Uterine/Implantation Data: No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Areolae/Nipple Retention in Pups: The male pups did not exhibit areola/nipple retention on PND 13.
Oestrous Cycle Prior to Sacrifice: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed;

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested. The lower gestation length at high dose was considered not treatment related as the difference was minimal (̴ 3%).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

TABLE 12. Summary of Oestrous Cycle Prior to Cohabitation

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Mean

Cycle

(days)

1 → 15 [a]

Mean

SD

N

4.6

0.5 10

4.5

0.4 10

4.6

0.3 10

4.7

0.3 10

[a] - Anova & Dunnett (Rank)

TABLE 17. Summary of Fertility Data

Sex: Both (Litter: A)			G1 0 mg/kg/day	G2 111 mg/kg/day	G3 333 mg/kg/day	G4 1000 mg/kg/day
Paired Males	-	N+ve	10	10	10	10
Paired Females	-	N+ve	10	10	10	10
Mated	-	N+ve	10	10	10	10
Pre-coital Interval (days)	[f]	Mean SD N	2.2 1.0 10	2.8 1.8 10	2.8 1.1 10	3.1 2.2 10
Male Mating Index	[f]	%	100.0	100.0	100.0	100.0
Female Mating Index	[f]	%	100.0	100.0	100.0	100.0
Littered	-	N+ve	8	9	9	9
Gest. Length (days)	[a]	Mean SD N	22.9 0.4 8	22.9 0.3 9	22.7 0.5 9	22.3 * 0.5 9
Fertile Male	-	N+ve	8	9	9	9
Fertile Female	-	N+ve	8	9	9	9
Mated/Pregnant/Littered	-	N+ve	8	9	9	9
Mated/Pregnant/Viable Litter	-	N+ve	8	9	9	9
Mated/Not Pregnant	-	N+ve	2	1	1	1
Male Fertility Index	[f]	%	80.0	90.0	90.0	90.0
Female Fertility Ind l ex	[f]	%	80.0	90.0	90.0	90.0

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

[a] - Anova & Dunnett (Rank): * = p < 0.05

TABLE 18. Summary of Litter Data

Sex: Both

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Total No of Pups Born

LD 0

[a]

Mean

SD

N Sum

14.0 4.0

8

112.0

12.2 4.3

9

110.0

13.3 2.0

9

120.0

12.9 3.8

9

116.0

Dead Pups on Day 0

LD 0

Sum

1

.

1

4

Live Female Pups on Day 0

LD 0

Sum

54

50

61

58

Live Male Pups on Day 0

LD 0

Sum

57

60

58

54

Live Pups on Day 0

LD 0

[a1]

Mean SD

N Sum

13.9 4.2

8

111.0

12.2 4.3

9

110.0

13.2 2.0

9

119.0

12.4 4.9

9

112.0

Live Pups

Day 1

LD 1

[a1]

Mean

SD

N Sum

13.9 4.2

8

111.0

12.2 4.3

9

110.0

13.2 2.0

9

119.0

12.4 4.9

9

112.0

Live Pups

Day 4

LD 4

[a1]

Mean

SD

N Sum

13.9 4.2

8

111.0

12.2 4.3

9

110.0

13.2 2.0

9

119.0

12.4 4.9

9

112.0

No. of Pups Culled on Day 4

4

Sum

48

43

47

No. of Live Pups Post Cull

LD 4

N

Sum

8

63

9

67

9

72

9

65

Dead/Cannibalized Pups 5-12

LD5-12

Sum

.

1

Live Pups

Day 13

LD 13

[a1]

Mean

SD

N Sum

7.9 0.4

8

63.0

7.4 1.7

9

67.0

8.0 0.0

9

72.0

8.0 0.0

8

64.0

Live Birth Index (%) Day 0

LD 0

[f]

Mean

SD

N

98.4

4.4

8

100.0

0.0

9

99.1

2.6

9

91.1

26.7

9

Sex Ratio Day 0 (%)

LD 0

[a]

Mean

SD

N

51.0

14.6

8

58.6

19.3

9

49.2

10.5

9

48.9 7.0

9

[a] - Anova & Dunnett

[a1] - Anova & Dunnett (Rank)

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

TABLE 8. Summary of Body Weight Gains- Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Absolute Weight

Gain

(g)

1 → 8 [a]

Mean

SD

N

13.95

6.62 10

11.42

6.96 10

15.93

5.00 10

17.65

9.01 10

8 → 14 [a]

Mean

SD

N

19.24

7.53 10

20.20 10.63

10

18.93 11.54

10

18.78 11.40

10

14 → 22 [a]

Mean

SD

N

8.30 11.72

10

3.03

7.40 10

3.68

9.45 10

1.16

8.05 10

22 → 29 [a]

Mean

SD

N

23.01 10.03

10

20.68

7.82 10

16.59

8.12 10

18.74

8.04 10

29 → 36 [a]

Mean

SD

N

12.04

7.95 10

14.23

5.75 10

15.17

8.44 10

13.16

8.42 10

36 → 43 [a]

Mean

SD

N

9.11

5.56 10

12.43

7.58 10

12.88

8.25 10

15.22

4.08 10

43 → 50 [a]

Mean

SD

N

8.08

7.30 10

8.59

7.44 10

8.36

7.67 10

12.58

8.61 10

50 → 57 [a]

Mean

SD

N

9.98

6.32 10

12.47

6.90 10

12.13

8.32 10

13.49

7.76 10

1 → 57 [a]

Mean

SD

N

103.69 31.49

10

103.04 30.48

10

103.66 20.76

10

110.77 20.42

10

Total % Weight Gain

1 → 57 [a1]

Mean

SD

N

24.02

6.69 10

24.08

7.05 10

24.32

4.80 10

25.94

4.29 10

[a] - Anova & Dunnett

[a1] - Anova & Dunnett

TABLE 8 contd. Summary of Body Weight Gains- Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Absolute Weight

Gain

(g)

1 → 8 [p]

Mean

SD

N

17.01

10.30

5

16.82

12.76

5

8 → 14 [p]

Mean

SD

N

19.13 7.49

5

17.43 9.24

5

14 → 22 [p]

Mean

SD

N

11.07 5.85

5

17.46

12.20

5

22 → 29 [p1]

Mean

SD

N

11.07 2.06

5

12.28 7.04

5

29 → 36 [p]

Mean

SD

N

19.72 6.32

5

22.26 5.63

5

36 → 43 [p]

Mean

SD

N

12.65 6.48

5

11.04 2.42

5

43 → 50 [p]

Mean

SD

N

7.22

12.12

5

13.58 9.45

5

50 → 52 [p]

Mean

SD

N

5.78 6.56

5

1.56 5.83

5

1 → 52 [p]

Mean

SD

N

103.65

15.46

5

112.44

22.26

5

52 → 59 [p]

Mean

SD

N

9.59 7.27

5

16.13 3.72

5

[p] - T-Test: [G1RvsG4R]

[p1] – Wilcoxon (Rank): [G1RvsG4R]

TABLE 8 contd. Summary of Body Weight Gains- Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Absolute Weight

Gain

(g)

59 → 65 [p]

Mean

SD

N

4.92 3.59

5

5.20 7.19

5

52 → 65 [p]

Mean

SD

N

14.51 9.88

5

21.33 9.68

5

Total % Weight Gain

1 → 52 [p]

Mean

SD

N

24.18 3.84

5

26.52 5.33

5

52 → 65 [p]

Mean

SD

N

3.38 2.34

5

5.00 2.24

5

[p] - T-Test: [G1RvsG4R]

TABLE 9. Summary of Body Weight Gains- Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Absolute Weight

Gain

(g)

1 → 8 [a]

Mean

SD

N

10.16

6.81 10

5.95

5.73 10

9.96

6.57 10

9.68

3.36 10

8 → 14 [a1]

Mean

SD

N

6.36

2.93 10

6.75

6.45 10

10.82

8.02 10

10.00

3.80 10

1 → 14 [a]

Mean

SD

N

16.53

7.17 10

12.69

8.44 10

20.78

8.03 10

19.68

4.92 10

Total % Weight Gain

1 → 14 [a]

Mean

SD

N

6.53

2.75 10

5.11

3.53 10

8.32

3.24 10

7.92

1.99 10

[a] - Anova & Dunnett

[a1] - Anova & Dunnett (Rank)

TABLE 9 contd. Summary of Body Weight Gains- Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Absolute Weight

Gain

(g)

1 → 8 [p]

Mean

SD

N

8.84 6.63

5

6.72 7.97

5

8 → 14 [p1]

Mean

SD

N

5.24 6.33

5

7.62 6.80

5

14 → 22 [p1]

Mean

SD

N

9.01

6.34

5

8.91 3.52

5

22 → 29 [p1]

Mean

SD

N

5.73 7.25

5

5.64 6.08

5

29 → 36 [p1]

Mean

SD

N

5.54 7.76

5

6.15 7.05

5

36 → 43 [p1]

Mean

SD

N

3.46 6.49

5

13.49 8.61

5

43 → 50 [p1]

Mean

SD

N

5.22 8.48

5

1.17 3.52

5

50 → 52 [p2]

Mean

SD

N

0.36 5.75

5

-1.24 4.42

5

1 → 52 [p1]

Mean

SD

N

43.41

18.86

5

48.45

17.39

5

52 → 59 [p1]

Mean

SD

N

4.29 2.65

5

-0.54 *

3.28

5

[p] - T-Test (Log): [G1RvsG4R]

[p1] - T-Test: [G1RvsG4R]

{52-59} G1RvsG4R: * = p < 0.05

[p2] – Wilcoxon (Rank): [G1RvsG4R]

TABLE 9 contd. Summary of Body Weight Gains- Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Absolute Weight

Gain

(g)

59 → 65 [p]

Mean

SD

N

0.65 3.99

5

2.48 4.71

5

52 → 65 [p]

Mean

SD

N

4.94 4.74

5

1.93 5.93

5

Total % Weight Gain

1 → 52 [p]

Mean

SD

N

17.39 7.36

5

19.25 6.70

5

52 → 65 [p]

Mean

SD

N

1.90 1.90

5

0.76 2.36

5

[p] - T-Test: [G1RvsG4R]

TABLE 13. Summary of Maternal Body Weights and Weight Gain during Gestation Period

Sex: Female

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Body

Weight

(g)

0 [a]

Mean

SD

N

270.36

22.23

8

263.55

16.06

9

273.29

13.78

9

264.19

12.38

9

7 [a]

Mean

SD

N

303.65

27.01

8

299.53

17.84

9

306.96

15.75

9

294.18

11.91

9

14 [a]

Mean

SD

N

337.35

36.37

8

339.93

21.63

9

347.91

20.42

9

333.07

20.98

9

20 [a]

Mean

SD

N

429.82

36.96

8

425.81

26.05

9

434.19

28.09

9

412.67

34.54

9

Absolute Weight

Gain

(g)

0 → 7 [a]

Mean

SD

N

33.29 7.58

8

35.97 9.83

9

33.67 8.61

9

29.98

10.73

9

7 → 14 [a]

Mean

SD

N

33.71

12.69

8

40.40 6.54

9

40.95 7.03

9

38.89

13.08

9

14 → 20 [a]

Mean

SD

N

92.47

19.86

8

85.89

13.09

9

86.28

10.87

9

79.60

16.27

9

0 → 20 [a]

Mean

SD

N

159.46

21.58

8

162.26

20.03

9

160.90

18.23

9

148.48

35.92

9

[a] - Anova & Dunnett

TABLE 15. Summary of Maternal Body Weights and Weight Gain during Lactation Period

Sex: Female

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Body

Weight

(g)

0 [a]

Mean

SD

N

333.56

35.70

8

322.84

29.70

9

331.00

24.45

9

311.74

27.34

9

4 [a]

Mean

SD

N

344.88

30.18

8

338.75

24.48

9

343.22

23.14

9

325.59

25.09

9

13 [a]

Mean

SD

N

354.02

23.66

8

353.82

24.49

9

355.57

14.96

9

348.92

24.93

8

Absolute Weight

Gain

(g)

0 → 4 [a]

Mean

SD

N

11.33

15.80

8

15.91

11.98

9

12.22 7.60

9

13.85

13.70

9

4 → 13 [a]

Mean

SD

N

9.13 7.66

8

15.07 9.42

9

12.35

10.86

9

17.39

14.97

8

0 → 13 [a]

Mean

SD

N

20.46

17.06

8

30.98

16.16

9

24.57

15.54

9

34.51

12.98

8

[a] - Anova & Dunnett

APPENDIX 5. Individual Body Weights- Males

Sex: Male Day(s) Relative to Start Date

G1 0 mg/kg/day	Body Weight (g)
	1	8	14	22	29	36	43	50	57
Rx7481	458.65	473.74	497.26	499.57	525.33	548.32	562.28	582.20	597.22
Rx7482	391.94	397.36	408.50	415.60	434.48	447.21	461.17	466.32	472.78
Rx7483	446.19	462.23	488.89	486.16	511.65	524.19	529.10	531.18	541.56
Rx7484	402.95	408.95	431.09	429.94	452.32	458.94	462.37	460.19	478.69
Rx7485	441.77	449.71	470.92	477.12	511.01	513.23	517.06	517.40	522.18
Rx7486	476.38	495.33	527.59	530.83	550.59	571.09	575.87	581.84	598.41
Rx7487	403.25	412.26	421.42	440.78	456.01	460.89	465.64	483.73	485.51
Rx7488	414.78	435.76	449.42	471.55	477.16	481.44	500.54	509.54	517.98
Rx7489	428.43	443.75	464.52	460.55	481.24	491.16	500.40	513.09	514.71
Rx7490	431.30	456.03	467.86	498.39	540.81	564.48	577.61	587.30	603.52
G2 111 mg/kg/day
Rx7501	446.01	458.90	483.14	490.80	504.37	523.09	533.41	548.29	567.87
Rx7502	438.07	460.97	486.42	493.91	513.35	533.66	537.85	557.79	574.75
Rx7503	388.95	391.01	395.84	389.54	402.78	417.41	427.24	422.81	437.98
Rx7504	453.65	472.86	492.31	494.82	509.40	520.56	526.81	528.10	543.28
Rx7505	473.71	483.24	504.61	495.34	534.14	540.52	565.01	574.30	571.87
Rx7506	399.58	416.80	436.49	439.18	459.98	478.89	482.71	498.46	508.60
Rx7507	426.74	432.72	452.23	456.29	484.42	505.17	529.07	540.87	551.75
Rx7508	419.67	426.99	452.53	460.86	483.04	487.43	496.34	499.12	514.34
Rx7509	409.57	412.94	415.11	412.79	429.24	440.93	459.00	468.23	472.96
Rx7510	413.27	426.96	466.67	482.13	501.77	517.10	531.63	536.97	556.23
G3 333 mg/kg/day
Rx7521	453.70	465.34	485.32	493.19	520.14	531.52	547.26	548.84	573.53
Rx7522	411.69	425.80	439.07	456.26	463.57	471.32	481.38	487.11	490.76
Rx7523	392.78	415.35	435.01	434.20	461.12	468.82	487.68	489.10	506.58
Rx7524	397.33	409.29	422.29	430.04	446.00	456.07	464.93	471.35	475.47
Rx7525	446.87	471.69	486.26	471.72	496.71	515.46	529.27	538.91	557.47
Rx7526	462.00	472.99	491.98	489.32	506.51	523.40	515.55	532.17	541.65
Rx7527	429.11	446.56	496.92	500.41	516.27	527.82	545.41	558.97	565.01
Rx7528	429.94	449.42	462.25	473.98	476.66	512.89	534.77	531.13	548.11
Rx7529	423.86	439.08	455.81	451.16	467.94	480.69	496.64	518.40	538.12
Rx7530	419.00	430.01	439.94	451.41	462.67	481.26	495.17	505.66	506.20
G4 1000 mg/kg/day
Rx7541	421.60	445.09	461.02	469.62	475.63	500.72	520.18	524.79	534.75
Rx7542	477.90	498.02	515.55	526.08	553.62	577.47	596.77	608.77	617.69
Rx7543	452.22	449.39	452.38	463.41	491.67	496.91	511.53	518.77	537.00
Rx7544	410.54	436.95	456.60	445.10	466.58	483.81	499.03	512.83	521.89
Rx7545	445.93	465.22	484.24	487.54	512.00	520.98	528.57	557.53	576.58
Rx7546	397.57	409.10	446.60	442.18	449.93	451.53	462.22	467.90	469.54
Rx7547	404.05	416.57	450.62	457.02	482.26	489.24	505.47	505.30	520.07
Rx7548	400.20	415.83	442.04	435.00	452.73	464.94	482.68	502.49	513.68
Rx7549	425.13	447.93	458.03	452.02	468.87	476.86	496.31	514.91	526.57
Rx7550	429.05	456.57	461.37	462.12	474.22	496.65	508.53	523.77	554.15

APPENDIX 5 contd. Individual Body Weights- Males

Sex: Male Day(s) Relative to Start Date

G1R 0 mg/kg/day				Body Weight (g)
	1	8	14	22	29	36	43	50	52
Rx7561	437.95	458.17	479.66	491.69	502.85	523.74	533.29	547.01	548.55
Rx7562	409.60	434.07	445.45	449.45	460.04	474.06	486.44	486.04	493.24
Rx7563	452.19	477.50	488.46	496.22	505.70	531.26	542.93	541.46	547.70
Rx7564	432.17	432.28	458.61	470.47	480.06	505.87	512.09	537.70	536.03
Rx7565	414.80	429.74	455.22	474.92	489.46	501.77	525.20	523.86	539.45

Sex: Male Day(s) Relative to Start Date

G4R 1000 mg/kg/day				Body Weight (g)
	1	8	14	22	29	36	43	50	52
Rx7571	415.42	433.52	461.34	490.66	494.96	516.88	524.43	530.57	540.77
Rx7572	432.70	462.19	466.07	465.95	472.24	502.77	514.46	517.40	520.40
Rx7573	393.47	413.11	431.95	452.64	467.23	482.06	491.71	510.98	508.25
Rx7574	437.33	432.71	455.83	466.40	480.99	501.78	514.93	528.37	530.50
Rx7575	445.41	466.91	480.42	507.26	528.91	552.16	565.31	591.40	586.61

G4R 1000 mg/kg/day	Body Weight (g)
	59	65
Rx7571	557.19	573.52
Rx7572	532.60	531.37
Rx7573	521.16	521.10
Rx7574	548.39	551.22
Rx7575	607.86	615.98

G1R 0 mg/kg/day	Body Weight (g)
	59	65
Rx7561	552.51	554.37
Rx7562	494.89	496.97
Rx7563	556.74	561.73
Rx7564	549.76	560.52
Rx7565	559.01	563.91

APPENDIX 6. Individual Body Weights- Females

Sex: Female Day(s) Relative to Start Date

	Body Weight (g)
G1, 0 mg/kg/day	1	8	14
Rx7491	256.63	273.57	279.45
Rx7492	247.42	255.36	267.34
Rx7493	238.44	248.77	258.25
Rx7494	257.33	268.47	270.32
Rx7495	263.86	278.72	286.17
Rx7496	232.47	234.66	242.54
Rx7497	256.77	257.79	262.27
Rx7498	247.57	249.13	253.61
Rx7499	250.26	266.63	272.53
Rx7500	269.84	289.13	293.37
G2, 111 mg/kg/day
Rx7511	267.01	276.89	287.65
Rx7512	259.07	255.10	267.18
Rx7513	239.13	246.17	242.14
Rx7514	273.88	284.25	285.03
Rx7515	255.27	256.84	263.89
Rx7516	228.65	233.89	249.31
Rx7517	235.88	251.87	259.67
Rx7518	254.22	255.66	254.56
Rx7519	252.43	255.21	260.95
Rx7520	248.61	257.74	270.70
G3, 333 mg/kg/day
Rx7531	253.37	261.27	282.93
Rx7532	258.14	268.53	275.96
Rx7533	268.53	280.96	293.15
Rx7534	229.82	239.49	247.92
Rx7535	263.20	272.41	278.78
Rx7536	248.63	256.46	271.42
Rx7537	240.86	257.54	264.13
Rx7538	261.43	261.75	263.40
Rx7539	237.99	261.08	263.78
Rx7540	251.31	253.39	279.63
G4, 1000 mg/kg/day
Rx7551	266.78	278.20	288.01
Rx7552	249.52	260.36	266.54
Rx7553	251.15	259.80	274.63
Rx7554	258.90	272.27	278.87
Rx7555	227.79	234.41	243.94
Rx7556	258.39	261.47	269.80
Rx7557	254.66	262.62	279.41
Rx7558	246.28	259.78	273.12
Rx7559	230.13	243.05	251.98
Rx7560	247.46	255.93	261.57

APPENDIX 6 contd. Individual Body Weights- Females

Sex: Female Day(s) Relative to Start Date

G1R 0 mg/kg/day				Body Weight (g)
	1	8	14	22	29	36	43	50	52
Rx7566	258.08	273.95	275.22	289.60	292.77	310.00	304.90	314.71	311.43
Rx7567	250.56	256.44	270.20	282.42	277.24	273.79	285.85	282.58	281.31
Rx7568	253.11	269.20	275.71	289.21	301.62	308.60	314.27	323.93	324.23
Rx7569	261.48	265.43	262.60	262.23	274.27	275.20	280.18	275.80	285.96
Rx7570	225.89	228.30	235.81	241.15	247.34	253.33	253.04	267.34	263.22

G1R 0 mg/kg/day	Body Weight (g)
	59	65
Rx7566	317.49	318.18
Rx7567	289.00	289.56
Rx7568	325.12	327.63
Rx7569	289.75	294.90
Rx7570	266.25	260.59

Sex: Female Day(s) Relative to Start Date

G4R 1000 mg/kg/day				Body Weight (g)
	1	8	14	22	29	36	43	50	52
Rx7576	258.54	258.88	260.67	268.24	266.00	277.23	293.27	292.51	287.94
Rx7577	251.98	252.06	270.78	283.13	293.61	289.71	308.86	310.91	310.06
Rx7578	256.84	273.92	277.77	287.30	295.72	309.27	327.75	327.35	322.14
Rx7579	236.83	239.48	243.87	247.43	247.96	255.53	253.87	260.79	266.70
Rx7580	250.46	263.90	273.26	284.78	295.77	298.05	313.47	311.53	310.07

G4R 1000 mg/kg/day	Body Weight (g)
	59	65
Rx7576	291.25	292.78
Rx7577	309.59	306.23
Rx7578	316.75	320.55
Rx7579	265.18	266.08
Rx7580	311.43	320.94

APPENDIX 7. Individual Body Weight Gain- Males

Sex: Male Day(s) Relative to Start Date

G1 0 mg/kg/day	Absolute Weight Gain (g)									Total % Weight Gain
	1 → 8	8 → 14	14 → 22	22 → 29	29 → 36	36 → 43	43 → 50	50 → 57	1 → 57	1 → 57
Rx7481	15.09	23.52	2.31	25.76	22.99	13.96	19.92	15.02	138.57	30.21
Rx7482	5.42	11.14	7.10	18.88	12.73	13.96	5.15	6.46	80.84	20.63
Rx7483	16.04	26.66	-2.73	25.49	12.54	4.91	2.08	10.38	95.37	21.37
Rx7484	6.00	22.14	-1.15	22.38	6.62	3.43	-2.18	18.50	75.74	18.80
Rx7485	7.94	21.21	6.20	33.89	2.22	3.83	0.34	4.78	80.41	18.20
Rx7486	18.95	32.26	3.24	19.76	20.50	4.78	5.97	16.57	122.03	25.62
Rx7487	9.01	9.16	19.36	15.23	4.88	4.75	18.09	1.78	82.26	20.40
Rx7488	20.98	13.66	22.13	5.61	4.28	19.10	9.00	8.44	103.20	24.88
Rx7489	15.32	20.77	-3.97	20.69	9.92	9.24	12.69	1.62	86.28	20.14
Rx7490	24.73	11.83	30.53	42.42	23.67	13.13	9.69	16.22	172.22	39.93
G2 111 mg/kg/day
Rx7501	12.89	24.24	7.66	13.57	18.72	10.32	14.88	19.58	121.86	27.32
Rx7502	22.90	25.45	7.49	19.44	20.31	4.19	19.94	16.96	136.68	31.20
Rx7503	2.06	4.83	-6.30	13.24	14.63	9.83	-4.43	15.17	49.03	12.61
Rx7504	19.21	19.45	2.51	14.58	11.16	6.25	1.29	15.18	89.63	19.76
Rx7505	9.53	21.37	-9.27	38.80	6.38	24.49	9.29	-2.43	98.16	20.72
Rx7506	17.22	19.69	2.69	20.80	18.91	3.82	15.75	10.14	109.02	27.28
Rx7507	5.98	19.51	4.06	28.13	20.75	23.90	11.80	10.88	125.01	29.29
Rx7508	7.32	25.54	8.33	22.18	4.39	8.91	2.78	15.22	94.67	22.56
Rx7509	3.37	2.17	-2.32	16.45	11.69	18.07	9.23	4.73	63.39	15.48
Rx7510	13.69	39.71	15.46	19.64	15.33	14.53	5.34	19.26	142.96	34.59
G3 333 mg/kg/day
Rx7521	11.64	19.98	7.87	26.95	11.38	15.74	1.58	24.69	119.83	26.41
Rx7522	14.11	13.27	17.19	7.31	7.75	10.06	5.73	3.65	79.07	19.21
Rx7523	22.57	19.66	-0.81	26.92	7.70	18.86	1.42	17.48	113.80	28.97
Rx7524	11.96	13.00	7.75	15.96	10.07	8.86	6.42	4.12	78.14	19.67
Rx7525	24.82	14.57	-14.54	24.99	18.75	13.81	9.64	18.56	110.60	24.75
Rx7526	10.99	18.99	-2.66	17.19	16.89	-7.85	16.62	9.48	79.65	17.24
Rx7527	17.45	50.36	3.49	15.86	11.55	17.59	13.56	6.04	135.90	31.67
Rx7528	19.48	12.83	11.73	2.68	36.23	21.88	-3.64	16.98	118.17	27.49
Rx7529	15.22	16.73	-4.65	16.78	12.75	15.95	21.76	19.72	114.26	26.96
Rx7530	11.01	9.93	11.47	11.26	18.59	13.91	10.49	0.54	87.20	20.81
G4 1000 mg/kg/day
Rx7541	23.49	15.93	8.60	6.01	25.09	19.46	4.61	9.96	113.15	26.84
Rx7542	20.12	17.53	10.53	27.54	23.85	19.30	12.00	8.92	139.79	29.25
Rx7543	-2.83	2.99	11.03	28.26	5.24	14.62	7.24	18.23	84.78	18.75
Rx7544	26.41	19.65	-11.50	21.48	17.23	15.22	13.80	9.06	111.35	27.12
Rx7545	19.29	19.02	3.30	24.46	8.98	7.59	28.96	19.05	130.65	29.30
Rx7546	11.53	37.50	-4.42	7.75	1.60	10.69	5.68	1.64	71.97	18.10
Rx7547	12.52	34.05	6.40	25.24	6.98	16.23	-0.17	14.77	116.02	28.71
Rx7548	15.63	26.21	-7.04	17.73	12.21	17.74	19.81	11.19	113.48	28.36
Rx7549	22.80	10.10	-6.01	16.85	7.99	19.45	18.60	11.66	101.44	23.86
Rx7550	27.52	4.80	0.75	12.10	22.43	11.88	15.24	30.38	125.10	29.16

APPENDIX 7 contd. Individual Body Weight Gain- Males

Sex: Male Day(s) Relative to Start Date

G1R 0 mg/kg/day				Absolute Weight Gain (g)
	1 → 8	8 → 14	14 → 22	22 → 29	29 → 36	36 → 43	43 → 50	50 → 52	1 → 52	52 → 59
Rx7561	20.22	21.49	12.03	11.16	20.89	9.55	13.72	1.54	110.60	3.96
Rx7562	24.47	11.38	4.00	10.59	14.02	12.38	-0.40	7.20	83.64	1.65
Rx7563	25.31	10.96	7.76	9.48	25.56	11.67	-1.47	6.24	95.51	9.04
Rx7564	0.11	26.33	11.86	9.59	25.81	6.22	25.61	-1.67	103.86	13.73
Rx7565	14.94	25.48	19.70	14.54	12.31	23.43	-1.34	15.59	124.65	19.56

G1R 0 mg/kg/day	Absolute Weight Gain (g)		Total % Weight Gain
	59 → 65	52 → 65	1 → 52	52 → 65
Rx7561	1.86	5.82	25.25	1.33
Rx7562	2.08	3.73	20.42	0.91
Rx7563	4.99	14.03	21.12	3.10
Rx7564	10.76	24.49	24.03	5.67
Rx7565	4.90	24.46	30.05	5.90

Sex: Male Day(s) Relative to Start Date

G4R 1000 mg/kg/day					Absolute Weight Gain (g)
	1 → 8	8 → 14	14 → 22	22 → 29	29 → 36	36 → 43	43 → 50	50 → 52	1 → 52	52 → 59
Rx7571	18.10	27.82	29.32	4.30	21.92	7.55	6.14	10.20	125.35	16.42
Rx7572	29.49	3.88	-0.12	6.29	30.53	11.69	2.94	3.00	87.70	12.20
Rx7573	19.64	18.84	20.69	14.59	14.83	9.65	19.27	-2.73	114.78	12.91
Rx7574	-4.62	23.12	10.57	14.59	20.79	13.15	13.44	2.13	93.17	17.89
Rx7575	21.50	13.51	26.84	21.65	23.25	13.15	26.09	-4.79	141.20	21.25

G4R 1000 mg/kg/day	Absolute Weight Gain (g)		Total % Weight Gain
	59 → 65	52 → 65	1 → 52	52 → 65
Rx7571	16.33	32.75	30.17	7.88
Rx7572	-1.23	10.97	20.27	2.54
Rx7573	-0.06	12.85	29.17	3.27
Rx7574	2.83	20.72	21.30	4.74
Rx7575	8.12	29.37	31.70	6.59

APPENDIX 8. Individual Body Weight Gain- Females

Sex: Female Day(s) Relative to Start Date

	Absolute Weight Gains(g)			Total % Weight Gain
G1, 0 mg/kg/day	1 → 8	8 → 14	1 → 14	1 → 14
Rx7491	16.94	5.88	22.82	8.89
Rx7492	7.94	11.98	19.92	8.05
Rx7493	10.33	9.48	19.81	8.31
Rx7494	11.14	1.85	12.99	5.05
Rx7495	14.86	7.45	22.31	8.46
Rx7496	2.19	7.88	10.07	4.33
Rx7497	1.02	4.48	5.50	2.14
Rx7498	1.56	4.48	6.04	2.44
Rx7499	16.37	5.90	22.27	8.90
Rx7500	19.29	4.24	23.53	8.72
G2, 111 mg/kg/day
Rx7511	9.88	10.76	20.64	7.73
Rx7512	-3.97	12.08	8.11	3.13
Rx7513	7.04	-4.03	3.01	1.26
Rx7514	10.37	0.78	11.15	4.07
Rx7515	1.57	7.05	8.62	3.38
Rx7516	5.24	15.42	20.66	9.04
Rx7517	15.99	7.80	23.79	10.09
Rx7518	1.44	-1.10	0.34	0.13
Rx7519	2.78	5.74	8.52	3.38
Rx7520	9.13	12.96	22.09	8.89
G3, 333 mg/kg/day
Rx7531	7.90	21.66	29.56	11.67
Rx7532	10.39	7.43	17.82	6.90
Rx7533	12.43	12.19	24.62	9.17
Rx7534	9.67	8.43	18.10	7.88
Rx7535	9.21	6.37	15.58	5.92
Rx7536	7.83	14.96	22.79	9.17
Rx7537	16.68	6.59	23.27	9.66
Rx7538	0.32	1.65	1.97	0.75
Rx7539	23.09	2.70	25.79	10.84
Rx7540	2.08	26.24	28.32	11.27
G4, 1000 mg/kg/day
Rx7551	11.42	9.81	21.23	7.96
Rx7552	10.84	6.18	17.02	6.82
Rx7553	8.65	14.83	23.48	9.35
Rx7554	13.37	6.6	19.97	7.71
Rx7555	6.62	9.53	16.15	7.09
Rx7556	3.08	8.33	11.41	4.42
Rx7557	7.96	16.79	24.75	9.72
Rx7558	13.50	13.34	26.84	10.9
Rx7559	12.92	8.93	21.85	9.49
Rx7560	8.47	5.64	14.11	5.70

APPENDIX 8 contd. Individual Body Weight Gain- Females

Sex: Female Day(s) Relative to Start Date

G1R 0 mg/kg/day				Absolute Weight Gain (g)
	1 → 8	8 → 14	14 → 22	22 → 29	29 → 36	36 → 43	43 → 50	50 → 52	1 → 52	52 → 59
Rx7566	15.87	1.27	14.38	3.17	17.23	-5.10	9.81	-3.28	53.35	6.06
Rx7567	5.88	13.76	12.22	-5.18	-3.45	12.06	-3.27	-1.27	30.75	7.69
Rx7568	16.09	6.51	13.50	12.41	6.98	5.67	9.66	0.30	71.12	0.89
Rx7569	3.95	-2.83	-0.37	12.04	0.93	4.98	-4.38	10.16	24.48	3.79
Rx7570	2.41	7.51	5.34	6.19	5.99	-0.29	14.30	-4.12	37.33	3.03

G1R 0 mg/kg/day	Absolute Weight Gain (g)		Total % Weight Gain
	59 → 65	52 → 65	1 → 52	52 → 65
Rx7566	0.69	6.75	20.67	2.62
Rx7567	0.56	8.25	12.27	3.29
Rx7568	2.51	3.40	28.10	1.34
Rx7569	5.15	8.94	9.36	3.42
Rx7570	-5.66	-2.63	16.53	-1.16

Sex: Female Day(s) Relative to Start Date

G4R 1000 mg/kg/day					Absolute Weight Gain (g)
	1 → 8	8 → 14	14 → 22	22 → 29	29 → 36	36 → 43	43 → 50	50 → 52	1 → 52	52 → 59
Rx7576	0.34	1.79	7.57	-2.24	11.23	16.04	-0.76	-4.57	29.40	3.31
Rx7577	0.08	18.72	12.35	10.48	-3.90	19.15	2.05	-0.85	58.08	-0.47
Rx7578	17.08	3.85	9.53	8.42	13.55	18.48	-0.40	-5.21	65.30	-5.39
Rx7579	2.65	4.39	3.56	0.53	7.57	-1.66	6.92	5.91	29.87	-1.52
Rx7580	13.44	9.36	11.52	10.99	2.28	15.42	-1.94	-1.46	59.61	1.36

G4R 1000 mg/kg/day	Absolute Weight Gain (g)		Total % Weight Gain
	59 → 65	52 → 65	1 → 52	52 → 65
Rx7576	1.53	4.84	11.37	1.87
Rx7577	-3.36	-3.83	23.05	-1.52
Rx7578	3.80	-1.59	25.42	-0.62
Rx7579	0.90	-0.62	12.61	-0.26
Rx7580	9.51	10.87	23.80	4.34

Appendix 23, TABLE 1. Summary of Haematology and Coagulation Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

RBC

(10^12/L)

15 [a]

Mean

SD

N

%Diff

8.85

0.64

5

-

8.57

0.20

5 -3.17

8.97

0.42

5

1.38

8.92

0.64

5

0.86

HGB

(g/L)

15 [a]

Mean

SD

N

%Diff

156

6

5

-

154

6

5

-2

164

7

5

158

5

1

HCT

(L/L)

15 [a]

Mean

SD

N

%Diff

0.494

0.025

5

.

0.487

0.010

5 -1.377

0.510

0.015

5

3.239

0.491

0.015

5 -0.526

MCV

(fL)

15 [a]

Mean

SD

N

%Diff

55.9

1.2

5

-

56.9

1.5

5

1.8

56.9

1.5

5

1.8

55.3

2.7

5 -1.1

MCH

(pg)

15 [a]

Mean

SD

N

%Diff

17.7

0.9

5

-

17.9

0.5

5

1.1

18.4

0.5

5

3.8

17.7

1.0

5

0.1

MCHC

(g/L)

15 [a]

Mean

SD

N

%Diff

316

11

5

-

315

10 5

0

323

7

5

2

321

7

5

1

Retic A (10^12/L)

15 [a]

Mean

SD

N

%Diff

0.197 0.028

5

-

0.202 0.043

5

2.642

0.201 0.009

5

1.931

0.238 0.028

5

21.037

[a] - Anova & Dunnett

Appendix 23, TABLE 1 contd. Summary of Haematology and Coagulation Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

PLT

(10^9/L)

15 [a]

Mean

SD

N

%Diff

1893

89

5

-

1910

144

5

1

1750

169

5

-8

1838

205

5

-3

WBC

(10^9/L)

15 [a1]

Mean

SD

N

%Diff

10.35

1.14

5

-

11.64

4.69

5

12.49

10.19

0.15

5 -1.49

9.40

2.28

5 -9.14

Neut A (10^9/L)

15 [a]

Mean

SD

N

%Diff

2.77

0.65

5

-

2.95

1.02

5

6.51

2.74

0.64

5 -0.87

2.63

0.35

5 -4.84

Lymp A

(10^9/L)

15 [a1]

Mean

SD

N

%Diff

6.79

1.00

5

-

7.98

3.82

5

17.43

6.85

0.74

5

0.82

6.20

2.26

5 -8.80

Mono A

(10^9/L)

15 [a]

Mean

SD

N

%Diff

0.25

0.04

5

-

0.25

0.07

5

2.42

0.25

0.05

5

0.00

0.22

0.07

5 -12.10

Baso A (10^9/L)

15 [a1]

Mean

SD

N

%Diff

0.03

0.01

5

-

0.04

0.03

5

37.50

0.03

0.00

5 -6.25

0.03

0.02

5 -18.75

Eosi A (10^9/L)

15 [a]

Mean

SD

N

%Diff

0.16 0.04

5

-

0.11 0.02

5 -28.75

0.16 0.04

5 -1.25

0.15 0.07

5 -3.75

Appendix 23, TABLE 1 contd. Summary of Haematology and Coagulation Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Prothrombin

Time

(seconds)

15 [a]

Mean

SD

N

%Diff

18.9

1.0

5

.

19.0

0.6

5

0.3

19.8

0.7

5

4.3

19.3

1.0

5

1.9

APTT

(seconds)

15 [a]

Mean

SD

N

%Diff

10.8

1.3

5

-

10.5

2.7

5 -2.8

11.7

1.1

5

8.7

10.6

2.8

5 -1.9

Appendix 23, TABLE 2. Summary of Haematology and Coagulation Parameters – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

RBC

(10^12/L)

66 [p]

Mean

SD

N

%Diff

9.00

0.27

5

-

9.07

0.23

5

0.78

HGB

(g/L)

66 [p]

Mean

SD

N

%Diff

156

5

-

161

3

5

3

HCT

(L/L)

66 [p]

Mean

SD

N

%Diff

0.486

0.019

5

.

0.500

0.006

5

3.007

MCV

(fL)

66 [p]

Mean

SD

N

%Diff

54.0

2.6

5

-

55.2

1.5

5

2.2

MCH

(pg)

66 [p]

Mean

SD

N

%Diff

17.4

0.7

5

-

17.8

0.7

5

2.6

MCHC

(g/L)

66 [p]

Mean

SD

N

%Diff

321

7

5

-

323

6

5

0

Retic A (10^12/L)

66 [p]

Mean

SD

N

%Diff

0.197 0.041

5

-

0.193 0.019

5 -2.432

[p] - T-Test: [G1RvsG4R]

Appendix 23, TABLE 2 contd. Summary of Haematology and Coagulation Parameters – Recovery Males

Sex: Male

Da

y(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

PLT

(10^9/L)

66 [p]

Mean

SD

N

%Diff

1614

83

5

-

1654

80 5

2

WBC

(10^9/L)

66 [p1]

Mean

SD

N

%Diff

9.12

1.66

5

-

9.16

1.23

5

0.48

Neut A (10^9/L)

66 [p1]

Mean

SD

N

%Diff

2.33

0.58

5

-

2.07

0.68

5 -10.91

Lymp A

(10^9/L)

66 [p1]

Mean

SD

N

%Diff

6.30

1.18

5

-

6.60

0.91

5

4.83

Mono A

(10^9/L)

66 [p1]

Mean

SD

N

%Diff

0.27

0.10

5

-

0.26

0.14

5 -2.24

Baso A (10^9/L)

66 [p1]

Mean

SD

N

%Diff

0.03

0.01

5

-

0.02

0.01

5 -28.57

Eosi A (10^9/L)

66 [p1]

Mean

SD

N

%Diff

0.14 0.06

5

-

0.17 0.06

5

16.67

[p] - Wilcoxon(Rank): [G1RvsG4R]

[p1] - T-Test: [G1RvsG4R]

Appendix 23, TABLE 2 contd. Summary of Haematology and Coagulation Parameters – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Prothrombin

Time

(seconds)

66 [p]

Mean

SD

N

%Diff

20.8

1.4

5

.

18.4 * 1.1

5 -11.6

APTT

(seconds)

66 [p1]

Mean

SD

N

%Diff

11.1

0.3

5

-

11.0

1.7

5 -0.9

[p] - T-Test: [G1RvsG4R]

G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05 [p1] - Wilcoxon(Rank): [G1RvsG4R]

Appendix 23, TABLE 3. Summary of Haematology and Coagulation Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

RBC

(10^12/L)

15 [a]

Mean

SD

N

%Diff

8.14

0.31

5

-

8.11

0.21

5 -0.34

8.23

0.26

5

1.15

8.12

0.07

5 -0.20

HGB

(g/L)

15 [a1]

Mean

SD

N

%Diff

152

7

5

-

153

6

5

1

153

5

0

153

4

5

0

HCT

(L/L)

15 [a]

Mean

SD

N

%Diff

0.478

0.011

5

.

0.484

0.018

5

1.129

0.481

0.010

5

0.585

0.487

0.003

5

1.839

MCV

(fL)

15 [a1]

Mean

SD

N

%Diff

58.8

1.7

5

-

59.6

1.0

5

1.4

58.5

1.8

5 -0.5

60.0

0.5

5

2.0

MCH

(pg)

15 [a1]

Mean

SD

N

%Diff

18.7

0.6

5

-

18.9

0.4

5

1.2

18.5

0.5

5 -1.0

18.8

0.3

5

0.3

MCHC

(g/L)

15 [a1]

Mean

SD

N

%Diff

318

8

5

-

317

4

5

0

317

5

0

313

6

5

-2

Retic A (10^12/L)

15 [a1]

Mean

SD

N

%Diff

0.193 0.032

5

-

0.220 0.042

5

14.330

0.227 0.043

5

17.653

0.234 0.027

5

21.703

[a] - Anova & Dunnett(Rank)

[a1] - Anova & Dunnett

Appendix 23, TABLE 3 contd. Summary of Haematology and Coagulation Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

PLT

(10^9/L)

15 [a]

Mean

SD

N

%Diff

1961

112

5

-

2026

272

5

3

2109

170

5

8

1881

130

5

-4

WBC

(10^9/L)

15 [a]

Mean

SD

N

%Diff

7.31

2.12

5

-

6.52

2.25

5 -10.75

7.59

2.81

5

3.89

7.26

0.81

5 -0.66

Neut A (10^9/L)

15 [a]

Mean

SD

N

%Diff

1.43

0.30

5

-

1.29

0.25

5 -9.68

1.51

0.69

5

6.17

1.86

0.44

5

30.15

Lymp A

(10^9/L)

15 [a]

Mean

SD

N

%Diff

5.41

2.01

5

-

4.91

1.97

5 -9.13

5.73

2.28

5

5.92

4.99

0.48

5 -7.69

Mono A

(10^9/L)

15 [a]

Mean

SD

N

%Diff

0.19

0.06

5

-

0.13

0.06

5 -31.18

0.13

0.05

5 -29.03

0.15

0.04

5 -18.28

Baso A (10^9/L)

15 [a]

Mean

SD

N

%Diff

0.02

0.01

5

-

0.02

0.01

5 -11.11

0.02

0.01

5 -11.11

0.02

0.01

5

11.11

Eosi A (10^9/L)

15 [a1]

Mean

SD

N

%Diff

0.12 0.06

5

-

0.09 0.04

5 -22.95

0.09 0.03

5 -24.59

0.12 0.05

5 -3.28

[a] - Anova & Dunnett

[a1] - Anova & Dunnett(Log)

Appendix 23, TABLE 3 contd. Summary of Haematology and Coagulation Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Prothrombin

Time

(seconds)

15 [a]

Mean

SD

N

%Diff

19.0

1.2

5

.

19.9

0.6

5

5.0

19.3

1.2

5

1.5

20.8

0.9

5

9.4

APTT

(seconds)

15 [a]

Mean

SD

N

%Diff

9.9

3.1

5

-

12.3

1.9

5

23.9

10.6

2.3

5

6.4

9.7

2.3

5 -2.0

Appendix 23, TABLE 4. Summary of Haematology and Coagulation Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

RBC

(10^12/L)

66 [p]

Mean

SD

N

%Diff

8.22

0.25

5

-

8.31

0.18

5

1.07

HGB

(g/L)

66 [p]

Mean

SD

N

%Diff

152

3

5

-

155

7

5

2

HCT

(L/L)

66 [p]

Mean

SD

N

%Diff

0.475

0.015

5

.

0.487

0.023

5

2.612

MCV

(fL)

66 [p]

Mean

SD

N

%Diff

57.8

2.2

5

-

58.6

1.8

5

1.5

MCH

(pg)

66 [p]

Mean

SD

N

%Diff

18.5

0.7

5

-

18.7

0.6

5

0.9

MCHC

(g/L)

66 [p]

Mean

SD

N

%Diff

320

6

5

-

319

3

5

0

Retic A (10^12/L)

66 [p]

Mean

SD

N

%Diff

0.169 0.021

5

-

0.175 0.037

5

3.306

[p] - T-Test: [G1RvsG4R]

Appendix 23, TABLE 4 contd. Summary of Haematology and Coagulation Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

PLT

(10^9/L)

66 [p]

Mean

SD

N

%Diff

1671

186

5

-

1760

98 5

5

WBC

(10^9/L)

66 [p]

Mean

SD

N

%Diff

6.26

1.23

5

-

5.28

0.81

5 -15.62

Neut A (10^9/L)

66 [p]

Mean

SD

N

%Diff

1.15

0.48

5

-

0.95

0.18

5 -17.53

Lymp A

(10^9/L)

66 [p]

Mean

SD

N

%Diff

4.79

0.91

5

-

4.09

0.63

5 -14.61

Mono A

(10^9/L)

66 [p]

Mean

SD

N

%Diff

0.09

0.02

5

-

0.09

0.04

5

4.44

Baso A (10^9/L)

66 [p]

Mean

SD

N

%Diff

0.01

5

-

0.01

5 -28.57

Eosi A (10^9/L)

66 [p1]

Mean

SD

N

%Diff

0.19 0.19

5

-

0.11 0.06

5 -41.67

[p] - T-Test: [G1RvsG4R]

[p1] T-Test(Log): [G1RvsG4R]

Appendix 23, TABLE 4 contd. Summary of Haematology and Coagulation Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Prothrombin

Time

(seconds)

66 [p]

Mean

SD

N

%Diff

19.6

0.7

5

.

18.0 * 0.8

5 -8.2

APTT

(seconds)

66 [p]

Mean

SD

N

%Diff

10.8

0.2

5

-

9.1 * 0.6

5 -15.2

[p] - T-Test: [G1RvsG4R]

{Prothrombin} G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05

{APTT} G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05

Appendix 23, TABLE 5. Summary of Clinical Chemistry Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Glu (mmol/L)

15 [a]

Mean

SD

N

%Diff

4.72

0.50

5

.

5.33

0.66

5

12.84

5.68

1.51

5

20.25

6.28

1.03

5

33.09

BUN

(mmol/L)

15 [a1]

Mean

SD

N

%Diff

4.86

0.39

5

.

4.13 * 0.32

5 -14.99

4.61

0.17

5 -5.11

4.18 * 0.34

5 -13.96

Creat (µmol/L)

15 [a1]

Mean

SD

N

%Diff

34 2

5

-

36 4

5

6

34 6

5

-1

38 4

5 11

AST

(U/L)

15 [a2]

Mean

SD

N

%Diff

82

12

5

.

85 7

5

4

85 7

5

4

89

20 5

8

ALT

(U/L)

15 [a1]

Mean

SD

N

%Diff

30 7

5

.

34 4

5 15

37 8

5 24

32 6

5

8

GGT

(U/L)

15 [a]

Mean

SD

N

%Diff

4

1

5

.

5 *

0

5 39

5 *

0

5 33

5

1

5 28

Alp (U/L)

15 [a]

Mean

SD

N

%Diff

94

16

5

.

114

21

5 20

97 7

5

3

99

30 5

5

[a] - Anova & Dunnett(Rank): *: Statistically significant difference from the control group at p < 0.05

[a1] - Anova & Dunnett: *: Statistically significant difference from the control group at p < 0.05 [a2] Anova & Dunnett(Log)

Appendix 23, TABLE 5 contd. Summary of Clinical Chemistry Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

CK

(U/L)

15 [a]

Mean

SD

N

%Diff

249

57

5

.

207

35

5 -17

410

233

5 65

516 *

205

5 107

T.Bil (µmol/L)

15 [a1]

Mean

SD

N

%Diff

1.80

0.38

5

.

1.80

0.82

5

0.11

1.32

0.54

5 -26.97

1.83

0.39

5

1.33

T.Chol (mmol/L)

15 [a]

Mean

SD

N

%Diff

2.12

0.46

5

.

2.07

0.21

5 -2.45

2.11

0.12

5 -0.85

2.01

0.16

5 -5.18

BA

(µmol/L)

15 [a1]

Mean

SD

N

%Diff

19.2

3.0

5

.

17.7

2.5

5 -7.5

19.0

4.2

5 -1.0

18.3

8.3

5 -4.3

Trig (mmol/L)

15 [a1]

Mean

SD

N

%Diff

0.76

0.20

5

.

0.74

0.28

5 -2.63

0.63

0.29

5 -16.58

0.70

0.25

5 -7.63

T.Pro (g/L)

15 [a1]

Mean

SD

N

%Diff

66.3

4.1

5

-

67.1

2.9

5

1.1

67.7

2.3

5

2.1

65.6

1.0

5 -1.1

ALB

(g/L)

15 [a1]

Mean

SD

N

%Diff

34.6

1.5

5

-

34.2

1.3

5 -1.3

34.5

1.5

5 -0.3

32.4

1.3

5 -6.4

[a] - Anova & Dunnett(Log): *: Statistically significant difference from the control group at p < 0.05 [a1] - Anova & Dunnett

Appendix 23, TABLE 5 contd. Summary of Clinical Chemistry Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

CK

(U/L)

15 [a]

Mean

SD

N

%Diff

249

57

5

.

207

35

5 -17

410

233

5 65

516 *

205

5 107

T.Bil (µmol/L)

15 [a1]

Mean

SD

N

%Diff

1.80

0.38

5

.

1.80

0.82

5

0.11

1.32

0.54

5 -26.97

1.83

0.39

5

1.33

T.Chol (mmol/L)

15 [a]

Mean

SD

N

%Diff

2.12

0.46

5

.

2.07

0.21

5 -2.45

2.11

0.12

5 -0.85

2.01

0.16

5 -5.18

BA

(µmol/L)

15 [a1]

Mean

SD

N

%Diff

19.2

3.0

5

.

17.7

2.5

5 -7.5

19.0

4.2

5 -1.0

18.3

8.3

5 -4.3

Trig (mmol/L)

15 [a1]

Mean

SD

N

%Diff

0.76

0.20

5

.

0.74

0.28

5 -2.63

0.63

0.29

5 -16.58

0.70

0.25

5 -7.63

T.Pro (g/L)

15 [a1]

Mean

SD

N

%Diff

66.3

4.1

5

-

67.1

2.9

5

1.1

67.7

2.3

5

2.1

65.6

1.0

5 -1.1

ALB

(g/L)

15 [a1]

Mean

SD

N

%Diff

34.6

1.5

5

-

34.2

1.3

5 -1.3

34.5

1.5

5 -0.3

32.4

1.3

5 -6.4

[a] - Anova & Dunnett(Log): *: Statistically significant difference from the control group at p < 0.05 [a1] - Anova & Dunnett

Appendix 23, TABLE 5 contd. Summary of Clinical Chemistry Parameters – Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

GLOB

(g/L)

15 [a]

Mean

SD

N

%Diff

31.7

2.8

5

-

32.9

2.2

5

3.8

33.2

1.4

5

4.7

33.2

0.7

5

4.6

A/G

(ratio)

15 [a]

Mean

SD

N

%Diff

1.09

0.06

5

-

1.04

0.07

5 -4.91

1.04

0.06

5 -4.96

0.98

0.06

5 -10.73

Pi

(mmol/L)

15 [a1]

Mean

SD

N

%Diff

1.98

0.05

5

.

2.05

0.09

5

3.33

2.18

0.11

5

10.19

2.01

0.24

5

1.51

Ca (mmol/L)

15 [a]

Mean

SD

N

%Diff

3.02

0.08

5

.

3.02

0.12

5

0.07

3.02

0.05

5

0.07

2.89

0.13

5 -4.17

Na (mEq/L)

15 [a]

Mean

SD

N

%Diff

145.9

1.0

5

-

144.8

0.9

5 -0.8

145.3

0.7

5 -0.4

144.1 *

0.5

5 -1.2

K (mEq/L)

15 [a]

Mean

SD

N

%Diff

3.65

0.15

5

-

3.59

0.37

5 -1.81

3.87

0.42

5

5.80

4.01

0.33

5

9.80

Cl (mEq/L)

15 [a]

Mean

SD

N

%Diff

96.3

1.3

5

-

96.5

0.8

5

0.2

96.0

0.5

5 -0.3

96.4

0.2

5

0.2

[a] - Anova & Dunnett: *: Statistically significant difference from the control group at p < 0.05 [a1] Anova & Dunnett(Log)

Appendix 23, TABLE 6. Summary of Clinical Chemistry Parameters – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Glu (mmol/L)

66 [p]

Mean

SD

N

%Diff

7.67

0.89

5

.

7.75

0.33

5

1.04

BUN

(mmol/L)

66 [p]

Mean

SD

N

%Diff

5.35

0.42

5

.

5.59

0.40

5

4.37

Creat (µmol/L)

66 [p]

Mean

SD

N

%Diff

36 6

5

-

45 *

4

5 24

AST

(U/L)

66 [p1]

Mean

SD

N

%Diff

124

59

5

.

93 7

5 -25

ALT

(U/L)

66 [p1]

Mean

SD

N

%Diff

82

65

5

.

40 *

4

5 -51

GGT

(U/L)

66 [p1]

Mean

SD

N

%Diff

4

1

5

.

4

1

5

0

Alp (U/L)

66 [p]

Mean

SD

N

%Diff

77

33

5

.

76 7

5

-2

[p] - T-Test: [G1RvsG4R]

{Creat} G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05

[p1] - Wilcoxon(Rank): [G1RvsG4R]

{ALT} G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05

Appendix 23, TABLE 6 contd. Summary of Clinical Chemistry Parameters – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

CK

(U/L)

66 [p]

Mean

SD

N

%Diff

194

43

5

.

209

26 5

8

T.Bil (µmol/L)

66 [p]

Mean

SD

N

%Diff

1.80

0.79

5

.

2.09

0.58

5

16.59

T.Chol (mmol/L)

66 [p]

Mean

SD

N

%Diff

2.46

0.67

5

.

2.05

0.38

5 -16.57

BA

(µmol/L)

66 [p1]

Mean

SD

N

%Diff

12.4

3.3

5

.

17.2

6.2

5

38.4

Trig (mmol/L)

66 [p]

Mean

SD

N

%Diff

0.72

0.29

5

.

0.73

0.12

5

0.55

T.Pro (g/L)

66 [p]

Mean

SD

N

%Diff

67.5

2.1

5

-

69.6

1.5

5

3.2

ALB

(g/L)

66 [p2]

Mean

SD

N

%Diff

30.3

1.7

5

-

31.6

0.6

5

4.3

[p] - T-Test: [G1RvsG4R]

[p1] - T-Test(Log): [G1RvsG4R]

[p2] Wilcoxon(Rank): [G1RvsG4R]

Appendix 23, TABLE 6 contd. Summary of Clinical Chemistry Parameters – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

GLOB

(g/L)

66 [p]

Mean

SD

N

%Diff

37.2

0.8

5

-

38.0

1.4

5

2.3

A/G

(ratio)

66 [p]

Mean

SD

N

%Diff

0.81

0.04

5

-

0.83

0.04

5

2.05

Pi

(mmol/L)

66 [p]

Mean

SD

N

%Diff

1.88

0.20

5

.

1.89

0.25

5

0.53

Ca (mmol/L)

66 [p]

Mean

SD

N

%Diff

2.94

0.08

5

.

3.08 * 0.12

5

5.04

Na (mEq/L)

66 [p1]

Mean

SD

N

%Diff

145.8

1.5

5

-

146.5

0.8

5

0.5

K (mEq/L)

66 [p]

Mean

SD

N

%Diff

3.82

0.21

5

-

3.88

0.21

5

1.41

Cl (mEq/L)

66 [p]

Mean

SD

N

%Diff

95.6

0.8

5

-

95.5

0.4

5 -0.1

[p] - T-Test: [G1RvsG4R]

Appendix 23, TABLE 7. Summary of Clinical Chemistry Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Glu (mmol/L)

15 [a]

Mean

SD

N

%Diff

5.31

0.33

5

.

5.59

0.56

5

5.24

5.75

0.62

5

8.36

6.51 * 0.81

5

22.72

BUN

(mmol/L)

15 [a1]

Mean

SD

N

%Diff

4.49

0.38

5

.

4.83

0.80

5

7.48

5.80

1.27

5

29.09

4.85

0.39

5

8.11

Creat (µmol/L)

15 [a2]

Mean

SD

N

%Diff

36 5

5

-

44 3

5 22

43 3

5 18

51 *

8

5 40

AST

(U/L)

15 [a]

Mean

SD

N

%Diff

79

10

5

.

94

21

5 19

85

11 5

8

80

14 5

2

ALT

(U/L)

15 [a1]

Mean

SD

N

%Diff

32

11

5

.

32 2

5

1

30 2

5

-8

28 3

5 -13

GGT

(U/L)

15 [a1]

Mean

SD

N

%Diff

4

1

5

.

5 *

0

5 39

4

1

5 11

5 *

1

5 28

Alp (U/L)

15 [a]

Mean

SD

N

%Diff

60 9

5

.

56

11

5

-7

77

13

5 28

56

14

5

-7

[a] - Anova & Dunnett: *: Statistically significant difference from the control group at p < 0.05

[a1] - Anova & Dunnett(Rank): *: Statistically significant difference from the control group at p < 0.05

[a2] Anova & Dunnett(Log): *: Statistically significant difference from the control group at p < 0.05

Appendix 23, TABLE 7 contd. Summary of Clinical Chemistry Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

CK

(U/L)

15 [a]

Mean

SD

N

%Diff

206

39

5

.

420 *

192

5 104

202

47

5

-2

311

168

5 51

T.Bil (µmol/L)

15 [a1]

Mean

SD

N

%Diff

2.49

0.30

5

.

1.74

0.61

5 -30.07

2.58

1.20

5

3.61

2.04

0.30

5 -18.04

T.Chol (mmol/L)

15 [a2]

Mean

SD

N

%Diff

2.46

0.33

5

.

2.36

0.24

5 -3.98

2.48

0.19

5

0.65

2.36

0.33

5 -4.22

BA

(µmol/L)

15 [a2]

Mean

SD

N

%Diff

29.0

13.5

5

.

21.9

6.6

5 -24.3

21.3

5.8

5 -26.6

19.2

4.8

5 -33.7

Trig (mmol/L)

15 [a2]

Mean

SD

N

%Diff

0.45

0.09

5

.

0.44

0.16

5 -1.79

0.63

0.24

5

41.07

0.47

0.07

5

4.02

T.Pro (g/L)

15 [a2]

Mean

SD

N

%Diff

65.8

1.9

5

-

68.8

0.5

5

4.5

69.1

2.9

5

5.0

67.8

3.6

5

2.9

ALB

(g/L)

15 [a2]

Mean

SD

N

%Diff

35.2

1.6

5

-

36.9

0.9

5

4.9

36.7

2.2

5

4.3

35.7

1.7

5

1.4

[a] - Anova & Dunnett(Log): *: Statistically significant difference from the control group at p < 0.05

[a1] - Anova & Dunnett(Rank)

[a2] Anova & Dunnett

Appendix 23, TABLE 7 contd. Summary of Clinical Chemistry Parameters – Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

GLOB

(g/L)

15 [a]

Mean

SD

N

%Diff

30.7

0.8

5

-

31.9 * 0.7

5

4.1

32.5 * 0.9

5

5.9

32.1

1.9

5

4.7

A/G

(ratio)

15 [a1]

Mean

SD

N

%Diff

1.15

0.06

5

-

1.16

0.05

5

0.78

1.13

0.05

5 -1.60

1.11

0.02

5 -3.16

Pi

(mmol/L)

15 [a1]

Mean

SD

N

%Diff

1.92

0.26

5

.

1.65

0.08

5 -14.05

1.84

0.08

5 -4.37

1.72

0.17

5 -10.51

Ca (mmol/L)

15 [a1]

Mean

SD

N

%Diff

2.81

0.24

5

.

2.85

0.08

5

1.57

2.87

0.10

5

2.28

2.90

0.13

5

3.35

Na (mEq/L)

15 [a]

Mean

SD

N

%Diff

143.6

0.3

5

-

142.1

1.3

5 -1.0

144.6

2.3

5

0.7

144.0

0.3

5

0.3

K (mEq/L)

15 [a1]

Mean

SD

N

%Diff

3.34

0.18

5

-

3.57

0.55

5

7.13

3.55

0.68

5

6.47

3.89

0.31

5

16.55

Cl (mEq/L)

15 [a]

Mean

SD

N

%Diff

95.6

0.6

5

-

94.8

0.7

5 -0.8

96.2

2.6

5

0.6

96.4

0.3

5

0.8

[a] - Anova & Dunnett(Rank): *: Statistically significant difference from the control group at p < 0.05 [a1] - Anova & Dunnett

Appendix 23, TABLE 8. Summary of Clinical Chemistry Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Glu (mmol/L)

66 [p]

Mean

SD

N

%Diff

6.94

0.71

5

.

7.47

0.38

5

7.70

BUN

(mmol/L)

66 [p]

Mean

SD

N

%Diff

6.30

0.54

5

.

7.83 * 0.44

5

24.35

Creat (µmol/L)

66 [p]

Mean

SD

N

%Diff

49 6

5

-

44 3

5 -11

AST

(U/L)

66 [p1]

Mean

SD

N

%Diff

122

55

5

.

102

30

5 -16

ALT

(U/L)

66 [p1]

Mean

SD

N

%Diff

41

23

5

.

37 9

5

-9

GGT

(U/L)

66 [p]

Mean

SD

N

%Diff

3

1

5

.

2

1

5 -25

Alp (U/L)

66 [p]

Mean

SD

N

%Diff

32 4

5

.

29

10

5 -10

[p] - T-Test: [G1RvsG4R]

Appendix 23, TABLE 8 contd. Summary of Clinical Chemistry Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

CK

(U/L)

66 [p]

Mean

SD

N

%Diff

243

59

5

.

203

79

5 -16

T.Bil (µmol/L)

66 [p]

Mean

SD

N

%Diff

2.86

0.93

5

.

2.68

0.70

5 -6.50

T.Chol (mmol/L)

66 [p1]

Mean

SD

N

%Diff

2.46

0.07

5

.

2.55

0.56

5

3.66

BA

(µmol/L)

66 [p2]

Mean

SD

N

%Diff

16.4

5.7

5

.

22.5

8.3

5

37.7

Trig (mmol/L)

66 [p]

Mean

SD

N

%Diff

0.49

0.13

5

.

0.48

0.12

5 -2.04

T.Pro (g/L)

66 [p]

Mean

SD

N

%Diff

68.7

2.5

5

-

67.1

4.0

5 -2.2

ALB

(g/L)

66 [p]

Mean

SD

N

%Diff

33.9

1.8

5

-

33.5

3.3

5 -1.2

[p] - T-Test: [G1RvsG4R]

[p1] - Wilcoxon(Rank): [G1RvsG4R]

[p2] T-Test(Log): [G1RvsG4R]

Appendix 23, TABLE 8 contd. Summary of Clinical Chemistry Parameters – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

GLOB

(g/L)

66 [p]

Mean

SD

N

%Diff

34.7

1.2

5

-

33.6

1.1

5 -3.2

A/G

(ratio)

66 [p]

Mean

SD

N

%Diff

0.98

0.05

5

-

1.00

0.08

5

2.02

Pi

(mmol/L)

66 [p]

Mean

SD

N

%Diff

1.52

0.24

5

.

1.51

0.19

5 -0.26

Ca (mmol/L)

66 [p]

Mean

SD

N

%Diff

2.96

0.07

5

.

2.95

0.09

5 -0.20

Na (mEq/L)

66 [p]

Mean

SD

N

%Diff

145.5

0.8

5

-

145.7

1.2

5

0.2

K (mEq/L)

66 [p]

Mean

SD

N

%Diff

3.57

0.41

5

-

3.64

0.30

5

1.91

Cl (mEq/L)

66 [p1]

Mean

SD

N

%Diff

95.6

0.6

5

-

95.3

1.7

5 -0.3

[p] - T-Test: [G1RvsG4R]

[p1] Wilcoxon(Rank): [G1RvsG4R]

TABLE 19. Summary of Pup Weight and Ano- Genital Distance Measurement

Sex: Female

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Litter Mean

Pup

Body weights

Males

(g)

0 [a]

Mean

SD

N

6.82 0.35

8

6.95 0.81

9

7.00 0.50

9

6.33 1.08

9

4 [a1]

Mean

SD

N

11.03 1.21

8

10.99 1.91

9

11.38 1.03

9

9.97 2.08

9

13 [a]

Mean

SD

N

31.79 3.00

8

30.48 5.06

9

32.87 2.69

9

30.02 3.34

8

Litter Mean

Pup

Body weights

Females

(g)

0 [a1]

Mean

SD

N

6.46 0.41

8

6.77 0.60

8

6.72 0.57

9

6.27 0.80

8

4 [a1]

Mean

SD

N

10.29 1.25

8

10.88 1.39

8

10.97 1.12

9

9.64 2.09

8

13 [a1]

Mean

SD

N

30.76 3.65

8

30.67 2.49

8

32.54 2.80

9

29.47 3.29

8

Litter Mean

Pup

Body weights

(g)

0 [a]

Mean

SD

N

6.64 0.34

8

6.78 0.79

9

6.86 0.55

9

6.19 1.02

9

4 [a1]

Mean

SD

N

10.68 1.08

8

10.74 1.90

9

11.16 1.06

9

9.71 2.03

9

13 [a]

Mean

SD

N

31.27 3.19

8

29.86 4.88

9

32.71 2.72

9

29.75 3.24

8

Mean of pup

AGD males

(mm)

[a]

Mean

SD

N

3.70 0.03

8

3.70 0.07

9

3.72 0.03

9

3.66 0.06

9

Mean of pup

AGD females

(mm)

[a1]

Mean SD

N

1.66 0.04

8

1.71 *

0.04

8

1.69 0.03

9

1.64 0.04

8

Mean of pup

AGR males

LD0 [a]

Mean

SD

N

1.95 0.03

8

1.94 0.05

9

1.95 0.04

9

1.99 0.09

9

[a] - Anova & Dunnett (Rank)

[a1] - Anova & Dunnett: * = p < 0.05

TABLE 19 contd. Summary of Pup Weight and Ano- Genital Distance Measurement

Sex: Female

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Mean of pup AGR females

LD0 [a]

Mean

SD

N

0.89 0.02

8

0.91 0.02

8

0.90 0.02

9

0.89 0.02

8

Mean of pup AGI males

LD0 [a1]

Mean

SD

N

2.0 0.0

8

1.9 0.1

9

1.9 0.0

9

2.0 0.1

9

Mean of pup

AGI females

LD0 [a]

Mean

SD

N

0.9 0.0

8

0.9 0.0

8

0.9 0.0

9

0.9 0.0

8

[a] - Anova & Dunnett

[a1] - Anova & Dunnett (Rank)

APPENDIX 19. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G1 0 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7491 Rx7492 Rx7493 Rx7494 Rx7495	Male Male Male Male Male	M01 M02 M03 M08 M02 M03 M07 M08 M01 M02 M05 M09 M01 M02 M03 M04 M01	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G1 0 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7495 Rx7496 Rx7498 Rx7500	Male Male Male Male	M02 M07 M08 M01 M02 M03 M01 M02 M03 M05 M01 M03 M04 M05	0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G2 111 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7511 Rx7512 Rx7513 Rx7514 Rx7515	Male Male Male Male Male	M01 M02 M05 M07 M01 M02 M03 M04 M01 M02 M04 M05 M03 M05 M06 M07 M01	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G2 111 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7515 Rx7517 Rx7518 Rx7519 Rx7520	Male Male Male Male Male	M02 M03 M01 M02 M06 M07 M01 M03 M05 M06 M01 M02 M04 M05 M01 M03 M04	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G2 111 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7520	Male	M05	0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G3 333 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7531 Rx7532 Rx7533 Rx7534 Rx7535	Male Male Male Male Male	M01 M06 M07 M10 M01 M02 M05 M06 M02 M04 M05 M07 M01 M02 M04 M05 M01	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

G3 333 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7535 Rx7537 Rx7538 Rx7539 Rx7540	Male Male Male Male Male	M03 M05 M07 M01 M02 M03 M06 M01 M02 M03 M04 M01 M04 M06 M07 M01 M02	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

APPENDIX 19 contd. Individual Observation of Aerolae/Nipple Retention in Male Pups on Lactation Day 13

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

G3 333 mg/kg/day Dam	Pup Sex	Pup
			Nipple Retention
			13
Rx7540	Male	M03 M05	0 0

Appendix 23, APPENDIX 13. Individual Animal Thyroid Hormones Profile – Males

G1 0 mg/kg/day	TSH (ng/mL) Day 57	T4 (ng/mL) Day 57	G3 333 mg/kg/day	TSH (ng/mL) Day 57	T4 (ng/mL) Day 57
Rx7481	1.46	29.17	Rx7521	0.78	28.01
Rx7482	1.63	29.99	Rx7522	3.03	33.79
Rx7483	0.62	26.78	Rx7523	1.36	29.66
Rx7484	1.29	24.83	Rx7524	0.78	26.84
Rx7485	1.82	18.11	Rx7525	2.31	40.32
Rx7486	2.26	26.51	Rx7526	1.91	31.52
Rx7487	1.83	29.66	Rx7527	3.77	34.00
Rx7488	3.82	28.56	Rx7528	1.17	24.33
Rx7489	2.44	29.44	Rx7529	3.32	22.16
Rx7490	2.28	26.90	Rx7530	2.55	24.22
G2 111 mg/kg/day			G4 1000 mg/kg/day
Rx7501	0.94	25.06	Rx7541	3.16	31.14
Rx7502	3.95	30.76	Rx7542	2.44	35.86
Rx7503	2.97	33.25	Rx7543	0.90	40.95
Rx7504	1.73	26.45	Rx7544	1.36	31.52
Rx7505	1.88	27.34	Rx7545	4.04	26.84
Rx7506	2.05	27.06	Rx7546	0.96	24.05
Rx7507	3.20	40.74	Rx7547	2.32	48.15
Rx7508	3.10	25.00	Rx7548	1.52	28.73
Rx7509	1.41	28.34	Rx7549	2.07	27.17
Rx7510	2.19	25.45	Rx7550	1.29	21.66

Appendix 23, APPENDIX 14.Individual Animal Thyroid Hormones Profile – Females

G1 0 mg/kg/day	TSH (ng/mL) LD 13	T4 (ng/mL) LD 13
Rx7491	2.62	11.07
Rx7492	1.91	11.20
Rx7493	3.41	10.74
Rx7494	2.10	13.05
Rx7495	2.44	11.95
Rx7496	1.91	13.19
Rx7498	3.20	11.55
Rx7500	2.65	12.59
G2 111 mg/kg/day
Rx7511 Rx7512	4.34 2.09	9.88 8.83
Rx7511 Rx7512	4.34 2.09	9.88 8.83	Rx7513	0.73	12.96
Rx7514	1.21	12.31
Rx7515	2.66	26.51
Rx7517	2.82	15.05
Rx7518	1.16	13.67
Rx7519	2.50	11.07
Rx7520	1.68	10.78

G3 333 mg/kg/day	TSH (ng/mL) LD 13	T4 (ng/mL) LD 13
Rx7531	1.89	8.08
Rx7532	3.54	11.68
Rx7533	3.01	12.96
Rx7534	2.87	10.99
Rx7535	2.14	14.45
Rx7537	3.64	16.43
Rx7538	1.52	17.90
Rx7539 Rx7540	1.46 3.13	8.21 11.77
Rx7539 Rx7540	1.46 3.13	8.21 11.77	G4 1000 mg/kg/day
Rx7551	1.86	13.67
Rx7552	3.17	14.25
Rx7554	2.31	12.31
Rx7555	1.26	16.74
Rx7556	3.64	16.32
Rx7558	2.51	19.90
Rx7559	1.41	9.96
Rx7560	1.83	11.25

Appendix 23, APPENDIX 15. Individual Animal Thyroid Hormones Profile – Pup Lactation Day 4

G1 0 mg/kg/day	TSH (ng/mL) LD 4	T4 (ng/mL) LD 4
Rx7491	0.31	11.00
Rx7492	0.20	7.11
Rx7493	0.18	7.83
Rx7494	0.17	8.69
Rx7495	0.12	9.35
Rx7498	0.59	14.32
Rx7500	0.41	7.34
G2 111 mg/kg/day
Rx7511	0.18	7.69
Rx7512	0.42	13.09
Rx7513	0.28	4.75
Rx7514	0.18	8.79
Rx7517	LLOQ	17.14
Rx7518	LLOQ	11.81
Rx7519	0.12	24.39
Rx7520	0.31	8.15

G3 333 mg/kg/day	TSH (ng/mL) LD 4	T4 (ng/mL) LD 4
Rx7531	0.26	7.96
Rx7532	0.22	11.33
Rx7533	0.20	4.20
Rx7534	0.20	8.15
Rx7535	LLOQ	8.20
Rx7537	0.14	9.52
Rx7538	0.22	8.37
Rx7539	0.23	7.67
Rx7540	0.23	7.76
G4 1000 mg/kg/day
Rx7551	LLOQ	9.57
Rx7552	LLOQ	6.19
Rx7554	0.14	8.92
Rx7555	LLOQ	9.19
Rx7556	0.17	9.29
Rx7558	0.12	8.13
Rx7559	LLOQ	7.03
Rx7560	0.44	7.69

LLOQ: Lower Limit of Quantification for TSH is below 0.1 ng/mL

Appendix 23, APPENDIX 16. Individual Animal Thyroid Hormones Profile – Pup Lactation Day 13

G1 0 mg/kg/day	TSH (ng/mL) LD 13	T4 (ng/mL) LD 13
Rx7491	0.10	27.26
Rx7492	1.11	29.27
Rx7493	0.61	30.03
Rx7494	0.70	42.10
Rx7495	0.20	37.64
Rx7496	0.42	45.42
Rx7498	0.26	27.66
Rx7500	0.28	34.71
G2 111 mg/kg/day
Rx7511 Rx7512	0.15 0.64	12.27 23.95
Rx7511 Rx7512	0.15 0.64	12.27 23.95	Rx7513	1.05	19.97
Rx7514	0.36	21.58
Rx7515	0.41	10.43
Rx7517	0.23	11.40
Rx7518	0.25	9.66
Rx7519	0.41	12.86
Rx7520	0.61	37.05

G3 333 mg/kg/day	TSH (ng/mL) LD 13	T4 (ng/mL) LD 13
Rx7531	0.67	25.56
Rx7532	0.61	45.66
Rx7533	0.79	41.04
Rx7534	0.28	22.48
Rx7535	0.26	30.90
Rx7537	0.14	40.28
Rx7538	0.44	43.05
Rx7539 Rx7540	0.59 0.88	20.28 26.69
Rx7539 Rx7540	0.59 0.88	20.28 26.69	G4 1000 mg/kg/day
Rx7551	0.70	33.42
Rx7552	0.36	34.65
Rx7554	1.15	25.90
Rx7555	0.34	21.68
Rx7556	0.25	26.92
Rx7558	0.62	18.42
Rx7559	0.15	19.26
Rx7560	0.17	16.86

Appendix 23, TABLE 17. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs - Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Terminal

Fasting BW

(g)

58 [a]

Mean

SD

N

%Diff

508.90

49.17

10

-

505.88

44.99

10 -0.59

506.20

31.55

10 -0.53

515.46

37.99

10

1.29

Epididymides

(g)

58 [a1]

Mean

SD

N

%Diff

1.7205

0.1178

10

-

1.5823

0.1423

10 -8.0281

1.6668

0.0474

10 -3.1166

1.6616

0.1981

10 -3.4218

Prostate (g)

58 [a]

Mean

SD

N

%Diff

1.4906

0.1419

10

.

1.5395

0.2427

10

3.2833

1.6640

0.2722

10

11.6344

1.5476

0.1561

10

3.8274

Seminal vesicles

& coagulating glands

(g)

58 [a]

Mean

SD

N

%Diff

1.9499

0.3619

10

-

1.9499

0.2946

10 -0.0005

1.9789

0.2776

10

1.4868

2.0680

0.3489

10

6.0589

Testes (g)

58 [a]

Mean

SD

N

%Diff

4.0445

0.3124

10

-

4.0290

0.4498

10 -0.3837

4.0093

0.3393

10 -0.8688

3.9906

0.5788

10 -1.3322

Levator ani bulbocavernosus

muscle complex

(g)

58 [a]

Mean

SD

N

%Diff

1.5210

0.1671

10

.

1.5070

0.1784

10 -0.9146

1.4791

0.1405

10 -2.7516

1.5347

0.1690

10

0.9047

Cowpers glands

(g)

58 [a]

Mean

SD

N

%Diff

0.1396

0.0179

10

.

0.1387

0.0310

10 -0.6518

0.1366

0.0167

10 -2.1344

0.1339

0.0342

10 -4.0968

Anova & Dunnett

Appendix 23, TABLE 17 contd. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs - Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Glans penis (g)

58 [a]

Mean

SD

N

%Diff

0.0963

0.0058

10

.

0.1024

0.0075

10

6.3006

0.0981

0.0113

10

1.8476

0.0952

0.0077

10 -1.2352

Thyroid with parathyroids

(g)

60^b [a]

Mean

SD

N

%Diff

0.0292

0.0048

10

-

0.0338

0.0027

10

15.9808

0.0308

0.0058

10

5.5898

0.0341

0.0061

10

17.0096

Appendix 23, TABLE 18. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs - Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Terminal

Fasting BW

(g)

58 [a]

Mean

SD

N

%Diff

508.90

49.17

10

-

505.88

44.99

10 -0.59

506.20

31.55

10 -0.53

515.46

37.99

10

1.29

Epididymides (%)

58 [a1]

Mean

SD

N

%Diff

0.3406

0.0356

10

.

0.3133

0.0180

10 -8.0087

0.3301

0.0154

10 -3.0830

0.3248

0.0487

10 -4.6183

Prostate (%)

58 [a]

Mean

SD

N

%Diff

0.2962

0.0455

10

.

0.3039

0.0348

10

2.6264

0.3309

0.0633

10

11.7246

0.3033

0.0500

10

2.3976

Seminal vesicles

& coagulating glands

(%)

58 [a]

Mean

SD

N

%Diff

0.3847

0.0697

10

.

0.3851

0.0418

10

0.1117

0.3909

0.0496

10

1.6193

0.4056

0.0856

10

5.4203

Testes (%)

58 [a]

Mean

SD

N

%Diff

0.8028

0.1065

10

.

0.7982

0.0763

10 -0.5719

0.7942

0.0785

10 -1.0706

0.7808

0.1356

10 -2.7492

Levator ani bulbocavernosus

muscle complex

(%)

58 [a1]

Mean

SD

N

%Diff

0.2990

0.0188

10

.

0.3001

0.0434

10

0.3806

0.2927

0.0271

10 -2.1170

0.2991

0.0393

10

0.0526

Cowper glands

(%)

58 [a]

Mean

SD

N

%Diff

0.0276

0.0042

10

.

0.0275

0.0060

10 -0.6834

0.0271

0.0039

10 -1.9932

0.0260

0.0066

10 -5.8786

Anova & Dunnett

Appendix 23, TABLE 18 contd. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs - Males

Sex: Male

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Glans penis

(%)

58 [a]

Mean

SD

N

%Diff

0.0191

0.0026

10

.

0.0203

0.0018

10

6.2906

0.0195

0.0029

10

1.8337

0.0186

0.0027

10 -2.7453

Thyroid with parathyroids

(%)

60^b [a]

Mean

SD

N

%Diff

0.0058

0.0012

10

.

0.0067

0.0006

10

16.1533

0.0061

0.0010

10

4.9000

0.0066

0.0012

10

14.5994

[a] - Anova & Dunnett; ^b: Weighed after fixation

Appendix 23, TABLE 22. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Terminal

Fasting BW

(g)

66 [p]

Mean

SD

N

%Diff

518.02

32.61

5

-

528.98

30.98

5

2.11

Epididymides

(g)

66 [p]

Mean

SD

N

%Diff

1.6820

0.1553

5

-

1.6957

0.1502

5

0.8145

Prostate (g)

66 [p]

Mean

SD

N

%Diff

1.4343

0.1814

5

.

1.4008

0.1080

5

-2.3356

Seminal vesicles

& coagulating glands

(g)

66 [p]

Mean

SD

N

%Diff

2.1069

0.3072

5

-

2.2126

0.1675

5

5.0168

Testes (g)

66 [p]

Mean

SD

N

%Diff

3.9836

0.5477

5

-

3.8483

0.3727

5

-3.3950

Levator ani

Bulbocavernosus muscle complex

(g)

66 [p]

Mean

SD

N

%Diff

1.4862

0.0824

5

.

1.5574

0.1149

5

4.7880

Cowpers glands

(g)

66 [p]

Mean

SD

N

%Diff

0.1751

0.0263

5

.

0.1412 * 0.0169

5

-19.3283

[p] - T-Test: [G1RvsG4R]

{Cowpers}{66} G1RvsG4R: *: Statistically significant difference from the control group at p < 0.05

Appendix 23, TABLE 22 contd. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Glans penis (g)

66 [p]

Mean

SD

N

%Diff

0.0958

0.0116

5

.

0.1021

0.0070

5

6.6193

Thyroid with parathyroids

(g)

71^b [p]

Mean

SD

N

%Diff

0.0399

0.0041

5

-

0.0448

0.0035

5

12.1805

; ^b: Weighed after fixation

Appendix 23, TABLE 23. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Terminal

Fasting BW

(g)

66 [p]

Mean

SD

N

%Diff

518.02

32.61

5

-

528.98

30.98

5

2.11

Epididymides (%)

66 [p]

Mean

SD

N

%Diff

0.3248

0.0228

5

.

0.3206

0.0211

5

-1.2924

Prostate (%)

66 [p]

Mean

SD

N

%Diff

0.2780

0.0412

5

.

0.2648

0.0137

5

-4.7577

Seminal vesicles

& coagulating glands

(%)

66 [p]

Mean

SD

N

%Diff

0.4091

0.0741

5

.

0.4188

0.0296

5

2.3541

Testes (%)

66 [p]

Mean

SD

N

%Diff

0.7693

0.0941

5

.

0.7267

0.0428

5

-5.5268

Levator ani bulbocavernosus

muscle complex

(%)

66 [p]

Mean

SD

N

%Diff

0.2873

0.0148

5

.

0.2948

0.0216

5

2.5895

Cowpers glands

(%)

66 [p]

Mean

SD

N

%Diff

0.0340

0.0061

5

.

0.0268

0.0038

5

-21.1709

Appendix 23, TABLE 23 contd. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs – Recovery Males

Sex: Male

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Glans penis

(%)

66 [p]

Mean

SD

N

%Diff

0.0186

0.0029

5

.

0.0193

0.0008

5

3.9241

Thyroid with parathyroids

(%)

71^b [p]

Mean

SD

N

%Diff

0.0077

0.0008

5

.

0.0085

0.0009

5

10.0141

^b: Weighed after fixation

Appendix 23, TABLE 27. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs - Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Terminal

Fasting BW

(g)

LD 14 [a]

Mean

SD

N

%Diff

324.40

27.62

10

-

318.32

31.75

10 -1.87

330.33

17.53

10

1.83

310.33

28.40

10 -4.34

Ovaries (g)

LD 14 [a]

Mean

SD

N

%Diff

0.1387

0.0227

10

-

0.1304

0.0182

10 -5.9697

0.1315

0.0134

10 -5.1983

0.1327

0.0140

10 -4.3259

Uterus with cervix

(g)

LD 14 [a1]

Mean

SD

N

%Diff

0.7818

0.5819

10

.

0.6528

0.1217

10 -16.4899

0.6300

0.1295

10 -19.4077

0.7521

0.1979

10 -3.7902

Thyroid with parathyroids

(g)

64^b [a2]

Mean

SD

N

%Diff

0.0317

0.0048

10

-

0.0258

0.0035

10 -18.6553

0.0290

0.0101

10 -8.4912

0.0267

0.0070

10 -15.5934

[a] - Anova & Dunnett; ^b: Weighed after fixation

[a1] - Anova & Dunnett(Rank)

[a2] Anova & Dunnett(Log)

Appendix 23, TABLE 28. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs - Females

Sex: Female

Day(s) Relative to Start Date

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Terminal

Fasting BW

(g)

LD 14 [a]

Mean

SD

N

%Diff

324.40

27.62

10

-

318.32

31.75

10 -1.87

330.33

17.53

10

1.83

310.33

28.40

10 -4.34

Ovaries (%)

LD 14 [a]

Mean

SD

N

%Diff

0.0432

0.0093

10

.

0.0410

0.0047

10 -5.0461

0.0400

0.0052

10 -7.5349

0.0431

0.0056

10 -0.4127

Uterus with cervix

(%)

LD 14 [a1]

Mean

SD

N

%Diff

0.2468

0.1950

10

.

0.2048

0.0300

10 -17.0100

0.1923

0.0475

10 -22.0622

0.2478

0.0867

10

0.4340

Thyroid with parathyroids

(%)

64^b [a]

Mean

SD

N

%Diff

0.0098

0.0015

10

.

0.0081

0.0012

10 -16.9891

0.0088

0.0029

10 -10.7046

0.0087

0.0024

10 -11.5055

[a] - Anova & Dunnett; ^b: Weighed after fixation

[a1] - Anova & Dunnett(Rank)

Appendix 23, TABLE 32. Summary of Terminal Fasting Body Weights and Organ Weights – Reproductive Organs – Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Terminal

Fasting BW

(g)

66 [p]

Mean

SD

N

%Diff

274.28

22.68

5

-

276.35

21.77

5

0.76

Ovaries (g)

66 [p]

Mean

SD

N

%Diff

0.1087

0.0229

5

-

0.1013

0.0210

5

-6.7709

Uterus with cervix

(g)

66 [p1]

Mean

SD

N

%Diff

0.7228

0.2099

5

.

0.7139

0.2763

5

-1.2423

Thyroid with parathyroids

(g)

71^b [p]

Mean

SD

N

%Diff

0.0308

0.0094

5

-

0.0380

0.0071

5

23.4568

[p] - T-Test: [G1RvsG4R]; ^b: Weighed after fixation

[p1] T-Test(Log): [G1RvsG4R]

Appendix 23, TABLE 33. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios – Reproductive Organs Recovery Females

Sex: Female

Day(s) Relative to Start Date

G1R

0 mg/kg/day

G4R

1000 mg/kg/day

Terminal

Fasting BW

(g)

66 [p]

Mean

SD

N

%Diff

274.28

22.68

5

-

276.35

21.77

5

0.76

Ovaries (%)

66 [p]

Mean

SD

N

%Diff

0.0395

0.0066

5

.

0.0365

0.0058

5

-7.6334

Uterus with cervix

(%)

66 [p]

Mean

SD

N

%Diff

0.2641

0.0775

5

.

0.2589

0.0963

5

-1.9917

Thyroid with parathyroids

(%)

71^b [p1]

Mean

SD

N

%Diff

0.0113

0.0038

5

.

0.0137

0.0016

5

20.4665

[p] - T-Test: [G1RvsG4R]; ^b: Weighed after fixation

[p1] - Wilcoxon(Rank): [G1RvsG4R]

Appendix 23, TABLE 37. Summary of Terminal Body Weights, Organ Weights and Organ to Body Weight Ratios – Pups

Sex: Female

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

G1

0 mg/kg/day

G2

111 mg/kg/day

G3

333 mg/kg/day

G4

1000 mg/kg/day

Litter Mean

Pup TBwt-M

13 [a]

Mean

SD

N

%Diff

32.24 3.69

8 .

30.40 5.01

9

-5.69

32.85 2.63 9

1.91

31.16 3.87

8

-3.36

Litter Mean Pup TBwt-F

13 [a]

Mean

SD

N

%Diff

30.71 3.15

8 .

30.81 2.02 8

0.33

31.36 3.03 9

2.09

29.22 3.88

8

-4.87

Litter Mean Pup Thy-M

14 [a1]

Mean

SD

N

%Diff

0.0058 0.0014

8 .

0.0066 0.0019

9

14.0933

0.0064 0.0007

9

11.5921

0.0065 0.0012

8

11.9048

Litter Mean Pup Thy-F

14 [a1]

Mean

SD

N

%Diff

0.0078 0.0020

8 .

0.0059 0.0012

8

-23.9165

0.0067 0.0015

9

-14.5354

0.0070 0.0029

8

-10.1124

Bwt/Pup

Thyroid - M (%)

13 [a]

Mean

SD

N

%Diff

0.0179 0.0037

8 .

0.0217 0.0045

9

20.9584

0.0196 0.0018

9

9.6132

0.0210 0.0048

8

17.4232

Bwt/Pup

Thyroid - F

(%)

13 [a2]

Mean

SD

N

%Diff

0.0253 0.0055

8 .

0.0192 0.0034

8

-24.0724

0.0211 0.0035

9

-16.5182

0.0243 0.0109

8

-4.0052

[a] - Anova & Dunnett

[a1] - Anova & Dunnett(Log)

[a2] - Anova & Dunnett(Rank)

Conclusions:

To summarize, oral (gavage) administration of the test item C. I. Pigment Yellow 175 to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item. Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item C. I. Pigment Yellow 175 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle [0.5 % (w/v) of carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q^®water] and administered at the graduated dose levels of 111, 333 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg body weight/day. Each main group in the experiment comprised of 10 male and 10 female rats and each recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19468 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 4 (Day 23) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % variation from the claimed concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumedGestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 56 and towards the end of recovery period for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 58), parental females (LD14) and the recovery animals (Day 65) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development.

Tissues/organs collected from randomly selected 5 males and females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups.In the absence of test item related histopathological changes in any of the suspected tissues in the high dose group (G4), tissues from the lower dose groups were not evaluated. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.

Under the experimental conditions employed, the following results were obtained:

Clinical signs and Mortality:There were no treatment related clinical signs or mortality observed at any of the doses tested. The yellowish faeces were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the yellowish coloured faecal matter was due to physical nature of the test item.

There were no abnormalities observed in pups.

Functional Observation Battery:No treatment-related neurological abnormalities were observed at any of the doses tested.

Body weights:The mean body weights and body weight gainswere unaffected by the treatment at all the tested doses in both sexes.

Food consumption:Treatment did not affect the food consumption at any of the tested doses in either sex.

Maternal body weights and food consumption:The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.

Fertility parameters:Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

Litter parameters:There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

Haematology, Coagulation, Clinical chemistry and Urine Parameters:No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.

Hormone analysis:The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

Terminal fasting body weights, organ weights and its ratios:There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.

Gross and histopathology:

Grossly noted yellow contents in intestinal segments ≥333 mg/kg bwt/day were attributed to the test item colour.

There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar ratsfor the test itemHostaperm-Gelb H6G (C. I. Pigment Yellow 175) is determined to be 1000 mg/kg bwt/dayunder the test conditions and doses employed.