Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Fertility:

Oral (gavage) administration of the test item 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 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Development:

In a study according to OECD TG 414 in rats the NOAEL for:

• Maternal toxicity was 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity was 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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%).
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Key result
Critical effects observed:
no
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
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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     



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  


5  



158  


5  


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  


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  


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  


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  


0.01  


5  


-  



0.01  


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





















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


















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





















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





















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





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





















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





















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






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


 

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


 

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


 

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


 

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)



60b [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


(%)



60b [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)



71b [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


(%)



71b [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)



64b [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


(%)



64b [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)



71b [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


(%)



71b [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.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
13 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please, see chapter 13: Read across justification
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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%).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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.

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
reliable
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park,
Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment for the first batch of rats and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.
Details on mating procedure:
The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day to confirm mating.
Duration of treatment / exposure:
Gestation day 5 to gestation day 19
Frequency of treatment:
Daily from gestation day 5 to gestation day 19
Duration of test:
Upto gestation day 20
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
111 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24 day 0 pergnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz221 Rz244
G2 Low dose 111 10 11.1 24 Rz245 Rz268
G3 Mid dose 333 10 33.3 24 Rz269 Rz292
G4 High dose 1000 10 100 24 Rz293 Rz316
Maternal examinations:
CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included
Ovaries and uterine content:
The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta
Fetal examinations:
• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.
Indices:
Refer Table 6 of the final report
Historical control data:
Refer Annexure 8 of the final report
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Grossly, orange contents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions in the study
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical biochemistry
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
TABLE 1.     Summary of Maternal Survival, Pregnancy Status and Fetus Disposition






























































































Group No.  


Parameters   


Dose (mg/kg/day)



G1


0



G2


111



G3


333



G4 1000



Total No. of rats found sperm positive / group



24



24



24



24



Duration of treatment  



 



GD  5 to 19 (total 15 days)



 



Caesarean section


(day of presumed gestation)



 



GD 20



 



Number of rats sacrificed at caesarean section



24



24



24



24



Number. of rats non-pregnant at caesarean section



1



3



0



1



Number of rats pregnant at caesarean section



23



21



24



23



Number of litters examined



23



21



24



23



Total number of fetuses



315



284



352



332



Total number of dead fetuses



0



0



0



0



 


Number of fetuses evaluated


a. External examination



 


 


315



 


 


284



 


 


352



 


 


332



b. Visceral examination



152



137



172



159



c. Skeletal examination



163



147



180



173



 


TABLE 3.   Summary of Maternal Body Weight of Pregnant Rats 














































































Sex: Female



Day(s) Relative to Mating



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


%Diff



251.75   16.44  


23        -  



253.71   16.08  


21       


0.78  



252.67   18.29  


24       


0.37  



253.98   16.09  


23       


0.89  



3 [a]



Mean


SD


N


%Diff



269.53   18.24  


23        -  



268.36   17.20  


21       


-0.44  



270.00   15.98  


24       


0.17  



270.84   16.72  


23       


0.48  



5 [a]



Mean


SD


N


%Diff



278.82   18.87  


23        -  



278.00   17.29  


21       


-0.29  



277.08   14.82  


24       


-0.62  



278.75   15.92  


23       


-0.03  



8 [a]



Mean


SD


N


%Diff



290.09   18.58  


23        -  



288.73   19.12  


21       


-0.47  



288.48   16.17  


24       


-0.56  



290.29   16.40  


23       


0.07  



11 [a]



Mean


SD


N


%Diff



305.64   20.35  


23        -  



303.84   19.22  


21       


-0.59  



306.62   16.97  


24       


0.32  



308.14   17.92  


23       


0.82  



14 [a]



Mean


SD


N


%Diff



321.97   20.18  


23        -  



322.06   20.98  


21       


0.03  



324.71   17.77  


24       


0.85  



325.03   18.73  


23       


0.95  



17 [a]



Mean


SD


N


%Diff



354.81   24.31  


23        -  



355.93   21.77  


21       


0.31  



359.51   19.59  


24       


1.32  



360.57   19.33  


23       


1.62  



20 [a]



Mean


SD


N


%Diff



406.10   29.81  


23        -  



405.36   27.69  


21       


-0.18  



408.60   24.29  


24       


0.62  



411.19   21.33  


23       


1.26  



 


TABLE 4.   Summary of Maternal Body Weight Gain of Pregnant Rats






































































Sex: Female



Day(s) Relative to Mating



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute Wei ght Gain


(g)



0 → 3 [a]



Mean


SD


N


%Diff



17.78  


7.37  


23       


.  



14.65  


4.29  


21       


-17.65  



17.33  


6.81  


24       


-2.56  



16.85  


3.97  


23       


-5.23  



3 → 5 [a]



Mean


SD


N


%Diff



9.28  


7.48  


23       


.  



9.64  


3.47  


21       


3.82  



7.08  


4.79  


24       


-23.71  



7.91  


5.10  


23       


-14.80  



5 → 8 [a]



Mean


SD


N


%Diff



11.27  


3.96  


23       


.  



10.73  


3.53  


21       


-4.78  



11.40  


4.27  


24       


1.10  



11.54  


4.68  


23       


2.39  



8 → 11 [a]



Mean


SD


N


%Diff



15.55  


4.70  


23       


.  



15.10  


4.00  


21       


-2.88  



18.14  


4.52  


24       


16.63  



17.85  


4.51  


23       


14.80  



11 → 14 [a]



Mean


SD


N


%Diff



16.32  


4.54  


23       


.  



18.23  


3.86  


21       


11.65  



18.09  


5.56  


24       


10.83  



16.89  


5.39  


23       


3.44  



14 → 17 [a]



Mean


SD


N


%Diff



32.84  


7.99  


23       


.  



33.87  


7.05  


21       


3.12  



34.79  


9.40  


24       


5.94  



35.54   12.79  


23       


8.20  



17 → 20 [a]



Mean


SD


N


%Diff



51.28  


9.75  


23       


.  



49.43  


8.61  


21       


-3.62  



49.10   11.98  


24       


-4.26  



50.63   11.05  


23       


-1.28  



 

























































Sex: Female



Day(s) Relative to Mating



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight Gain


(g)



0 → 5 [a]



Mean


SD


N


%Diff



27.07   10.81  


23       


.  



24.29  


4.70  


21       


-10.28  



24.41  


7.69  


24       


-9.82  



24.76  


5.36  


23       


-8.51  



5 → 20 [a]



Mean


SD


N


%Diff



127.28   17.71  


23       


.  



127.36   17.31  


21       


0.06  



131.52   16.57  


24       


3.33  



132.45   12.40  


23       


4.06  



0 → 20 [a]



Mean


SD


N


%Diff



154.35   21.67  


23       


.  



151.64   19.49  


21       


-1.75  



155.93   18.28  


24       


1.03  



157.21   14.81  


23       


1.86  



Adjusted


Body weight


(g)



GD20 - GU WT


[a]



Mean


SD


N


%Diff



321.91   19.51  


23       


.  



319.50   21.72  


21       


-0.75  



319.77   20.49  


24       


-0.67  



323.23   17.42  


23       


0.41  



Adjusted


Body weight 


Gain


(g)



ADJ BWT-


GD5BWT [a]



Mean


SD


N


%Diff



43.10   11.26  


23       


.  



41.51  


6.50  


21       


-3.69  



42.69   12.01  


24       


-0.95  



44.48  


8.44  


23       


3.21  



GU WT: Gravid Uterus Weight  ADJ BWT: Adjusted Body Weight


 


TABLE 6.          Summary of Maternal Data






































































































































































Sex: Female


Day(s) Relative to Mating



 



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Group size



 



     N



24



24



24



24



Pregnant at C/S



 



    N



23



21



24



23



Gravid Uterus Weight



[a]



Mean


SD



84.183


19.582



85.852


17.355



88.837


15.053



87.965


14.543



Number of CorporaLutea



[k]



Mean


SD


Sum



16.4


1.8


377



16.2


2.0


340



17.3


2.1


414



16.7


2.3


384



No. of Implantation



[k]



Mean


SD


Sum



14.7


2.4


338



14.7


2.9


308



15.8


2.1


380



15.2


2.6


349



Dams with Early Resorption



 



   N



10



13



10



12



Number of Early Resorptions



[k]



Mean


SD


Sum



0.8


1.4


19



0.8


0.7


17



0.9


1.4


22



0.7


0.9


17



% Early


Resorption


/Animal



[k]



Mean


SD



6.88


15.80



6.24


6.14



5.72


9.00



4.92


5.92



Dams with Late Resorption



 



  N



3



5



5



0



Number of Late Resorptions



[k]



Mean


SD


Sum



0.2


0.5


4



0.3


0.7


7



0.3


0.5


6



0.0


0.0


0



% Late Resorption /Animal



[k]



Mean


SD



1.16


3.27



2.15


4.81



1.76


3.72



0.00


0.00



Dams with Resorptions



[f]



  N



11



15



13



12



Total Number of


Resorption (Early + Late)



[f]



Mean


SD


Sum



1.0 1.5


23



1.1 1.2


24



1.2 1.7


28



0.7


0.9


17



Pre-implantation Loss/Animal



[k]



Mean


SD



1.70


1.64


 



1.52


1.78


 



1.42


1.32


 



1.52


1.62


 



% Preimplantation


Loss



[k]



Mean


SD



10.4


10.7



9.9


13.3



8.1


7.1



9.1


10.3



Postimplantation


Loss/Animal



[k]



Mean


SD



1.00


1.51


 



1.14


1.20


 



1.17


1.74


 



0.74


0.86


 



% Postimplantation


Loss (%)



[k]



Mean SD



8.0


16.1



8.4


8.4



7.5


11.2



4.9


5.9



Note: Gross evaluation of placenta revealed no findings


[a] - Anova & Dunnett(Log)


[k] - Kruskal-Wallis & Wilcoxon


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


 


TABLE 7.        Summary of Litter Data



































































































































Sex: Female


Day(s) Relative to M



ating 



 



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Total Number of fetuses



 



Sum



315



284



352



332



Total Number of Dead Fetuses



 



Sum



0



0



0



0



Total Number of Live fetuses



 



Sum



315



284



352



332



Live Male fetus



 



Sum



173



158



187



159



% Male Fetus



 



 



54.9



55.6



53.1



47.9



Mean Fetal


Weight- Male


(g)



[c]



Mean


SD



4.01


0.33



4.29* 


0.33



4.10


0.24



4.08


0.32



Mean AGD- Male (mm)



[c]



Mean


SD



2.74


0.11



2.75


0.11



2.71


0.16



2.78


0.20



Live Female fetus



 



Sum



142



126



165



173



% Female Fetus



 



 



45.1



44.4



46.9



52.1



Mean Fetal


Weight- Female


(g)



[c1]



Mean


SD



3.78


0.31



4.03*


0.27



3.85


0.23



3.79


0.27



Mean AGD- Female (mm)



[c1]



Mean


SD



1.07


0.07



1.08


0.08



1.07


0.10



1.06


0.06



Mean Fetal


Weight -Male+Female


(g)



[c2]



Mean


SD



3.91


0.30



4.17* 


0.28



3.99


0.23



3.94


0.31



Mean AGD  Male + Female (mm)



[c2]



Mean


SD



1.98


0.24



2.02


0.19



1.95


0.26



1.87


0.30



[c] - Ancova/Anova & Dunnett; {Covariate(s): Number of Live Male Fetuses} 


[c1] - Ancova/Anova & Dunnett; Covariate(s): Number of Live Female Fetuses}


[c2] - Ancova/Anova & Dunnett; Covariate(s): Number of Live Fetuses}


*: Statistically significant different from vehicle control at p < 0.05


 


TABLE 8.         Summary of Fetal External Observations













































Exam Type: External



        


Number of Live Fetuses Examined:                                                         



           G1      


0 mg/kg/day



           G2      


111 mg/kg/day



           G3      


333 mg/kg/day



          G4      


1000 mg/kg/day



315  



284  



352  



          332     



 



         Number of Litters Evaluated:                                                   



           23      



            21      



           24      



           23      



Limbs


Tail, Rudimentary - Anomaly



 



Fetuses N(%)



1(0.3) 



0(0.0) 



1(0.3) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



[Fetuses N]


 


TABLE 9.         Summary of Fetal External Observations































































Exam Type: Fresh Visceral-Body Only



        


Number of Live Fetuses Examined:                                                    



           G1      


0 mg/kg/day



           G2      


111 mg/kg/day



           G3      


333 mg/kg/day



          G4      


1000 mg/kg/day



152  



137  



172  



          159     



 



          Number of Litters Ev aluated:                            



           23      



            21      



           24      



           23      



Abdomen


Kidney, both, Renal pelvis dilation, Moderate - Anomaly



 



Fetuses N(%)



0(0.0) 



0(0.0) 



1(0.6) 



3(1.9) 



 



 



Litters N(%)



0(0.0) 



0(0.0) 



1(4.2) 



2(8.7) 



Kidney, Right, Renal pelvis dilation, Moderate - Anomaly



 



Fetuses N(%)



0(0.0) 



0(0.0) 



0(0.0) 



1(0.6) 



 



 



Litters N(%)



0(0.0) 



0(0.0) 



0(0.0) 



1(4.3) 



[Fetuses N]


 


TABLE 10.       Summary of Fetal Skeletal Observations





























































































































































































Exam Type: Skeletal-Entire



        


Number of Live Fetuses Examined:                                                         



           G1      


0 mg/kg/day



           G2      


111 mg/kg/day



           G3      


333 mg/kg/day



          G4      


1000 mg/kg/day



163  



147  



180  



          173     



 



         Number of Litters Evaluated:                                                   



           23      



            21      



           24      



           23      



Caudal vertebrae


4th caudal centrum, Absent - Malformation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



3rd caudal centrum, Absent - Malformation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



2nd caudal centrum, Absent - Malformation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



1st caudal centrum, Absent - Malformation



 



Fetuses N(%)



0(0.0) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



0(0.0) 



0(0.0) 



1(4.2) 



0(0.0) 



2nd caudal arch, Absent - Malformation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



1st caudal arch, Absent - Malformation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



1(4.2) 



0(0.0) 



sacral vertebrae


4th sacral arch, Incomplete ossification - Variation



 



Fetuses N(%)



1(0.6) 



0(0.0) 



0(0.0) 



0(0.0) 



 



 



Litters N(%)



1(4.3) 



0(0.0) 



0(0.0) 



0(0.0) 



Lumbar vertebrae


1st lumbar centrum, Dumbbell-shaped - Anomaly



 



Fetuses N(%)



0(0.0) 



1(0.7) 



0(0.0) 



0(0.0) 



 



 



Litters N(%)



0(0.0) 



1(4.8) 



0(0.0) 



0(0.0) 



thoracic vertebrae


13th thoracic centrum, Split - Anomaly



 



Fetuses N(%)



0(0.0) 



1(0.7) 



1(0.6) 



0(0.0) 



 



 



Litters N(%)



0(0.0) 



1(4.8) 



1(4.2) 



0(0.0) 



[Fetuses N]

Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

• Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Orange 36 to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of C.I. Pigment Orange 36 to pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.


 


A total of 96Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:














































Group Nos.



Groups



Dose


(mg/kg/day)



Dosage volume (mL/kg)



Concentration (mg/mL)



No. of Day 0 pregnant rats



G1



Vehicle control*



0



10



0



24



G2



Low dose



111



10



11.1



24



G3



Mid dose



333



10



33.3



24



G4



High dose



1000



10



100



24



*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water


 


The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half of the fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (GD20).


 


Results of the study are summarized below:


 


·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected light to dark orange coloured faeces were observed in the test item treated groups which can be accounted for the physical nature of the test item.


Grossly, orangecontents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.


·        Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of
1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no remarkable findings.


·        Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.


·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to
1000 mg/kg/day.


·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Orange 36 up to the highest dose of
1000 mg/kg/day.


 


Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for


 


·           Maternal toxicity is1000 mg/kg/dayas the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.


 


Fetal developmental toxicity and Teratogencity is1000 mg/kg/dayas fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day





[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.


 



Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please see chapter 13: Read across justification
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park,
Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment for the first batch of rats and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.
Details on mating procedure:
The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day to confirm mating.
Duration of treatment / exposure:
Gestation day 5 to gestation day 19
Frequency of treatment:
Daily from gestation day 5 to gestation day 19
Duration of test:
Upto gestation day 20
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
111 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24 day 0 pergnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz221 Rz244
G2 Low dose 111 10 11.1 24 Rz245 Rz268
G3 Mid dose 333 10 33.3 24 Rz269 Rz292
G4 High dose 1000 10 100 24 Rz293 Rz316
Maternal examinations:
CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included
Ovaries and uterine content:
The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta
Fetal examinations:
• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.
Indices:
Refer Table 6 of the final report
Historical control data:
Refer Annexure 8 of the final report
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Grossly, orange contents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Orange HL (C.I. Pigment Orange 36) up to the highest dose of 1000 mg/kg/day
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions in the study
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

• Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Orange 36 to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of C.I. Pigment Orange 36 to pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

 

A total of 96Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.

Groups

Dose

(mg/kg/day)

Dosage volume (mL/kg)

Concentration (mg/mL)

No. of Day 0 pregnant rats

G1

Vehicle control*

0

10

0

24

G2

Low dose

111

10

11.1

24

G3

Mid dose

333

10

33.3

24

G4

High dose

1000

10

100

24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half of the fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected light to dark orange coloured faeces were observed in the test item treated groups which can be accounted for the physical nature of the test item.

Grossly, orangecontents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

·        Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of
1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no remarkable findings.

·        Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to
1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Orange 36 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

 

·           Maternal toxicity is1000 mg/kg/dayas the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

 

Fetal developmental toxicity and Teratogencity is1000 mg/kg/dayas fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day


[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
reliable
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification

The test material did not cause any adverse effects in reproductive and developmental toxicity studies in rats.

Additional information