Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
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 and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: (P) 13-14 weeks
- Weight at study initiation: (P) Males:330.41 to 390.80 g; Females: 200.11 to 230.68 g;

- Housing: Pre-mating: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.

Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term (GD 20).

Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects during pre-mating period and post-mating period for males and pre-mating period for females. Enrichment was changed along with cage at least once a week.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet-Pellet (Certified) manufactured by Envigo, P.O. Box 44220, Madison, WI 53744-4220, was provided ad libitum to animals.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: Start: 26 May 2017 End: 30 May 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20−24°C
- Humidity (%): 59 – 68 %
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours’ light and 12 hours’ dark cycle
IN-LIFE DATES: From: 31 May 2017 To: 11August 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Required quantities of the test item was weighed in to a pre-calibrated beaker* and small volume of vehicle [0.5 % (w/v) of carboxymethyl cellulose in Milli-Q® water] was added and mixed using glass rod. The volume was made up with the vehicle to attain desired concentrations of 11, 33 and 100 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test, the test item forms a suspension in 0.5% carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q® water, while it could not be dissolved/suspended in Milli-Q® water. Hence 0.5% carboxymethyl cellulose sodium salt (medium viscosity) was used as vehicle for dose formulation preparation
- Concentration in vehicle: G2: 11; G3: 33 and G4: 100 mg/mL
- Amount of vehicle (if gavage): 10.0 g of Sodium carboxy methyl cellulose was added to 1800 mL of Milli-Q® water in a 2000 mL pre-marked beaker* and stirred on a magnetic stirrer.
- Lot/batch no. (if required): MKBQ8416V
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how): One per cage
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and Day 35 (2nd month) of the treatment and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn.
The analysis was done as per the method validated under Advinus Study No.: G14745. One set of samples were analysed for concentration (a.i) analysis.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to specific groups of rats at approximately the same time each day (varying by ± 3 hours), for a period of 36 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed after overnight fasting.
Females: The dose formulation was administered orally by gavage to the specific groups of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and continued through mating, pregnancy and up to LD 13. On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.
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 group at an equivolume of 10 mL/kg Bwt.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: [15 to 16] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
110 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 110 (G2), 330 (G3) and 1000 (G4) mg/kg/day were selected for this by the Sponsor based on the outcome of a 28-day repeated dose study.
In addition to the test doses, vehicle control group was included. Animals in the vehicle control group were handled in a manner similar to the treatment groups except for the test item administration.

- Rationale for animal assignment (if not random): Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5-day cycle) was weighed and the corresponding body weights was recorded. The data was transferred to EDP (Electronic Data Processing) for data input (temporary identification number and body weight) into an excel spread sheet. The body weight recorded was stratified in ascending order.
Statistical analysis and ensured that the weight variation was minimal and inter group variation did not exceed ± 20% of the mean body weight in each sex. Rats with extreme body weights were discarded. Grouping was done one day prior to initiation of the treatment.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [Yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:] Yes

GROSS EXAMINATION OF DEAD PUPS:
[Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in [Section 9.9.2 in Main report] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.9.3 in Main report] were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, ano-genital index and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). When the data found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test is found significant.
Post implantation loss (%), no. of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was performed for testing the differences found significant.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group
Reproductive indices:
a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100

b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100

c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100

d. Female fertility index (%) = [Number of pregnant females (confirmed at necropsy) / Number of females used for mating] x 100

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 / Number of implantations) x 100
Offspring viability indices:
a. Mean litter size per group = (Total Number of pups / Total Number of littered animals)

b. Mean viable litter size = (No. of viable pups on Day 1 / No. of females littered)

c. Live birth index (%) = [No. of viable pups born (at first observation) / Total no. of pups born (at first observation)] x 100

d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100

e. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100

f. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were observed at any of the doses tested in both sexes. Light yellow coloured faeces were observed at 110 and 330 mg/kg Bwt/day dose, whereas at 1000 mg/kg Bwt/day dose, the dark yellow coloured faeces were observed in both sexes. The yellow coloured faeces observed are considered to be due to physical nature of the test item.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed at any of the tested doses in both sexes.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not altered throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treatment groups.
Decreased thyroid stimulating hormone (TSH) level noted in adult males (39%) at 1000 mg/kg Bwt/day dose was considered incidental, as it was not accompanied by any changes in thyroxin (T4) level and/or microscopic correlation.
Decreased thyroxin (T4) level noted in pups on lactation Day 4 at all dose level (G2: 27%, G3: 34% and G4: 20%) was considered incidental, as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and also lacked the dose progression and/or microscopic correlation.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes observed in the terminal fasting body weights in adult male and female rats.
Statistically significant increase in absolute and relative thyroid gland weight at all doses in adult male rats considered as test item-related non adverse change in absence of correlating microscopic changes in thyroid gland at 1000 mg/kg Bwt/day.
There were no test item-related changes observed in organ weight in adult female rats.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related adverse gross pathological changes observed in reproductive organs in both males and females rats. Gross pathological examination of dead pups and pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.
Grossly, yellow intestinal content at ≥330 mg/kg Bwt/day in males and at 1000 mg/kg Bwt/day in females was observed. In addition, yellow cecum content at 330 mg/kg Bwt/day in males and yellow stomach content at 1000 mg/kg Bwt/day in females were observed. All these gross pathology changes were attributed to the physical nature of test item and considered as test item-related non-adverse change.
Single incidence of small sized female pup (Dam: Ru1628) was observed at 330 mg/kg Bwt/day considered as incidental finding and not related to test item administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related histopathological changes noted (including reproductive tissues) in any of the adult male and female rats. The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats.

There were no test item-related microscopic changes in thyroid gland of pups of all the dose levels.

Few randomly distributed microscopic findings observed in some of the rats were considered as incidental background findings commonly noted at this age group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
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.
Pre-coital time was determined (in days) from oestrous cycle evaluation from the day of Cohabitation to the evidence of mating.
The calculated mean oestrous cycle length was 4.00, 4.05, 4.00 and 4.05 days in vehicle control, 110, 330 and 1000 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive performance:
no effects observed
Description (incidence and severity):
The duration of gestation (gestation length), pre-coital time, fertility indices were not affected at any of the doses tested, when compared to vehicle control group.

Details on results (P0)

The purpose of this study in Wistar Rats was to generate information concerning the effects of PV-Echtgelb H9G VP 2430 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.
The test item was weighed and suspended in vehicle i.e., 0.5% Carboxy Methylcellulose Sodium salt (medium viscosity) in Milli-Q Water and administered by oral gavage at the dose levels of 110 (G2), 330 (G3) and 1000 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 males and 10 female rats. The prepared dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females.
The identity of PV-Echtgelb H9G VP 2430 was provided by the study sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 1 and 110 mg/mL under Advinus Study No. G14745. Based on the results, the test item was stable and homogenous in the vehicle (0.5 % Carboxy Methylcellulose Sodium salt (medium viscosity) in Milli-Q® water) up to 24 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd month (Day 35) of the treatment period. The results indicated that the analysed concentrations were within ± 15% of variations from the theoretical concentrations.
All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period where food consumption was not measured. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day 13 (PND 13). Blood samples were collected for thyroid hormone analysis prior to necropsy from males and females (LD 13), on LDs 4 and 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. Gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.
Tissues collected from all animals in the control and high dose groups 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 any test item-related findings at the high dose, the low and mid 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:
• There were no treatment-related clinical signs observed at any of the doses tested in both sexes.
• The body weights and food consumption were unaffected by the treatment at all the tested doses in both sexes.
• The maternal body weights and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses tested.
• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, fertility indices of sires and dams and survival indices at all the tested doses.
• Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.
• 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 and ratio to the cube root of body weight were observed at any of the doses tested when compared to the vehicle control group.
• No areola/nipple retention was observed in male pups on PND 13 at any of the doses tested.
• There were no test item related changes in the terminal body weights at any of the doses tested when compared to the vehicle control group. Significant increase in the absolute and relative weight of thyroid gland was observed at all the tested doses in adult males. This change was considered as test item-related non-adverse as there were no associated microscopic changes in thyroid gland at all the tested doses.
• Decrease in the thyroid stimulating hormone (TSH) level in adult males (39%) at 1000 mg/kg Bwt/day dose was considered incidental, as it was not accompanied by any changes in thyroxin (T4) level and/or microscopic correlation. Decreased thyroxin (T4) level noted in pups on lactation Day 4 at all dose level (G2: 27%, G3: 34% and G4: 20%) was considered incidental, as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and also lacked the dose progression and/or microscopic correlation.
• Grossly, yellow intestinal content at ≥330 mg/kg Bwt/day in males and at 1000 mg/kg Bwt/day in females, yellow stomach content at 1000 mg/kg Bwt/day in females were observed. All these gross pathology changes were attributed to the physical nature of test item and considered test item-related non-adverse effects. There were no test item-related histopathological changes noted (including reproductive tissues) in any of the adult male and female rats. The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats. There were no test item-related microscopic changes in thyroid gland of pups at all the doses tested.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treatment groups.
Decreased thyroid stimulating hormone (TSH) level noted in adult males (39%) at 1000 mg/kg Bwt/day dose was considered incidental, as it was not accompanied by any changes in thyroxin (T4) level and/or microscopic correlation.
Decreased thyroxin (T4) level noted in pups on lactation Day 4 at all dose level (G2: 27%, G3: 34% and G4: 20%) was considered incidental, as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and also lacked the dose progression and/or microscopic correlation.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No treatment-related changes in the ano-genital distance and ratio to the cube root of body weight were observed at any of the doses tested when compared to the vehicle control group.
No areola/nipple retention was observed in male pups on PND 13 at any of the doses tested.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant changes in terminal body weights and thyroid weights in male/female pups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross pathological examination of dead pups and pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.
Single incidence of small sized female pup (Dam: Ru1628) was observed at 330 mg/kg Bwt/day considered as incidental finding and not related to test item administration.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic changes in thyroid gland of pups of all the dose levels.
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
body weight and weight gain
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

 

Table 1: Summary of Body Weights (g) and Net Body Weight Gains (g) – Males

 

Group

 

Week

Net

Dose

Day 1

1

2

3

4

5

 

Bwt. gain

(mg/kgBwt/day)

 

 

 

 

 

 

 

 

Week 1-5

G1

Mean

359.97

365.95

375.13

379.16

389.81

404.37

 

44.40

0

SD

16.67

18.70

23.40

22.36

22.86

23.29

 

11.23

N

10

10

10

10

10

10

 

10

 

 

G2

Mean

357.54

364.27

373.49

377.03

388.37

400.14

 

42.59

110

SD

16.76

18.41

18.48

19.18

20.76

19.53

 

8.59

N

10

10

10

10

10

10

 

10

 

 

G3

Mean

358.85

367.54

376.84

380.07

390.18

403.36

 

44.51

330

SD

16.84

18.72

19.56

22.08

24.69

27.83

 

12.56

N

10

10

10

10

10

10

 

10

 

 

G4

Mean

358.04

362.08

371.88

376.83

388.84

401.43

 

43.39

1000

SD

16.71

20.24

23.57

25.44

28.95

31.29

 

19.08

 

N

10

10

10

10

10

10

 

10

N: No. of rats


 

Table 2: Summary of Body Weights (g) and Net Body Weight Gains (g) – Females

 

Group

 

Week

Net

Dose

Day 1

1

2

Bwt. gain

(mg/kgBwt/day)

 

 

 

 

Week 1-2

G1

Mean

218.21

219.94

224.64

6.43

0

SD

8.97

8.01

7.41

6.89

N

10

10

10

10

G2

Mean

219.28

220.08

221.78

2.50

110

SD

7.76

7.62

7.41

6.35

N

10

10

10

10

G3

Mean

220.53

220.73

222.32

1.78

330

SD

7.53

6.17

9.85

5.73

N

10

10

10

10

G4

Mean

219.46

218.42

223.57

4.11

1000

SD

8.10

6.30

8.13

5.23

 

N

10

10

10

10

N: No. of rats

 

 

 

 

 

 


 

Table 3: Summary of Food Consumption (g/rat/day) – Males

 

 

Week

Dose

1

2

5

 

(mg/kgBwt/day)

 

 

 

 

 

G1

Mean

22.68

21.53

22.82

 

0

SD

1.04

1.18

0.95

 

N

5

5

5

 

 

G2

Mean

21.87

21.31

22.01

 

110

SD

1.12

1.33

1.45

 

N

5

5

5

 

 

G3

Mean

23.19

21.85

23.37

 

330

SD

2.74

1.28

1.52

 

N

5

5

5

 

 

G4

Mean

22.96

21.99

22.40

 

1000

SD

1.25

1.27

1.27

 

 

N

5

5

5

 

N: No. of cages


 

Table 4: Summary of Food Consumption (g/rat/day) – Females

 

Group

 

Week

Dose

1

2

(mg/kgBwt/day)

 

 

 

G1

Mean

16.67

16.54

0

SD

1.56

1.05

N

5

5

G2

Mean

16.78

16.12

110

SD

1.51

0.86

N

5

5

G3

Mean

16.72

16.24

330

SD

1.22

1.48

N

5

5

G4

Mean

16.62

16.09

1000

SD

0.51

0.42

N

5

5

N: No. of cages

 

 

 


 

Table 5: Summary ofOestrousCycle Length Prior to Cohabitation Period

 

Group No.

 

 

 

Dose

No. of rats

Oestrouscycle length (Days)

(mg/kgBwt/day)

 

 

 

G1

10

Mean

4.00

0

SD

0.14

 

 

G2

10

Mean

4.05

110

SD

0.25

 

 

 

G3

10

Mean

4.00

330

SD

0.14

 

 

 

G4

10

Mean

4.05

1000

SD

0.16

 

 

 

 

 

 


 

Table 6: Summary of Maternal Body Weights (g) and Weight Change During Gestation Period

 

Group

 

Body weight on gestation day

Body weight change during gestation days

Dose

0

7

14

20

 0-7

 7-14

 14-20

 0-20

(mg/kgBwt/day)

 

 

 

 

 

 

 

 

 

G1

Mean

226.64

246.69

272.12

325.41

20.05

25.43

53.29

98.77

0

SD

10.56

10.05

11.44

14.76

3.23

3.99

7.33

11.40

N

10

10

10

10

10

10

10

10

G2

Mean

228.79

249.97

276.78

329.27

21.18

26.80

52.50

100.48

110

SD

11.33

15.06

15.75

17.61

5.97

5.38

5.45

12.29

N

10

10

10

10

10

10

10

10

G3

Mean

235.98

256.04

279.86

333.22

20.06

23.83

53.36

97.24

330

SD

10.88

11.76

13.30

18.32

4.45

8.08

5.65

14.69

N

10

10

10

10

10

10

10

10

G4

Mean

223.91

248.13

274.22

323.05

24.21

26.09

48.83

99.13

1000

SD

5.86

8.38

10.85

20.41

7.74

3.50

11.93

18.35

N

10

10

10

10

10

10

10

10

N: No.of Dams


 

Table 7: Summary of Maternal Food Intake (g) During Gestation Period

 

Group

 

Total food intake during gestation days

Per day food intake during gestation

Dose

 0-7

 7-14

 14-20

 0-20

 0-7

 7-14

 14-20

 0-20

(mg/kgBwt/day)

 

 

 

 

 

 

 

 

 

G1

Mean

132.75

152.01

139.63

424.39

18.96

21.72

23.27

21.22

0

SD

7.31

12.36

14.88

30.72

1.05

1.76

2.48

1.53

N

10

10

10

10

10

10

10

10

G2

Mean

139.13

154.21

140.54

433.87

19.88

22.03

23.42

21.69

110

SD

16.40

16.26

12.58

40.97

2.34

2.32

2.10

2.05

N

10

10

10

10

10

10

10

10

G3

Mean

137.10

155.41

142.22

434.73

19.59

22.20

23.70

21.74

330

SD

14.36

17.88

17.20

42.31

2.05

2.56

2.87

2.11

N

10

10

10

10

10

10

10

10

G4

Mean

136.68

156.25

137.44

430.37

19.53

22.32

22.91

21.52

1000

SD

8.74

8.14

11.88

17.44

1.25

1.16

1.98

0.87

N

10

10

10

10

10

10

10

10

N: No.of Dams

 

 

 

 

 

 

 

 

 

 


Table 8: Summary of Maternal Body Weights (g) and Weight Change During Lactation Period

 

Group

 

Body weight on Day

Weight change during

Dose

0

4

13

0-4

4-13

0-13

(mg/kgBwt/day)

 

 

 

 

 

 

 

G1

Mean

244.75

250.50

256.10

5.76

5.60

11.36

0

SD

14.84

15.02

18.32

5.39

5.39

7.29

N

10

10

10

10

10

10

+

G2

Mean

240.33

254.55

267.04

14.22

12.48

26.71

110

SD

20.35

16.04

16.99

10.43

6.17

6.58

N

10

10

10

10

10

10

G3

Mean

248.26

258.77

268.30

10.50

9.53

20.04

330

SD

18.36

21.17

14.55

9.74

9.38

7.81

N

10

10

10

10

10

10

+

G4

Mean

239.50

240.88

257.90

1.37

17.20

17.11

1000

SD

14.56

14.31

14.04

8.41

10.94

11.44

N

10

10

9

10

9

9

N: No.of Dams; +Significantly higher than the vehicle control group


 

Table 9: Summary of Maternal Food Intake (g) During Lactation Period

 

Group

 

Total food intake during lactation days

Per day food intake during lactation days

Dose

 0-4

 4-13

 0-13

 0-4

 4-13

 0-13

(mg/kgBwt/day)

 

 

 

 

 

 

 

G1

Mean

116.20

418.62

534.82

29.05

46.52

41.14

0

SD

11.70

27.49

26.78

2.93

3.06

2.06

N

10

10

10

10

10

10

G2

Mean

126.21

415.05

541.26

31.55

46.12

41.64

110

SD

25.54

61.30

66.89

6.38

6.81

5.14

N

10

10

10

10

10

10

G3

Mean

121.05

442.83

563.88

30.26

49.20

43.38

330

SD

13.36

28.32

29.37

3.34

3.15

2.26

N

10

10

10

10

10

10

G4

Mean

109.97

409.87

520.40

27.49

45.54

40.03

1000

SD

9.52

29.38

37.22

2.38

3.26

2.86

N

10

9

9

10

9

9

 

N: No.of Dams

 

Applicant's summary and conclusion

Conclusions:
To summarize, daily oral (gavage) administration of test item “PV-Echtgelb H9G VP 2430” to Wistar rats at the dose levels 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females had no effects on general health, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters, survival indices and terminal body weights. The significant increase in absolute and relative of thyroid gland weight at all the tested doses in adult males was considered as test item-related non-adverse change as there were no associated microscopic changes observed in thyroid gland.

There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treatment groups. Decreased thyroid stimulating hormone (TSH) level was observed in adult males (39%) at 1000 mg/kg Bwt/day dose and was considered incidental, as there were no associated changes in thyroxin (T4) level and/or microscopic correlation. Decreased thyroxin (T4) level observed in pups on lactation Day 4 at all dose level (G2: 27%, G3: 34% and G4: 20%) was considered incidental, as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and also lacked the dose progression and/or microscopic correlation

Grossly, yellow intestinal content at ≥330 mg/kg Bwt/day in males and at 1000 mg/kg Bwt/day in females, yellow stomach content at 1000 mg/kg Bwt/day in females were observed. All these gross pathology changes were attributed to the physical nature of test item and considered as test item-related non-adverse effects.
There were no test item-related histopathological changes noted (including reproductive tissues) in any of the adult male and female rats.

The qualitative assessment of stages of spermatogenesis in testes did not reveal any test item associated findings in adult rats.

There were no test item-related microscopic changes in thyroid gland of pups at all the doses tested.
No Observed Adverse Effect Level
Daily oral (gavage) administration of PV-Echtgelb H9G VP 2430 to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, thyroid hormones, reproductive performance and on the offspring parameters.

Hence, the No Observed Adverse Effect Level (NOAEL) of PV-Echtgelb H9G VP 2430 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.
Executive summary:

The purpose of this study in Wistar Rats was to generate information concerning the effects of PV-Echtgelb H9G VP 2430on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was weighed and suspended in vehiclei.e.,0.5% Carboxy Methylcellulose Sodium salt (medium viscosity) in Milli-Q Waterand administered by oral gavage at the dose levels of 110 (G2), 330 (G3) and 1000 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 males and 10 female rats. The prepared dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females.

The identity ofPV-Echtgelb H9G VP 2430was provided by the study sponsor by aCertificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 1 and 110 mg/mL under Advinus Study No. G14745. Based on the results, the test item was stable and homogenous in the vehicle (0.5 % Carboxy Methylcellulose Sodium salt (medium viscosity) in Milli-Q®water) up to 24 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2ndmonth (Day 35) of the treatment period. The results indicated that the analysed concentrations were within ± 15% of variations from the theoretical concentrations. 

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period where food consumption was not measured.Female rats were also weighed onGestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day 13 (PND 13). Blood samples were collected for thyroid hormone analysis prior to necropsy from males and females (LD 13), on LDs 4 and 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. Gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.

Tissues collected from all animals in the control and high dose groups 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 any test item-related findings at the high dose, the low and mid 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:

·        There were no treatment-related clinical signs observed at any of the doses tested in both sexes.

·        The body weights and food consumption were unaffected by the treatment at all the tested doses in both sexes.

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

·        Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, fertility indices of sires and dams and survival indices at all the tested doses.

·        Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.

·        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 and ratio to the cube root of body weight were observed at any of the doses tested when compared to the vehicle control group.

·        No areola/nipple retention was observed in male pups on PND 13 at any of the doses tested.

·        There were no test item related changes in the terminal body weightsat any of the doses tested when compared to the vehicle control group.Significant increase in the absolute and relative weight of thyroid gland was observed at all the tested doses in adult males. This change was considered as test item-related non-adverse as there were no associated microscopic changes in thyroid gland at all the tested doses.

·        

·        thyroid gland of pups at all the doses tested.

No Observed Adverse Effect Level

Daily oral (gavage) administration of PV-Echtgelb H9G VP 2430 to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, thyroid hormones, reproductive performance and on the offspring parameters. Hence, the No Observed Adverse Effect Level (NOAEL) of PV-Echtgelb H9G VP 2430 forreproductive and developmental toxicityis considered to be          1000 mg/kg Bwt/day.