Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 04, 2016 to June 21, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD and GLP

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
- Premating exposure duration for parental (P0) animals
Two weeks

- Basis for dose level selection
Dose levels for the study were selected based on the observation and results of Dose Range Finding study (RCC Study No. 118). As per Dose Range Finding study, mild histopathological findings in low dose group were observed. Hence as per the sponsor, three dose groups were included in this study i.e. 30, 100 and 300 mg/kg.
- Route of administration
Oral

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recommended by the guidelines
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Animal Breeding
RCC Laboratories India Private Limited

- Females (if applicable) nulliparous and non-pregnant: [yes/no]
Yes

- Age at study initiation: (P) x wks; (F1) x wks
13-14 weeks

- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
Males = 308.6 to 383.3 g, Females = 212.4 to 271.7 g

- Housing:

Accommodation
In groups of two animals per cage in Polycarbonate cages (Approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding for acclimatization. Pregnant females were caged individually with corn cob bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.

- Diet (e.g. ad libitum):
ad libitum
- Water (e.g. ad libitum):
ad libitum
- Acclimation period:
Six days under laboratory conditions, after veterinary examination
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21.0 to 22.4°C
- Humidity (%):
57 to 64 %
- Air changes (per hr):
more than 10
- Photoperiod (hrs dark / hrs light):
12 hours light and 12 hours dark.
IN-LIFE DATES: From: May 18, 2016 To: August 05, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
3.0 mg/ml - Low dose (corresponds to 5.3 mg test material (reaction mass))
30 mg/ml - mid dose (corresponds to 53 mg test material (reaction mass))
100 mg/ ml - High dose (corresponds to 177 mg test material (reaction mass))
- Amount of vehicle (if gavage):
10 ml/ kg

all doses relate to the active ingredient of the reaction mass, trisodium trioxalatoferrate. The quantity of the test material was chosen accordingly
Details on mating procedure:
- M/F ratio per cage:
- Length of cohabitation:
14 days

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
sperm in vaginal smear] referred to as day 0 of pregnancy

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
14 days
- Further matings after two unsuccessful attempts: [no / yes (explain)]
No
- After successful mating each pregnant female was caged (how):
Individually
- Any other deviations from standard protocol:
No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluent (vehicle) on treatment start date and treatment end date for homogeneity (mean of homogeneity is given as dose concentration) analyses. On week 7, samples of all dose formulations were analysed for dose concentrations by analysing triplicate samples
The recovery content was found homogeneously distributed in the suspension at concentrations 3.0 mg/mL, 10 mg/mL and 30 mg/mL for the test item respectively. (i.e. % coefficient of variation (RSD) ranged in between 0.153 to 0.578)
Duration of treatment / exposure:
Males were dosed for a day 0 to day 30. This includes two weeks prior to mating, mating and post mating period
Females were dosed throughout the study this includes two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery
Frequency of treatment:
once daily
Details on study schedule:
Administration / Treatment
Males
Premating period June 07, 2016 to June 20, 2016
Mating & Post mating period June 21, 2016 to July 01, 2016
Females
Premating period June 07, 2016 to June 20, 2016
Mating period* June 21, 2016 to July 01, 2016
Gestation period* June 22, 2016 to July 23, 2016
Lactation period* July 13, 2016 to August 05, 2016

Blood collection for T4 & TSH July 07, 2016, July 18, 2016 to August 05, 2016
Necropsy
Males July 07, 2016
Females July 27, 2016 to August 05, 2016
Pups July 18, 2016 to August 05, 2016
Experimental Completion Date January 27, 2017

Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day
Remarks:
Intermediate dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
High Dose
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes
Details on study design:
- Dose selection rationale:


Dose levels for this study were selected based on the observation and results of Dose Range Finding study (RCC Study No. 118). As per Dose Range Finding study, mild histopathological findings in low dose group were observed. Hence as per the sponsor, three dose groups were included in this study i.e. 30, 100 and 300 mg/kg (active ingredient)
- Rationale for animal assignment (if not random):
- Other:

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: YES
- Cage side observations checked in table [No.?] were included. YES

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: YES

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: YES

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
YES
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
NO

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: NO

OTHER:
Oestrous cyclicity (parental animals):
Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study
Sperm parameters (parental animals):
Histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) and epididymides in male animals
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Yes
[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:]

GROSS EXAMINATION OF DEAD PUPS: YES
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: NO

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: NO
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

Animals survived till the end of the treatment period were euthanized by carbon dioxide asphyxiation and necropsied.


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

All the macroscopic abnormalities were recorded. The ovaries, uterus, cervix, testes, epididymides, prostate, seminal vesicles with coagulating glands and thyroid of all adult animals were preserved in 10% neutral buffered formalin (NBF) solution except testes and epididymides which were preserved in modified Davidson’s fixative for 24 hours and transferred to NBF.
Histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) and epididymides in male animals and ovaries in female animals of high dose (G4) and control (G1) group animals

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Weight of the following organs was recorded on scheduled dates of necropsy. Wet weights were taken immediately after dissection to avoid drying for adult animals.
Epididymides
Testes
Uterus
Ovaries
Prostate gland
Seminal vesicles
Levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis
Thyroid (after fixation)



Postmortem examinations (offspring):
on day 13 All surviving pups will be subjected for the gross examination
Statistics:
The statistical methods (Normality, ANOVA/ T-test)were used to analyze the body weight, feed consumption and organ weights and biochemical parameters.
Data are summarized in tabular form. Statistical analysis was performed using statplus program. Statistical analysis of AGD was based on individual pup data, taking litter effects into account. Where appropriate, the litter was the unit of analysis. Statistical analysis of pup body weight was based on individual pup data, taking litter size into account.
Reproductive indices:
Mating Index (%)

Pregnancy Index (%)

Parturition Index (%)

Pre–natal loss



Mating Index (%)

Pregnancy Index (%)

Parturition Index (%)




Offspring viability indices:

Pre–natal loss

Post– natal loss

Sex Ratio (% males)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in the control group (0 mg/kg bodyweight), low dose group (30 mg/kg body weight, active ingredient), mid dose group (100 mg/kg body weight, active ingredient) and high dose groups (300 mg/kg body weight) in both male and female animals
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant findings in thyroxine (T4) and thyroid stimulating hormone (TSH) levels were observed in parent animals (males, females)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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):
Microscopic examination revealed no abnormality attributable to the test item in high dose (G4) group. The tissues evaluated were within normal histological limits. Microscopically, degeneration/atrophy of testes and aspermia with necrotic debris and giant cells in epididymides was observed in one male animal of high dose group. The changes observed were considered to be incidental findings.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
the value relates to trisodium trioxalatoferrate and not to the test material (reaction mass)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: P1 (second parental generation)

General toxicity (P1)

Clinical biochemistry findings:
no effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
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:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
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:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Remarks:
the value relates to trisodium trioxalatoferrate and not to the test material (reaction mass)
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology

Overall reproductive toxicity

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

Any other information on results incl. tables

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

 

Parameters

Group 1

Group 2

Group 3

Group 4

Pairs started (N)

12

12

12

12

Females showing evidence of copulation (N)

12

12

12

12

Females achieving pregnancy (N)

12

12

12

12

Conceiving Days 1 - 5 (N)

11

11

8

8

Conceiving Days ≥ 6 (N)

1

1

4

4

Pregnancy = 20 Days (N)

0

0

0

0

Pregnancy = 21 Days (N)

0

0

1

2

Pregnancy = 22 Days (N)#

9

11

10

7

Pregnancy = 23 Days (N)#

3

1

1

3

Dams with live young born (N)

12

12

12

12

Dams with live young at Day 4 pp (N)

10

11

10

10

Implants/dam (mean)

8.08

9.00

8.75

9.92

Live pups/dam at birth (mean)

7.20

7.55

6.50

7.00

Live pups/dam at Day 4 (mean)

7.20

7.55

6.50

7.00

Sex ratio % males at Day 1 post partum

60.62

53.17

54.67

46.06

Sex ratio % males at Day 4 post partum

60.62

53.17

54.67

46.06

Pup weight at Day 1 -Combined (g, mean)

3.87

4.01

3.84

3.83

Pup weight at Day 4 - Combined (g, mean)

7.79

8.31

8.16

8.13

Pup weight at Day 1 Male (g, mean)

4.13

4.35

4.14

4.17

Pup weight at Day 4 Male ((g, mean)

8.21

8.92

8.75

8.75

Pup weight at Day 1 Female (g, mean)

3.48

3.63

3.48

3.53

Pup weight at Day 4 Female (g, mean)

7.20

7.45

7.42

7.53

Male with nipple retention on day 13

0

0

0

0

ABNORMAL PUPS (Based on gross finding) 

Dams with 0 (N)

10

11

10

10

Dams with 1 (N)

10

11

10

10

Dams with 2 (N)

10

11

10

10

Dams with more than 2 abnormal pups (N)

10

11

10

10

 

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

 

Parameters

Group 1

Group 2

Group 3

Group 4

LOSS OF OFFSPRING  

Pre-natal (Implantations minus live births) 

Females with 0 (N)

5

1

3

0

Females with 1 (N)

1

4

3

4

Females with 2 (N)

1

2

2

2

Females with 3 (N)

3

3

1

3

Females with >4 (N)

0

1

1

1

Post-natal (live births minus alive at post natal Day 4) 

Females with 0 (N)

10

11

10

10

Females with 1 (N)

10

11

10

10

Females with 2 (N)

10

11

10

10

Females with 3 (N)

10

11

10

10

Females with >4 (N)

10

11

10

10

Post-natal (live births minus alive at post natal Day 13) 

Females with 0 (N)

10

11

10

10

Females with 1 (N)

10

11

10

10

Females with 2 (N)

10

11

10

10

Females with 3 (N)

10

11

10

10

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The administration of the test substance to Wistar rats by oral gavage, at dose levels of 30, 100 and 300 mg/kg/day (active ingredient), resulted in no treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals. No treatment-related effects on reproduction/ development such as mating index, fertility index, gestation length, pre-natal loss, post-natal loss, sex ratio and offspring growth and development were observed. Therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity is considered to be the highest dose employed in the studyi.e.300 mg/kg/day.
No Observed Effect Level (NOEL) for Reproduction / Developmental Toxicity : 300 mg/kg body weight (the value relates to trisodium trioxalatoferrate and not to the test material (reaction mass))
Executive summary:

The administration of the test substance to Wistar rats by oral gavage, at dose levels of 30, 100 and 300 mg/kg/day, resulted in no treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals. No treatment-related effects on reproduction/ development such as mating index, fertility index, gestation length, pre-natal loss, post-natal loss, sex ratio and offspring growth and development were observed. Therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity is considered to be the highest dose employed in the studyi.e.300 mg/kg/day.

No Observed Effect Level (NOEL) for Reproduction / Developmental Toxicity : 300 mg/kg body weight (the value relates to the active ingredient trisodium trioxalatoferrate and not to the test material (reaction mass))