Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 January 2017 to 28 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:


NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Genamin DMG 75 (N, N-Dimethyl-D-Glucamine)

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)
Version / remarks:
OECD Guideline for Testing of Chemicals, Number 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29 July 2016
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN:

- Premating exposure duration for parental (P0) animals: Yes (14 days)

- Basis for dose level selection: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected based on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Genamin DMG 75 by Oral Gavage in Sprague Dawley Rats (Study No.: BIO-TX 1986).

These doses were selected as the test item Genamin DMG 75 when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 39 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups and females (dams) were sacrificed on lactation day 14 did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight). .

- Route of administration: oral (gavage) route

- Other considerations:
choice of species and strain: Rat (Rattus norvegicus) and Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities)

vehicle: distilled water was used as vehicle for the preparation of formulations as the test item is clearly miscible with distilled water as evidenced by the in-house miscibility test results

number of animals: 96 (48 Males + 48 Females); 12 Males + 12 Females/group as per OECD 421 guideline specification

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Genamin DMG 75


Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Global Amines Germany GmbH
84504 Burgkirchen, Germany and RAK-KRS-00022

- Expiration date of the batch: 01 April 2018
- Purity test date: 24 September 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: 6 hours at Ambient (21 to 29°C) conditions as per Study No.: BIO-ANM 374 at concentrations of 10 and 100 mg/mL.

- Solubility of the test substance in the solvent/vehicle: Distilled water was used as vehicle for the preparation of formulations as the test item is clearly miscible with distilled water as evidenced by the in-house miscibility test results.

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 wks
- Weight at study initiation: Males: 200 to 300 g; Females: 200 to 300 g
- Housing: standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube.

Premating: two animals of same sex and group per cage.
Mating: two animals (one male and one female) of same group .

- Diet (ad libitum): Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet - Pellet (Manufactured by Envigo) ad libitum.
- Water (ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period (Including Oestrus Cycle Evaluation): 25 January 2017 to 12 February 2017
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 to 23.6oC
- Humidity (%): 40 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 25 January 2017; To: 23 April 2017 (till end of last female necropsy)

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Remarks on MMAD:
not apllicable
Vehicle:
not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Distilled water was used as vehicle for the preparation of formulations as the test item is clearly miscible with distilled water as evidenced by the in-house miscibility test results.
- Concentration in vehicle: G1 (vehicle control): 0 mg/mL; G2 (low dose): 10 mg/mL; G3 (mid dose): 30 mg/mL; G4 (high dose): 100 mg/mL
- Amount of vehicle: 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 20 days (including remating of females with proven male of same group after 14 days)
- Proof of pregnancy: 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: individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For active ingredient (a.i.) concentration analysis, the prepared formulations were sampled in duplicate sets (5 mL for each set from middle layer) from each dose level during weeks 1 and 5 of the treatment period. The samples (both sets) were sent to Analytical Department of Bioneeds India Private Limited for concentration analysis. The analysis was done as per the validated method (Bioneeds Study No.: BIO-ANM 374).
The collected samples were transferred to Analytical Department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analysed. The second aliquot was stored as a backup purpose at room temperature. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) was equal to or less than 10.0%.

Duration of treatment / exposure:
Males: two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment).

Females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 14 to 15 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
12 animals per sex per dose (group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected based on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Genamin DMG 75 by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1986).
These doses were selected as the test item Genamin DMG 75 when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 39 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups and females (dams) were sacrificed on lactation day 14 did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+9.06 and -10.90 for males and +9.58 and -8.41 for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once were included in (table 1)

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. (table 2, 3, 9, 10, 12 & 13)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; weekly once; gestation days 0 to 7, 7 to 14 and 14 to 20 during pregnancy and day 1 to 4, 4 to 7, 7 to 13 during lactation period. (table 4, 11 & 14)

Oestrous cyclicity (parental animals):
two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days), daily from the beginning of the treatment period until evidence of mating and on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in 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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities


Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals [after completion of mating period (35 days of treatment)]
- Maternal animals: All animals [on lactation day 14]

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [26 and 27] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY
The tissues indicated in Table [26 and 27] were prepared for microscopic examination
Statistics:
The data was subjected to various statistical analysis using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05)
The statistical analysis was followed to the parameters as mentioned below table:
Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ weights, Anogenital distance, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss, Post implantation loss:- Parametric - One-way ANOVA with Dunnett’s post test.

Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight:- Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test.

Pregnancy rate. Dams with live pups, Dams with dead pups, No. of litters with/without dead pups, No. of litters with/without resorptions:- Cross Tabs - Chi-square test/ Fischer's Exact Test
Reproductive indices:
Male Fertility Index (%) = Number of Males Impregnating Females
------------------------------------------------- x 100
Number of Males used for Mating
Female Fertility Index (%) = Number of Females confirmed with Pregnancy
--------------------------------------------------------- x 100
Number of Females used for Mating
Parturition (%) = Number Of Parturitions
------------------------------- x 100
Number Of Pregnancies
Copulatory Interval (Days) = Date of Initiation of Cohabitation - Date of Confirmation of Mating
Gestation Length (days) = Date of Delivery - Date of Confirmation of Mating
Offspring viability indices:
Male/Female Sex Ratio = Number Male Pups Per Dam
-----------------------------------
Number Female Pups Per Dam
Live Birth Index (%) = Number Of Viable Pups Born
------------------------------------ x 100
Number Of Pups Born
Lactation Day 4
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 4
----------------------------------------------------- x 100
Number Of Pups Born
Lactation Day 7
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 7
------------------------------------------------------ x 100
Number Of Viable Pups on Lactation Day 4
Lactation Day 13 Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 13
------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 7

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
not applicable
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significant reduction in Percent change in body weight on days 8, 14 and 21 in G3 group males (mid dose) and reduction in Percent change in body weight on day 21 in G4 group males (high dose) when compared with vehicle control group animals. This change can be considered as incidental and not treatment related as there were no changes observed in feed consumption during this period and no effects observed in the mean body weights.

Statistical significant increase in Percent change in gestation body weight during GD 7 to 14 in G2 group animals (low dose) was noted when compared with vehicle control group animals. This change can be considered as incidental and not treatment related as there were no changes observed in feed consumption during this period and no effects observed in the mean gestation body weights.

Statistical significant decrease in Percent change in lactation body weight during LD 7 to 13 in G3 group animals (mid dose) was noted when compared with vehicle control group animals. This change can be considered as incidental and not treatment related as there were no changes observed in feed consumption during this period and no effects observed in the mean lactation body weights
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in serum T4 levels of lactation day 13 pups at all the tested groups were noted when compared with vehicle control group animals.
The variation observed in the T4 levels could not be attributed as treatment related as there were no changes observed in serum T4 levels of males, dams and lactation day 4 pups. The T4 levels obtained in lactation day 13 pups across the groups are within historical control range (41.773 ng/mL to 98.703 with a mean of 66.365 ng/mL) and no abnormal microscopic findings were noted in thyroid at all the tested dose groups. Hence, the variation observed in the T4 levels is considered to be incidental.
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
One animal from G4 group showed reduced sperm content in the lumen of epididymides, unilaterally. This lesion was considered as incidental finding and not the treatment related because of lack of consistency.
No treatment related histopathological findings noticed in adults animals and thyroid of lactation day 13 pups.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
One animal from G4 group showed reduced sperm content in the lumen of epididymides, unilaterally. This lesion was considered as incidental finding and not the treatment related because of lack of consistency. Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations
Reproductive performance:
no effects observed
Description (incidence and severity):
not applicable

Details on results (P0)

no treatment elated effects were noted in reproductive and developmental end points at all the tested dose groups

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effect found up to the highest dose tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The reproduction toxicity of the registration substance was investigated according to the Guideline OECD 421. Rats were treated at dose of up to 1000 mg/kg/day. No effect was observed. The NOAEL of 1000 mg/kg/day was obtained for systemic toxicity and reproduction toxicity.
Executive summary:

The registration substance was evaluated for possible adverse effects following repeated oral dosing to males for 35 days and to females, two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development.

A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[Distilled water], theanimals in G2, G3 and G4groups were administered withtest itemat the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).  

The dose formulation analysis for active ingredient (a.i.) concentration analysis was performed during week 1 and 5 of treatment period and the results were within acceptable limits.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. Body weights of male and female pups were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The number of nipples/areolae in male pups were counted on lactation day 13.

Gross pathology and organ weighing were performed on day 35 for males and on lactation day 14 for dams. The gross pathology was performed on lactation day 13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

The dams were observed for body weight and feed consumption during gestation and lactation.

The animals did not reveal any clinical signs of toxicity. No mortalityor morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption and in organ weights (both absolute and relative) were observed in both the sex of all the group animals.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, pup weights, anogenital distance of pups.

The adults and lactation day 13 pups did not reveal any treatment related gross pathological and histopathological findings.

The serumT4 levels were estimated for males, dams, lactation day 4 and 13 pups. There were no treatment related changes observed in serum T4 levels of males, dams and in serum T4 levels of lactation day 4 and 13 pups at any of the tested dose group animals when compared with vehicle control group animal.

The NOAEL of 1000 mg/kg/day was obtained for the systemic toxicity and for the reproduction toxicity.