2,2'-(octylimino)bisethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

On the basis of this reproduction/ developmental toxicity screening test with Genamin 3920 in male and female Wistar rats (according to OECD TG 421) with dose levels of 20, 80, and 320 mg/ kg body weight/ day the following conclusions can be made:

At dose levels of 80 and/ or 320 mg/kg body weight/ day there were incidences of slight to moderate or severe salivation, piloerection and

abnormal breathing in males and/ or females. These findings were attributed to discomfort or local irritative effects of the treatment.

There was a tendency towards a reduced body weight gain and food intake in males and/ or females at 320 mg/ kg body weight/ day.

These findings were minimal and transient in appearance.

There was no effect of toxicity noted at any dose levels for reproductive toxicity parameters.

There were slightly higher percent pre and post implantation loss (without statistical significance) at 80 and 320 mg/ mg body weight/ day,

but the mean values were within the historical control data range.

Thus, the NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was considered to be 320 mg/kg body weight/ day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-04 to 2014-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female;
the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 277 - 312 g (mean: 296.55 g, ± 20% = 237.24 – 355.86 g)
females: 174 - 205 g (mean: 190.90 g, ± 20% = 152.72 – 229.08 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act
on Animal Welfare the animals were bred for experimental purposes.


Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female was paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 060613)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. All animals received at BSL were healthy.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most
homogenous variation in body weight throughout the groups of males and females.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

Preparation of the Test Item Formulation
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (aqua ad iniectabilia) was added to give
the appropriate final concentration of the test item. The formulation was placed on vortex machine for short period to ensure proper
homogenistation of the formulation. The vehicle was selected as per dose range finding study (BSL study 120520).
The test item formulation was prepared freshly on each administration day before the administration procedure.

Experimental Groups and Doses
14-day dose range finding (DRF) oral toxicity study (BSL project no. 120520) was performed with Genamin 3920. In this study clinical signs
indicative of toxicity occurred at 500 mg/kg body weight/ day. In a 28 days repeated dose toxicity study in rats of the same strain
(BSL study no. 134233), at a daily oral dose of 500 mg/kg bw mortality occurred during the treatment period. Based on the results of the DRF
and the 28 days repeated dose toxicity studies and also in consultation with the sponsor the following doses (Table 1) were selected for the 3
dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating
and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were
dosed after the mating period until the minimum total dosing period of 28 days were completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50)
LD 20 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 80 mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 320 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Animal no. 27 was inadvertently administered with a higher Dose volume (1.8 mL instead of 1.6 mL application volume) for 4 days
(day 10 till day 13 of the study period). The animals in the control group were handled in an identical manner to the dose group subjects
and received the vehicle using the same dose volume (5 mL/ kg) as used for the high dose group.

Administration of Doses
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was
5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured
(measured once a week).

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start
of the mating period to confirm the evidence of mating. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration were analysed with respect to the target nominal concentration. Homogeneity of the test item in the vehicle were
analysed for the dose levels 20 and 320 mg/kg body weight/ day.
Samples for the nominal concentration verification were taken in the study week 1 (first week of pre-mating period), 3 (first week of mating),
5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the dose levels 20 and 320 mg/ kg body weight/ day in study
week 1 and 5 (12 samples).
For investigation of stability an appropriate method was developed at the analytical laboratory of the Clariant. It was planned to investigate
stability (nonGLP) in water for injection at two concentrations (4 and 100 mg/mL) for storage at room temperature for at least 6 hours using a
GC-FID method.
All formulation samples were analysed on the day of sample collection and were stored at -20° C. These samples were analysed after the
completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 134237.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 54 days, i.e. during 14 days of
pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 26 January 2012
Study Initiation Date: 29 August 2013
1st Amendment to Study Plan: 31 November 2013
2nd Amendment to Study Plan: 02 December 2013
3rd Amendment to Study Plan: 29 January 2014
Experimental Starting Date: 19 September 2013
Experimental Completion Date: 12 November 2013
Completion Date of Delegated Phase (Histopathology): 13 July 2014
Completion Date of Delegated Phase (Formulation Analysis): 18 February 2014

Remarks:

Doses / Concentrations:
0, 20, 80, 320 mg/kg Body weight/day
Basis:
nominal conc.

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made once a day approximately at the same time each day after dosing. The health condition of the animals
was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were
made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea,
changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response
to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.


Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly
during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20
and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose
administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after
the delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups
were identified by tattooing on paw. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals
were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24432, expiry date: 01/2015 and Serumwerk,
lot no: 00512, expiry date: 07/2014). All surviving pups were killed on PND 4 by decapitation.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females were sacrificed on study day 26 from the day of evidence of mating.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices
and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina and all organs showing macroscopic
lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were preserved in
modified Davidson’s Solution and then transferred in 70% ethanol. 
Prostate (seminal vesicles with coagulating glands) as a whole of all male animals were inadvertently preserved in 70% ethanol for 24 hours
and then transferred to 10 % neutral buffered formalin. This was discussed with the Principal investigator- histopathology (Dr. Yoshimasa Okazaki)
and according to him this would not impact the tissue processing and evaluation.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed together. Organ weights of animal found dead (animal no. 54, LD group) were not taken.
Organs of animal which died during the course of the study were preserved and examined histopathologically (all gross lesions, lung, brain,
urinary bladder, stomach, lymph nodes (mesenteric and axillary), small and large intestines, (including Peyer´s patches), trachea, liver
kidneys, thymus, adrenal glands, spleen, heart, ovaries, testes, accessory sex organs (prostate, seminal vesicle with coagulating gland) , vagina
uterus with cervix, epididymides) in order to determine the cause of death.

Histopathology
A full histopathology was carried out on the preserved organs and tissues of an animal which died during the study.
Sections from the following organs and tissues (Epididymides (preserved in modified Davidson’s Solution), Testes
(preserved in modified Davidson’s Solution), Ovaries, Uterus with Cervix, Prostate gland, Vagina, Seminal vesicles with Coagulating glands and
All gross lesions) from animals of control and dose group 320 mg/kg body weight/ day, as well as all gross lesions from all groups,
were examined by light microscopy. Ovaries, uterus with cervix and vagina from female no. 67 (80 mg/ kg body weight/ day)
were processed and examined as well, because this animal failed to produce a pregnancy.
In addition to the reproductive organs and tissues described above, the following organs and tissues were also processed and examined in
female no. 54 (dose level 20 mg/ kg body weight/ day) which died prematurely during the treatment period: brain, stomach, small and large
intestines (including Peyer’s patches), liver, thymus, spleen, lung, urinary bladder, lymph nodes (mesenteric and axillary), trachea, kidneys,
adrenal glands and heart. On the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified
contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). Blocking, embedding, cutting,
H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for tissue processing issued a detailed phase plan which was attached to the study plan per amendment.
The principal histopathological investigator provided the histopathology results to the study director by e-mail and sent a pathology phase
report to the study director upon the completion of the study.

Postmortem examinations (offspring):

All surviving pups were sacrificed by decapitation on post-natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed
for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test.
These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

There were no changes of toxicological relevance for reproductive and viability indices in the dose groups when compared to the corresponding
control.
The fertility index (number of pregnant females/ number of copulated females X 100) in control and dose groups were as follows, 100% pregnancy
rate at 0, 20 and 320 mg/ kg body weight/ day and 90% at 80 mg/ kg body weight/ day. The slight decrease in fertility index at 80 mg/kg
body weight/ day had no toxicological or biological relevance.

Offspring viability indices:

The viability index was slightly reduced, not statistically significant, at the dose level 320 mg/ kg body weight/ day when compared to control group.
This was due to 3 missing pups (assumed to be cannibalized by dam) of single isolated female animal (Animal 78). This was considered to be
spontaneous and incidental in origin.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and females, there were no adverse changes of toxicological relevance for the body weight and body weight gain during the study
period in the dose group. In males there was statistically significant decrease in body weight gain at 320 mg/kg body weight/ day after the 1st
week of treatment, but with the progress of the study the weight gain improved after the 2nd and 3rd week of treatment. At the end of the
treatment there was a mild, but not statistically significant decrease in body weight gain at 320 mg/ kg body weight/ day. These decreases
were transient and the findings were not considered to be an adverse effect of treatment.
In females there was statistically significant increase in body weight at 20 mg/kg body weight/ day during the lactation day 0.
There was also considerable decrease in weight gain at 20 mg/ kg body weight/ day between lactation days 0-4 but without statistical significance.
This decrease had no biological relevance. There was a slight but not statistically significant decrease in body weight gain noted during the
gestation and lactation period at 320 mg/ mg body weight/ day. In the absence of statistical significance the findings were not considered to
be an adverse effect of treatment.

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

Description (incidence and severity):

no administration via drinking water

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Mortality
There was no mortality due to test item treatment occurred during the study.
One female (animal no. 54) treated at 20 mg/ kg body weight/ day was found dead prematurely on premating day 5. The cause of animal’s
death was considered to be accidental influx (regurgitation or aspiration) into the respiratory tract, and this was considered not to be the toxic
event caused by systemic exposure of the test item.

Clinical Observations
In males and females, there were no adverse clinical signs caused due to the systemic exposure of test item during the study.
There were clinical signs including slight to severe salivation and slight to moderate piloerection in few animals treated at 20 or 80 mg/ kg
body weight/ day, but observed in most animals at 320 mg/ kg body weight/ day. There were also signs of moving the bedding in most
animals at 80 and 320 mg/ kg body weight/ day and slight to severe abnormal breathing in most animals at 320 mg/ kg body weight/ day.
Abnormal breathing was also noted in single isolated animals of 20 and 80 mg/ kg body weight/ day.
The salivation and moving the bedding observed in animals was considered to be due to the discomfort caused due to treatment.
The abnormal breathing was considered to be an effect associated with the test item treatment, but had no toxicological significance.
This finding corroborated 28 day toxicity study (BSL study no. 134233) performed with Genamin 3920, where there were necrotic inflammatory
lesions and the relating reactive changes observed in trachea and lung or peribronchial inflammation accompanying bronchial/ bronchiolar
luminal contents, hypertrophy of intrapulmonary bronchial epithelium and regenerated tracheal mucosal epithelium recorded at or above
100 mg/ kg body weight/ day. These histopathological lesions were attributable to the treatment with the test item and were considered to
be the results of local irritative effects due to the physicochemical character of the test item.
There were also lower incidences of eschar, nasal discharge, half eyelid closure, reduced spontaneous activity, exophthalmos and diarrhea in
animals of control or dose groups. These were transient and were in few isolated animals. Therefore, these were not considered to be of
toxicological relevance.

Body Weight Development
In males and females, there were no adverse changes of toxicological relevance for the body weight and body weight gain during the study
period in the dose group. In males there was statistically significant decrease in body weight gain at 320 mg/kg body weight/ day after the 1st
week of treatment, but with the progress of the study the weight gain improved after the 2nd and 3rd week of treatment. At the end of the
treatment there was a mild, but not statistically significant decrease in body weight gain at 320 mg/ kg body weight/ day. These decreases
were transient and the findings were not considered to be an adverse effect of treatment.
In females there was statistically significant increase in body weight at 20 mg/kg body weight/ day during the lactation day 0.
There was also considerable decrease in weight gain at 20 mg/ kg body weight/ day between lactation days 0-4 but without statistical significance.
This decrease had no biological relevance. There was a slight but not statistically significant decrease in body weight gain noted during the
gestation and lactation period at 320 mg/ mg body weight/ day. In the absence of statistical significance the findings were not considered to
be an adverse effect of treatment.

Food Consumption
In males and females, there were no adverse changes of toxicological relevance for food consumption caused due to test item treatment
during the study. In males there was no statistically significant change in food consumption. In females there was statistically significant
decrease in food consumption at 320 mg/ kg body weight/ day after the 2nd week of gestation, but the decrease was minimal and transient.

Pathology- Macroscopic Findings
In a decedent (female no. 54), fluid content of trachea, red spots of thymus and discoloured red of axillary lymph node were recorded.
The tracheal fluid content was associated with accidental influx (regurgitation or aspiration) of the dosing solution into the respiratory tract.
Red spots of thymus and discoloured red of axillary lymph node, both of which correlated microscopically with congestion, were non-specific
changes which are commonly recorded in the dead animals.
In the survivors, there were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded (including
epididymides yellow spots in few males of control and dose groups) were considered to be within the range of normal background alterations
which may be recorded in animals of this strain and age.

Organ Weight
In males and females, there were no changes of toxicological relevance for absolute and relative (to terminal body weight) reproductive
organ weights. In males, there were no statistically significant changes, but in females there was statistically significant increase in relative
ovary weight at 320 mg/ kg body weight. There was also slight but not statistically significant increase in absolute ovary weight at 320 mg/ kg
body weight/ day. In the absence of microscopic changes in the ovaries, the increase in ovary weight was considered to have no biological relevance.

Histopathology
Histopathological evaluation revealed tracheal mucosal necrosis, as well as flocculent and fluid content in the bronchi and bronchiole of the
lung and in alveoli surrounding the bronchiolar/alveolar duct area of the lung in female no. 54 which died prematurely. These were considered
to be the causes of death. However, the animal’s death was unlikely to be the toxic event caused by systemic exposure of the test item, and it was
considered that the animal’s death had happened by the accidental influx (regurgitation or aspiration) of dosing solution into the respiratory tract.
Congestion was recorded in lung, thymus and spleen. These were considered to be non-specific findings that are commonly found in the dead
animals. There were not treatment-related effects on the completeness of stages or cell populations of the testes.
Minimum to slight tubular degeneration/atrophy and/or minimum spermatid retention were recorded in a few animals from both of the control
and high-dose males. These were considered to be within the range of normal background lesions which are occasionally observed in males of
this strain and age. Nothing special was observed in ovaries, uterus with cervix and vagina from female no. 67 which was not pregnant.
All microscopic findings recorded in the survivors were within the range of normal background lesions which may be recorded in animals
of this strain and age.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries
observed at dose level 20, 80 and 320 mg/ kg body weight/ day were 102.2%, 106.0% and 113.3% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as mean of measured concentration did not differ from nominal concentration by
more than 15%. Homogeneity of formulation samples was determined in study week 1 and 5 for the dose levels 20 and 320 mg/ kg body weight/ day. The mean recovery observed for dose level 20 mg/ kg body weight was between 90.1% and 104.8% of the nominal value and between 91.7%
and 115.4% of the nominal value for dose level 320 mg/ kg body weight/ day. The coefficients of variation of the different sampling locations
(top, middle and bottom) were between 0.2% and 2.2% for dose level 20 mg/ kg body weight/ day and between 0.2% and 3.0% for dose level
320 mg/ kg body weight/ day. All samples were homogenous, as COV was below or equal 10%. The stability analysis for 6 hours (RT) was
performed at client’s facility as non-GLP study (Clariant Report 14-163122). The test substance is not sufficiently water soluble.
A concentration of 4 mg/l results in a turbid solution. No additional signals could be detected after 6 hours. Therefore it can be concluded
that the test substance is stable in water over a period of 6 hours.

Precoital Interval and Duration of Gestation
There were no changes of toxicological relevance for the duration of precoital and the duration of gestation days in the dose group when
compared to the corresponding control. There were no statistically significant changes between the control and dose groups.
There was a slight but not statistically significant increase in the duration of precoital interval at the dose level 80 and 320 mg/ kg body weight/ day. This increase was considered to be within the normal range and had no toxicological relevance.

Pre- and Post-Natal Data
There were no adverse changes of toxicological relevance for pre and post natal data including number of corpora lutea, number of
implantation sites, percent pre and post implantation loss and number of live pups (PND 0 and 4). There were no statistically significant
changes for pre and post natal data between the control and dose groups.
There were increases, not statistically significant, in percent pre- and post- implantation loss at the dose level 80 and 320 mg/ kg body weight/ day. Taking into account the number of corpora lutea and number of implantation sites being comparable between the control and dose groups
and in addition the difference in live pups being very little between the control and the dose groups, the increase in percent pre and post
implantation loss were not considered to be an adverse effect of treatment. In addition the mean values of pre and post natal data were
within the historical control data range.

Reproductive Indices
There were no changes of toxicological relevance for reproductive and viability indices in the dose groups when compared to the
corresponding control. The fertility index (number of pregnant females/ number of copulated females X 100) in control and dose groups
were as follows, 100% pregnancy rate at 0, 20 and 320 mg/ kg body weight/ day and 90% at 80 mg/ kg body weight/ day. The slight decrease
in fertility index at 80 mg/kg body weight/ day had no toxicological or biological relevance. The viability index was slightly reduced, not
statistically significant, at the dose level 320 mg/ kg body weight/ day when compared to control group. This was due to 3 missing pups
(assumed to be cannibalized by dam) of single isolated female animal (Animal 78). This was considered to be spontaneous and incidental in origin.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 320 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Thus, the NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was considered to be 320 mg/kg body weight/ day.

Remarks on result:

other: Generation: P + F1

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Litter Data
There were no changes of toxicological relevance for litter data including total number of pups born, still birth and runts on PND 0 and number
of live pups, number of male and female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes for litter data
between the control and dose groups. There was a slight but not statistically significant decrease in the number of female pups at 20 and
80 mg/ kg body weight/ day compensated by slight increase in male pups. The mean values of litter data were within the historical control data range.

Litter Weight Data
There were no changes of toxicological relevance for litter weight data including pup mean weight, total litter weight and male and female
litter weight on PND 0 and 4. There were no statistically significant changes between the control and dose groups.
There was a slight but not statistically significant decrease in female litter weight on PND 0 and PND 4 in all dose groups. These changes were
corresponding to the lower number of female pups and in addition did not show dose response relation. The mean values of litter weight data
in control and dose groups were within the historical control data range and slight decrease in female litter weight was not of biological relevance.
 
Pup Survival Data
There was no effect of toxicological relevance on survival of the pups from PND 0 to PND 4 observed in any of the dose groups when
compared with controls. Three pups (Pup no. 5, 8 and 9) from animal 78 treated at 320 mg/ kg body weight/ day were missing on PND 1 and
were considered to cannibalized by the dam. As this was observed in single isolated female, it was considered spontaneous and incidental in origin.

Pup External Findings
There were no treatment related gross external findings observed in any of the dose groups.
There were few incidences of external findings observed at 0 mg/ kg body weight/ day (black spot on lumbar region, Pup no. 1, dam no. 46),
20 mg/ kg body weight/ day (wound on thoracic region, Pup no. 7, Dam no. 57; small and discoloured, Pup no. 1, Dam no. 60; small, Pup no. 15,
Dam no. 60), 80 mg/ kg body weight/ day (dark spot on abdomen, Pup no. 1, Dam no. 63; small, Pup no. 1, Dam no. 64)
and 320 mg/ kg body weight/ day (injury on head, Pup no. 1, Dam no. 74; injury on abdomen, Pup no. 7, Dam no. 78), which
were considered to be spontaneous and not related to the test item treatment.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 320 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed

Key result

Reproductive effects observed:

no

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with Genamin 3920 in male and female Wistar rats with dose levels of 20, 80, and 320 mg/ kg body weight/ day the following conclusions can be made:
At dose levels of 80 and/ or 320 mg/kg body weight/ day there were incidences of slight to moderate or severe salivation, piloerection and
abnormal breathing in males and/ or females. These findings were attributed to discomfort or local irritative effects of the treatment.
There was a tendency towards a reduced body weight gain and food intake in males and/ or females at 320 mg/ kg body weight/ day.
These findings were minimal and transient in appearance.
There was no effect of toxicity noted at any dose levels for reproductive toxicity parameters.
There were slightly higher percent pre and post implantation loss (without statistical significance) at 80 and 320 mg/ mg body weight/ day,
but the mean values were within the historical control data range.
Thus, the NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was considered
to be 320 mg/kg body weight/ day.

Executive summary:

The aim of this study was to assess the possible effects of Genamin 3920 on male and female fertility and embryofetal development after repeated dose administrationin Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days,

i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal

day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but receivedaqua ad iniectabilia(water for injection), the vehicle used in

this study. The 4 groups comprised 10 male and 10 femalewistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animal that died (animal no. 54) was examined macroscopically. At the conclusion of the test, all surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after the delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on GD 26 from the day of evidence of mating.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups killed (by decapitataion) on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues (reproductive organs) was performed on animals of dose levels 0 and 320 mg/kg body weight/ day and in non pregnant female animal (no. 67) of the dose level 80 mg/ kg body weight/ day.All gross lesions macroscopically identified were examined microscopically in all animals.

The following doses were evaluated:

Control:                        0        mg/kg body weight/ day

Low Dose:                    20       mg/kg body weight/ day

Medium Dose:              80       mg/kg body weight/ day

High Dose:                   320     mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was suspended inaqua ad iniectabiliaand administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

Summary Results

There was no mortality due to test item treatment occurred during the study.However, one female (animal no. 54) treated at

20 mg/ kg body weight/ day was found dead prematurely on premating day 5 and wasconsidered to be accidental influx

(regurgitation or aspiration) into the respiratory tract.

In males and females, there were no adverse clinical signs caused due to systemic exposure of test item during the study. There were clinical signs including salivation, piloerection and abnormal breathing in most animals treated at 80 or 320 mg/ kg body weight/ day. These clinical signs were considered to be due to discomfort or caused by the local irritative effect of the test item.

In males and females, there were no adverse changes of toxicological relevance for the body weight, body weight gain and food consumption during the study period in the dose group. The decreases in body weight gain and food consumption in males and/or females at

320 mg/ kg body weight/ day were minimal and transient in appearance.

There were no changes of toxicological relevance for litter data including total number of pups born, still birth and runts on PND 0 and number

of live pups, number of male and female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes between the control and dose groups.

There were no changes of toxicological relevance for litter weight data including pup mean weight, total litter weight and male and female litter weight on PND 0 and 4. There were no statistically significant changes between the control and dose groups.

There were no changes of toxicological relevance for the duration of precoital and the duration of gestation days in the dose groups when compared the corresponding control. There were no statistically significant changes between the control and dose groups.

There were no adverse changes of toxicological relevance for pre and post natal data including number of corpora lutea, number of implantation sites, percent pre and post implantation loss and number of live pups (PND 0 and 4). There were no statistically significant changes for pre and post natal data between the control and dose groups.There were slightly higher percent pre and post implantation loss (without statistical significance) at 80 and 320 mg/ mg body weight/ day, but the mean values were within the historical control data range.

There were no changes of toxicological relevance for reproductive and viability indices in dose groups when compared to the corresponding control.

There was no effect of toxicological relevance on the survival of the pups from PND 0 to PND 4 in any treatment group when compared with control. There were no treatment related gross external pup findings observed in any of the dose groups.

In males and females, there were no changes of toxicological relevance for absolute and relative (to terminal body weight) reproductive

organ weights. At the necropsy, fluid content of trachea, red spots of thymus and discoloured red of axillary lymph node were recorded in a decedent (female no. 54). The tracheal fluid content was associated with accidental influx (regurgitation or aspiration) of the dosing solution into the respiratory tract. Red spots of thymus and discoloured red of axillary lymph node, both of which correlated microscopically with congestion, were non-specific changes which are commonly recorded in the dead animals.In the survivors, there were no gross lesions that could be attributed to treatment with the test item. No histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina.There were no treatment-related effects

on the testicular histomorphology including spermatogenesis and interstitial cell structure.

No treatment related changes was observed in the reproductive organs of one female (no. 67, mid-dose group) which was not pregnant.

Conclusion

On the basis of this reproduction/ developmental toxicity screening test with Genamin 3920 in male and female Wistar rats with dose levels of

20, 80, and 320 mg/ kg body weight/ day the following conclusions can be made:

At dose levels of 80 and/ or 320 mg/kg body weight/ day there were incidences of slight to moderate or severe salivation, piloerection and abnormal breathing in males and/ or females. These findings were attributed to discomfort or local irritative effects of the treatment.

There was a tendency towards a reduced body weight gain and food intake in males and/ or females at 320 mg/ kg body weight/ day.

These findings were minimal and transient in appearance.

There was no effect of toxicity noted at any dose levels for reproductive toxicity parameters.

There were slightly higher percent pre and post implantation loss (without statistical significance) at 80 and 320 mg/ mg body weight/ day,

but the mean values were within the historical control data range.

Thus, the NOAEL for both systemic toxicity of adult animals (male and female) and the reproductive and developmental toxicity was considered to be 320 mg/kg body weight/ day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

320 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 reliable without restriction as the data originate from a recent screening study according to OECD TG 421 performed under GLP-regulations.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Pregnant Wistar rats when treated with ‘Genamin 3920’ at 75, 150 and 350/300 mg/kg/day did not reveal any foetal toxicity. No teratogenic effects attributable to treatment were observed. Significant decrease in body weight and feed consumption along with significant decrease in uterine weights were observed in dams treated at 350/300 mg/kg.

Based on findings, the NOAEL of ‘Genamin 3920’ in pregnant Wistar rats is 150 mg/kg for maternal toxicity and 300 mg/kg for foetal toxicity under the tested conditions.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FEBRUARY 2021 to JUNE 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 25 June, 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

This study was audited by the Quality Assurance, Vimta Labs Ltd., in compliance with the Organisation for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice (GLP) ENV/MC/CHEM (98) 17, and the findings were reported to the

Limit test:

no

Specific details on test material used for the study:

Name Genamin 3920
Chemical Name 2,2’-(Octylimino)bisethanol
Manufacturer’s / Sponsor’s Name and Address Clariant Produkte (Deutschland) GmbH, Corporate Product Stewarship on behalf of BU ICS
Bruningstr. 50, 65926 Frankfurt, Germany
Molecular Formula C12H27NO2
CAS-No. 15520-05-5
Molecular weight 217.35 g/mol
Physical Appearance Transparent liquid
Storage Conditions Ambient (Room Temperature 20-25 °C)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species : Rat (Rattus norvegicus)
Strain : Wistar
Age : 10 - 12 weeks at start of treatment
Sex : Pregnant females
Body Weight : 217.23 – 341.45 g at start of treatment
Number of Animals : 96 pregnant females (dams)
Animals were maintained under the following environmental conditions:
Temperature : 20.6 – 24.3°C
Relative humidity : 38 – 67%
Light/dark cycle (photoperiod) : 12-hour light and 12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Genamin 3920 in the dose formulations were in the range between 97.5 – 105.1 which were within the acceptance criteria (85% - 115%). No degraded impurity was observed during the 48h stability test period. The results revealed homogenous distribution of Genamin 3920 in the dose formulation.

Duration of treatment / exposure:

Dose formulations of vehicle and test item were administered to respective groups by oral route once daily from GD 5 to GD 19 of presumed gestation. Dose was administered using a calibrated disposable syringe attached to a 16 and18-gauge stainless steel ball tipped oral gavage needle. Individual animal dose volumes were calculated based on their most recent body weight. Homogeneity of the dose formulations during sampling and dosing were maintained by constant stirring using a magnetic stirrer.

Frequency of treatment:

Once daily from Gestation Day 5 to Gestation Dat 19

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Remarks:

Animals were initially dosed at 350 mg/kg. As overt toxicity was observed, dose wad reduced to 300 mg/kg/day.

No. of animals per sex per dose:

24 presumed pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose range finding study (Vimta Study No. VLL/1020/NG/T108) pregnant dams were treated at dose levels of 50, 150 and 300 mg/kg b.w./day. In this study, a slight but not statistically significant decrease in body weight and feed consumption was observed in the highest dose (300 mg/kg). In another repeated dose 90-day oral toxicity study (Vimta Study No. VLL/1020/G/T107), no treatment related clinical signs and mortality occurred at the highest dose of 350 mg/kg b.w./day, after 75 days of treatment. The doses for the current study are selected taking into consideration, the outcome of the above-mentioned studies. In a subacute repeated dose oral toxicity study (according to OECD 407) (Study No. 134233) male and female Wistar rats were treated with 20, 100, and 500 mg/kg b.w./day of the test item for 28 days. Since at a daily oral dose of 500 mg/kg b.w./day, 5/10 female animals died during the treatment period, 350 mg/kg per day was selected as the highest tolerable dose for pregnant dams.
- Rationale for animal assignment (if not random): Pregnant females received on a particular day were randomly allocated to 4 groups (G1- G4; 1 vehicle control group and 3 test groups) ensuring minimum differences in mean body weight between the groups. This procedure was continued till each group gets the required number of females (dams).

Maternal examinations:

The following maternal data was recorded.
• Pregnancy status
• Uterine weight
• No. of corpora lutea
• Total/viable/dead foetuses
• No. of implantation sites
• Total/early/late resorptions
Based on the uterine observations the following parameters were calculated as per
Formulas mentioned in Appendix.10
• Adjusted Maternal Weight
• Relative Uterus Weight
• Pre and Post Implantation Loss
• Percentage live and dead implants
• Percentage live offspring

Ovaries and uterine content:

Following termination, as soon as possible after death, the uteri were removed (Caesarean section) and the pregnancy status of the animals ascertained. Uteri that appear non-gravid was examined with ammonium sulphide staining to confirm the non-pregnant status. The gravid uterus along with cervix was weighed immediately.

Blood sampling:

On gestation day 20, blood (approximately 2 ml) was collected from all surviving dams through retro-orbital plexus in tubes (without anticoagulant) under mild isoflurane anaesthesia.

Fetal examinations:

Foetal Examination: All the foetuses were removed sequentially and were identified by serial numbers as per SOP.
The following litter data was recorded:
• Sex identification
• Body Weight
• Anogenital distance
• Individual foetus and placenta weight (g) (placenta was discorded)
All the foetuses were examined for external malformations.

Visceral and Head Razor Examination: Approximately half of the live foetuses per litter were selected. A detailed soft tissue examination was performed as per in-house SOP, which included observation of all the organs and structures of the head, neck, thorax and abdomen. Reproductive tract was examined for signs of altered development. External foetal sex (as determined by gross examination) was compared internally (gonadal observations). The heads of foetuses were decapitated and preserved in Bouin’s fixative for head razor examination.

Skeletal Examination: Foetuses selected for skeletal examination were eviscerated and fixed in alcohol. Detailed examinations of the skeleton (bone + cartilage) were performed after staining with alizarin red S and alcian blue. Examination included observation of all the bone structures and cartilage of the head, spine, rib cage, pectoral, pelvis and limbs.

Statistics:

The data was analysed using Graphpad Prism, version 4.00. Statistical comparisons were carried out between treatment and control groups using parametric (one-way analysis of variance (ANOVA), Dunnett's t-Test) or non-parametric (Kruskal-Wallis test, Mann Whitney’s Test) test procedures. The choice of parametric or non-parametric test was based on whether the groups satisfy the homogeneity of variance as evaluated by Bartlett’s test. Statistical significance was evaluated at p≤0.05 and/or p≤0.01. All quantitative data were summarized and expressed as Mean ± SD.

Historical control data:

Historical data provided in the Study Report to allow comparison to control data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three dams were found dead between GD 9-15 with clinical signs/symptoms at 350 mg/kg body weight/day. Animals treated at 350 mg/kg/day displayed salivation, hyperactivity, aggression until reduction of dose to 300 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three dams were found dead between GD 9-15

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight was significantly reduced from GD 8-20 in dams treated with Genamin 3920 at the dose of 350/300 mg/kg. All dams belonging to the control, low and mid dose treated groups displayed normal body weight gain throughout the study period. Adjusted maternal weight (after exclusion of uterine weight) of treated dams derived on GD 20 was significantly reduced in the 350/300 mg/kg group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption was significantly reduced from GD 8-20 in dams treated with Genamin 3920 at the dose of 350/300 mg/kg.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes in T3, T4 and TSH attributable to treatment were observed. Further, no histopathological changes in thyroid/parathyroid was observed in any of the treated groups as compared to the control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the found dead dams treated at 350 mg/kg/day, gross pathological evaluations revealed red discoloration in lung and mottled liver

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological observations of the found dead animals revealed kidneys with necrosis, bilateral, multifocal (3+) in 2 dams and kidneys with necrosis, bilateral, multifocal (2+), hyperplasia with squamous cell, non-glandular stomach, diffuse (3+) in 1 dam in the high dose treated group of 350 mg/kg body weight/day.

Number of abortions:

no effects observed

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

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Surviving dams of the high dose group treated at 350/300 mg/kg/day revealed significant decrease in the gravid uterine weights as compared to the control group.
No other abnormalities were observed in the evaluated maternal parameters viz., placental weight, number of corpora lutea, implantations, resorptions and percent implantation loss in treated (75, 150 and 350/300 mg/kg/day) dams as compared to control group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

necropsy findings

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Body weight and gravid uterine weight

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no significant changes in the mean number of male and female foetuses and total number of foetuses in any of the treated groups (75, 150 and 350/300 mg/kg/day) as compared to the control group. Foetal weight was comparable between treated and control groups. No malformations related to treatment were detected during external examination of the pups. Few incidences of haematoma in right pinna and lower mandible (jaw) observed in Genamin 3920 treated dams at 75 mg/kg and 350/300 mg/kg (0.3% and 0.4%), were within normal historical ranges and hence considered as common spontaneous lesions.

No malformations/abnormalities were detected in any of the treated and control group pups during the visceral and head razor examinations except for a few incidental findings in control and treated groups (like haemorrhages in lungs, adrenals, kidney and in lateral ventricle of brain). The incidence and severity of the observations were within the normal historical control ranges.

Foetal skeletal examination did not reveal any treatment related malformations/ abnormalities in any of the treated and control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Summary of Maternal and Litter Data

Parameters	Group & Dose (mg/kg b.wt/day)
	G1 (0)	G2 (75)	G3 (150)	G4 (350/300)
No. of rats / group	24	24	24	24
Duration of treatment (GD5-GD19)	15 days	15 days	15 days	15 days
Caesarean section carried out on	GD 20	GD 20	GD 20	GD 20
No. of rats found non-pregnant / group	2	1	1	2
No. of rats found pregnant / group	22	23	23	20
No. of rats found dead / group	0	0	0	3
No. of litters examined	22	23	23	20
No. of resorptions (early)	16	28	11	24
No. of resorptions (late)	4	7	7	5
Total no. of foetuses	340	331	360	281
Mean litter size	15	14	15	14
No. of foetuses evaluated
a.  External examination	340	331	360	281

Summary of Clinical Signs

Group & Dose (mg/kg)	Clinical Signs	No. of Dams showing Clinical Signs
		Gestational Day
		0	1	2	3	4	5	6	7	8	9	10
G1 (0)	Normal	24	24	24	24	24	24	24	24	24	24	24
G2 (75)	Normal	24	24	24	24	24	24	24	24	24	24	24
G3 (150)	Normal	24	24	24	24	24	24	24	24	24	24	24
G4 (350/300)	Normal	24	24	24	24	24	21	10	4	7	8	9
	Burrowing Behaviour	0	0	0	0	0	4	14	20	17	14	13
	Salivation	0	0	0	0	0	0	0	2	8	14	13
	Hyperactivity	0	0	0	0	0	0	0	0	0	1	5
	Aggression	0	0	0	0	0	0	0	0	0	0	1
	Found Dead	0	0	0	0	0	0	0	0	0	2	0

Group & Dose (mg/kg)	Clinical Signs	No. of Animals showing Clinical Signs
		Gestational Day
		11	12	13	14	15	16	17	18	19	20
G1 (0)	Normal	24	24	24	24	24	24	24	24	24	24
G2 (75)	Normal	24	24	24	24	24	24	24	24	24	24
G3 (150)	Normal	24	24	24	24	24	24	24	24	24	24
G4 (350/300)	Normal	9	11	11	11	13	14	17	21	21	21
	Burrowing Behaviour	11	11	11	9	7	4	1	0	0	0
	Salivation	11	11	11	9	7	4	1	0	0	0
	Hyperactivity	7	5	5	6	5	4	2	0	0	0
	Aggression	4	5	5	6	4	6	2	0	0	0
	Found Dead	0	0	0	0	1	0	0	0	0	0

Group & Dose (mg/kg)	Clinical Signs	No. of Animals showing Clinical Signs
		Gestational Day
		11	12	13	14	15	16	17	18	19	20
G1 (0)	Normal	24	24	24	24	24	24	24	24	24	24
G2 (75)	Normal	24	24	24	24	24	24	24	24	24	24
G3 (150)	Normal	24	24	24	24	24	24	24	24	24	24
G4 (350/300)	Normal	9	11	11	11	13	14	17	21	21	21
	Burrowing Behaviour	11	11	11	9	7	4	1	0	0	0
	Salivation	11	11	11	9	7	4	1	0	0	0
	Hyperactivity	7	5	5	6	5	4	2	0	0	0
	Aggression	4	5	5	6	4	6	2	0	0	0
	Found Dead	0	0	0	0	1	0	0	0	0

 

Summary of Maternal Body Weight (g)

Gestational Day	Group & Dose (mg/kg b.w.)
	G1 (0)			G2 (75)			G3 (150)			G4 (350/300)
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0	241.37	22.98	22	238.89	19.23	23	246.56	18.79	23	235.99	18.71	20
3	256.12	24.73	22	252.38	18.78	23	259.14	19.27	23	249.99	17.99	20
5	265.03	27.64	22	261.13	18.40	23	269.80	18.23	23	258.21	19.34	20
8	276.40	28.73	22	272.68	20.04	23	277.70	19.55	23	250.62↓↓	25.81	20
11	292.89	29.27	22	287.61	20.45	23	293.80	18.99	23	266.99↓↓	24.79	20
14	310.27	28.94	22	303.76	19.82	23	310.52	19.11	23	283.36↓↓	21.74	20
17	345.04	31.21	22	336.66	20.31	23	344.82	20.18	23	312.51↓↓	26.65	20
20	393.18	34.44	22	385.65	22.17	23	394.24	22.63	23	355.42↓↓	32.97	20
20*	301.95	29.49	22	299.77	21.08	23	302.22	19.14	23	276.85↓↓	25.00	20

Key: N = Number of Dams, SD = Standard Deviation, * = Adjusted Maternal body Weight, ↓↓ = significantly high at P ≤ 0.01

 

Summary of Dam Body Weight Gain (g)

Gestational Day	Group & Dose (mg/kg b.w.)
	G1 (0)			G2 (75)			G3 (150)			G4 (350/300)
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0-3	6.12	1.98	22	5.70	1.31	23	5.17	3.22	23	6.00	1.93	20
3-5	3.42	1.33	22	3.51	1.77	23	4.20	3.05	23	3.28	1.40	20
5-8	4.33	2.36	22	4.41	1.58	23	2.92	1.67	23	-3.03↓↓	5.36	20
8-11	6.05	3.04	22	5.51	2.24	23	5.85	1.72	23	6.72↓↓	4.37	20
11-14	6.02	2.48	22	5.67	1.51	23	5.73	1.65	23	6.33↓↓	3.73	20
14-17	11.25	1.85	22	10.91	2.92	23	11.08	2.00	23	10.27↓↓	3.53	20
17-20	14.01	2.80	22	14.61	3.08	23	14.35	1.86	23	13.76↓↓	5.04	20
0-5	9.75	2.36	22	9.41	1.90	23	9.51	2.08	23	9.47	2.31	20
5-20	48.73	6.58	22	47.95	6.72	23	46.29	4.94	23	37.93↓↓	11.79	20
0-20	63.19	7.20	22	61.92	8.77	23	60.24	7.21	23	51.02↓↓	13.45	20

Key: N = Number of Dams, SD = Standard Deviation, ↓↓ = significantly high at P ≤ 0.01, Note: Body weight gain considered only for pregnant dams

 

Summary of T3, T4, TSH Values

Parameters	Group & Dose (mg/kg b.w.)
	G1 (0)			G2 (75)			G3 (150)			G4 (350/300)
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T3	25.90	6.02	22	29.99	7.71	23	31.34	7.23	23	28.97	9.43	20
T4	1.08	0.24	22	1.12	0.25	23	1.08	0.21	23	1.04	0.33	20
TSH	2.21	0.91	22	2.61	1.25	23	2.36	1.16	23	2.60	1.37	20

Key: N = Number of Dams, SD = Standard Deviation

Note: Values of pregnant dams alone were considered.

 

Summary of Dam Examination and Derived Uterine Parameters

Parameters	Group & Dose (mg/kg b.w.)
	G1 (0)			G2 (75)			G3 (150)			G4 (350/300)
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Uterine Weight (g)	91.24	10.17	22	85.88	14.41	23	92.02	12.11	23	78.57↓↓	16.71	20
Relative Uterine Weight (%)	23.24	2.12	22	22.26	3.54	23	22.38	5.96	23	21.99	3.94	20
Placental Weight (g)	0.55	0.08	22	0.54	0.07	23	0.53	0.08	23	0.51	0.08	20
Total No. of Corpora Lutea	18.45	3.33	22	17.52	2.17	23	18.09	2.43	23	17.40	1.88	20
Total No. of Implantations	16.36	1.50	22	15.91	1.76	23	16.43	1.65	23	15.50	2.26	20
Total No. of live Implantations	15.45	1.79	22	14.39	2.59	23	15.65	2.27	23	14.05	2.86	20
Total No. of dead Implantations	0.00	0.00	22	0.00	0.00	23	0.00	0.00	23	0.00	0.00	20
Early Resorptions (No.)	0.73	0.88	22	1.22	1.78	23	0.48	0.67	23	1.20	1.99	20
Late Resorptions (No.)	0.18	0.50	22	0.30	0.70	23	0.30	0.70	23	0.25	0.55	20
Total Resorptions (No.)	0.91	0.97	22	1.52	2.29	23	0.78	1.09	23	1.45	2.14	20
Pre-implantation Loss (%)	9.36	12.85	22	8.50	10.41	23	8.38	8.99	23	10.74	10.27	20
Post Implantation Loss (%)	5.60	6.24	22	9.29	14.25	23	5.04	7.49	23	9.32	13.84	20
Live Foetus (%)	94.40	6.24	22	90.71	14.25	23	94.96	7.49	23	90.68	13.84	20
Dead Foetus (%)	0.00	0.00	22	0.00	0.00	23	0.00	0.00	23	0.00	0.00	20

Key: N = Number of Dams, SD = Standard Deviation, ↓↓ = significantly high at P ≤ 0.01

 

Summary of Litter data

Parameters		Group & Dose (mg/kg b.w.)
		G1 (0)	G2 (75)	G3 (150)	G4 (350/300)
Total No. of litters		22	23	23	20
Total No. of foetuses		340	331	360	281
Mean litter size		15	14	15	14
Total No. Dead foetuses		0	0	0	0
Total No. of live foetuses		340	331	360	281
Total weight of live foetuses	Mean	3.74	3.76	3.80	3.62
	SD	0.07	0.07	0.10	0.12
No. of live male foetuses		164	165	172	121
Weight of live male foetuses	Mean	3.83	3.86	3.90	3.70
	SD	0.07	0.07	0.22	0.22
No. of live female foetuses		176	166	188	160
Weight of live female foetuses	Mean	3.64	3.66	3.70	3.50
	SD	0.07	0.21	0.22	0.23
Anogenital Distance (mm)	Mean	2.64	2.70	2.62	2.47
	SD	0.96	0.94	0.90	0.85
Crown Rump Length (cm)	Mean	3.74	3.78	3.78	3.73
	SD	0.27	0.23	0.25	0.32
Sex ratio- Male Female		0.93	0.99	0.91	0.76

 

Summary of Gross Pathological Findings of Dams

No. of Animals/Group: 24

Gross Pathological Findings	Group & Dose (mg/kg)
	G1 (0)	G2 (75)	G3 (150)	G4* (350/300)
No abnormalities detected	22	23	23	20
Lungs: Red discolouration	0	0	0	3
Liver: Mottled	0	0	0	1

Note: * = 1 dam will have more number of observations

Summary of Foetal Observations (Incidence and Percentage)

Observations		Group & Dose (mg/kg b.w.)
		G1 (0)	G2 (75)		G3 (150)	G4 (350/300)
No. of litters examined		22	23		23	20
No. of foetuses examined		340	331		360	281
External Examination
No abnormality Detected	340			330	360	280
Hematoma on right Pinna	0			1(0.3%)	0	0
Lower mandible-Hematoma (Jaw)	0			0	0	1(0.4%)
Visceral Examination
No. of foetus examined for examination		175	171		186	146
No abnormality Detected		173	169		185	142
Pin point Haemorrhages on Lungs		1(0.6%)	0		1(0.5%)	1(0.7%)
Kidney-Pale		1(0.6%)	0		0	0
Kidneys-Haemorrhagic		0	1(0.6%)		0	0
Adrenals-Haemorrhagic		0	1(0.6%)		0	0
Right Kidney- Haemorrhagic		0	0		0	1(0.7%)
Brain – Lateral ventricle Haemorrhagic		0	0		0	2(1.4%)

 

Summary of Foetal Skeletal Observations (Incidence and Percentage)

Skeletal Parameters	G1 (0)		G2 (75)		G3 (150)		G4 (350/300)
Skull:	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected
Parietal incomplete ossification	6	3.6	5	3.1	7	4.0	12	8.9
Interparietal incomplete ossification	1	0.6	1	0.6	0	0	2	1.5
Interparietal Unossified	1	0.6	0	0	0	0	0	0
Supra occipital	0	0	0	0	0	0	1	0.7
Ribs-Thorax:
Wavy ribs in 11^thto14^th	1	0.6	1	0.6	0	0	0	0
Knobby (nodulated) ribs	1	0.6	0	0	0	0	1	0.7
14^th Rib ossification Site(SN)	4	2.4	11	6.9	3	1.7	3	2.2
Thoracic Centrum:
11^th Thoracic centrum dumbbell shaped	1	0.6	0	0	0	0	1	0.7
12^th Thoracic centrum dumbbell shaped	2	1.2	1	0.6	0	0	1	0.7
12^th Thoracic centrum Bipartite ossification	0	0	1	0.6	0	0	0	0
11th Thoracic centrum Bipartite ossification	0	0	0	0	0	0	1	0.7
Vertebral arches:
5th Sternebra dumb bell Shaped	11	6.7	12	7.5	6	3.4	8	5.9
2nd Sternebra UO	10	6.1	7	4.4	6	3.4	4	2.9
2nd Sternebra dumb bell Shaped	3	1.8	4	2.5	3	1.7	3	2.2
Manubrium UO	2	1.2	1	0.6	0	0	0	0
Sternebra UO	1	0.6	2	1.3	0	0	2	1.5
4th Sternebra UO	1	0.6	0	0	1	0.6	2	1.5
5th Sternebra IO	24	14.5	21	13.1	28	16.1	19	14.1
2nd Sternebra IO	8	4.8	5	3.1	11	6.3	6	4.4
4th Sternebra IO	1	0.6	1	0.6	1	0.6	0	0
Sternebra Misaligned	3	1.8	4	2.5	4	2.3	3	2.2
5th Sternebra Bipartite ossification	3	1.8	0	0	3	1.7	1	0.7
6th Sternebra IO	4	2.4	6	3.8	13	7.5	3	2.2
6th Sternebra UO	4	2.4	4	2.5	1	0.6	8	5.9
5th Sternebra UO	16	9.7	14	8.8	11	6.3	11	8.1
Sternebra IO	1	0.6	0	0	1	0.6	4	2.9

 

Skeletal Parameters	G1 (0)		G2 (75)		G3 (150)		G4 (350/300)
Vertebral arches:	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected	Foetal. Incidence.	% foetus affected
5th Sternum IO	1	0.6	0	0	0	0	0	0
6th Sternum IO	1	0.6	0	0	0	0	0	0
6th Sternebra dumb bell Shaped	1	0.6	0	0	1	0.6	0	0
5th Sternebra Misaligned	0	0	1	0.6	0	0	0	0
2nd Sternebra Misaligned	0	0	1	0.6	0	0	0	0
Sternebra dumb bell Shaped	0	0	1	0.6	0	0	0	0
Metatarsal UO	0	0	1	0.6	0	0	0	0
Metatarsal IO	0	0	1	0.6	0	0	0	0
pelvic ventebral arch ossification (extra)	0	0	1	0.6	0	0	0	0
Xiphoid P Sternebra UO	0	0	0	0	3	1.7	1	0.7
Right Ischium UO	0	0	0	0	1	0.6	0	0
4th Sternebra dumb bell Shaped	0	0	0	0	1	0.6	0	0
3rd Sternebra UO	0	0	0	0	1	0.6	0	0
2nd Sternebra B O	0	0	0	0	1	0.6	0	0
2nd Sternebra dumb bell ossification	0	0	0	0	0	0	1	0.7
3rd Sternebra dumb bell ossification	0	0	0	0	0	0	1	0.7
4th Sternebra dumb bell ossification	0	0	0	0	0	0	1	0.7
Pubis IO	0	0	0	0	0	0	1	0.7

 

 

Historical Range

Caesarean Section Data – Gestation Day 20

Parameters	Mean	SD	Min	Max
Females/Study	22.11	5.60	18	25
Pregnant Females	19.33	5.15	16	24
% Pregnant	94.20	6.39	89	98
# All Resorbed	0.00	0.00	0	0
% of Preg. All resorbed	0.00	0.00	0	0
# Corpora Lutea	17.88	4.43	11	18
# Implants	13.88	2.76	9	15
# Resorptions	0.26	0.66	0	4
% Resorptions	3.08	8.56	0	15
# Live Foetuses	13.59	5.91	9	14
# Dead Foetuses	0.01	0.08	0	1
% Dead + Resor./Implants	0.03	0.09	0.0	0.5
Sex Ratio (% Males)	51.43	18.76	0	100
Live Foetal Weights (g)
All foetuses	3.62	0.31	2.79	4.97
Male Foetuses	3.68	0.43	0.00	5.10
Female Foetuses	3.49	0.41	0.00	4.75

Maternal Data – Gestation Day 20

Parameters	Mean	SD	Min	Max
Females/Study	22.18	5.60	18.00	25.00
Pregnant Females	21.03	5.15	16.00	24.00
% Pregnant	94.81	6.39	80.00	96.00
Corrected Maternal Body Weight (g)	235.56	19.24	184.60	295.40
Relative uterus weight (%)	18.31	4.52	2.55	25.07
% Pre implantation loss	5.95	13.53	0.00	26.00
% Post implantation loss	2.01	5.22	0.00	31.00

Foetal External Anomalies

Observations	Foetal Incidence			Litter Incidence
	Incidence		Average %	Incidence	Average %
Anasarca	0.30		0.01	0.20	0.12
Conjoined twins	1.00		0.04	0.03	0.02
Hematoma	1.00		0.04	0.26	0.15
Local edema	0.50		0.02	0.14	0.08
Pallid fetus	0.00		0.00	0.06	0.03
SKULL
Cephalocele	0.03		0.00	0.03	0.02
Craniorachischisis	0.40		0.01	0.20	0.11
Domed head	0.03		0.00	0.03	0.02
Encephalomeninogocele	0.50		0.02	0.03	0.02
Exencephaly	1.00		0.04	0.31	0.18
Meningocele	0.03		0.00	0.03	0.02
Microcephaly	0.03		0.00	0.03	0.02
EYES
Ablepharia	0.22		0.01	0.08	0.05
Anophtalmia	0.52		0.02	0.35	0.21
Cyclopia	0.03		0.00	0.03	0.02
Exophthalmos	0.03		0.00	0.06	0.03
Microphthalmia	2.03		0.07	0.75	0.44
Open eyelid	0.33		0.01	0.17	0.10
EAR
Ear, displaced	0.13		0.00	0.11	0.07
Microtia	0.03		0.00	0.03	0.02
NOSE
Nasal atresia		0.06	0.00	0.06	0.03
Nasal dysplasia		0.10	0.00	0.08	0.05
Rhinocephaly		0.06	0.00	0.06	0.03
MOUTH AND JAW
Aglossia		0.10	0.00	0.08	0.05
Agnathia		0.41	0.01	0.28	0.16
Astomia		0.19	0.01	0.17	0.10
Cleft face		0.06	0.00	0.06	0.03
Cleft lip		0.03	0.00	0.03	0.02
Cleft palate		0.29	0.01	0.25	0.15
High-arched palate		0.13	0.00	0.06	0.03
Microglossia		0.06	0.00	0.06	0.03
Micrognathia		0.31	0.01	0.34	0.20
Microstomia		0.13	0.00	0.11	0.07
Protruding tongue		0.21	0.01	0.31	0.18

Observations	Foetal Incidence		Litter Incidence
	Incidence	Average %	Incidence	Average %
TORSO
Atresia ani	0.29	0.01	0.25	0.15
Gastroschisis	0.22	0.01	0.20	0.11
Holorachischisis	0.03	0.00	0.03	0.02
Omphalocele	0.16	0.01	0.14	0.08
Scoliosis	0.03	0.00	0.03	0.02
Shortened torso	0.06	0.00	0.06	0.03
Spina bifida aperta	0.13	0.00	0.11	0.07
Umbilical hernia	0.34	0.01	0.34	0.20
EXTERMITIES
Clubbed forefoot	0.06	0.00	0.06	0.03
Clubbed hindfoot	0.03	0.00	0.03	0.02
Extrodactyly	0.03	0.00	0.03	0.02
Extromelia	0.03	0.00	0.03	0.02
Forelimb Flexion	0.13	0.00	0.06	0.03
Micromelia	0.10	0.00	0.08	0.05
Phocomelia	0.03	0.00	0.03	0.02
Tail malformation	0.76	0.03	0.99	0.58
ADDITIONAL FINDINGS
Abdominal hernia	0.03	0.00	0.03	0.02
partial twinning	0.03	0.00	0.03	0.02
Shiny	0.10	0.00	0.08	0.05
Polymelia	0.03	0.00	0.03	0.02
Growth retarded	0.86	0.03	0.51	0.30
Fused placenta	0.13	0.00	0.06	0.03
Vibrissal pad, malf.	0.03	0.00	0.03	0.02
Upper jaw missing	0.03	0.00	0.03	0.02
Hard palate missing	0.03	0.00	0.03	0.02

 

Foetal Head Razor Section Anomalies

Observations	Foetal Incidence		Litter Incidence
	Incidence	Average %	Incidence	Average %
Third Ventricle: Slightly Dilated	11	2.50	18	2.48
Third Ventricle: Dilated	6	1.17	8	1.45
Third Ventricle: Misshapen	1	0.39	1	0.56
Lateral Ventricles: Slightly Dilated	18	5.28	49	7.91
Lateral Ventricles: Dilated	7	3.32	20	4.06
Lateral Ventricles: Misshapen	1	0.19	1	0.56
Cerebral Hemisphere: Hemorrhagic	1	0.38	1	0.42
Lateral Ventricles: Hemorrhagic	1	0.25	2	0.37
Nasal Septum: Misshapen	1	0.25	1	0.14
Nasal Conchae: Small	1	0.38	3	0.68
Nasal Cavity: Hemorrhagic	1	0.13	3	0.71
Nasal Cavity: Large	1	0.13	1	0.37
Nasal Cavity: Dilated	1	0.13	1	0.08
Nasal Cavity: Misshapen	1	0.13	1	0.09
Nasal Septum: Hemorrhagic	1	0.13	1	0.12
Eye: Anopthalmia	1	0.13	1	0.37
Eye: Small	1	0.13	1	0.14
Olfactory Lobes: Large	2	0.25	2	0.37
Olfactory Lobes: Misshapen	1	0.13	1	0.19
Palate: Hemorrhagic	1	0.13	1	0.08
Retina (U): Misshapen	1	0.13	2	0.31

Foetal Visceral Anomalies

Observations	Foetal Incidence (%)						Litter Incidence (%)
	Mean		SD		Max		Mean		SD		Max
CORONAL SECTIONS
Cerebrum: Misshapen	0.011		0.06		0.84		0.053		0.57		4.21
Cerebral ventricular enlargement	0.110		0.27		2.58		0.561		2.27		22.02
Hydrocephalus	0.016		0.09		0.73		0.086		0.61		5.73
Lens: Agenesis	0.026		0.18		0.57		0.126		0.54		3.72
Lens: Hypoplastic	0.013		0.07		0.79		0.122		0.84		5.61
Nasal cavity: Enlarged	0.002		0.02		0.48		0.022		0.28		3.93
Retina: Agenesis	0.006		0.06		0.68		0.051		0.43		4.46
Retina: Folded	0.019		0.13		1.47		0.179		0.58		7.03
Microphthalmia	0.019		0.13		1.27		0.207		1.08		9.02
GENERAL
Abdominal cavity: Fluid-filled	0.003		0.04		0.61		0.027		0.38		3.51
Situs inversus	0.009		0.18		0.63		0.118		0.75		3.07
BLOOD VESSLES
Aortic arch: Enlarged	0.002		0.02		0.58		0.032		0.29		2.75
Aortic arch: Interrupted	0		0		0		0		0		0
Aortic arch: Retroesophageal	0.001		0.00		0.26		0.027		0.41		2.89
Azygos vein: Doubled	0.039		0.27		1.72		0.165		0.89		9.27
Azygos vein: Right-sided	0.003		0.02		0.59		0.013		0.24		4.17
Carotid: Abnormal origin	0.004		0.07		0.82		0.027		0.46		2.81
Ductus arteriosus: Hypoplastic	0.004		0.08		0.49		0.037		0.46		3.72
Innominate: Agenesis	0.054		0.40		2.21		0.276		1.27		12.54
Innominate: Hypoplastic	0.009		0.05		1.03		0.046		0.45		5.13
Pulmonary trunk: Stenosis	0.003		0.03		0.74		0.034		0.45		3.81
Truncus arteriosus: Persistent	0.006		0.04		0.81		0.044		0.38		4.74
HEART
Atrium: Enlarged		0.005		0.04		0.57		0.043		0.36		2.98
Cardiomegaly		0.005		0.03		0.52		0.031		0.27		5.33
Dextrocadia		0.003		0.06		0.48		0.026		0.43		4.21
Double apex		0.004		0.04		0.41		0.036		0.25		5.12
Misshapen heart		0.017		0.18		0.73		0.127		0.55		3.91
Situs inversus, cardio-vascular		0.008		0.04		0.56		0.088		0.41		4.49
Ventricular hypertrophy		0.005		0.04		0.68		0.039		0.27		3.00
Ventricular hypoplasia		0.004		0.08		0.53		0.018		0.29		3.12
Ventricular septal defect (VSD)		0.012		0.16		0.71		0.116		0.53		4.93
LUNG AND TRACHEA
Lung: Abnormal lobe formation		0.009		0.08		0.53		0.061		0.52		3.31
Lung: Agenesis		0.006		0.05		0.59		0.048		0.56		4.78
Lung: Diff. hemorrh. (mottled)		0.005		0.04		0.66		0.031		0.46		2.81
Lung: Enlarged		0.003		0.03		0.59		0.021		0.43		3.00
Trachea: Short		0.005		0.05		0.84		0.031		0.27		3.81
DIAPHRAGM
Diaphragmatic hernia		0.009		0.04		0.51		0.063		0.41		5.27

Observations	Foetal Incidence (%)						Litter Incidence (%)
	Mean		SD		Max		Mean	SD	Max
DIGESTIVE TRACT
Esophagus: Short	0.007		0.06		0.87		0.033	0.42	4.81
Stomach: Situs inversus	0.006		0.05		0.48		0.061	0.36	4.00
SPLEEN
Spleen: Hypoplastic	0.004		0.05		0.56		0.031	0.27	3.89
ADRENAL
Focally hemorrhagic	0.009		0.08		0.71		0.065	0.54	4.73
KIDNEY
Distended renal pelvis	0.581		1.22		5.83		5.017	6.22	41.81
Hydronephrosis	0.161		0.47		5.36		0.631	2.18	19.75
Kidney: Diffuse hemorrhagic	0.019		0.17		1.59		0.062	0.84	7.30
Kidney: Displaced	0.006		0.05		0.38		0.059	0.48	4.68
Kidney: Hypoplas. (red. Papilla)	0.822		2.13		12.27		2.167	7.81	43.02
Kidney: Supernumerary	0.003		0.04		0.49		0.021	0.58	3.89
URETER AND BLADDER
Hydroureter	0.347		73.00		3.86		1.223	3.84	17.83
Ureter: Convoluted	0.581		1.99		18.27		3.193	6.93	97.28
Ureter: Distended	1.284		3.71		37.25		6.315	21.84	81.92
Urinary bladder: Enlarged	0.165		0.71		6.73		0.573	1.88	31.36
MALE REPRODUCTIVE TRACT
Testis: Agenesis	0.005		0.10		2.17		0.039	0.44	3.92
Testis: Displaced	0.426		1.89		12.63		2.113	6.83	69.82
Testis: Supernumerary	0.003		0.04		0.81		0.061	0.73	4.93
SKULL
Bone island		0.002		0.02		0.38	0.021	0.28	4.81
Frontals: Contain holes		0.006		0.09		1.16	0.041	0.58	6.13
Frontals: Hypoplastic		0.004		0.05		0.57	0.029	0.37	4.21
Frontals: Misshapen		0.004		0.04		0.56	0.041	0.82	3.68
Frontals: Bifid		0.002		0.03		0.51	0.029	0.36	3.49
Mandible: Agenesis		0.017		0.06		0.62	0.051	0.37	3.28
Mandible: Hypoplastic		0.003		0.02		0.71	0.051	0.28	3.29
Mandible: Misshapen		0.004		0.02		0.52	0.061	0.20	4.04
Maxilla: Agenesis		0.005		0.08		0.83	0.029	0.42	3.29
Maxilla: Misshapen		0.009		0.04		0.52	0.058	0.62	4.74
Nasals: Misshapen		0.006		0.05		0.78	0.059	0.73	5.82
Orbits: Hypoplastic		0.007		0.09		0.65	0.034	0.54	4.71
Orbits: Misshapen		0.033		0.18		0.98	0.339	1.34	7.83
Parietals: Misshapen		0.003		0.02		0.47	0.031	0.31	2.48
SPINAL COLUMN
Vertebral Scrambling		0.018		0.13		1.92	0.078	0.58	9.02
Cervical vertebrae: Fused		0.013		0.17		1.21	0.089	0.53	3.71
Cervical vertebrae: Hypoplastic		0.005		0.03		0.41	0.062	0.47	3.81
Cervical vertebrae: Misaligned		0.004		0.06		0.58	0.027	0.36	4.00
Cervical vertebrae: Misshapen		0.002		0.02		0.34	0.032	0.41	4.86

Observations	Foetal Incidence (%)			Litter Incidence (%)
	Mean	SD	Max	Mean	SD	Max
SPINAL COLUMN
Cervical centra: Split	0.003	0.05	0.79	0.031	0.47	4.89
Thoracic vertebrae: Agenesis	0.061	0.28	3.79	0.223	1.37	9.63
Thoracic vertebrae: Fused	0.024	0.16	1.29	0.126	0.68	4.82
Thoracic vertebrae: Misaligned	0.005	0.06	0.42	0.037	0.51	5.21
Thoracic vertebrae: Misshapen	0.017	0.05	0.61	0.169	0.71	6.82
Thoracic centra: Dumbbell-shaped	0.397	1.07	17.24	1.212	5.81	57.33
Thoracic centra: Split	0.537	1.00	7.82	2.116	4.84	23.69
Thoracic centra: Misshapen	0.010	0.03	0.73	0.067	0.52	3.62
Lumbar vertebrae: Agenesis	0.014	0.13	0.96	0.005	0.03	0.23
Lumbar vertebrae: Fused	0.008	0.07	1.27	0.057	0.59	6.17
Lumbar vertebrae: Misaligned	0.008	0.06	0.80	0.058	0.63	2.27
Lumbar centra: Dumbbell-shaped	0.016	0.07	0.82	0.053	0.58	3.25
Lumbar centra: Split	0.021	0.11	1.89	0.213	0.88	4.82
Sacral vertebrae: Agenesis	0.003	0.04	0.37	0.039	0.03	3.29
Sacral vertebrae: Fused	0.181	0.53	9.67	0.314	2.17	27.73
Sacral vertebrae: Misaligned	0.026	0.18	0.76	0.206	1.45	9.03
Caudal vertebrae: Agenesis	0.007	0.04	0.44	0.063	0.55	3.26
Caudal vertebrae: Fused	0.081	1.45	10.60	0.154	1.02	23.27
Lumbosacral shift	0.037	0.19	1.41	0.312	1.56	9.81
Missing vertebra	0.011	0.04	0.52	0.161	0.49	3.28
Vertebral count > or < normal	0.137	0.25	1.22	0.761	1.39	10.52
STERNEBRAE
Multiple fusions	0.069	0.33	1.23	0.537	1.69	3.82
Agenesis	0.009	0.10	0.27	0.096	0.53	2.18
Split	0.036	0.33	0.82	0.216	1.33	9.54
Misaligned	0.361	1.47	3.81	2.367	2.81	22.17
Asymmetric	0.053	0.18	1.37	0.531	1.67	13.89
Misshapen	0.037	0.11	0.84	0.167	1.84	4.21
RIBS
Agenesis	0.021	0.17	0.29	0.425	1.81	3.72
Cervical	0.516	1.27	4.04	3.482	5.63	17.03
Fused	0.081	0.11	0.37	0.483	1.07	3.29
Hypoplastic	1.005	2.63	16.92	3.760	9.53	53.72
Misshapen	0.884	2.16	5.61	2.190	2.15	37.83
Supernumerary	2.167	3.27	16.83	23.445	19.94	96.81
Wavy	0.213	0.27	1.31	1.067	1.13	9.83
Bent	0.037	0.44	1.21	0.387	2.13	9.52
PELVIC GIRDLE
Ischium: Hypoplastic	0.023	0.04	0.29	0.171	0.59	3.79
SHOULDER GIRDLE
Scapula: Misshapen	0.053	0.038	0.47	0.169	0.733	3.45

 

Observations	Foetal Incidence (%)			Litter Incidence (%)
	Mean	SD	Max	Mean	SD	Max
APPENDICULAR SKELETON
Fore limb
Long bone: Hypoplastic	0.033	0.11	0.83	0.211	1.23	10.01
Long bone: Misshapen	0.002	0.02	0.13	0.063	0.42	3.76
Phalanx: Misaligned	0.007	0.07	0.76	0.059	0.74	3
Phalanx: Clubbed	0.023	0.13	1.27	0.231	0.72	6.03
Hind limb
Long bone: Small	0.034	0.11	1.27	0.236	0.87	5.92
Long bone: Misshapen	0.006	0.03	0.34	0.237	0.59	2.47
Phalanx: Agenesis	0.104	0.61	2.74	0.481	3.22	21.63
Phalanx: Supernumerary	0.019	0.12	1.05	0.116	2.17	4.72
Phalanx: Misaligned	0.011	0.11	0.87	0.053	0.27	3.37
Talus: Advanced	0.025	0.23	1.81	0.095	0.52	2.91

Foetal Skeletal Anomalies

Observations	Foetal Incidence (%)			Litter Incidence (%)
	Mean	SD	Max	Mean	SD	Max
SKULL
Bone island	0.002	0.02	0.38	0.021	0.28	4.81
Frontals: Contain holes	0.006	0.09	1.16	0.041	0.58	6.13
Frontals: Hypoplastic	0.004	0.05	0.57	0.029	0.37	4.21
Frontals: Misshapen	0.004	0.04	0.56	0.041	0.82	3.68
Frontals: Bifid	0.002	0.03	0.51	0.029	0.36	3.49
Mandible: Agenesis	0.017	0.06	0.62	0.051	0.37	3.28
Mandible: Hypoplastic	0.003	0.02	0.71	0.051	0.28	3.29
Mandible: Misshapen	0.004	0.02	0.52	0.061	0.20	4.04
Maxilla: Agenesis	0.005	0.08	0.83	0.029	0.42	3.29
Maxilla: Misshapen	0.009	0.04	0.52	0.058	0.62	4.74
Nasals: Misshapen	0.006	0.05	0.78	0.059	0.73	5.82
Orbits: Hypoplastic	0.007	0.09	0.65	0.034	0.54	4.71
Orbits: Misshapen	0.033	0.18	0.98	0.339	1.34	7.83
Parietals: Misshapen	0.003	0.02	0.47	0.031	0.31	2.48
SPINAL COLUMN
Vertebral Scrambling	0.018	0.13	1.92	0.078	0.58	9.02
Cervical vertebrae: Fused	0.013	0.17	1.21	0.089	0.53	3.71
Cervical vertebrae: Hypoplastic	0.005	0.03	0.41	0.062	0.47	3.81
Cervical vertebrae: Misaligned	0.004	0.06	0.58	0.027	0.36	4.00
Cervical vertebrae: Misshapen	0.002	0.02	0.34	0.032	0.41	4.86
Cervical centra: Split	0.003	0.05	0.79	0.031	0.47	4.89
Thoracic vertebrae: Agenesis	0.061	0.28	3.79	0.223	1.37	9.63
Thoracic vertebrae: Fused	0.024	0.16	1.29	0.126	0.68	4.82
Thoracic vertebrae: Misaligned	0.005	0.06	0.42	0.037	0.51	5.21
Thoracic vertebrae: Misshapen	0.017	0.05	0.61	0.169	0.71	6.82
Thoracic centra: Dumbbell-shaped	0.397	1.07	17.24	1.212	5.81	57.33
Thoracic centra: Split	0.537	1.00	7.82	2.116	4.84	23.69
Thoracic centra: Misshapen	0.010	0.03	0.73	0.067	0.52	3.62
Lumbar vertebrae: Agenesis	0.014	0.13	0.96	0.005	0.03	0.23
Lumbar vertebrae: Fused	0.008	0.07	1.27	0.057	0.59	6.17
Lumbar vertebrae: Misaligned	0.008	0.06	0.80	0.058	0.63	2.27
Lumbar centra: Dumbbell-shaped	0.016	0.07	0.82	0.053	0.58	3.25
Lumbar centra: Split	0.021	0.11	1.89	0.213	0.88	4.82
Sacral vertebrae: Agenesis	0.003	0.04	0.37	0.039	0.03	3.29
Sacral vertebrae: Fused	0.181	0.53	9.67	0.314	2.17	27.73
Sacral vertebrae: Misaligned	0.026	0.18	0.76	0.206	1.45	9.03
Caudal vertebrae: Agenesis	0.007	0.04	0.44	0.063	0.55	3.26
Caudal vertebrae: Fused	0.081	1.45	10.60	0.154	1.02	23.27
Lumbosacral shift	0.037	0.19	1.41	0.312	1.56	9.81
Missing vertebra	0.011	0.04	0.52	0.161	0.49	3.28
Vertebral count > or < normal	0.137	0.25	1.22	0.761	1.39	10.52
STERNEBRAE
Multiple fusions	0.069	0.33	1.23	0.537	1.69	3.82
Agenesis	0.009	0.10	0.27	0.096	0.53	2.18

Observations	Foetal Incidence (%)			Litter Incidence (%)
	Mean	SD	Max	Mean	SD	Max
STERNEBRAE
Split	0.036	0.33	0.82	0.216	1.33	9.54
Misaligned	0.361	1.47	3.81	2.367	2.81	22.17
Asymmetric	0.053	0.18	1.37	0.531	1.67	13.89
Misshapen	0.037	0.11	0.84	0.167	1.84	4.21
RIBS
Agenesis	0.021	0.17	0.29	0.425	1.81	3.72
Cervical	0.516	1.27	4.04	3.482	5.63	17.03
Fused	0.081	0.11	0.37	0.483	1.07	3.29
Hypoplastic	1.005	2.63	16.92	3.760	9.53	53.72
Misshapen	0.884	2.16	5.61	2.190	2.15	37.83
Supernumerary	2.167	3.27	16.83	23.445	19.94	96.81
Wavy	0.213	0.27	1.31	1.067	1.13	9.83
Bent	0.037	0.44	1.21	0.387	2.13	9.52
PELVIC GIRDLE
Ischium: Hypoplastic	0.023	0.04	0.29	0.171	0.59	3.79
SHOULDER GIRDLE
Scapula: Misshapen	0.053	0.038	0.47	0.169	0.733	3.45
APPENDICULAR SKELETON
Forelimb
Long bone: Hypoplastic	0.033	0.11	0.83	0.211	1.23	10.01
Long bone: Misshapen	0.002	0.02	0.13	0.063	0.42	3.76
Phalanx: Misaligned	0.007	0.07	0.76	0.059	0.74	3
Phalanx: Clubbed	0.023	0.13	1.27	0.231	0.72	6.03
Hindlimb
Long bone: Small	0.034	0.11	1.27	0.236	0.87	5.92
Long bone: Misshapen	0.006	0.03	0.34	0.237	0.59	2.47
Phalanx: Agenesis	0.104	0.61	2.74	0.481	3.22	21.63
Phalanx: Supernumerary	0.019	0.12	1.05	0.116	2.17	4.72
Phalanx: Misaligned	0.011	0.11	0.87	0.053	0.27	3.37
Talus: Advanced	0.025	0.23	1.81	0.095	0.52	2.91

Conclusions:

Pregnant Wistar rats when treated with ‘Genamin 3920’ at 75, 150 and 350/300 mg/kg/day did not reveal any foetal toxicity. No teratogenic effects attributable to treatment were observed. Significant decrease in body weight and feed consumption along with significant decrease in uterine weights were observed in dams treated at 350/300 mg/kg.
Based on findings, the NOAEL of ‘Genamin 3920’ in pregnant Wistar rats is 150 mg/kg for maternal toxicity and 300 mg/kg for foetal toxicity under the tested conditions.

Executive summary:

This study was conducted to estimate the prenatal developmental toxicity and maternal toxicity of Genamin 3920, when administered by oral route to pregnant Wistar rats during Gestation Days (GD) 5 to 19. The study was carried out in accordance with the OECD Guideline for Testing of Chemicals No. 414 and United States EPA Health Effects Test Guideline OCSPP 870.3700.

A total of four groups (G1, G2, G3 and G4) of pregnant Wistar rats were used in the study out of which, Group 1 (G1) served as the vehicle control. Group 2 (G2), Group 3 (G3) and Group 4 (G4) were administered the test item at the dose levels of 75, 150 and 350/300 mg/kg/day respectively, through oral route, once daily, from gestation days (GD) 5 to 19. As mortality was observed at the dose level of 350 mg/kg/day (high dose) during different gestation days, the dose was reduced to 300 mg/kg/day for this group. The control group was administered the vehicle alone (corn oil).

Test item formulations were prepared in corn oil. Dose formulation analysis was performed on formulation samples prepared on the first day of dose administration. The concentration of Genamin 3920 in the dose formulations were in the range between 97.5% - 105.1%, which were within the acceptance criteria (85% - 115%). All the formulations met the acceptance criteria and confirmed that the animals received the intended dose of test item. 

In-life observations included morbidity, mortality, clinical signs of toxicity, maternal body weight and food consumption. A Caesarean section was performed on Day 20 of gestation followed by which, visceral examination of all the dams was performed. Litter data, sex and weight of individual foetuses were recorded followed by which, external malformations, visceral, head razor and skeletal examinations were performed on foetuses.

All dams belonging to control, low and mid dose were normal and were free from all visible clinical signs throughout the treatment period. Three dams (Dam No. T018/074, T018/084 and T018/086) treated at 350 mg/kg body weight/day were found dead between GD 9-15. Animals treated at 350 mg/kg/day displayed salivation, burrowing behaviour, hyperactivity and aggression. The observed clinical signs disappeared when the high dose was reduced to 300 mg/kg/day.

Maternal body weight, body weight gain and feed consumption data were significantly reduced from GD 8-20 at the dose of 350/300 mg/kg. All dams belonging to the control, low and mid dose treated groups displayed normal body weight gain throughout the study period. Adjusted maternal weight (after exclusion of uterine weight) of treated dams derived on GD 20 was significantly reduced in the 350/300 mg/kg group. No significant changes were observed in the low and mid dose and was comparable to control group animals.

There were no changes in T3, T4 and TSH values attributable to treatment. No pathological observations in thyroid/parathyroid were evident in the control, low, mid and high dose treated dams.

In the found dead dams belonging to high dose, gross pathological evaluations revealed red discoloration (3/3) in lung and mottled liver (1/3) which were considered to be treatment related. All other dams belonging to high dose group following reduction of dose from 350 to 300 mg/kg/day survived the entire treatment period and displayed no gross pathological abnormalities. No abnormalities were detected in the mid and low dose treated dams as well as the control animals during gross pathological examinations.

Histopathological observations of the found dead animals revealed kidneys with necrosis, bilateral, multifocal (3+) in 2 dams and kidneys with necrosis, bilateral, multifocal (2+), hyperplasia with squamous cell, non-glandular stomach, diffuse (3+) in one dam in the high dose treated group of 350 mg/kg body weight/day. No histopathological lesions in thyroid/parathyroid were recorded in the survived animals of high dose as well as control, low and mid dose.

No abnormalities were observed in the evaluated maternal parameters viz., mean gravid absolute and relative uterine weights, placental weight, number of corpora lutea, No of implantations, resorptions and implantation losses in treated (75 and 150mg/kg/day) and control group. Significant changes were observed in the gravid uterine weights of high dose treated group (350/300 mg/kg/day) as compared to the control group.

There were no significant changes in the mean number of male and female foetuses and total number of foetuses in any of the treated groups (75, 150 and 300 mg/kg/day) as compared to the control group. Foetal weight was comparable between treated and control groups. No malformations related to treatment were detected during external examination of the pups.

No malformations/abnormalities were detected in any of the treated and control groups during the visceral and head razor examinations.

Foetal skeletal examination did not reveal any treatment related malformations/ abnormalities in any of the treated and control groups.

Conclusion

Pregnant Wistar rats when treated with ‘Genamin 3920’ at 75, 150 and 350/300 mg/kg/day did not reveal any foetal toxicity. No teratogenic effects attributable to treatment were observed. Significant decrease in body weight and feed consumption along with significant decrease in uterine weights were observed in dams treated at 350/300 mg/kg.

Based on findings, the NOAEL of ‘Genamin 3920’ in pregnant Wistar rats is 150 mg/kg for maternal toxicity and 300 mg/kg for foetal toxicity under the tested conditions.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

1 reliable without restriction as the data originate from a recent screening study according to OECD TG 414 performed under GLP-regulations.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on experiment data obtained from a OECD TG 421 and OECD 414 studies in the absence of toxicological relevant effects on reproductive and developmental endpoints, the test item is considered to be non reproductive/develpmental toxic.