2,2'-(octylimino)bisethanol

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(octylimino)bisethanol

EC Number:

239-555-0

EC Name:

2,2'-(octylimino)bisethanol

Cas Number:

15520-05-5

Molecular formula:

C12H27NO2

IUPAC Name:

2-[(2-hydroxyethyl)(octyl)amino]ethan-1-ol

Test material form:

other: liquid

Test animals

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.

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.