Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-14 to 2015-12-03
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

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

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: 5-6 weeks old, females: 5-6 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 147 - 182 g (mean: 163.34 g, ± 20% = 130.67 – 196.01 g)
females: 120 - 150 g (mean: 133.93 g, ± 20% = 107.15 – 160.72 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. 1526, 1239) during the 70 days premating period. With the onset
of mating the animals were provided with Altromin 1310 P breeding diet for rats and mice (lot no. 1512, 0727). After the completion of the
mating period all males had access to Altromin 1324 maintenance diet (lot no. 1526, 1239).
- 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 in groups of 5 in type IV cages except during the mating period when one female was paired with one male.
During the gestation the females were housed individually. After parturition females were housed together with the litter until weaning.
All cages are made of polysulphone and were provided with saw fibre bedding. (lot no. 02102140627, 02102140831, 02102141114,
02102150227). Females of both generations were provided with nesting material on GD 18.
- 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 all animals were acclimated to laboratory conditions for minimum 5 days. Detailed clinical observation
outside the home cage was made on P Animals. There were no adverse clinical signs observed in the animals. All animals used for the study were
weighed and randomly assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups
of males and females, respectively. The P Animals were assigned a unique identification number (numbering with ear-tattoo).
For the F1 and F2 generation, the individual identification (tattoo mark on paw) of pups soon after birth was made. In addition the unique
identification number (numbering with ear-tattoo) was provided at weaning for animals selected for mating (F1 Generation).

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose)
and 1 control group (C):
Control: 0 mg/kg body weight/day
Low Dose: 100 mg/kg body weight/day
Medium Dose: 300 mg/kg body weight/day
High Dose: 1000 mg/kg body weight/day

The parental P animals were treated with the test item formulation or vehicle on 7 days per week for 10 weeks premating (males and females),
a maximum of 2 weeks mating (males and females), during pregnancy and lactation up to weaning (females). On PND21, 1 pup/sex/litter were
selected for dosing of F1. These animals will be dosed over 10 weeks prior to mating, through mating, gestation, and lactation until weaning of F2.
Parental males of P and F1 were killed after the mating when no longer required for further reproductive assessment.

Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/day corresponding to a limit
dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and
a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle.

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.
All animals were not dosed with the vehicle or test item formulation on 05 October 2014. Dose application for 1 animal (no. 192) was not made
on lactation day 17 (date 20 Dec 2014). Inadvertently the dose administration was missed. The premating treatment period was extended to 71
days instead of 70 days to compensate the missing dose during the premating period.
During the first week a higher dose volume was applied to animal no. 121 (0.9 mL instead of 0.7 mL). This error was due to miscalculation.

Details on mating procedure:
P Generation:
Mating of P animals was performed after 10 weeks dosing period.
F1 Generation:
On PND21, 1 pup/sex/litter was selected from F1 generation for mating to produce F2. For litters 102 and 106 of Control; litters 151 and 153 of MD;
litter 136 of HD group 2 pups/ sex/ litter were chosen. For litters 184 and 196, 2 female pups and 2 males pups per litter, respectively were chosen.
This deviation was due to non-availability of enough pups for mating (25 F1 pairs/ group).
Mating of 25 F1 pairs/ group to produce F2 was performed after a 10 weeks dosing period, which started after weaning. Mating was performed in the ratio of 1:1 (male to female) within each group. The subsequent morning and the next morning there onwards the vaginal smear of females were
checked to confirm evidence of mating. Day of vaginal plug and/or sperm was considered as day ‘0’ of gestation. After mating was confirmed the
mated pairs were separated.
Maximum duration of cohabitation was 2 weeks. Cages were arranged in such a way that possible effects due to cage placement were minimized.
Females showing no evidence of copulation or pregnancy were sacrificed 26 days after the evidence of mating or from the last day of the mating
period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the dosing formulations for 0 hour and 6 hours at room temperature were tested in week 1 from formulation samples of LD and HD
groups. The samples were stored between -15 and -35 °C (4 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high and low dose formulations in the first and last week of
study. The samples were stored between -15 and -35 °C (12 samples).
For determination of the concentration in dosing formulations, samples were retained from all groups once in the first and last week of study,
once during mating of P, once during mating of F1 and last week of study. The samples were stored between -15 and -35 °C (16 samples).
Each sample was retained twice and was labelled sample ‘A’ and sample ‘B’.
At the end of the treatment period all ‘A’ samples of dosing formulations were shipped on dry ice and protected from light to:
CIP
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of test item concentration in the dosing formulations was performed by CIP GmbH, in accordance with GLP.
The procedures followed were described in a detailed analytical phase plan. CIP GmbH issued an analytical phase report which was integrated in
the report of this study.
Duration of treatment / exposure:
The parental P animals were treated with the test item formulation or vehicle on 7 days per week for 10 weeks premating (males and females),
a maximum of 2 weeks mating (males and females), during pregnancy and lactation up to weaning (females). On PND21, 1 pup/sex/litter were
selected for dosing of F1. These animals were dosed over 10 weeks prior to mating, through mating, gestation, and lactation until weaning of F2.
Parental males of P and F1 were killed after the mating when no longer required for further reproductive assessment.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 02 May 2014
Study Initiation Date: 14 August 2014
Amendment to Study Plan: 21 October 2014
2nd Amendment to Study Plan: 17 December 2014
Experimental Starting Date: 18 August 2014
Experimental Completion Date: 21 April 2015
Completion Date of Delegated Phase (Histopathology): 22 September 2015
Completion Date of Delegated Phase (Formulation Analysis): 20 November 2015
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
0, 100, 300, 1000 mg/kg bw
No. of animals per sex per dose:
200 animals (100 males and 100 females) were included in the study (25 male and 25 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
Body Weight
The parental P animals (not in gestation and lactation) were weighed once before the assignment to the experimental groups, on the first day of
dosing and weekly thereafter (to adjust the dose volume to most recently measured body weight) as well as at the end of the study (at necropsy).
The F1 animals selected for mating were weighed on PND 21, weekly thereafter, and on the day of sacrifice. The weight of F1 males and females
was also measured on the day of balanopreputial separation and vaginal opening, respectively.
During gestation and lactation, the P and F1 females were weighed on GD 0, 7, 14, and 20 and on PND 0, 4, 7, 14, and 21.

Food Consumption
Food consumption was measured for the P animals in weekly intervals and for the F1 animals’ weekly beginning at weaning.
Food consumption was not measured during mating period.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Check for morbidity and mortality was made twice a day for dosed animals (except during weekend and holidays where observation was made
daily once) every day until sacrifice for all animals. Litters (not directly dosed) were checked for morbidity and mortality once a day.
A detailed clinical observation (in-cage and out of cage) of the P animals was performed prior to the first dose and weekly thereafter until sacrifice.
A detailed clinical observations (in-cage and out of cage) of the F1 was performed at weaning, and weekly thereafter until sacrifice.
Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and at least once a week thereafter. Changes in gait, posture,
response to handling, as well as the presence of clonic and tonic movements, stereotypy or bizarre behaviour were recorded.
Oestrous cyclicity (parental animals):
Daily over a period of 3 weeks prior to the mating period, the estrous cycle of all P and F1 female animals was examined. In addition,
the estrous cycle was recorded during the whole cohabitation period to confirm the mating.
Sperm parameters (parental animals):
At necropsy, one testis and epididymis was used for the measurement of homogenization resistant spermatid numbers and cauda epididymal
sperm reserves, respectively. Sperm motility was evaluated using the sperms from cauda epididymis of surviving male animals. The evaluation
was made in surviving male animals from P and F1 generation using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0).
Sperm morphology from proximal vas deferens was evaluated (conducted visually by slide reading) from of P and F1 generation males which
survived until the end of the treatment. This evaluation was made in the animals of control and HD groups.
The testes of males from P and F1 generation were stored at -80 °C. Cauda sperm reserves were obtained from the concentration and volume of
sperm in the suspension used to complete the qualitative evaluations, and the number of sperm recovered by subsequent mixing and/or
homogenizing the remaining cauda tissue. Sperm in the concentrated suspension was frozen for subsequent evaluation of cauda epididymal sperm
numbers. The sperm counts were reported in relation to the weight of the epididymis.
The slides meant for sperm morphology reading was also prepared for the same animals on the day of necropsy and stored at ambient temperature. The sperm morphology was not extended to LD and MD groups as there were no treatment related findings identified at the time of evaluation in
HD group.
Litter observations:
The duration of gestation was recorded and was calculated from day ‘0’ of pregnancy.
Each F1 and F2 litter was examined as soon as possible after delivery of the dam (PND 0) to establish the number and sex of pups, stillbirths,
live births, runts, presence of gross abnormalities and body weight.
Anogenital distance was measured on PND 0 in F2 pups.
From all F1 and F2 pups, sex and body weight were determined again on PND 4, 7, 14, and 21.
All F1 pups were evaluated for general development at the appropriate time points as described in:

Parameter Begin of Check End of Check
Pinna unfolding PND 0 PND 16
Incisor eruption PND 6 PND 12
Onset of coat development* PND 3 PND 16
Opening of Eyes PND 10 PND 21
* for F1 pups only day of hair growth was recorded. For F2 pups both days of hair growth and onset of coat development were recorded.

F1 pups selected for mating were evaluated for general development related to sexual maturation at the appropriate time points as described:

Parameter Begin of Check End of Check
Descensus of Testes PND 16 PND 30
Balanopreputial separation PND 25 PND 36
Opening of Vagina PND 30 PND 44
The F2 litter was evaluated for pinna unfolding, incisor eruption and onset of coat development.
F1 (not selected for mating) and F2 pups were also examined for righting reflex, startle reflex and motor activity once before weaning
and/or terminal sacrifice. Dead pups were necropsied.
Postmortem examinations (parental animals):
Terminal Sacrifice
P Males were sacrificed after completion of mating period or mating of all females using an anaesthesia (ketamine/xylazin, 2:1; ketamine:
Pharmanovo, lot no: 24863, expiry date: 10/2015 and lot no. 25175, expiry date: 06/2016; xylazin: Serumwerk, lot no: 01213, expiry date: 10/2015
and Rebo Pharm, lot no. 400260/1, expiry date: 01/2017).
P Females were sacrificed after weaning of F1.
For all animals, a gross necropsy was performed. A specific examination of the uterus for implantation was performed.
At the time of termination or death during the study, the female was examined macroscopically for any structural abnormalities or pathological
changes which may have influenced the pregnancy.
The number of corpora lutea and the implantation sites was counted for P females.
All organs showing macroscopic lesions of all adult animals were preserved. Tissues were fixed in 4% neutral-buffered formaldehyde with the
exception of testes and epididymides which were fixed in modified Davidson's solution for 24 hours and then transferred to 70% Ethanol.

Organ Weight
At the end of the treatment period, organ weights of all P parental animals were taken from brain, pituitary gland, adrenal glands, liver, kidneys,
spleen, thyroid gland, uterus with cervix, ovaries, testes, prostate, seminal vesicles with coagulating and epididymides (total and cauda).
The epididymides, which were chosen for evaluation of sperm motility, were weighed (total epididymal and cauda epididymis). Paired Organs
were weighed together.
Postmortem examinations (offspring):
Terminal Sacrifice
F1 Males were sacrificed after completion of mating period or mating of all females using an anaesthesia (ketamine/xylazin, 2:1; ketamine:
Pharmanovo, lot no: 24863, expiry date: 10/2015 and lot no. 25175, expiry date: 06/2016; xylazin: Serumwerk, lot no: 01213, expiry date: 10/2015
and Rebo Pharm, lot no. 400260/1, expiry date: 01/2017).
F1 Females were sacrificed after weaning of F1.
For all animals, a gross necropsy was performed. A specific examination of the uterus for implantation was performed.
At the time of termination or death during the study, the female was examined macroscopically for any structural abnormalities or pathological
changes which may have influenced the pregnancy.
The number of corpora lutea and the implantation sites was counted for F1 females.
All organs showing macroscopic lesions of all adult animals were preserved. Tissues were fixed in 4% neutral-buffered formaldehyde with the
exception of testes and epididymides which were fixed in modified Davidson's solution for 24 hours and then transferred to 70% Ethanol.
On PND 21 of the F1, 1 pup / sex / litter and of the F2 at weaning 1 pup / sex / litter was examined macroscopically for structural abnormalities
or changes. Special attention was paid to the reproductive organs.
F1 Pups not selected randomly for mating or detailed macroscopic observation were killed after gross external observation on or after PND 21.

Organ Weight
At the end of the treatment period, organ weights of all F1 parental animals were taken from brain, pituitary gland, adrenal glands, liver, kidneys,
spleen, thyroid gland, uterus with cervix, ovaries, testes, prostate, seminal vesicles with coagulating glands and epididymides (total and cauda).
The epididymides, which were chosen for evaluation of sperm motility, were weighed (total epididymal and cauda epididymis). Paired Organs were
weighed together.
F1 and F2 weanlings:
Brain, thymus, and spleen were weighed from F1 and F2 weanlings selected for macroscopic observation. Paired organs were weighed together.
Statistics:
Whenever possible, a statistical assessment of the results was performed for each gender by comparing values of treated with control animals
using a one-way ANOVA and a post-hoc Dunnett Test. Sperm morphology was statistically assessed by Unpaired ‘t’ test. These statistics were
performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Reproductive indices:
Reporductive Indices [%]
Offspring viability indices:
Viability Indices [%]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There was no mortality in the parental generation.

Clinical Observations
In both males and females of P Generation, there were no adverse test item treatment related clinical findings.
However, in P generation there were signs of alopecia, crust, bloody ear and salivation in male and/or females of control and/or test item
treated groups. There were no clinical signs of toxicological relevance observed during the detailed clinical observations.

Body Weight Development
In P Generation, there were no adverse effects of test item treatment on weight development of male and female animals.
However, in males there was statistically significantly higher or lower weight gain in LD group on last weeks of premating (day 57-64 and days 64-71)
and 3rd week of mating/ postmating (day 14-21). There was also higher weight gain on mating/ post mating days 7-14 in LD, MD and HD groups.
In females there was statistically significantly lower weight on premating day 50 in MD and HD groups; statistically significantly lower weight on
lactation day 21 in MD group; statistically significantly higher weight gain between premating days 50-57 in MD and HD groups; statistically
significantly lower weight gain was noted during the lactation days 0-21 in MD and HD groups which was due to transient changes in the lactation
days 0-4 or 4-7. These changes were either transient and/or without dose response relationship.

Food Consumption
In P Generation, there were no adverse effects on food consumption in both males and females. However, in males there was statistically
significantly lower food intake in MD group during the premating days 43-50. In females there was statistically significantly lower food intake
on premating days 64-71 in MD group and statistically lower food intake in LD, MD and HD groups on lactation days 14-21.
These changes were not dose response related.

Sperm Analysis
In P generation, there were no effects on the weights of testes, tunica albuginea and parenchyma. There were no effects on sperm motility,
epididymal sperm count and testicular sperm count. However, there was slightly lower no. of sperm count in HD group without statistical significance.The lower count in HD group was due to lower counts in animals 83, 84 and 90 (3 of 25 males). This finding was limited to 3 animals and also there
was no statistically significant difference, therefore was not considered to be an adverse effect of test item treatment.

Estrous Cyclicity
In P females, there was no statistically significantly difference in the number of total estrous cycles and normal and abnormal cycles in the test item
treated groups compared to corresponding controls.

Pathology
The following macroscopic/gross findings were randomly or sporadically observed in each generation of animals, but all were within the range
of gross lesions that may be recorded in this strain of rats or were incidental or agonal changes which can happen during the procedure for sacrifice,and hence, were deemed to be not treatment-related.
In P generation, the macroscopic findings in the males were yellow spot on epididymides (in MD), pale focus on kidney (in LD) and rough surface of
spleen in (in LD). In females the macroscopic findings were enlarged or dilated adrenal glands (in LD and HD), dilated GI tract (in LD), enlarged
mesenteric lymph node (in LD), small thyroid and parathyroid, fluid distended uterus and alopecia (in HD). In F1 parent males there were yellow
spot on epididymides (in C, LD, MD), Lung discoloured white (in HD), tumor (in HD). In females there was yellow focus on adrenal glands (in LD),
fluid distended uterus (in C, MD, HD).

Organ Weight
In P generation males, There was statistically significantly lower absolute spleen weight in HD group (-9.7 %). There was also statistically significantly
lower relative (to brain weight) spleen weight in in MD and HD groups (approx. -10 %). In the absence of adverse organ weight changes in the
subsequent F1 generation, this finding was not considered to be an adverse effect of test item treatment. No adverse effect was also considered
on the basis of 90 days repeated dose toxicity study (BSL no. 142118) with the test substance [7], wherein there were no histopathological changes
in spleen and other organs of males and females at the end of the treatment period.
In P generation females, There was statistically significantly lower absolute and relative liver weight in LD, MD and/ or HD groups
(ranging from -6 % to -10 %). There was no dose response relationship. The finding was not considered to be an adverse effect of test item treatment.
There was also lower absolute and relative (to body and brain weight) thyroid weight in HD group (ranging from -12.9 % to -15.8 % compared to
controls); higher relative (to body and brain weight) uterus weight (10.3 % to 12.2 %) in HD group and higher relative ovary weight (to body weight)
(15.2 %) in MD group compared to corresponding control. In the absence of statistical significance, these findings were not considered to
be an adverse effect.

Histopathology
Under the conditions of this study, the test item produced no histological evidence of toxicological properties in the organs and
tissues examined in this study, especially of male and female reproductive organs of P generation generation animals.
All findings recorded in the examination of male and female reproductive organs (which included histopathology, detailed examination of
ovaries including quantitative analyses and detailed examination of testis and epididymis including testicular sperm staging) were of incidental
character or within the range of normal background lesions which may be recorded in animals of this strain and age, and histologic findings that
could be attributed to treatment with the test item were not observed in any animals examined in this study.
In conclusion, based on pathology evaluation, the test item produced no histomorphological evidence of toxicological properties
in any organs and tissues examined in this study, and the histomorphological no-observed effect level (NOEL) and the no-observed adverse
effect level (NOAEL) regarding male and female reproductive organs were established at 1000 mg/kg bw/day in both sexes under the condition
of this study.

Dose Formulation Analysis
The analytical method was validated successfully according to guideline SANCO/3029/99 rev. 4 (11/07/2000) in the analytical phase
“Dose Formulation Analysis of Total Phosphorus“ of BSL study “28-Day Repeated Dose Oral Toxicity Study in Wistar Rats including a 14-Day Recovery Period”. The study was performed under directorship of Dr. Carsten Schleh, BSL, with BSL study code 116478.
CIP Phase ID was 12B05086-01-RARW. For further details see respective analytical phase report of CIP, dated 26 November 2012.
For the analysis of phosphorus in dose formulation, the volume of the samples was determined and the samples were completely digested
with nitric acid. The digested samples were analyzed with ICP-OES.
Samples for procedural recoveries and control samples were carried along with the samples of this study. Procedural recoveries were performed
at 2 concentration levels with 2 replicates at each level. The results of the procedural recovery experiments as well as the results of linearity test
showed that SANCO requirements were fulfilled.
Test substance concentration, stability and homogeneity in dosing formulations were obtained by ICP-OES.
The analytical results obtained for the individual dose groups were consistent with the analysis of the % of nominal of the test item for the
concentration, stability and homogeneity analyses.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation P
Remarks:
Generation: P, F1 and F2 (migrated information)

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation P1
Remarks:
Generation: P, F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects
Histopathological findings:
no effects observed

Details on results (F1)

Mortality
In F1 parental animals there were mortalities in both sexes of HD group.
Male No. 283 (HD group) was euthanized for animal welfare reasons because of the development of subcutaneous spontaneous tumor mass.
Male No. 298 (HD group) died in accident.
Male No. 300 (HD) was found dead. Because of advanced autolysis, the tissues were not collected/ preserved, and therefore, the histopathological
examination for clarifying the cause of death was not performed. Female No. 392 (HD) was found dead. Details are unknown because of cannibalism. Given no test item treatment related microscopic changes in this study, the death of animals 300 and 392 were not considered to be of toxicological
relevance. One male animal (no. 235) of LD group was revealed female after being assigned to the study, and then this animal was euthanized
without further examination. The remainder of animals survived the each scheduled study period.

Clinical Observations
In both males and females of F1 Generation, there were no adverse test item treatment related clinical findings.
In F1 generation, there were signs of abnormal breathing, crust, alopecia, nasal discharge, piloerection, reduced spontaneous activity,
deformed snout, hyperaesthesia, kyphosis and swelling on flank. These findings in P and F1 generations were either transient in appearance
or seen in isolated animals or were without dose response. There were no clinical signs of toxicological relevance observed during
the detailed clinical observations.

Body Weight Development
In F1 Generation, there were no adverse effects of test item treatment on weight development of male and female animals. However, in males there
was statistically lower weight in LD group on premating days 50 and 64. There was a statistically significantly lower weight gain in HD group
between days 29-36. In females, there was statistically significantly lower weight in LD group on day 50 (-4.3%); statistically significantly lower
weight gain between premating days 57 to 64 and higher weight gain between day 64-70 in MD group; statistically significantly lower weight gain
in MD and HD groups during lactation days 0-4. With the progress of the study there were no statistically significant changes between the control
and test item treated groups, therefore the findings were not considered to be an adverse effect of test item treatment.
There was no effect on body weight measured during the day of balanopreputial separation and vaginal opening in male and female animals,
respectively of F1 generation.

Food Consumption
In F1 Generation, there were no adverse effects on food consumption in both males and females.

Litter Data
In F1 generation there were no effects of test item treatment on litter data. In F2 generation, there was statistically significantly higher no. of male
pups in HD group (PND 0, 4, 7, 14, 21). The higher male pups in HD group were compensated by lower number of female pups. In addition,
there was no dose response relationship. Therefore, the finding was not considered to be an adverse effect of test item treatment.

Litter Weight Data
In F1 generation, there was no effect of test item treatment. However, there was statistically significantly higher pup mean weight on PND 4 in
MD group. This finding was not dose response related.
In F2 generation, there was statistically significantly lower pup mean weight on PND 21 in HD group and statistically significantly higher male
litter weight in HD group on PND 0. These findings did not show dose response relationship and in addition were within the historical control
data range. Therefore, the findings were not considered to be an adverse effect of test item treatment.

Precoital Interval and Duration of Gestation
In both P and F1 generation there were no effects on the precoital interval and the duration of gestation.

Pre- and Post-Natal Data
There were no effects of test item treatment on pre and post- natal data in both generations.

Developmental Landmark
In F1 and F2 Pups, the days of developmental landmarks were within the normal range in test item treated groups compared to the control.
There was statistically significantly higher relative anogenital distance in F2 female pups at HD level. The increase in anogenital distance in HD
female pups did not show dose response relationship and were within the historical control data range. Hence, the statistical significance was not
considered to have any toxicological relevance. There were no effect on the days of balanopreputial separation and the days of vaginal opening.

Reproductive Indices
In both generations there were no effect on reproductive indices including implantation index, male and female mating and fertility indices,
pregnancy index, gestation index, live birth index and viability index. However, there were reduced mating, fertility and pregnancy indices in LD
and/or HD groups of male and/or females of F1 generation. In the absence of dose response relation, the findings were not considered adverse.

Pup Survival Data
In F1 generation, there were no effects on pup survival between PND 0-21. In F2 generation, there was no statistically significant effect on the
survival of pups. However, 4 pups of female 389 (HD group) were found dead or missing on PND 1. This was in an isolated female and was
considered incidental.

Pup External Findings
In F1 and F2 pups, there were no test item treatment relevant external findings when compared to the controls. However there were few incidences
which were observed in few isolated pups or litters of test item treated and/ or control groups between the PND 0 and 21. The external findings in
F1 and/or F2 pups observed in test item treated and/ or control groups were hematoma, pale, cold, no sign of suckling, small size, dark snout,
alopecia, spot on ear or below eye, bent tail and missing tail or hind limb phalanges, dry skin, crust, injury, discolored skin, alopecia, stained
pups with intact umbilical cord. Most of these findings in F1 and F2 pups were transient.
The alopecia was observed in pups (F1 and F2) during PND 9 to PND 21.This is generally observed in pups housed in groups.

Pre weaning Neurology
In both generations, there were no effects on neurological behavior of pups.

Sperm Analysis
In F1 generation, there was no effect on the weights of testes, tunica albuginea and parenchyma.
There was no effect on sperm motility, testicular sperm count and epididymal sperm count.
In F1 generations, there were no statistically significant differences on the effect of sperm morphology in HD group compared to
corresponding control group. The examination was not extended to LD and MD groups.

Estrous Cyclicity
In F1 females, there was no statistically significantly difference in the number of total estrous cycles and normal and abnormal
cycles in the test item treated groups compared to corresponding controls.

Pathology
The following macroscopic/gross findings were randomly or sporadically observed in each generation of animals, but all were within the range
of gross lesions that may be recorded in this strain of rats or were incidental or agonal changes which can happen during the procedure for
sacrifice, and hence, were deemed to be not treatment-related.
In F1 pups killed on PND 21, there were no macroscopic findings of toxicological relevance. In F2 Pups did not show the macroscopic findings
of toxicological relevance. However, there were incidences of thymus red spots in both sexes (C, LD, MD and HD groups), dilated prostate (in LD)
and kidney hydronephrosis and dilated ureter in male pup (in LD group).

Organ Weight
In F1 generation males, There was statistically significantly lower absolute and relative (to brain weight) spleen weight in LD (-10.3%) and/ or MD
(approx -10%). In the absence of dose response relationship, the finding was not considered to be an adverse effect of test item treatment.
In F1 generation females, There was statistically significantly lower absolute ovary weight in HD group (-13.5%). There was also lower relative ovary
(to body and brain weight) weight in HD group (-12% to 13%), but without statistical significance. In the absence of histopathological changes,
this finding was not considered to be an adverse effect of test item treatment.
In F1 male and female pups (killed on PND 21), there were no effects on absolute and relative (to body and brain weight) organ weight. However,
there was lower absolute and relative thymus weight (to brain weight) in female LD group (-11%) without statistical significance and this was not
considered to be related to test item treatment.
In F2 male pups (killed on PND 21), there was lower absolute and relative (to brain weight) spleen weight (-15.4 to -18.4%) in HD group and higher
relative thymus (to body weight) weight (12.1%) in HD group. In the absence of statistical significance, the findings were not considered to be an
adverse effect of test item treatment.
In female pups, there was lower absolute and relative (to brain weight) spleen weight (-11.7 to -13.3%) in HD group. in the absence of statistical
significance, the finding was not considered to be an adverse effect of test item treatment. There was statistically significantly higher relative
(to body weight) brain weight in LD (10.3%) and HD (13.9%) groups. This finding did not show dose response relationship and therefore was
not considered to be an adverse effect of test item treatment.

Histopathology
Under the conditions of this study, the test item produced no histological evidence of toxicological properties in the organs
and tissues examined in this study, especially of male and female reproductive organs of parental F1 generation animals.
All findings recorded in the examination of male and female reproductive organs (which included histopathology, detailed examination of ovaries
including quantitative analyses and detailed examination of testis and epididymis including testicular sperm staging) were of incidental character
or within the range of normal background lesions which may be recorded in animals of this strain and age, and histologic findings that could
be attributed to treatment with the test item were not observed in any animals examined in this study.
In conclusion, based on pathology evaluation, the test item produced no histomorphological evidence of toxicological properties
in any organs and tissues examined in this study, and the histomorphological no-observed effect level (NOEL) and the no-observed adverse effect
level (NOAEL) regarding male and female reproductive organs were established at 1000 mg/kg bw/day in both sexes under the condition of
this study.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Details on results (F1)'
Remarks on result:
other: see 'Details on results (F1)'
Remarks:
see 'Details on results (F1)'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, in the present study, dosages of 100, 300 and 1000 mg test substance / kg body weight /day did not reveal adverse effect of test item treatment in P, F1 and F2 generations.
Based on the data generated from this study:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity, reproductive toxicity (mating and fertility) and developmental toxicity
was considered to be 1000 mg/kg/day.
Executive summary:

The aim of this study was to assess the possible health hazards likely to arise from repeated exposure over two generations.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia, the vehicle used in this study. The 4 groups comprised 25 male and 25 female Wistar rats for each P and F1 generations.

The following doses were evaluated:

Control:                        0         mg/kg body weight/day

Low Dose:                    100     mg/kg body weight/day

Medium Dose:              300    mg/kg body weight/day

High Dose:                   1000  mg/kg body weight/day

The test item formulation was prepared freshly on each administration day before the administration procedure. The test item was dissolved in aqua ad iniectabilia.The application volume for all groups was 5 mL/kg body weight and for each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

The parental P animals were treated with the test item formulation or vehicle on 7 days per week for10 weeks premating (males and females), a maximum of 2 weeks mating (males and females), post mating up to termination (males), during pregnancy and lactation up to weaning (females).On PND 21, 1 pup/sex/litter was selected for dosing of F1. These animals were dosed over 10 weeks prior to mating, through mating, gestation, and lactation until weaning of F2.

Mating of P animals was performed after 10 weeks dosing period. On PND 21, 1 pup/sex/litter was selected from F1 generation for mating to produce F2.

Mating of 25 F1 pairs/ group to produce F2 was performed after a 10 weeks dosing period, which started after weaning. Mating was performed in the ratio of 1:1 (male to female) within each group.Maximum duration of cohabitation was 2 weeks.

Body weight and food consumption was measured in the parental animals of both generations. Clinical observations and pathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring.

The litters (F1 and F2) were examined as soon as possible after delivery and on special days for developmental landmarks. Fertility parameters (sperm analysis and estrous cyclicity) of males or females of P and F1 parental animals were evaluated.

At necropsy of P and F1 parental animals and selected weanlings were subjected to detailed macroscopic observations and organs collected, subset of organs weighed and preserved for the histopathological examination.


Summary Results

There was no mortality in the parental generation. In F1 parental animals there were mortalities in both sexes of HD group. Male No. 283 was euthanized for animal welfare reasons because of the development of subcutaneous spontaneous tumor mass; Male No. 298 died in accident; male No. 300 and female No. 392 were found dead. Given no test item treatment related microscopic changes in this study, the deaths were not considered to be due to test item toxicity. The remainder of animals survived the each scheduled study period.

In both males and females of P and F1 Generation, there were no adverse test item treatment related clinical findings.

In both P and F1 Generations, there were no adverse effects of test item treatment on body weight development and food consumption of

male and female animals.

There were no effects on the duration of precoital and gestation days in both P and F1 generations.

There were no effects of test item treatment on pre and post- natal data including number of corpora lutea, number of implantation sites and percent implantation losses (pre and post) in both generations.

There were no adverse effects of test item treatment on litter data including number of pups born, still births and runts on PND 0 and number

of live pups, male and female pups, sex ratio, pup mean weight, total litter weight and male and female litter weights (PND 0 to PND 21) of both generations.

In both generations there were no adverse effect on reproductive indices including implantation index, male and female mating and fertility indices, pregnancy index, gestation index, live birth index and viability index.

In F1 and F2 Pups, the days of developmental landmarks were within the normal range in test item treated groups compared to the control. There were no effect on the days of balanopreputial separation and the days of vaginal opening.

There was statistically significantly higher relative anogenital distance in F2 female pups at HD level, but there was no dose response relationship. Hence, this statistical significance was not considered to have any toxicological relevance.

In F1 generation, there were no effects on pup survival between PND 0-21. In F2 generation, there was no statistically significant effect on the survival of pups. However, 4 pups of female 389 (HD group) were found dead or missing on PND 1. This was in an isolated female and was considered incidental.

In F1 and F2 pups, there were no external findings of toxicological relevance when compared to the controls. However there were few incidences which were observed in few isolated pups or litters of test item treated and/ or control groups between the PND 0 and 21. The external findings in F1 and/or F2 pups observed in test item treated and/ or control groups were hematoma, pale, cold, no sign of suckling, small size, dark snout, alopecia, spot on ear or below eye, bent tail and missing tail or hind limb phalanges, dry skin, crust, injury, discolored skin, alopecia, stained pups with intact umbilical cord. Most of these findings in F1 and F2 pups were transient.

In both generations, there were no effects on neurological behavior of pups.

In both P and F1 generations, there were no effects on the weights of testes, tunica albuginea and parenchyma. There were no adverse effects on sperm motility, epididymal sperm count, testicular sperm count and sperm morphology. There were no effects on the estrous cyclicity of females in the dose groups of both generation compared to corresponding control.

There were macroscopic/gross findings randomly or sporadically observed in each generation of animals, but all were within the range of gross lesions that may be recorded in this strain of rats or were incidental or agonal changes which can happen during the procedure for sacrifice, and hence, were deemed to be not treatment-related.

In P generation males, there was statistically significantly lower absolute spleen weight in HD group (-9.7%); statistically significantly lower relative (to brain weight) spleen weight in in MD and HD groups (approx. -10%). In the absence of adverse organ weight changes in the HD group of subsequent F1 generation, this finding was not considered to be an adverse effect of test item treatment. In P generation females, There was statistically significantly lower absolute and relative liver weight in LD, MD and/ or HD groups (ranging from -6% to -10%). In the absence of dose response relationship, the finding was not considered to be an adverse effect of test item treatment.

In F1 generation males, there was statistically significantly lower absolute and relative (to brain weight) spleen weight in LD (-10.3%) and/ or MD (approx -10%). In the absence of dose response relationship, the finding was not considered to be an adverse effect of test item treatment. In F1 generation females, There was statistically significantly lower absolute ovaries weight in HD group (-13.5%). There was also lower relative ovaries (to body and brain weight) weight in HD group (-12% to 13%), but without statistical significance. In the absence of histopathological changes, this finding was not considered to be an adverse effect of test item treatment.

In male and female pups of F1 and F2 (killed on PND 21), there were no adverse effects on absolute and relative organ weights in test item treated group compared to corresponding control.

Under the conditions of this study, the test item produced no histological evidence of toxicological properties in the organs and tissues examined in this study, especially of male and female reproductive organs of P generation and parental F1 generation animals.

All findings recorded in the examination of male and female reproductive organs (which included histopathology, detailed examination of ovaries including quantitative analyses and detailed examination of testis and epididymis including testicular sperm staging) were of incidental character or within the range of normal background lesions which may be recorded in animals of this strain and age, and histologic findings that could be attributed to treatment with the test item were not observed in any animals examined in this study.

In conclusion, based on pathology evaluation, the test item produced no histomorphological evidence of toxicological properties in any organs and tissues examined in this study, and the histomorphological no-observed effect level (NOEL) and the no-observed adverse effect level (NOAEL) regarding male and female reproductive organs were established at 1000 mg/kg bw/day in both sexes under the condition of this study.

Dose formulation analysis:Test substance concentration, stability and homogeneity in dosing formulations were obtained by ICP-OES.

The analytical results obtained for the individual dose groups were consistent with the analysis of the % of nominal of the test item for theconcentration, stability and homogeneity analyses.


Conclusion

In conclusion, in the present study, dosages of 100, 300 and 1000 mg test substance / kg body weight /day did not reveal adverse effect of test item treatment in P, F1 and F2 generations.

Based on the data generated from this study:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity, reproductive toxicity (mating and fertility) and developmental toxicity was considered to be 1000 mg/kg/day.