222 OP

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Available studies:

1. OECD 416

- NOAEL (OECD 416, systemic toxicity, reproductive toxicity): 1000 mg/kg bw/d

- NOAEL (OECD 416, developmental toxicity): 1000 mg/kg bw/d (highest dose tested)

2. OECD 421

- NOAEL (OECD 421, P): 1000 mg/kg bw/d

- NOEL (OECD 421, F1): 1000 mg/kg bw/d (highest dose tested)

Short Description of OECD 416 (key study):

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group (0, 100, 300 and 1000 mg/kg bw/d). The 4 groups comprised 25 male and 25 female Wistar rats for each P and F1 generations. 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.

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.

Link to relevant study records

Reference

Reference 1

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source : Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 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).

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

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

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 [%]

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: 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

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.

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)

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)

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

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.

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)'

Reproductive effects observed:

not specified

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System

Species/strain: healthy 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: 284 - 321 g (mean: 300.45 g, ± 20% = 240.36 – 360.54 g)
females: 196 - 209 g (mean: 197.13 g, ± 20% =157.70 - 236.55 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 [7] 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. 0939)
- 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 will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030512)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs
before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
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, respectively.

Experimental Groups and Doses
According to the results of the dose range finding study (BSL project no. 116477) and 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).
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of
pre-mating and 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.
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. The doses were selected on the basis of data
from a Dose Range Finding Study (BSL study no. 116477).
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same
volume 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.

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 pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group.
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. Stability and homogeneity of the test item in
the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating),
5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study
week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the
preparation (at room temperature), from high and low dose formulations (4 samples).
At the end of the treatment period all 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 Chemisches Institut Pforzheim GmbH,
in accordance with GLP. The procedures followed were described in a detailed analytical phase plan that was attached to the study plan per
amendment. CIP GmbH issued an analytical phase report which was integrated in the report of this study.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle. daily 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.

Frequency of treatment:

daily

Details on study schedule:

Arrival of the Test Item: 23 December 2011
Study Initiation Date: 26 July 2012
1st Amendment to Study Plan: 11 December 2012
Experimental Starting Date: 03 August 2012
Experimental Completion Date: 26 September 2012
Completion Date of Delegated Phase (Histopathology): 04 April 2013
Completion Date of Delegated Phase (Formulation Analysis): 06 March 2013
Date of Draft Report (BSL):20 December 2012

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
0, 100, 300, 1000 mg/kg bw

No. of animals per sex per dose:

10 male and 10 female animals per group.

Control animals:

yes

Details on study design:

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.

Positive control:

None.

Parental animals: Observations and examinations:

Clinical Observations

General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects 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.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. 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.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined.

Litter observations:

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 delivery of the dam 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 writing numbers on the back with the help of a permanent marker or by tattooing. 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 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Medistar, lot no: 00212, expiry date: 03/2014
and Serumwerk, lot no: 00711, expiry date: 08/2013). Inadvertently, one female was sacrificed one day in advance.

Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an 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, testes, epididymides, accessory sex
organs (prostate, seminal vesicles with coagulating glands as a whole), 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.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and
implantation sites was recorded for any females sacrificed 24 to 26 days after the end of the pairing period with no evidence of mating and for
any females sacrificed on day 25 post-coitum due to non-delivery.

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 separately.
 
Histopathology
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs
showing gross lesions were examined in Control and HD animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of
additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court,
Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking,
embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.
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):

Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.
The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities,
including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight
changes, effects on mortality and any other toxic effects.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to
the next. Mean body weights and food consumption were also presented as figures. The relative organ weights were calculated in relation to the
body weight (measured at necropsy) and are presented as percentage.
Findings which were categorised into slight, moderate, marked and severe are not related to levels of statistical significance, but related to levels of
biological relevance in consideration of the normal variation of the respective parameter (historical data) and also taking into account the respective values of the control animals of the study.
All results were reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results and histopathological findings were presented in separate phase reports attached to this report.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation
and clinical biochemistry and 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 5.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

Viability Indices [%]

Offspring viability indices:

Viability Indices [%]

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see details on results

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Mortality
No mortalities were recorded at any dose levels.

Clinical Observations
Few clinical symptoms could be recorded in male and female animals of all groups and some could be found increased in HD group.
Attributed to the test item application can be slight piloerection (6/10 male HD animals animals; 1/10 male MD animals; 4/10 female MD animals;
1/10 male LD animals; 1/10 male C animals) and salivation (2/10 male HD animals; 1/10 male MD animals; 1/10 female MD animals).
These two findings, attributed to the test item, do not have a direct toxicological relevance as individual findings but are symptoms of discomfort.

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study in the C and treatment groups.
Body weights of male animals in HD group were slightly decreased during the whole time of application. During pre-mating days 7-14 the body
weight gain of the HD animals was 13.00 g and hence significantly decreased (p<0.05) to C group which exhibited 18.90 g. However,
the attenuated body weight gain was just seen within one week and no other statistical significant changes could be measured.
The overall body weight gain was still in the normal range of variation of this rat strain. Hence, a test item relation is possible but no
toxicological relevance can be mentioned. For female animals, body weight development in treatment groups was comparable to the C group.

Food Consumption
Food consumption was significantly increased for female HD animals during pre-mating days 7-14 (p<0.01).
This increase is not considered to be of toxicological relevance.
No considerable effect of the test item on food consumption was found in any of the other groups or time points of
male animals and in female animals.

Litter Data
No treatment-related effect of the litter data was observed such as the total number of pups born, number of males and females, sex ratio,
live pups on PND 0 and PND 4.
One still birth could be observed in MD group but this is considered to be incidental
The number of total live pubs on PND 4 was slightly below the number at PND0. However, this is not assumed to dead pubs but to a mistake
in the laboratory which led to missing values at PND 4 for animal No. 55. However, since all pubs were alive and normal at PND3 and since all
other animals from the LD group, as well as MD and HD groups, did not show any dead pubs during PND0-4 these missing data does not affect
the validity and scientific outcome of the study.

Litter Weight Data
No considerable change could be observed in litter weight data for treatment groups when compared to C groups.
All mean values were comparable with the C group.

Precoital Interval and Duration of Gestation
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.
The values were comparable between the groups. All pregnancies resulted in normal births.
Successful mating resulted in 10 pregnancies in the control and low dose groups as well as 9 pregnancies in the medium and high dose
group animals. These lower pregnancy rates are considered to be incidental and not test item related.

Pre- and Post-Natal Data
A statistical significant increase in the percentage of pre-implantation loss was observed for the LD group when compared to C group.
55.4 % and 46.0 % could be observed, respectively. In addition, the HD group showed a tendency to an increased percentage of pre-implantations
loss, too. Since this increase is rather slight (although statistical significant in LD group) and since no dose dependency could be found,
this is not assumed to be test item related. Furthermore, a general high percentage of pre-implantation loss could be observed – also in the C group. Hence, this is assumed to be incidental.
The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of post-implantation
loss remained unaffected due to the treatment with test item when compared with the control group.
 
Reproductive Indices
A reduced copulation index could be mentioned for MD and HD group. Respective 90% were evaluated instead of 100%.
This is assumed to be incidental and not test item related.
Fertility index, copulation index, and delivery index were comparable among all groups.


Pathology
Few specific gross pathological changes were recorded for the male and female animals and were not considered to be
treatment-related but incidental. 

Organ Weight
In males and females, there were no considerable differences in the absolute and relative organ weights of the treatment groups
when compared with the control group.

Histopathology
No test item-related histological findings were noted in the male and female reproductive organs. Female reproductive organs showed
similar post-partum histomorphology in the control and high dose group. The number of large ovarian corpora lutea was not essentially
different between control animals and animals treated at 1000 mg/kg/day.
One female treated at 300 mg/kg/day and one female treated at 1000 mg/kg/day were found not to be pregnant at terminal sacrifice,
but this was not considered to be test item-related.
As a conclusion, under the conditions of the present study and based on the limited extent of histopathological evaluation as defined by
the study plan, the NOEL (No Observed Effect Level) for pathology is considered to be 1000 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slight clinical symptoms and a tendency towards an attenuated body weight gain at 1000 mg/kg bw/d

Clinical signs:

no effects observed

Description (incidence and severity):

see details on results

Mortality / viability:

no mortality observed

Description (incidence and severity):

see details on results

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see details on results

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

see details on results

Histopathological findings:

not examined

Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
One female animals of the LD group (No. 55) could not be evaluated at PND4 since it was accidentally sacrificed on PND3. However, up till PND 3, all
pubs were alive and all other female animals of the LD group did not show any pub mortality. Hence, the missing values do not affect the validity
and scientific outcome of the study.

Pup External Findings
No treatment-related gross external findings were observed in any of the treated groups. Few incidences of external findings were observed
which were considered to be spontaneous and not related to the test item.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: no effects

Reproductive effects observed:

not specified

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with the substance in male and female Wistar rats with dose levels
of 100, 300, and 1000 mg/kg body weight / day the following conclusions can be made:
No effects of the substance were found at dose levels of 300 mg/kg body weight. The NOEL of the substance in this study is considered to be 300 mg/kg body weight.
At a dose level of 1000 mg/kg body weight slight clinical symptoms occurred in few more animals than in control animals and a tendency towards
an attenuated body weight gain was observed. However, these effects are not considered to be in the respective toxic range.
Thus, the NOAEL in this study is considered to be 1000 mg/kg body weight.

Executive summary:

Summary

The aim of this study was to assess the possible effects of the substance on male and female fertility and embryofetal development after repeated dose administration in 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 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

received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity.

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 delivery of the dam 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 28 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.

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

Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

The following doses were evaluated:

Control:                       0        mg/kg body weight

Low Dose:                   100     mg/kg body weight

Medium Dose:             300     mg/kg body weight

High Dose:                  1000   mg/kg body weight

 

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injectionem (sterile water) and 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 the most recently taken body weight measurements. The administration volume was 5 mL/kg body weight.

Summary Results

No mortalities were recorded at any dose levels.

Few clinical symptoms could be recorded in male and female animals of all groups and some could be found increased in HD group. Attributed to the test item application can be slight piloerection and salivation.

During pre-mating days 7-14 the body weight gain of the HD animals was 13.00 g and hence significantly decreased (p<0.05) to C group which exhibited 18.90 g.

Food consumption was significantly increased for female HD animals during pre-mating days 7-14 (p<0.01).

No treatment-related effect of the litter data was observed such as the total number of pups born, number of males and females, sex ratio, live pups on PND 0 and PND 4. One still birth could be observed in MD group but this is considered to be incidental

No considerable change could be observed in litter weight data for treatment groups when compared to C groups. 

No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.

A statistical significant increase in the percentage of pre-implantation loss was observed for the LD group when compared to C group. In addition, the HD group showed an increased tendency to an increased percentage of pre-implantations sites, too. Since this increase is rather slight (although statistical significant in LD group) and since no dose dependency could be found, this is not assumed to be test item related or of toxicological relevance.

A reduced copulation index could be mentioned for MD and HD group. Respective 90% were evaluated instead of 100%. This is assumed to be incidental and not test item related.

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

No treatment-related gross external findings were observed in any of the treated groups.

Few specific gross pathological changes were recorded for the male and female animals and were not considered to be treatment-related but incidental.

In males and females, there were no considerable differences in the absolute and relative organ weights of the treatment groups when compared with the control group.

No test item-related histological findings were noted in the male and female reproductive organs.

 

Conclusion

On the basis of this reproduction/ developmental toxicity screening test with the substance in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight / day the following conclusions can be made:

No effects of the substance were found at dose levels of 300 mg/kg body weight. The NOEL of the substance in this study is considered to be 300 mg/kg body weight.

At a dose level of 1000 mg/kg body weight slight clinical symptoms occurred in few more animals than in control animals and a tendency towards an attenuated body weight gain was observed. However, these effects are not considered to be in the respective toxic range. Thus, the NOAEL in this study is considered to be 1000 mg/kg body weight.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restrictions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Additional information

Descriprion of OECD 416:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group (0, 100, 300 and 1000 mg/kg bw/d). The 4 groups comprised 25 male and 25 female Wistar rats for each P and F1 generations. 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.

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.

Description of OECD 421:

The test item was administered daily in graduated doses (100, 300 or 1000 mg/kg bw/d) 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 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 received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. During the period of administration, the animals were observed each day for signs of toxicity. 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 delivery of the dam 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 28 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

 

No mortalities were recorded at any dose levels. Few clinical symptoms could be recorded in male and female animals of all groups and some could be found increased in HD group. Attributed to the test item application can be slight piloerection and salivation.During pre-mating days 7-14 the body weight gain of the HD animals significantly decreased to C group which exhibited. Food consumption was significantly increased for female HD animals during pre-mating days 7-14. No treatment-related effect of the litter data was observed such as the total number of pups born, number of males and females, sex ratio, live pups on PND 0 and PND 4. No considerable change could be observed in litter weight data for treatment groups when compared to C groups.  No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group. No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls. No treatment-related gross external findings were observed in any of the treated groups.In males and females, there were no considerable differences in the absolute and relative organ weights of the treatment groups when compared with the control group. No test item-related histological findings were noted in the male and female reproductive organs.

Thus, the NOAEL for P and F1 in this study is considered to be 1000 mg/kg body weight. There were no treatment related changes observed for reproductive and developmental parameters.

Effects on developmental toxicity

Description of key information

The developmental properties of the test item were studied in a developmental toxicity study in rats. 


  

Four groups of females were dosed with 0, 100, 300 and 1000 mg/kg bw/d from day 5 to day 19 post coitum. All females were sacrificed on day 20 post coitum and the fetuses were removed by Caesarean section. All dams survived. No maternal or developmental effects were observed up to 1000 mg/kg bw/d. There were no clinical signs or change in body weight development or food consumption reported. All prenatal parameters were unaffected. The macroscopic examination of the dams did not reveal gross pathological changes. Fetal parameters like total number of fetuses, mean fetal weight etc. was unaffected. There were no visceral or skeletal findings reported. Based on the reported observations the NOAEL(maternal) and NOAEL(developmental) was considered to exceed 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Species/strain: healthy 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 of the females at the arrival at BSL: approx. 11-12 weeks old
- Age of the males at the start of pairing: approx. 11-12 weeks old
- Body weight at the allocation of the animals to the experimental groups:
males: 302 - 356 g (mean: 328.44 g, ± 20% = 262.75 – 394.13 g)
females: 187 - 236 g (mean: 207.71 g, ± 20% = 166.17 – 249.25 g)
- The animals were derived from a controlled full-barrier maintained breeding system (SPF).

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)
- 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 pre-mating period when females were kept in groups of two animals and
mating period when two females were paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 290114)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua ad injectionem

Details on exposure:

PREPARATION OF THE TEST ITEM FORMULATION

The test item was weighed into a tarred plastic vial on a precision balance.
The test item was dissolved in Aqua ad injectionem.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
The test item formulations were prepared freshly on each administration day before the administration procedure.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

CHARACTERISATION OF THE VEHICLE
- Name: Aqua ad injectionem
- Manufacturer: AlleMan Pharma
- Batch No.: 311651
- Physical State: liquid
- Storage Conditions: at room temperature
- Expiry Date: 10/2016
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

ADMINISTRATION OF DOSES
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage.
The application volume for all groups was 5 mL/kg bw/day.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Concentration in vehicle: 0, 20, 60, 200 mg/ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the measured concentration of the test item in the vehicle was performed at
various intervals. Samples for analysis of the dose formulations of the test item in the vehicle (concentration) were taken in the first and last week of
the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples
were taken in the first and last week of the study. Samples for stability analysis were taken before in life initiation 0 hours after the preparation,
6 hours after the preparation (at room temperature) and another sample 10 days after the preparation (at room temperature) from high and low dose formulations. Each sample was retained twice (sample A, sample B, each of at least 50 mL). All formulation samples were stored at -15 to -35°C and
the A samples were shipped on dry ice after completion of the in-life phase of the toxicity study to:
CIP
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of the 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 (phase study no. 14B07113-01RARW) that was attached to the study plan per amendment. The results are reported in an analytical phase report which is attached to this report.
The B samples were retained at BSL until the completion of the final study report and will be discarded thereafter.

Details on mating procedure:

PREPARATION OF THE ANIMALS
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the
mating a detailed clinical observation outside the home cage was made.
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of
animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of each female was checked to confirm the
pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an
unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other.
Each animal was assigned a unique identification number. After getting 100 sperm positive females, the remaining females and males were discarded without any observations.

Duration of treatment / exposure:

The test item was orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5 to gestation day
(GD) 19.

Frequency of treatment:

Once per day. 7 days per week

Duration of test:

On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females were subjected to a caesarean section.

No. of animals per sex per dose:

Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of
sperm positive vaginal smears (GD 0). The 4 groups comprised 25 female Wistar rats in each the control, the LD group, the MD group and the HD
group, respectively. 156 animals (52 males and 104 females) were included in the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- General clinical observations were made at least once a day, preferably at the same time each day. 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.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the
treatment. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20.
- Males were not weighed in this study except on the day of their arrival.


FOOD CONSUMPTION: Yes
- Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20.
- Food consumption was measured neither for males during the entire study nor for both males and females during the mating period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- On gestation day 20, sperm positive females no. 1-11 (control group), no. 26-36 (LD group), no. 51-62 (MD group) and 76-87 (HD group) were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally (Narcoren®, Merial; lot no.: 236014; expiry date: 30.01.2017) at a dosage of approximately 8 mL/kg bw. The remaining sperm positive females were subjected to a caesarean
section under ketamine/xylazine-anaesthesia (ketamine/xylazine, 2:1, Pharmanovo, lot no: 24863, expiry date: 10/2015 and Serumwerk,
lot no: 01213, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). After removing the uterus, blood from the abdominal aorta was collected in serum separator tubes and the females were exsanguinated. After completion of the experimental phase, serum samples were sent
to the sponsor for analysis. Results of the analysis will be reported separately by the sponsor and will not be part of this study.
- At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological
changes which may have influenced the pregnancy.
- After removing the uterus, pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
- Each gravid uterus with the cervix was weighed. The number of corpora lutea was counted for pregnant animals. The uterine contents were
examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
- Males were sacrificed without any observations any time after the mating.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

FOETAL EVALUATIONS
All foetuses from a particular dam were identified by using strings with numbered plates and were weighed and sexed based on the anogenital
distance. Each foetus was examined for external anomalies.
One half of each litter was processed by Alizarin red staining and examined for skeletal alterations. The remaining litter was examined for soft tissue anomalies by a microdissection technique.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination was performed for internal structure including the eyes,
brain, nasal passage and tongue by razor blade serial sectioning technique.

EXTERNAL EXAMINATION
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for
the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

SKELETAL EXAMINATION
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95% ethanol.
These foetuses were processed using the Alizarin red staining technique. After fixation in 95% ethanol, the foetuses were macerated with a
1% aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025% in 1% aqueous potassium hydroxide) for
1 day. After that the foetuses were again transferred to 1 % KOH. Alizarin stained foetuses were then cleared and dehydrated in a solution containing 2 parts of 70% ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and
tympanic ring (annulus). Similarly, the vertebral centers, ribs and sterna centers were also examined for size, shape and counted for the number of
ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles,
fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each
foetus.

VISCERAL EXAMINATION
Foetuses scheduled for the visceral examination were pinned to a paraffin covered petri dish with the ventral side up under a stereo microscope.
The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of
lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear
fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or
the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was
sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the
presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental
defect.
The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for
size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the
thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of
the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was
taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through
the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.
After the completion of the visceral examination by the microdissection technique, foetuses were transferred to plastic bottles containing
formalin-aceto-alcohol for later craniofacial examination by the razor-blade-serial-section technique.

CRANIOFACIAL EXAMINATION
Before initiating the serial sectioning with a razor blade, foetuses were transferred to the beaker containing tap water for deformalisation. After
deformalisation, a single foetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem.
Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.

Statistics:

A statistical assessment of the results of the body weight, food consumption, prenatal parameters and litter weight data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral,
craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and
interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITIY
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

CLINICAL OBSERVATIONS
There were no clinical signs of toxicological relevance in the dose groups when compared to the control group.
Low incidences of slight clinical signs like alopecia on various body parts, chromodacryorrhea and moving the bedding were noted in isolated females of the dose groups and/or the control group. As these findings were seen irrespective of the groups in isolated animals, they are considered to be incidental.
None of the females showed signs of abortion prior to the scheduled sacrifice.
After littering on study day 15 (8 pups), female no. 59 of the MD group was discarded without further observations, as apparently this animal had
already been pregnant before the first detection of sperm in the vaginal smear.

BODY WEIGHT DEVELOPMENT
The mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group.
There were no test item-related effects of toxicological relevance noted for body weight and body weight gain in the females. Throughout the
treatment period, body weights were within the normal range of variation for this strain.

FOOD CONSUMPTION
In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in the control, the LD, the MD and the HD group.
There were no test item-related effects of toxicological relevance on food consumption during the treatment period.

PRENATAL DATA
Prenatal parameters like group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation
sites, number of live and dead foetuses, number of late resorptions, number of male and female foetuses, sex ratio and postimplantation loss
remained unaffected in the dose groups when compared to the control group. However, the preimplantation loss was marginally lower in the HD
group (10%) and the LD group (9%) when compared to the control group (16%) and the MD group (16%). Due to lack of dose dependency and without achieving statistical significance, this is considered to be incidental and not related to the test item.
Successful mating resulted in 23/25 pregnancies in the LD group, 19/25 in the MD group and 23/25 in the HD group, compared to 24/25
pregnancies in the control group. In the MD group a moderately lower pregnancy rate (number of pregnancies / number of females mated or
sperm positive x 100) was observed compared to the control group (96% in control, 92% in the LD, 76% in the MD and 92% in the HD group).
This is considered to be incidental in nature.

PATHOLOGY
No gross pathological changes were observed during the macroscopic examination of the females of any group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with the test item at dose levels of 100, 300 and 1000 mg/kg body weight/day administered on gestation days 5 to 19, the following conclusions can be made:

Remarks:

No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER DATA

There were no test item-related effects of toxicological relevance noted for the total number of foetuses, number of male and female foetuses,
mean foetus weight, total litter weight and male and female litter weight. Slight differences in the parameters were within the normal range of
variation for this strain.

FOETAL EVALUATION
External examination
There were no external abnormalities considered to be of toxicological relevance noted in any of the dose groups. The statistical analysis showed no statistically significant changes.
One foetus of the HD group was observed with a red snout. As this finding was noted only in one single foetus, it is considered to be incidental in
nature and unrelated to the treatment with the test item.
Visceral examination
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including
control.
Extra tissue at the median hepatic lobe was noted with a statistically significantly lower litter incidence in the LD group compared to the control
group and supernumerary liver lobe was seen at a statistically higher frequency in the MD group compared to the control group.
Without dose-dependency, these findings are considered to be incidental.
The remaining visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls and were statistically insignificant. As observed findings are either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they are considered to be
spontaneous in nature.
Craniofacial examination
Craniofacial examination by a razor blade serial sectioning technique revealed few abnormalities in all groups including controls.
Statistical analysis of the data revealed no significant effect in any of the findings.
Retinal folds and slightly dilated third and lateral ventricles were observed at low frequencies comparable to the control group and are considered spontaneous in nature.
Anophthalmia was noted in one foetus of the HD group. Without achieving statistical significance and with just one single foetus being affected,
this finding is considered incidental and not related to the treatment with the test item.
Skeletal examination
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence comparable to or lower in the dose groups when compared to the control group.
A statistically significantly higher litter incidence of an extra ossification site at the 4th left sacral vertebral arch in the LD group compared to the
control group is considered to be incidental as there was no dose-dependent pattern.
There was no indication of a test item-related trend in the type and incidences of the skeletal findings and they were therefore considered to be
spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.

Remarks:

No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day.

Key result

Developmental effects observed:

no

DOSE FORMULATION ANALYSIS

Formulation analysis was performed on samples of all dose groups collected at various intervals during the study.

Concentration verification of samples of all dose groups was investigated in the first and the last week of the study. The recoveries were 99.8% and 101.5% in the LD group, 97.3% and 99.9% in the MD group and 97.2 and 98.7% in the HD group, respectively.

Stability of formulation samples was investigated for concentration of the LD and the HD group before in life initiation. After 6 hours storage at room temperature, recoveries were 96.3% for the HD group and 98.4% for the LD group. After 10 days storage at room temperature, recoveries were 95.1% for the HD group and 99.0% for the LD group.

Homogeneity of formulation samples was determined in the first and the last week of the study for the LD and the HD group. Recoveries for the different sampling locations (top, middle, bottom) were between 98.8% and 101.8% in the LD group and between 95.7% and 98.2% in the HD group. DEVIATIONS FROM THE STUDY PLAN There were the following deviations from the study plan:

Before:

A statistical assessment of the results of the body weight, food consumption will be performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters will be analysed using a Chi-square test.The statistics will be performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

New:

A statistical assessment of the results of the body weight, food consumption, prenatal parameters and litter weight data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation.The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reason:

It was decided to use Fisher’s Exact test instead of Chi-square test in order to calculate an exact p-value for more meaningful evaluation.

Concerning:

Foetal Evaluations,study plan, p. 15

Before:

All foetuses from a particular dam will be identified by using different colour strings and will be weighed and sexed based on the anogenital distance. Each foetus will be examined for external anomalies.

New:

All foetuses from a particular dam were identified by using strings with numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies.

Reason:

New foetal identification method with actual number written on plate has been introduced for quick identification of the foetuses.

These deviations did not influence the quality or integrity of the present study.

Conclusions:

On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with the test item at dose levels of 100, 300 and
1000 mg/kg body weight/day administered on gestation days 5 to 19, the following conclusions can be made:
No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study,
1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.

Executive summary:

This study was performed in order to be compliant with Chinese regulations for notification. The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of the test item oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised 25 female Wistar rats in each the control, the LD group, the MD group and the HD group, respectively.

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 day of administration. The test item was dissolved in Aqua ad injectionem and administered daily during gestation days 5 to 19 to the female animals. Dose volumes were adjusted individually based on the most recently measured body weight.

Animals of the control group were handled identically as the dose groups, but received Aqua ad injectionem, the vehicle used in this study.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. All female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead foetuses.

The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

Foetuses were identified by strings with numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities.

Body weight and food consumption were measured on gestation days 0, 5, 8, 11, 14, 17 and 20.

Summary Results

Maternal Findings

No mortality occurred in the control group or any of the dose groups during the treatment period of this study.

There were no clinical signs of toxicological relevance in the dose groups when compared to the control group. None of the females showed signs of abortion prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the low dose (LD), the medium dose (MD) and the high dose (HD) group. There were no test item-related effects of toxicological relevance noted for body weight and body weight gain in the females.

In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in the control, the LD, the MD and the HD group. There were no test item-related effects of toxicological relevance on food consumption during the treatment period.

Prenatal parameters like mean gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, number of live and dead foetuses, number of late resorptions, number of male and female foetuses, sex ratio and post- and pre-implantation loss remained unaffected in the dose groups when compared to the control group.

No gross pathological changes were observed during the macroscopic examination of the females of any group.

Foetal Findings

There were no test item-related effects of toxicological relevance noted for the total number of foetuses, number of male and female foetuses, mean foetus weight, total litter weight and male and female litter weight.

There were no external abnormalities considered to be of toxicological relevance noted in any of the dose groups. The statistical analysis showed no statistically significant changes.

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control. As observed findings are either minor variations and/or due to lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they are considered to be spontaneous in nature.

Craniofacial examination by a razor blade serial sectioning technique revealed few findings in all groups including control. Statistical analysis of the data revealed no significant effect in any of these findings.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence comparable to

or lower in dose groups when compared to the control group.There was no indication of a test item-related trend in the type and incidences of the skeletal findings and they were therefore considered to be spontaneous in nature.

Dose Formulation Analysis

Concentration verification of samples of all dose groups was investigated in the first and the last week of the study. The recoveries were 99.8% and 101.5% in the LD group, 97.3% and 99.9% in the MD group and 97.2 and 98.7% in the HD group, respectively.

Stability of formulation samples was investigated for concentration of the LD and the HD group before in life initiation. After 6 hours storage at room temperature, recoveries were 96.3% for the HD group and 98.4% for the LD group. After 10 days storage at room temperature, recoveries were 95.1% for the HD group and 99.0% for the LD group.

Homogeneity of formulation samples was determined in the first and the last week of the study for the LD and the HD group. Recoveries for the different sampling locations (top, middle and bottom) were between 98.8% and 101.8% in the LD group and between 95.7% and 98.2% in the HD group.

Conclusion

On the basis of this prenatal developmentaltoxicity study in pregnant female Wistar rats with the test item at dose levels of 100, 300 and 1000 mg/kg body weight/day administered on gestation days 5 to 19, the following conclusions can be made:

No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restriction

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

n.a.

Additional information

Effects on development:

- NOAEL (OECD 414, maternal): 1000 mg/kg bw/d

- NOEL (OECD 414, developmental): 1000 mg/kg bw/d (highest dose tested)

This study was performed in order to be compliant with Chinese regulations for notification.The developmental properties of the test item were studied in a developmental toxicity study in rats. Four groups of females were dosed with 0, 100, 300 and 1000 mg/kg bw/d from day 5 to day 19 post coitum. All females were sacrificed on day 20 post coitum and the fetuses were removed by Caesarean section. All dams survived. No maternal or developmental effects were observed up to 1000 mg/kg bw/d. There were no clinical signs or change in body weight development or food consumption reported. All prenatal parameters were unaffected. The macroscopic examination of the dams did not reveal gross pathological changes. Fetal parameters like total number of fetuses, mean fetal weight etc. was unaffected. There were no visceral or skeletal findings reported. Based on the reported observations the NOAEL(maternal) and NOAEL(developmental) was considered to exceed 1000 mg/kg bw/d.

Toxicity to reproduction: other studies

Description of key information

no further studies required

Additional information

n.a.

Mode of Action Analysis / Human Relevance Framework

No adverse reprotoxic or develpomental mode of action identified.

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive or developmental toxicity is appropriate.With reference to the OECD 416, OECD 421 and 414 studies performed with the test item and the lack of reproductive/developmental effects, it is concluded that the substance is not subject to classification and labeling according to Regulation 1272/2008/EC regarding reproductive toxicity.