Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-21 to 2016-05-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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
Body weight at the start of pairing:
males: 381 - 453 g (mean: 424.42 g, ± 20% = 339.54 – 509.31 g)
females: 196 - 230 g (mean: 211.24 g, ± 20% = 168.99 – 253.48 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. 210716/0631)
- 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 type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102150820) (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (4 animals / cage) in type IV cages.
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
PREPARATION OF THE TEST ITEM FORMULATION
The test item was dissolved in sesame oil.
The vehicle was selected based on the test item’s characteristics, testing guideline and due to use in previously performed toxicity tests.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing and or use of ultra turrax homogeniser for 2-3 minutes.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

CHARACTERISATION OF THE VEHICLE
Name: sesame oil
Manufacturer: Henry lamotte Oils GmbH
Batch No.: 5872001, 5872901
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 30.10.2016
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 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Concentration in vehicle: 0, 2.5, 7.5, 25 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 concentration of the test item in the vehicle was performed at various intervals, using a nonaqueous titration method with potentiometric determination of equivalence point.

For determination of the concentration of test item in dosing formulations, samples of approx. 35 mL in a 50 mL falcon tube were retained from all groups once in the first and last week of the study (in total 8 samples). Sample analysis was performed on the same day within 6 hours after preparation. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Samples for testing of homogeneity were taken from the top, middle and bottom of the HD and LD preparation and analysed on the same day (in total 12 samples). Samples were taken once in the first and last week of the study. All samples were homogenous, as coefficients of variation was below 10%.

All samples of dosing formulations were analysed at Eurofins Munich in accordance with GLP under the reference number 153604.
Details on mating procedure:
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Each animal was assigned a unique identification number.

Prior to the start of the mating a detailed clinical observation outside the home cage was made. No animal showed pathological signs before mating. Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Once mainting had occured, the males and females were separated. 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.
Duration of treatment / exposure:
The female animals were treated with the test item formulation or vehicle on 7 days per week basis between gestation day 5 and gestation day 19.
Frequency of treatment:
Once per day, 7 days per week.
Duration of test:
Exposure duration was 15 days for the females. On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females were subjected to a caesarean section.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg/day (2.5 mg/ml)
Basis:
other: nominal in Sesame oil
Remarks:
Doses / Concentrations:
30 mg/kg/day (7.5 mg/ml)
Basis:
other: nominal in Sesame oil
Remarks:
Doses / Concentrations:
100 mg/kg/day (25 mg/ml)
Basis:
other: nominal in Sesame oil
No. of animals per sex per dose:
In total, the 4 groups comprised of 33 female Wistar rats each in the control, the medium dose (MD) group, the high dose (HD) group and 32 female Wistar rats in the low dose (LD) group.

Initially 156 animals (52 males and 104 females) were included in the study. However, after the terminal sacrifice of first 8 females from each group on respective gestation day 20, it was revealed that number of non pregnant animals from each group (4/8 in control and LD and 3/8 in MD and HD) was increased.

At that time, there were still 17 females from each group supposed to be sacrificed and it was not clear out of those how many females will be non pregnant. In order to achieve 20 pregnant females per group as recommended in OECD 414 guideline, it was decided to include additional 10 females per group on 12 January 2016 before completing the necropsies of all initially allocated 25 females/group to avoid any further delay in the study.

Eleven females (numbers 24, 25, 49, 50, 74, 75, 98, 99, 100, 110 and 120) were not assigned to any dose group, as few females were not mated or apparently mated/pregnant but not detected sperm positive during the vaginal smear evaluation and eventually littered.These animals were therefore excluded from the study without any further observations.


Control animals:
yes, concurrent vehicle
Details on study design:
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.

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.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS
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. None of the mated and distributed female in various groups showed signs of premature delivery prior to the scheduled termination.

Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT AND FOOD CONSUMPTION
All animals were weighed once before initiation of pairing to ensure that the body weights were within + 20% variation. The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20. Food consumption was not measured for males during the entire study or for both male and females during the mating period.

POST-MORTEM EXAMINATION
On gestation day 20, sperm positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using an overdose of sodium pentobarbital injected intraperitoneally (Narcoren®, Merial; lot no.: 207015; expiry date: 31 January 2018 and lot no.: 250055; expiry date: 31 May 2018) at a dosage of approximately 8 mL/kg bw.)

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. Observed macroscopic findings were preserved in 10% neutral buffered formalin.

Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed.

Uteri that appeared 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 at any time after the completion of the mating of the requisite number of females.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination, and the examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
Fetal examinations:
The following fetal examinations were performed:
- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter
- Head examinations: half per litter

FOETAL EVALUATIONS
All foetuses from a particular dam were identified by using 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 examined for soft tissue anomalies by a microdissection technique. The remaining half of each litter was processed by Alizarin red staining. However, skeletal evaluation was performed for 20 litters from each group and remaining 8 from control (5, 8, 11, 13, 15, 16, 19 and 20) 7 from LD (30, 32, 34, 38, 39, 44 and 47), 9 from MD (52, 55, 59, 60, 61, 66, 69, 70 and 72 ) and 7 from HD (78, 82, 86, 89, 90, 92 and 97) were not evaluated.

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.

For interpretation of significant external, visceral and skeletal findings, they were described as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

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 centres, ribs and sterna centres 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 was 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 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 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).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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 and all animals survived until the scheduled necropsy.

CLINICAL OBSERVATIONS
No test item-related clinical signs of toxicological relevance were observed in any of the dose groups when compared to the control group. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice. Low incidences of the clinical signs like alopecia (1/33 in MD and HD), abnormal breathing (1/33 in HD) crust (1/33 in MD) and slight to moderate piloerection (3/33 in MD and HD) was noted in isolated females of the MD and HD dose groups. These clinical signs were considered to be incidental and not related to the treatment with test item.

BODY WEIGHT DEVELOPMENT
The mean body weight increased with the progress of the study in the control and treatment groups. There were no statistically significant and test item related effects of toxicological relevance noted for body weight. Throughout the treatment period, body weights were within the normal range of variation for this strain and marginal decrease in high dose group mean values throughout the study period was attributed to the initial low group mean body weights before initiation of the treatment on gestation day 0 and 5 compared to the control group.

However, Body weight gain was statistically significantly lower in the HD group during gestation day 8-11 when compared to the control group. Although body weight gain was statistically significantly lower during gestation day 8-11 in HD group, it was recovered as the study progressed and overall body weight gain from GD 0-20 was comparable with controls and therefore it was not considered as an adverse effect due to test item administration.

FOOD CONSUMPTION
In correlation to the body weight and body weight gain, food consumption on a few occasions in the HD group and on one occasion in MD group was noted to be lower after gestation day 11 compared to the control group. Statistical significance was achieved over the study period from day 11 -14, 14-17 in HD and day 17-20 in MD and HD when compared with the controls.

As differences in food consumption of the control group were marginal and did not have an impact on the mean body weights in either group and importantly no statistically significant effect was observed in overall food consumption (GD 0-20) in the treatment groups compared with the controls, this transiently reduced food consumption in the MD and HD group was not considered to be adverse.

PRENATAL DATA
Prenatal parameters like group mean terminal body weight, gravid uterus weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, No of female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post- implantation loss remained unaffected in the dose groups when compared to the control group. No dead foetuses were observed in any of the groups. There were no statistically significant differences observed for prenatal data except statistically significantly lower adjusted maternal weight in HD group and significantly higher number of male foetuses in the MD group when compared to the control group.

This reduction in adjusted maternal weight was marginal and attributed to the lower initial group mean body weight on gestation day 0 and as a result there was also lower terminal body weight observed in the HD group which is used for the calculation of adjusted maternal body weight. Therefore, this effect is not considered as adverse. This higher number of male foetuses in the MD group was correlated with significantly higher male litter weight and due to lack of dose dependency, it is not considered to be an adverse effect due to test item administration.

Successful mating resulted in 27/32 pregnancies in the LD group, 29/33 in the MD group and 27/33 in the HD group compared to 28/33 pregnancies in the control group. Slightly low pregnancy rate (no. of pregnancies / no. of females mated or sperm positive x 100) of 81-84 % in the LD and HD group was considered to be a biological variation of animals of this strain and age.

PATHOLOGY
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of any group at necropsy. One each incidence of supernumerary liver lobe (81), extra tissue at median liver lobe (92) and fluid filled uterus, oviduct and cervix (95) and 2 incidences of cleft at lateral liver lobe (92 and 96) in the females of the HD group was not assumed to be test item related and considered to be spontaneous background findings for this strain.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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 for the total number of foetuses, the number of female foetuses, total litter weight and female litter weight. However, statistically significantly higher number of male foetuses and male litter weight were observed in the MD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on the mean number of male foetuses and male litter weight in MD group was not considered to be test item related.

FOETAL EVALUATION
External Examination
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Low incidences of haematoma on various body parts were noted in isolated females of the control group and/or the dose groups without dose dependency. These findings were considered to be spontaneous in nature and not related to 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. 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 controls. As observed findings were 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 were considered to be spontaneous in nature. Litter incidences were statistically insignificant except discoloured dark lateral liver lobe which was observed at higher frequencies in the HD group. There was also focus (few spots) discoloured dark lateral liver lobe observed at significantly lower frequencies in HD group in comparison to the control group. The discoloration of organ is considered likely to reflect the consequence of a functional disorder and thus not strictly developmental anomalies and as an adverse effect of the treatment with the test item.

Craniofacial Examination
Craniofacial examination by razor blade serial sectioning technique revealed few abnormalities in all groups including control. Statistical analysis of litter incidences revealed no statistical significance of any of the findings. Dilated third and lateral ventricles were observed at frequencies comparable to the control group and were considered spontaneous in nature. Retinal fold (L) was noted in one foetus (12) of female no. 82 of the HD group and retinal fold (R) was noted in one foetus (8) of female no. 59 of the MD group. Small pituitary gland was noted in one each foetus of female no. 121 (2) and 135 (4) of MD and HD group, respectively. Furtheremore, small nasal cavity was noted in one foetus of female no. 126 (4) of MD group. With just one single foetus being affected in
these groups, these findings were considered to be 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 generally comparable to or slightly lower or higher in the dose groups when compared to the control group.A significantly higher litter incidence of left 4th sacral vertebral arch with the extra ossification site was seen without dose dependency in the MD group when compared to the control group. There was also statistically significantly higher litter incidence of left 14th rudimentary rib in the HD group observed without dose dependency when compared to the control group and was not considered as an effect of the test item. These alterations are classified as a variation. Therefore, it is believed that change is transient that is unlikely to adversely affect
survival or health of the individual. A statistically significant decrease in litter incidence for incomplete ossification of right parietal bone in the LD and MD group compared to the control group was considered to be an incidental as the frequencies were even less in numbers compared to controls.

A higher litter incidence of unossified 5th sternebra in the HD and incomplete ossification of 6th sternebra in the LD and HD group without achieving statistical significance were observed when compared to the control group. These sternebra findings are considered to be variations and not considered to be adverse effect due to test item administration.

There was no statistical significance and no indication of a test item-related trend in the type and incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

Formulation analysis was performed for nominal concentration on samples of all dose groups and homogeneity samples from LD and HD dose groups collected at various intervals during the study (first and last week of the study).

Concentration Analysis

Concentration analysis of formulation samples was determined at three concentrations, 2.5 mg/mL (LD), 7.5 mg/mL (MD), and 25 mg/mL (HD) in the first and last study weeks.The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 103.1%, 102.6% and 99.3% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

Homogeneity

Homogeneity of formulation samples was determined at two concentrations, 2.5 mg/mL and 25 mg/mL, in the first and last study weeks.The mean recoveries observed for the LD dose group was 108.3% and 98.7% of the nominal value of the LD dose group and 99.0% and 95.0% of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.5% and 0.2% in LD dose group, 0.8% and 3.1% in HD dose group.

All samples were homogenous, as COV was below or equal to 10%.

DEVIATIONS FROM THE STUDY PLAN

There were the following deviations from the study plan:

Concerning the Number and Sex of the Animals

Initially 156 animals (52 males and 104 females) were included in the study. However, afterthe terminal sacrifice of first 8 females from each group on respective gestation day 20, it was revealed that number of non pregnant animals from each group (4/8 in control and LD and 3/8 in MD and HD) was increased. At that time, there were still 17 females from each group supposed to be sacrificed and it was not clear out of those how many females will be non pregnant. In order to achieve 20 pregnant females per group as recommended in OECD 414 guideline, after consultation and in agreement with the study monitor, additional 40 females were included in the study.

The reason for this deviation is that more females were used for pairing than initially planned due to increased number of non-pregnant females.

Concerning Mating

Due to increased number of non pregnant animals in all groups, additional 40 females were mated and assigned to control and/or treated groups in order to obtain approximately 20 pregnant females per group. Males were discarded without any observations. Eleven female numbers (24, 25, 49, 50, 74, 75, 98, 99, 100, 110 and 120) were not assigned as few females were not mated or apparently mated/pregnant but not detected sperm positive during the vaginal smear evaluation and eventually littered and therefore excluded from the study without any further observations.The 4 groups comprised 33 female Wistarrats each in the control, the medium dose (MD) group, the high dose (HD) group and 32 femaleWistarrats in the low dose (LD) group.

The reason for this deviation was the increased number of non-pregnant females.

Concerning Foetal Evaluations

All foetuses from a particular dam were identified by using 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 examined for soft tissue anomalies by a microdissection technique. The remaining half of each litter was processed by Alizarin red staining. However, skeletal evaluation was performed from 20 litters from each group and remaining 8 from control (5, 8, 11, 13, 15, 16, 19 and 20) 7 from LD (30, 32, 34, 38, 39, 44 and 47), 9 from MD (52, 55, 59, 60, 61, 66, 69, 70 and 72 )and 7 from HD (78, 82, 86, 89, 90, 92 and 97) were not evaluated. 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.

The reason for this deviation is that after conclusion of all necropsies, pregnant females observed in each group were 28 in control, 27 in LD, 29 in MD and 27 in HD. As per OECD 414 guideline minimum 20 female per group are required in order to make the study valid from regulatory acceptance point of view. Therefore, 20 litters from each group were evaluated for skeletal findings and remaining 8 from control, 7 from LD, 9 from MD and 7 from HD were not evaluated due to technical and logistic reasons. These deviations did not influence the quality or integrity of the present study.

Applicant's summary and conclusion

Conclusions:
SUMMARY AND CONCLUSION
On the basis of this OECD 414 prenatal developmental toxicity study in pregnant female Wistar rats with Amines, C16-22-alkyl at dose levels of 10, 30 and 100 mg/kg bw/day administered on gestation days 5 to 19, the following conclusions can be made:
No mortality, test item-related clinical signs of toxicological relevance, effect on maternal body weight and food consumption was observed in any of the dose groups when compared to the control group.
No test item related effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level tested.
No adverse effects of Amines, C16-22-alkyl on females and fetuses were found at dose levels up to 100 mg/kg body weight/day.

The NOAEL for both maternal toxicity and fetal toxicity of Amines, C16-22-alkyl in this study is considered to be 100 mg/kg bw/day.

COMMENTS ON DOSE SELECTION
The selection of doses for the current OECD 414 study was based on the experiences from previously conducted repeated dose studies with Amines, C16-22-alkyl. In the 28-day oral toxicity test the following doses were used: 0 / 3.5 / 10 / 30 mg/kg bw. In this study it was not possible to detect a NOAEL due to pathological finings in all dose groups, but a general NOEL was 30 mg/kg bw. There is also an OECD 421 study with Amines, C16-22-alkyl available. Doses administrated in this study were 0 / 15 / 60 / 180 mg/kg bw. Based on the findings the NOAEL (parental) was set to 15 mg/kg bw and NOAEL (developmental toxicity) was set to 60 mg/kg bw. As some deaths occurred both in middle and high dose groups early in the OECD 421 study, it was decided not to go too high in the dose selection for the (potentially more sensitive) pregnant females in the OECD 414 study and the selected doses for this study were 0 / 10 / 30 / 100 mg/kg bw.
Executive summary:

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 Amines, C16-22-alkyl via oral administration (gavage) to female rats during gestation days 5 to 19 at dose volume of 4 ml/Kg bw. 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 33 female Wistar rats in each the control, the medium dose (MD) group, the high dose (HD) group and 32 femaleWistarrats in the low dose (LD) group.

The following doses were evaluated:

  • Control: 0 mg/kg bw/day
  • Low Dose: 10 mg/kg bw/day
  • Medium Dose: 30 mg/kg bw/day
  • High Dose: 100 mg/kg bw/day

The test item formulation was prepared freshly on each administration day before the administration procedure. The test item formulation was prepared with sesame oil. 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 sesame oil, the vehicle used in this study.

During the period of administration, the animals were observed each day for signs of toxicity and mortality. The females were sacrificed on their 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) and live and dead foetuses. 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 was processed by Alizarin red staining. However, skeletal evaluation was performed for 20 litters from each group.

Body weight and food consumption were measured on gestation days 0, 5, 8, 11, 14, 17 and 20. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

Summary Results

Maternal Findings

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

No test item-related clinical signs of toxicological relevance were observed in any of the dose groups when compared to the control group.

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control and treatment groups. There were no statistically significant and test item related effects of toxicological relevance noted for body weight. There was marginal decrease in high dose group mean values observed throughout the study period which could be attributed to the initial low group mean body weights before initiation of the treatment on gestation day 0 and 5 compared to the control group. However, body weight gain was statistically significantly lower in the HD group during gestation day 8-11 when compared to the control group. Although body weight gain was statistically significantly lower during gestation day 8-11 in HD group, it was recovered as the study progressed and overall body weight gain from GD 0-20 was comparable with controls and therefore it was not considered as an adverse effect due to test item administration.

In correlation to the body weight and body weight gain, food consumption on a few occasions in the HD group and on one occasion in MD group was noted to be lower after gestation day 11 compared to the control group. Statistical significance was achieved over the study period from day 11 -14, 14-17 in HD and day 17-20 in MD and HD when compared with the controls. As differences in food consumption of the control group were marginal and did not have an impact on the mean body weights in either groupand importantly no statistically significant effect was observed in overall food consumption (GD 0-20) in the treatment groups compared with the controls, this transiently reduced food consumption in the MD and HD group was not considered to be adverse.

Prenatal parameters like group mean terminal body weight, gravid uterus weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, no of female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. No dead foetuses were observed in any of the groups. There were no statistically significant differences observed for prenatal data except statistically significantly lower adjusted maternal weight in HD group and significantly higher number of male foetuses in the MD group when compared to the control group.

No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of any group at necropsy.

Foetal Findings

There were no test item related effects of toxicological relevance for the total number of foetuses, the number of female foetuses, total litter weight and female litter weight. However, statistically significantly higher number of male foetuses and male litter weight were observed in the MD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on the mean number of male foetuses and male litter weight in MD group was not considered to be test item related.

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Low incidences of haematoma on various body parts were noted in isolated females of the control group and/or the dose groups without dose dependency. These findings were considered to be spontaneous in nature and not related to the test item.

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.

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 controls. As observed findings were 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 were considered to be spontaneous in nature. Litter incidences were statistically insignificant except discoloured dark lateral liver lobe which was observed at higher frequencies in the HD group. There was also focus (few spots) discoloured dark lateral liver lobe observed at significantly lower frequencies in HD group in comparison to the control group. The discoloration of organ is considered likely to reflect the consequence of a functional disorder and thus not strictly developmental anomaliesas an adverse effect of the treatment with the test item.

Craniofacial examination by razor blade serial sectioning technique revealed few abnormalities in all groups including control. Statistical analysis of litter incidences revealed no significant effect for any of the findings.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. A significantly higher litter incidence of left 4th sacral vertebral arch with extra ossification site was seen without dose dependency in the MD group when compared to the control group. There was also statistically significantly higher litter incidence of left 14th rudimentary rib in the HD group observed without dose dependency when compared to the control group and was not considered as an effect of the test item. These alterations are classified as a variation. Therefore, it is believed that change is transient that is unlikely to adversely affect survival or health of the individual. A statistically significant decrease in litter incidence for incomplete ossification of right parietal bone in the LD and MD group compared to the control group was considered to be an incidental as the frequencies were even less in numbers compared to controls. There was no statistical significance and no indication of a test item-related trend in the type and incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.

Conclusion

On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with Amines, C16-22-alkyl at dose levels of 10, 30 and 100 mg/kg bw/day administered on gestation days 5 to 19, the following conclusions can be made:

  • No mortality, test item-related clinical signs of toxicological relevance, no macroscopic abnormalities or effect on maternal body weight and food consumption was observed in any of the dose groups when compared to the control group.
  • No test item realted effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level tested.
  • No adverse effects of Amines, C16-22-alkyl on females and fetuses were found at dose levels up to 100 mg/kg body weight/day.
  • The NOAEL for both maternal toxicity and fetal toxicity of Amines, C16-22-alkyl in this study is considered to be 100 mg/kg bw/day.