Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September, 2017 - 18 May, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: - EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Version / remarks:
July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Expiry date May 16, 2018

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
Species / Strain: Rat, Hsd.Han: of Wistar origin
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Rats were shipped in filtered cartons.
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Number of animals
involved in the study: 48 males, 48 females (nulliparous, non-pregnant females)
Number of groups: 4 (3 dose levels + 1 control group)
Number of animals/group 12 animals/sex in the control and dose groups except
Age of animals at start of Male animals: 82 – 92 days
the treatment: Female animals: 87 – 92 days
Body weights at start of 340 – 394 g for male animals
the study: 206 – 244 g for female animals
The weight variation did not exceed  20 per cent of the mean weight
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder (Appendix 19).
Acclimatization time: 27 days

Animal husbandry

Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder (Appendix 19).
Animal room no.: 21/A
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals/ cage
Cage type: Type III polypropylene/polycarbonate;
Size: 22 x 32 x 19 cm (width x length x height)

Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany; see Appendix 22).
The bedding is suitable as nesting material. Details of quality of bedding material were reported.
The cages and bedding were changed twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.

Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.

Food and water supply

Animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum. Food was changed at weekly intervals. Fresh drinking water was given daily.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier
provided an analytical certificate of the standard diet for the batch used. Contents of the standard diet for rats and mice guaranteed by the supplier are
presented in Appendix 20.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months
by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).
The quality control results are available at Toxi-Coop Zrt.’s archives.

Identification of animals

Animals were identified by unique numbers. The individual identification was performed by a marker pen on the tail for transient identification and by ear
punching for permanent animal numbers. Pre-study numbers were given for each animal on the basis of the master file of Toxi-Coop Zrt, which were
replaced with the final identification numbers (i.e. numbers used during the study) after randomization.

Randomization

All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation.
There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean
weight of animals from all test groups was as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and
variability between the groups according to the actual body weight.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
hydrogenated vegetable oil
Remarks:
sunflower oil
Details on exposure:
The test item was administered orally via gavage, once daily at 0 (vehicle only), 50, 100 and 300 mg/kg body weight/day (mg/kg bw/day) doses to four groups of Hsd.Han: of Wistar rats
The route of application was selected in compliance with international guidelines.
The oral route is the anticipated route of human exposure to the test item.

Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide was formulated in the vehicle (sunflower oil) in concentrations of 10, 20 and 100 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored in a
refrigerator (at 5±3 oC) until the administration.

Details on mating procedure:
Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage.
Females remained with the same male until copulation occurred.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. Mating pairs were clearly identified in the raw data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study.
Five aliquots of 5 mL of each formulation (10, 20 and 60 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken and analyzed. The samples
were stored at 5 ± 3 °C before the analysis.

Date of sampling: October 11, 2017 and November 27, 2017
Date of analysis: October 12, 2017 and November 28, 2017
Concentration of the test item in the dosing formulations varied between the range of 98 % and 107 % in comparison to the nominal values.
Results and details of analysis are attached to the Report.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of
the test item from the vehicle was within the acceptance criteria (106 % and 97% of the nominal concentrations of ~1 mg/mL and ~200 mg/mL, respectively).
Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide proved to be stable in sunflower oil at the intended concentrations at
room temperature for four hours and at 5 ± 3oC for three days.
A separate analytical report (Study no. 805-100-2235) provided these data.
Duration of treatment / exposure:
The experimental period involved 27 days of acclimatization (including 14 days for examination of estrous cycle) and 47-57 days treatment/observation period
(depending on the effectiveness of mating) and necropsy days. One male animal at 50 mg/kg bw/day was administered for 18 days and subjected to necropsy on Day 18 due to its early death.
The day of first treatment is considered as day 0 of examination.
Frequency of treatment:
once daily
Details on study schedule:
Study design

a) Males
A PM M ↓
Acclimatization period Pre-mating period Mating period Post mating period FOB
27 days 14 days 1-8 days 25-32 days Blood sampling
Necropsy
Organ weighing†††
on Day 47

b) Females
A PM M G ↓ PP/PN ↓
Acclimatization period Pre-mating period Mating period Gestation period Delivery Lactation period FOB
27 days† 14 days 1-8 days 21-24 days 3 – 17 days Blood sampling
Necropsy
Organ weighing
57†††
Necropsy:
Dams: L14-L18

Remarks:
† Pre-treatment examination of estrous cycle included
†† = For non-pregnant females necropsy was conducted on Day 47
†††= For animals selected for toxicity examinations

Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (vehicle only)
Dose / conc.:
50 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
48 males, 48 females (nulliparous, non-pregnant females)
Number of groups: 4 (3 dose levels + 1 control group)
Number of animals/group 12 animals/sex in the control and dose groups except
Control animals:
yes, concurrent vehicle
Details on study design:
Dosing of both sexes begun after 27 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and
including the day before the necropsy. Rats of this strain reach full sexual maturity at the age of 10 weeks. The mating phase started after 14-days
treatment (pre-mating) period.
The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours). Control animals were treated
concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Male animals were dosed for 47 days (14 days pre-mating and 1-8 days mating, plus 25 – 32 days of post-mating period; until the necessary number of
pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1 – 8 days mating period and throughout pregnancy and at least up to and including day 13
post-partum or the day before sacrifice (altogether for 51-58 days depending on mating day). The day of birth (viz. when parturition is complete) was
defined as day 0 post-partum. Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
All animals were subjected to a full detailed gross necropsy. The brain, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole of all male animals were weighed.
Five male and five female animals were randomly selected from each group for individual observations in a functional observation battery (FOB) according to SOP ATK 001, clinical pathology examinations, organ weighing and for a full histopathological investigation. Five dams and males these females cohabited
with from each groups were involved in these examinations.
F1 offspring were observed for clinical signs, litter weight, body weight, anogenital distance and nipple retention. Blood samples for serum T4 assessment were pooled from at least two pups per litter on postnatal day 4 and 13, where it was feasible. Remaining pups were euthanized on postnatal day 13 or shortly thereafter.

Mating procedure

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage.
Females remained with the same male until copulation occurred.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as
evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. Mating pairs were clearly identified in the raw data.

Examinations

Parental animals: Observations and examinations:
Mortality

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

Clinical observations

General clinical observations were made on parental animals once a day, after the administration at approximately the same time.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern,
occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and
response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group on Day 43. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and
Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

Body weight

All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after
parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment
volumes, but these data were not valuated statistically. Body weight was measured on day of necropsy for female animals subjected to organ weighing
(selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

Food consumption measurement

The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating
phase (pre-mating on Days 7, 13, and post-mating on Days 20, 27, 34, 41 and 46 for male animals), pre-mating on Days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals.

Examination of placental sign

All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the
examination was repeated on day 14 of gestation.

Observation of the delivery process

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestation day 21 onwards. All
observations and any evidence of abnormal deliveries were considered. The duration of gestation was recorded and was calculated from day 0 of
pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the
presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection.
Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Clinical pathology

Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous
plexus under Isofluran anesthesia.
Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for
clinical chemistry.
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4).

Hematology

Blood samples for hematology measurements were collected in tubes containing K3EDTA (spray-dried; MiniCollect® 0.25 mL, manufactured by Greiner
Bio-One International AG, Kremsmünster, Austria) and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 oC until
analysis by SYSMEX XT-2000iV.
The following parameters were measured in all selected animals and in recovery animals by SYSMEX XT-2000iV.

Blood coagulation

Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.8 % (MiniCollect® 1 mL;
manufactured by Greiner Bio-One International AG, Kremsmünster, Austria). Tubes were filled up to the final volume (marked on the tubes).
Blood were centrifuged at 2500 rpm for 15 minutes within 20 – 30 minutes after the sampling. Supernatant plasma samples were stored at 2-8°C and
measured.
The following blood coagulation parameters were determined in selected animals and in recovery animals by AMAX Destiny Plus.
Clinical chemistry

Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 mL Z Serum Sep C/A (no anticoagulant; manufactured by
Greiner Bio-One International AG, Kremsmünster, Austria). At least 1.0 mL blood was collected into clinical chemistry tubes. Samples were stored in a dark
place at room temperature for 30-40 minutes and then centrifuged at 4500 rpm for 15 minutes. Serum samples were stored at 2-8°C and measured.
The following parameters were measured in all selected animals and in recovery animals by Konelab 60i:

Determination of serum thyroid hormone (T4)

Blood samples for determination of serum levels of thyroid hormones (T4) were drawn as described at 4.4.9.3 in the morning hours (approximately between 8h and 10:30h a.m.).

Blood samples were collected from animals as follows:
 from at least pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter)
 from all dams and at least two pups per litter on post-partal/postnatal day 13, if it was feasible
 from all parent male animals at termination on Day 47
 from non-pregnant female animals on study Day 47

All samples for T4 determination were stored at -25 to -15°C until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (T4) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.

Necropsy

Gross necropsy was performed on each animal.
One male animal (no. 208) at 50 mg/kg bw/day died and was subjected to necropsy immediately after the death on Day 18.
All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”) and were subjected to gross necropsy as follows:
 Parental male animals: after the optionally extended post-mating period on Day 47.
 Dams on post-partum days 14 -18 (on Days 51-58)
 Non-pregnant females on Day 47
 Offspring: on postnatal day 13 or shortly thereafter.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was
observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the
reproductive system. The numbers of corpora lutea and implantation sites were recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, adrenal glands and
pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
The following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for dead animal and for five male and five female animals randomly selected from each group:

Organ weight

At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a
whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated
and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
Paired organs were weighed together.

Histopathology

Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination was also performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in non-pregnant females and males these females cohabited with in the low and middle dose groups.
Full histopathology examinations were performed on the preserved organs and tissues of the dead animal and of randomly selected animals in the control and high dose (300 mg/kg bw/day).

In addition, uterus in single female animals at 50 and 100 mg/kg bw/day, and the thymus in two male animals at 100 mg/kg bw/day were also processed and evaluated histologically on the basis of macroscopic observations at the necropsy.
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks.
Estrous cycle was evaluated and considered at randomization.

Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and
during the mating period until evidence of mating.
Vaginal smears were also prepared on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.
Sperm parameters (parental animals):
Detailed histological examination was performed on the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births,
runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0) and on day 13 post-partum
with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth
(dead pups).
All litters were checked and recorded daily for the number of viable and dead pups. Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore,
individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 and the litter weights were calculated for evaluation on
postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.
Postmortem examinations (parental animals):
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
Postmortem examinations (offspring):
There was no test item related effect on offspring’s extra uterine mortality.
Statistics:
The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Body weight and food consumption data of non-pregnant female animals were determined and recorded according to the study plan but these were not reported.
Reproductive indices:
Formulas for calculation of mating and fertility indices

Copulatory index: Measure of animals’ ability to mate
Males : (Number of males with confirmed mating/Total number of males cohabited)x100

Females: (Number of sperm positive females/Total number of females cohabited)x100

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males: (Number of males impregnating a female/ Total number of males with confirmed mating)x100
Females:( Number of pregnant females/ Number of sperm positive females)x100

Gestation index: Measure of pregnancy that provides at least one live pup

(Number of females with live born pups/Number of pregnant females)x100
Offspring viability indices:
Formulas for calculation of pup mortality and sex ratio indices

Intrauterine mortality
Pre-implantation mortality: (number of corpora lutea-number of implantations/ number of corpora lutea )x100


Post-implantation mortality:(number of implantations- number of liveborns/number of implantations)x100


Total intrauterine mortality: (number of corpora lutea-number of liveborns/ number of corpora lutea)x100

Post-natal mortality: (Number of liveborns-number of live pups on PN13/ number of liveborns)x100


Survival Index : (number of live pups on postnatal day13/ number of pups born)x100


Sex ratio: (number of pups examind- number of males (females)/ number of pups examined)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations nor at the functional observations.
Nuzzling up the bedding material noted for all male animals and for all female animals at 300 mg/kg bw/day shortly after the administration from Day 3 up to the last treatment day was not considered to be toxicologically relevant due to the short duration and animals showed normal behavior thereafter.
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality at 50, 100 or 300 mg/kg bw/day groups during the course of study (male and female).
One female animal at 50 mg/kg bw/day died on Day 18 due suffocation. Foamy content in the trachea and histopathological findings (alveolar emphysema and edema, congestion in the lungs) refer to para-gastric treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or in female animals at 50, 100 or 300 mg/kg bw/day during the entire treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was not affected at 50, 100 or 300 mg/kg bw/day groups in male animals (during the course of the pre-mating and post mating periods) and in female animals (during the pre-mating, gestation and lactation periods).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 50, 100 and 300 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The bile acid concentration was elevated in female animals at 300 mg/kg bw/day with respect to their control indicative some disturbances in the enterohepatic circulation or the hepatobiliary system.
The serum thyroid hormone (free T4) level was comparable to their control in parental male animals and in PN 13 day offspring at 50,100 and 300 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The behavior and physical condition of the animals was not impaired at each dose level (50, 100 or 300 mg/kg bw/day) during the entire treatment period.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 300 mg/kg bw/day.
There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 300 mg/kg bw/day groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not found at any dose level (50, 100 and 300 mg/kg bw/day).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive ability were comparable in the control and in 50, 100 and 300 mg/kg bw/day groups (male and female).
There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (50, 100 and 300 mg/kg bw/day).

Details on results (P0)

Mortality

There was no test item related mortality at 50, 100 or 300 mg/kg bw/day groups during the course of study (male and female).
One male animal (no.208) at 50 mg/kg bw/day was found dead on Day 18. There were no preceding clinical signs. Necropsy observation revealed foamy
content in the trachea. Histologically alveolar emphysema, congestion and alveolar edema was observed in the lungs in connection with a probably
suffocation caused by mis-gavage. No toxic lesions (degeneration, inflammation, or necrosis) were detectable in the other organs (liver, kidney etc.).

Clinical observations

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical
observations.
There were no clinical signs in any group, i.e. the animals (male and female) exhibited normal behavior and physical condition with no abnormalities at 50 and 100 mg/kg bw/day.
Nuzzling up the bedding material was noted for all male animals (12/12) and for all female animals at 300 mg/kg bw/day (12/12; pre-mating, mating, post-mating, gestation or lactation periods, respectively) shortly after the administration from Day 3 up to the last treatment day. The duration of nuzzling up the bedding
material was short and animals showed normal behavior thereafter.
Alopecia on the skin of the abdomen and neck (1/12) and on the forelimbs (1/12) was observed in two control male animals from Day 13 and 20, respectively, up to the end of the study. Similarly, this observation was also detected on these animals during the weekly detailed clinical observations from Day 13 and 20.
Alopecia is a common finding in experimental rats of this strain with similar age and was present only in control animals, therefore has no toxicological
meaning.

Functional observations

Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, 50, 100 or 300 mg/kg bw/day groups, on Day 43).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item
treated groups in the examined parameters during the course of the functional observations.
Alopecia on the forelimbs was also noted for male control animal (1/5) at the functional observations.


Body weight and body weight gain

The body weight development was not adversely affected by the test item in male or in female animals at 50, 100 or 300 mg/kg bw/day during the entire
treatment period.
Statistical significance with respect to the control was detected in male animals at 300 mg/kg bw/day at the slightly lower mean body weight gain between
Days 0 and 7. This slight reduction in the body weight gain did not result in significant change in the mean body weight or in the summarized body weight gain
of high dose treated male animals therefore was not considered to be toxicologically relevant.
There were no significant differences between the control and test item treated female animals in the mean body weight or in the mean body weight gain
during the entire treatment period: pre-mating, mating and lactation periods.

Food consumption

Compared to the control animals, toxicologically significant differences were not detected in the mean daily food consumption at 50, 100 or 300 mg/kg bw/day in male animals (during the course of the pre-mating and post mating periods) or in female animals (during the pre-mating, gestation and lactation periods).
The mean daily food consumption was similar to that of the control in male animals at 50 mg/kg bw/day during the entire treatment period and in female animals at 50 and 100 mg/kg bw/day during the pre-mating, gestation and lactation periods.
The mean food consumption of male animals at 100 mg/kg bw/day slightly exceeded the control between Days 34 and 41 and between Days 41 and 46.
In compliance with the slight body weight changes, statistically significant difference with respect to the control was noted for the slightly lower mean daily
food consumption in male and female animals at 300 mg/kg bw/day between Days 0 and 7.
These slight changes in the mean daily food consumption however were judged to be toxicologically not relevant due to the minor degree and transient
occurrence.

Estrous cycle

A test item influence on the estrous cycle was not detected at any dose level (50, 100 or 300 mg/kg bw/day).
Statistically significant difference with respect to the control was detected at the lower mean number of female animals with regular cycle at
50 mg/kg bw/day during the pre-treatment period. This difference was considered to be indicative of the biological variation.
There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular cycles, in the
mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-mating period.

Delivery data of dams

The evaluated delivery parameters of dams were not affected at 50, 100 or 300 mg/kg bw/day.
The number and percentage of pregnant females and dams delivered, the mean number of implantation, the number of post-implantation loss, the duration of
pregnancy, number of dams with prolonged pregnancy and pups births (total, live, viable, still birth) and live birth index were comparable in all groups
(50, 100 and 300 mg/kg bw/day).

Reproductive ability

The examined parameters of reproductive ability were comparable in the control and in 50, 100 and 300 mg/kg bw/day groups (male and female).
The number and percentage of fertile or infertile male animals, the copulatory and fertility indices were identical in all male groups.
The percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s), the copulatory, fertility and gestation indices
were comparable in female animals of control and test item treated groups.
Statistical difference with respect to the control was noted at the slightly higher mean number of days in the pre-coital interval and at the higher mean
number of conceiving days at 100 mg/kg bw/day. These findings were considered to be toxicologically not relevant because of the low degree and similar
changes was not observed at the high dose.

Hematology and blood coagulation

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 50, 100 or 300 mg/kg bw/day.
At 50 mg/kg bw/day, the examined hematological and blood coagulation parameters were comparable with their control in male and in female animals.
At 100 mg/kg bw/day, statistically significant differences with respect to the control were observed at the slightly shorter mean prothrombin time (PT) in
male animals and at the lower mean percentage of monocytes (MONO) and lower mean corpuscular hemoglobin concentration (MCHC) in female animals.
At 300 mg/kg bw/day, the mean platelet count (PLT) and mean activated partial thromboplastin time (APTT) were slightly above these of the control group in
male animals. In the female animals at 300 mg/kg bw/day, lower mean percentage of basophil granulocytes (BASO) and lower mean corpuscular hemoglobin concentration were observed.
All these slight changes were considered to have no toxicological relevance as these were with minor degree or there were no accompanying findings in the related parameters (MONO, BASO, MCHC, PLT, PT and APTT). The slight changes in MONO, MCHC or PT were present only in the mid dose treated animals.
Therefore slight differences in hematological parameters were judged to be indicative of biological variation and not related to the test item effect.

Clinical chemistry

There were no adverse changes in the examined clinical chemistry parameters at 50, 100 or 300 mg/kg bw/day (male and female).
The bile acid concentration was slightly elevated in female animals at 300 mg/kg bw/day with respect to their control indicative some disturbances in the
enterohepatic circulation or the hepatobiliary system. However there were no accompanying histopathological findings in the hepatobiliary system of these
animals. Therefore change in bile acid concentration was judged to be toxicologically not relevant.
In the male animals, statistical differences with respect to the control was noted for the lower mean aspartate aminotransferase activity (AST) at 50 and 300 mg/kg bw/day, for the lower mean concentration of potassium (K+) at 100 mg/kg bw/day and for the slightly elevated albumin concentration (ALB) at 300 mg/kg bw/day.
In the female animals, the concentration of bile acid (BAC at 300 mg/kg bw/day) and total protein (TPROT at 100 mg/kg bw/day) was above the control value.
Statistically significant differences with respect to their control in male and female animals at 50, 100 or 300 mg/kg bw/day (AST, K+, ALB and TPROT) were
considered to be toxicologically not relevant as mean values were close to their control ranges and there were not related findings, therefore these were
judged to have no toxicological relevance.

Serum thyroid hormone

The serum thyroid hormone (free T4) level was comparable to their control in parental male animals and in PN 13 day offspring at 50,100 and
300 mg/kg bw/day.

Necropsy

Specific macroscopic alterations indicative of test item effect were not observed in the organs or tissues at any dose levels (50, 100 or 300 mg/kg bw/day)
at the necropsy.
In dead male animal (1/12 at 50 mg/kg bw/day), foamy content was observed in the trachea as macroscopic indicative of cause of the death (suffocation)
Hemorrhage was detected in the thymus in two male animals at 100 mg/kg bw/day.
In dams, hydrometra was seen in the control (1/11) and in the low dose group (1/11). Hydrometra was also observed in one non-pregnant female animal at
100 mg/kg bw/day (1/1). The eye of one control dam (1/11) damaged at the blood sampling
Hemorrhage in the thymus was considered to be the consequence of circulatory disturbance developed during exsanguination procedure.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these were judged to be toxicologically not relevant.

Organ weight

There were no test item related adverse effects in the weights of examined organs in male or female animals at 50, 100 or 300 mg/kg bw/day.
The liver weights (absolute and relative to body or brain weights) exceeded their control in male animals at 50, 100 or 300 mg/kg bw/day and in female animals at 300 mg/kg bw/day. There were no related hepatic findings at the histological examinations therefore liver weight changes were considered to be
adaptive response to the altered demands.
Statistical significance with respect to the control was detected at the higher mean liver weights (absolute and relative to body and brain weights) in the male animals at 50, 100 and 300 mg/kg bw/day. In male animals, the mean kidney weight relative to brain weight at 100 mg/kg bw/day and mean weight of adrenal glands relative to body weight at 300 mg/kg bw/day also exceeded their control.
In the female animals, statistical difference with respect to their control was observed at the lower mean brain weight relative to body weight at
50 mg/kg bw/day and at the slightly higher mean liver weights (absolute and relative to body and brain weights) at 300 mg/kg bw/day.
The slight differences in weights of some organs (kidneys and brain) with respect to their control were judged to be toxicologically not relevant but indicative of individual variations. These findings were observed in the lower dose treated animals and there were no histopathological changes to support their
relevance.

Histopathology

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 300 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (300 mg/kg bw/day).
In dead male animal at 50 mg/kg bw/day, alveolar emphysema, congestion and alveolar edema was detected in the lungs in moderate degree in connection
with a probably suffocation as the probable cause of death. No toxic lesions (degeneration, inflammation, or necrosis) were detected in the other organs at the histological investigation.
In the male animals belonging to the test item treated and control groups (12/12 control; 2/2 at 50 mg/kg bw/day; 1/1 at 100 mg/kg bw/day; 12/12 at 300 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal
and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (dams: 11/11 control and 11/11 at 300 mg/kg bw/day; non-pregnant females: 1/1 control,
1/1 at 50 mg/kg bw/day, 1/1 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic
of the species, age and phase of the active sexual cycle in all cases of control and treated groups.

The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma was normal in all cases as well.
Dilatation of uterine horns observed in three female animals (1/11 control dam; 1/1 dam at 50 mg/kg bw/day, 1/1 non-pregnant female at 100 mg/kg bw/day)
is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance
as there were no inflammatory or other pathological lesions.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in control group; 1/5 male and
1/5 female at 300 mg/kg bw/day) occurred sporadically and these were considered as a consequence of hypoxia, dyspnea and circulatory disturbance
developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in control (1/5 male and 1/5 female) and 300 mg/kg bw/day (1/5 male and 1/5
female) groups. Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Acute hemorrhage in the thymus of two male animals at 100 mg/kg bw/day (2/2) was considered to be the consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines,
the liver, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female
reproductive system or the central, or peripheral nervous system in surviving animals. The quantity and quality of hemopoietic cells in the bone marrow,
and the cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for systemic toxicity of male/female rats
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for reproductive performance of male/ female rats

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring showing clinical signs was comparable in the control and test item treated groups.
The percentages of cyanotic and missing (cannibalized) pups were slightly higher than in the control group at 300 mg/kg bw/day. Based on these signs and body weight values, these clinical signs were considered to be indicative of the inadequate nursing by two dams and not related to test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
The percentage and litter mean number of dead offspring were higher (no statistical significance) at 300 mg/kg bw/day with respect to the control group due to the high number of missing (cannibalized) pups in two litters in the high dose groups. Clinical signs observed in offspring of these dams (no. 423, 427) refer to inadequate nursing (no milk in the stomach, cold, paleness and cyanosis). The mean body weight of pups in these litters was comparable with the control at the birth (423, 427) and on postnatal day 4 (427). Therefore test item influence on the high mortality rate of offspring is questionable.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item
treated groups (50, 100 and 300 mg/kg bw/day) on postnatal days 0, 4 and 13.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The anogenital distances (in male or female offspring) or nipple retention (male) were not affected by the test item at 50, 100 and 300 mg/kg bw/day).
The anogenital distance, both absolute and normalized, were consistent with the control in male offspring in all the treated groups.
Statistical significance was observed at the longer mean anogenital distances (absolute and normalized) in female pups at 50 and 300 mg/kg bw/day with
respect to their control. In the lack of dose relevance these slight differences with respect to their control were judged to be indicative of the biological
variation and not related to the test item.
Nipples/areoles were not visible in any of the examined male offspring in the control or 50, 100 or 300 mg/kg bw/day groups on postnatal day 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
The lung flotation test was negative in four pups i.e. these pups died intra uterine (2 at 50 mg/kg bw/day and 2 at 300 mg/kg bw/day) and there were no
macroscopic changes but one of them at 50 mg/kg bw/day was partially cannibalized.
One pup found dead at 300 mg/kg bw/day was partially cannibalized on postnatal day 0 thus it was not feasible to examine the gender and lung flotation test.
In one pup in control group (1/1), yellowish, foamy mucous content was found in the intestines.

There were no macroscopic findings in two dead pups (2/3) and one was partially cannibalized (1/3) at 50 mg/kg bw/day. The stomach was empty in one of these pups (1/3). In the dead pups at 100 mg/kg bw/day (2/2), no macroscopic changes were detected and the stomach contained milk.Four dead pups were subjected to necropsy at 300 mg/kg bw/day. No macroscopic findings (1/4), autolyzed visceral organs (2/4) or partial cannibalism (1/4) were noted for these pups.
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Mortality

There was no test item related effect on offspring’s extra uterine mortality.
The percentage and litter mean number of dead offspring were higher (no statistical significance) at 300 mg/kg bw/day with respect to the control group
due to the high number of missing (cannibalized) pups in two litters in the high dose groups. Clinical signs observed in offspring of these dams (no. 423, 427) refer to inadequate nursing (no milk in the stomach, cold, paleness and cyanosis). The mean body weight of pups in these litters was comparable with the
control at the birth (423, 427) and on postnatal day 4 (427). Therefore test item influence on the high mortality rate of offspring is questionable.


Sex Distribution

There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.


Clinical observations

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring showing clinical signs was comparable in the control and test item treated groups.
The percentages of cyanotic and missing (cannibalized) pups were slightly higher than in the control group at 300 mg/kg bw/day. Based on these signs
and body weight values, these clinical signs were considered to be indicative of the inadequate nursing by two dams and not related to test item.


Anogenital distance and nipple retention

The anogenital distances (in male or female offspring) or nipple retention (male) were not affected by the test item at 50, 100 and 300 mg/kg bw/day).
The anogenital distance, both absolute and normalized, were consistent with the control in male offspring in all the treated groups.
Statistical significance was observed at the longer mean anogenital distances (absolute and normalized) in female pups at 50 and 300 mg/kg bw/day with
respect to their control. In the lack of dose relevance these slight differences with respect to their control were judged to be indicative of the biological
variation and not related to the test item.
Nipples/areoles were not visible in any of the examined male offspring in the control or 50, 100 or 300 mg/kg bw/day groups on postnatal day 13.

Body weight

A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all
test item treated groups (50, 100 and 300 mg/kg bw/day) on postnatal days 0, 4 and 13.

Necropsy

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
The lung flotation test was negative in four pups i.e. these pups died intra uterine (2 at 50 mg/kg bw/day and 2 at 300 mg/kg bw/day) and there were no
macroscopic changes but one of them at 50 mg/kg bw/day was partially cannibalized.
One pup found dead at 300 mg/kg bw/day was partially cannibalized on postnatal day 0 thus it was not feasible to examine the gender and lung flotation test.
In one pup in control group (1/1), yellowish, foamy mucous content was found in the intestines.

There were no macroscopic findings in two dead pups (2/3) and one was partially cannibalized (1/3) at 50 mg/kg bw/day. The stomach was empty in one
of these pups (1/3).
In the dead pups at 100 mg/kg bw/day (2/2), no macroscopic changes were detected and the stomach contained milk.
Four dead pups were subjected to necropsy at 300 mg/kg bw/day. No macroscopic findings (1/4), autolyzed visceral organs (2/4) or partial cannibalism (1/4) were noted for these pups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for F1 Offspring

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, Reaction mass of N-butylphthalimide and
n-propylphthalimide and N-sec-butylphthalimide administered at 50, 100 or 300 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats.

The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats:300 mg/kg bw/day

NOAEL for F1 Offspring:300 mg/kg bw/day

Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 100 and 300 mg/kg bw/day compared to control animals according to OECD 422. The guideline is designed for use with the rat, which is the preferred rodent species for toxicity and reproduction toxicity testing.As a screening test, it was intended to provide initial informationon the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects onmale and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide wasadministered orally (by gavage) once daily at 0 (vehicle only),50, 100 and 300 mg/kg body weight/day(mg/kg bw/day) doses to four groups ofHsd.Han: of Wistar ratsconsisting of 12 animals per sex per groupin concentrations of10, 20 and 60 mg/mLcorresponding to a 5 mL/kg bw dosing volume.A group of vehicle (sunflower oil) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide was stable in sunflower

oilin concentrations of ~1 mg/mL and ~200 mg/mL at room temperature for fourhoursand in a refrigerator for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide concentrations in the dosing formulations varied within the range between98 % and 107 %of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 47 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 17, i.e. up to the day before necropsy (altogether for 51-58 day depending on the mating day; non-pregnant females were administered for 47 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring.Estrous cycle was monitored by examining vaginal smears before the treatmentfor two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation, and on the day of the necropsy.

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.


Blood samples were collected for determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter – if it was feasible – at

termination on post-partum/ post-natal day 13 and from all parent male animals at termination. Samples of parental male animals and pups on postnatal day 13 were measured and evaluated.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides,prostate and seminal vesicles with coagulating glands as a wholeof adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and

one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). In addition, these organs were processed histologically in non-pregnant females and males cohabited with at the mid dose group.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and full histopathology examination.

Full histopathology was performed in dead male animal at 50 mg/kg bw/day. In addition, uterus with vagina in single female animals at 50 and 100 mg/kg bw/day, and the thymus in two male animals at 100 mg/kg bw/daywere also processed and evaluated histologically on the basis of macroscopic observations at the necropsy.

 

The results of this study were summarized as follows:

Mortality

There was no test item related mortality at 50, 100 or 300 mg/kg bw/day groups during the course of study

(male and female).

One female animal at 50 mg/kg bw/day died on Day 18 due suffocation. Foamy content in the trachea and histopathological findings (alveolar emphysema and edema, congestion in the lungs) refer to para-gastric treatment.

 

Clinical and functional observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations nor at the functional observations. The behavior and physical condition of the animals was not impaired at each dose level (50, 100 or 300 mg/kg bw/day) during the entire treatment period.

Nuzzling up the bedding material noted for all male animals and for all female animals at 300 mg/kg bw/day shortly after the administration from Day 3 up to the last treatment day was not considered to be toxicologically relevant due to the short duration and animals showed normal behavior thereafter.

 

Body weight and body weight gain

The body weight development was not adversely affected by the test item in male or in female animals at 50, 100 or 300 mg/kg bw/day during the entire treatment period.


Food consumption

The mean daily food consumption was not affected at 50, 100 or 300 mg/kg bw/day groups in male animals (during the course of the pre-mating and post mating periods) and in female animals (during the pre-mating, gestation and lactation periods).

 

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (50, 100 and 300 mg/kg bw/day).

 

Reproductive performance and delivery data

The examined parameters of reproductive ability were comparable in the control and in 50, 100 and 300 mg/kg bw/day groups (male and female).

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (50, 100 and 300 mg/kg bw/day).

 

Hematology

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 50, 100 and 300 mg/kg bw/day.

 

Clinical chemistry

The bile acid concentration was elevated in female animals at 300 mg/kg bw/day with respect to their control indicative some disturbances in the enterohepatic circulation or the hepatobiliary system.

The serum thyroid hormone (free T4) level was comparable to their control in parental male animals and in PN 13 day offspring at 50,100 and 300 mg/kg bw/day.

 

Necropsy

Macroscopic findings related to the effect of the test item were not found in male and female animals at 50, 100 and 300 mg/kg bw/day.

 

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level.

The examined organ weights of animals selected for toxicity examinations were comparable in the control and50, 100 and 300 mg/kg bw/daygroups at the end of the treatment period.

 

Histopathology

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 300 mg/kg bw/day.

There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 300 mg/kg bw/day groups.

Offspring

Noadverseeffect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Conclusion

Under the conditions of the present study, Reaction mass of N-butylphthalimide and n-propylphthalimide and N-sec-butylphthalimide administered at 50, 100 or 300 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Hsd.Han: Wistar rats.

The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 300 mg/kg bw/day

 

NOAEL for reproductive performance of male/ female rats: 300 mg/kg bw/day

 

NOAEL for F1 Offspring:300 mg/kg bw/day