Registration Dossier

Administrative data

Description of key information

Main study OECD 422 in progress (05/2018). Based on available data (no adverse effects observed up to 300 mg/kg bw/day in pre study OECD 407) the substance is not classified with repeated dose toxicity according to the CLP regulation 1272/2008/EC.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Route of administration:
oral: gavage
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Key result
Dose descriptor:
NOAEL
Effect level:
> 0 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: preliminary results, study in progress
Key result
Critical effects observed:
not specified
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.
Executive summary:

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

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26. July - 29. September, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
03 October 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests is a well-known laboratory model with sufficient historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species / Strain: Rat, Hsd.Han: of Wistar origin
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Age of animals at start of Male animals: 58 – 63 days
the study: Female animals: 58 – 63 days
Body weights at start of 233 – 273 g for male animals
the study: 139 – 161 g for female animals
The weight variation did not exceed ¿ 20 per cent of the mean weight
Number and sex of animals: 25 naïve male and 25naïve female (nulliparous and non-pregnant animals) rats
Number of groups: 5 (4 dose levels + 1 control group)
Number of animals/group: 10 (5 male; 5 female)
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Acclimatization time: 13 days

Husbandry

Housing conditions

Animal room no.: 18/4
Housing: 5 animals of the same sex/ cage
Cage type: Type IV polypropylene/ polycarbonate

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 12). 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 produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum except overnight food deprivation before the blood sampling.
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 10.
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

Individual identification was performed by numbers on the tail of the animals written with a permanent marker. In the pre-treatment period, the numbers were
given on the basis of the laboratory master file of Toxi-Coop Zrt. and were be re-marked as necessary to ensure correct identification.
The cages were marked by identity cards, with information about the study number, name of the test item, dose and mode of administration, species and
strain, sex of animals, cage number and individual animal numbers, start of the administration, date of the necropsy. Boxes were arranged in such a way
that possible effects due to cage placement are minimized.

Randomization

Animals were randomly assigned to test groups. All animals were sorted according to body weight by computer and grouped according to weight ranges.
There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping was controlled
by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines. The oral route is the possible route of human exposure to the test item.

Dose levels
A control and for dose groups were involved in the study. Table below contains the group number, doses, dosing volume and number of animals.

Justification of dose level selection

The dose setting with 0, 50, 100, 300 and 450 mg/kg bw/day is based on the results of an acute oral toxicity study of Reaction mass of N-buthylphthalimide and
n-propylphthalimide and N-sec- butylphthalimide in rats and in agreement with the Sponsor. The LD50 value of the test item was between 300 and 2000 mg/kg
bw with an estimated cut off value of 500 mg/kg bw.
Doses are selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
Vehicle:
vegetable oil
Remarks:
Sunflower oil (Helianthi annui oleum raffinatum)
Details on oral exposure:
The test item was orally administered daily ;once (7 days per week) in graduated doses to four groups of experimental animals for a period of 14 days. Control animals were treated concurrently with the vehicle only. The actual treatment volume was calculated according to the most recent body weight. A treatment
volume of 5 mL/kg body weight was applied. Animals were not treated on the day of gross pathology.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations was performed once during this DRF study. Five aliquots of 5 mL of each formulation to be administered to the
animals (10, 20, 60 and 90 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken.
Date of sampling: August 01, 2017
Date of analysis: August 02, 2017
The Reaction mass of N-buthylphthalimide and N-propylphthalimide and N-sec-butylphthalimide concentrations in the samples varied within the range from 92 %
to 107 % in comparison to the nominal values. Details and results of the formulation analysis are presented in Appendix 8.2.
Reaction mass of N-buthylphthalimide 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 in a refrigerator (5 ± 3 oC) for three days. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 106 % at ca. 1 mg/mL and 97 % at ca. 200 mg/mL). Reaction mass of N-buthylphthalimide and N-Propylphthalimide and
N-sec-Butylphthalimide proved to be stable in a refrigerator (at 5 ± 3 °C) for three days and at room temperature for four hours.
A separate analytical report (Study no. 805-100-2235) provided these data.

Duration of treatment / exposure:
Five groups of Hsd.Han: of Wistar rats consisting of five animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 50, 100, 300 and 450 mg/kg body weight/day (mg/kg bw/day) doses in nominal concentrations of 0, 10, 20, 60 and 90 mg/mL corresponding to a 5 mL/kg bw dosing
volume. A group of vehicle (sunflower oil) treated animals (n= 5/sex) served as a control.
The experimental period involved 13 days of acclimatization, 14 days treatment and observation period and necropsy on the following day (study Day 14).
The day of first treatment was considered as Day 0 of examination.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 2.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3.
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 4.
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
Group 5.
No. of animals per sex per dose:
five animals per group and sex
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of animals

25 male and 25 female healthy rats were used in the study. Animals were selected for this study on the basis of adequate body weight, a body weight
within ± 20% of the mean within a sex and free from clinical signs of disease or injury. Selected rats were distributed by randomization according to
stratification by body weight so that there was no statistically significant difference among group body weight means within a sex.

The test item was orally administered daily (7 days per week) in graduated doses to four groups of experimental animals for a period of 14 days. Control
animals were treated concurrently with the vehicle only. The actual treatment volume was calculated according to the most recent body weight.
A treatment volume of 5 mL/kg body weight was applied. Animals were not treated on the day of gross pathology.

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


Detailed clinical observations were conducted once a day, after treatment at approximately the same time. 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.


Individual body weights were recorded on Day 0 (prior to study start) and once weekly (on Days 7, and 13) with a precision of 1 g. Individual body weight
changes were calculated according to the days of measurements and for the study overall.
The animals were also weighed immediately prior to sacrifice (on Day 14; fasted body weight) in order to calculate organ weight to body weight ratio.


Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g once weekly to coincide with body weight
measurements (given food on Days 0 and 7; remained food on Days 7 and 13). All animals were food deprived overnight prior to blood sampling.

Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted at termination of the treatment
(i.e. one day after the last treatment).
Animals were food deprived overnight (for approximately 16 hours) prior to blood collection. Blood samples were harvested from the retro orbital venous
plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology (MiniCollect®K3EDTA tubes, spray-dried, 0.25 mL), one for determination of blood clotting times (MiniCollect® 3.8 % sodium citrate, for APTT and PT 1.0 mL) and the third one (Vacuette 2.5 mL Z Serum Sep C/A) to obtain serum samples for clinical chemistry. All of the three tubes are manufactured by Greiner Bio-One International AG, Kremsmünster, Austria.
The tubes for hematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 ml blood was collected into the
clinical chemistry tubes.

Hematology parameters were measured in all animals by SYSMEX XT-2000iV.

Blood coagulation parameters were measured by AMAX Destiny Plus

Clinical chemistry parameters were measured in all surviving animals by Konelab 60i

Gross pathology was performed on every experimental animal at termination of the treatment i.e. one day after the last treatment, on Day 14.
Animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis.

The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
The following organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes, epididymides, which were preserved in modified
Davidson solution and then stored in 4 % formaldehyde solution for possible future histopathological examination:

Organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

Organ weight

The following organs were weighed and recorded. Paired organs were weighed together.
With precision of 0.01g: Liver, kidneys, testes, epididymides, seminal vesicles with coagulating gland and prostate as a whole, uterus with fallopian tubes,
thymus, spleen, brain and heart.
With precision of 0.001g: Adrenal glands

Histopathology

Histopathological examinations were not performed as dose selection for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental
Toxicity Screening Test in the Rat (main study) was possible with the results of this 14-day dose range finder study.
Observations and examinations performed and frequency:
Clinical observations

Detailed clinical observations were conducted once a day, after treatment at approximately the same time. 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.

Body weight and body weight gain

Individual body weights were recorded on Day 0 (prior to study start) and once weekly (on Days 7, and 13) with a precision of 1 g. Individual body weight
changes were calculated according to the days of measurements and for the study overall.
The animals were also weighed immediately prior to sacrifice (on Day 14; fasted body weight) in order to calculate organ weight to body weight ratio.

Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted at termination of the treatment
(i.e. one day after the last treatment).
Animals were food deprived overnight (for approximately 16 hours) prior to blood collection. Blood samples were harvested from the retro orbital venous
plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology (MiniCollect®K3EDTA tubes, spray-dried, 0.25 mL), one for determination of blood clotting times (MiniCollect® 3.8 % sodium citrate, for APTT and PT 1.0 mL) and the third one (Vacuette 2.5 mL Z Serum Sep C/A) to obtain serum samples for clinical chemistry. All of the three tubes are manufactured by Greiner Bio-One International AG, Kremsmünster, Austria.
The tubes for hematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 ml blood was collected into the clinical
chemistry tubes.
Sacrifice and pathology:
Pathology

4.8.1 Necropsy

Gross pathology was performed on every experimental animal at termination of the treatment i.e. one day after the last treatment, on Day 14.
Animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis.

The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
The following organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes, epididymides, which were preserved in modified Davidson solution and then stored in 4 % formaldehyde solution for possible future histopathological examination:
Organ weight

The following organs were weighed and recorded. Paired organs were weighed together.
With precision of 0.01g: Liver, kidneys, testes, epididymides, seminal vesicles with coagulating gland and prostate as a whole, uterus with fallopian tubes, thymus, spleen, brain and heart.
With precision of 0.001g: Adrenal glands

4.8.3 Histopathology

Histopathological examinations were not performed as dose selection for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study) was possible with the results of this 14-day dose range finder study.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
-Body weight
-Hematology
-Blood coagulation
-Clinical chemistry
-Organ weight
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reaction Mass of N-Buthylphthalimide and N-Propylphthalimide and N-sec-Butylphthalimide caused clinical signs at 100, 300 or 450 mg/kg bw/day in male or female animals during the two weeks treatment period. All signs were with minimal degree and short duration (approximately 15 minutes for salivation, nuzzling up or chewing the bedding material; 2 hours for activity decrease) and animals fully recovered thereafter.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain: The body weight gain was slightly reduced at 100 mg/kg bw/day in female animals and at 300 and 450 mg/kg bw/day
doses in male and female animals on week 1 and for study overall but this did not result in significant body weight change compared to control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was slightly and transiently less with respect to their control (male and female) at 300 and 450 mg/kg bw/day on week 1 in accordance with the body weight changes at these doses.
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 test item related significant changes in the examined parameters at 50, 100, 300 or 450 mg/kg bw/day (male and female).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters at 50, 100, 300 or 450 mg/kg bw/day (male and female). Slight elevation in ALT activity in female animals dosed with 450 mg/kg bw/day might be indicative a test item effect on hepatic function and histological
examination could reveal the nature of this change.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day
Male animals exhibited normal behavior and physical condition with no abnormalities during the course of 15-day observation period (including day of necropsy).
300 mg/kg bw/day
Decreased activity (2/5), salivation (3/5) and nuzzling up the bedding material (5/5) were observed in male animals from Days 1, 2, 6 or 7 up to and including Day 13.
Decreased activity (1/5) and salivation (4/5) were noted for female animals. The onset and duration of these signs were similar to that of male animals.
All male and female animals were normal on the day of the necropsy
450 mg/kg bw/day
Decreased activity (5/5male, 2/5 female), salivation (5/5male, 5/5 female), chewing the bedding material (3/5 male and 2/5 female) and nuzzling up the bedding material (5/5 male, 5/5 female) were observed in male animals mainly from Days 1, 2, or from Days 5, 6 (in two male animals) up to and including Day 13.
All male and female animals were normal on the day of the necropsy
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weights (absolute and relative to body and brain weights) were significantly elevated in male animals at 100 mg/kg bw/day and in male and female
animals at 300 and 450 mg/kg bw/day with respect to controls.
Statistically significant reduction of thymus weights (absolute and relative to body and brain weights) with respect to controls in female animals dosed with
450 mg/kg bw/day was probably due to an increased physiologic involution in the lack of any systemic changes referring to immunotoxic relevance.
Histological examination could reveal the nature of this change.

Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related pathologic findings were not detected in male or female animals at 50, 100, 300 or 450 mg/kg bw/day at the necropsy. Dark color of the liver in two male animals (2/5) probably was related to the test item effect and histological examination could reveal the nature of this change.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test item related pathologic findings were not detected in male or female animals at 50, 100, 300 or 450 mg/kg bw/day at the necropsy. Dark color of the liver in two male animals (2/5) probably was related to the test item effect and histological examination could reveal the nature of this change.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 - <= 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
other: liver weight gain
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
other: elevated AST
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
other: body weight
Organ:
other: slightly reduced
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
other: body weight
Organ:
other: slightly reduced
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
other: body weight
Organ:
other: reduced body weight

There was no mortalityin the control, 50, 100, 300 or 450 mg/kg bw/daygroups during the 14-day treatment period (male and female).

 

Reaction mass of N-buthylphthalimide and n-propylphthalimide and N-sec-butylphthalimide caused clinical signs in female animals at 100 mg/kg bw/day (salivation) and in male and female animals at 300 and 450 mg/kg bw/day (decreased activity, salivation, chewing the bedding material or nuzzling up the bedding material. The onset of signs was 2-3 minutes after the administration and signs were with short duration (salivation, nuzzling up or chewing the bedding material ceased approximately 10 minutes thereafter, activity decrease ceased approximately within two hours).

Control group and 50 mg/kg bw/day

There were no clinical signs in control male or female animals during the 14-day treatment period. The behavior and physical state of animals were considered to be normal on the day of necropsy, too.

100 mg/kg bw/day

Male animals exhibited normal behavior and physical condition with no abnormalities during the course of 15-day observation period (including day of necropsy).

Salivation was noted for two female animals (2/5) from Day 8 up to the last treatment day (Day 13). Salivation appeared 2-3 minutes after the administration and ceased approximately 10 minutes thereafter.

Animals were normal on the day of the necropsy.

300 mg/kg bw/day

Decreased activity (2/5), salivation (3/5) and nuzzling up the bedding material (5/5) were observed in male animals from Days 1, 2, 6 or 7 up to and including Day 13.

Decreased activity (1/5) and salivation (4/5) were noted for female animals. The onset and duration of these

signs were similar to that of male animals.

All male and female animals were normal on the day of the necropsy

450 mg/kg bw/day

Decreased activity (5/5male, 2/5 female), salivation (5/5male, 5/5 female), chewing the bedding material (3/5 male and 2/5 female) and nuzzling up the bedding material (5/5 male, 5/5 female) were observed in male

animals mainly from Days 1, 2, or from Days 5, 6 (in two male animals) up to and including Day 13.

All male and female animals were normal on the day of the necropsy

 

The body weight of the male and female animals were not adversely affected in any test item treated groups

(50, 100, 300 or 450 mg/kg bw/day) throughout the entire observation period.


The mean body weight gain was slightly reduced at 300 and 450 mg/kg bw/day on week 1 and as a consequence for the study overall. These slight changes in the body weight gain were transient and did not result in significant reduction in the mean body weight values. Similar findings were detected in the mean body weight gain of female animals at 100,300 and 450 mg/kg bw/day however the differences with respect to the control were not statistically significant and were with similar degree in each of these groups.

50 mg/kg bw/day

The mean body weight and body weight gain were similar in male and female animals to that of their control group.

100 mg/kg bw/day

There were no significant differences between the control and test item treated male animals in the mean

body weight or body weight gain.

The mean body weight gain was slightly lower – no statistical differences – in female animals between Day 0 and 7 as well as between Days 0 and 13.

300 and 450 mg/kg bw/day

The body weight gain was slightly lower than in the control group in male animals of mid-high and high

dose groups between Days 0 and 7 and for the study overall (between Days 0 and 13).

The mean body weight gain was slightly lower in female animals between Day 0 and 7 as well as between Days 0 and 13 but without statistical difference.

The mean daily food consumption of male and female animals was slightly reduced in male and female animals at

300 and 450 mg/kg bw/day between Days 0 and 7.

The mean daily food consumption was similar in the control and 50 mg/kg bw/day and 100 mg/kg bw/day test item treated animal (male and female) during the 14-day observation period.

 

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at

50, 100, 300 or 450 mg/kg bw/day in male and female animals.

50 mg/kg bw/day

The examined hematological parameters were comparable in the male control and test item treated groups.

In the female animals of low dose group, statistical significance was observed at the lower mean

percentage of basophil granulocytes (BASO) with respect to the control.

100 mg/kg bw/day

Slight but statistically significant differences with respect to their controls were noted in male and female animals for the

lower mean percentage of basophil granulocytes, lower mean hemoglobin concentration (HGB) and hematocrit value (HCT).


300 mg/kg bw/day

There were no significant differences between the control ad test item treated male animals in the examined hematological parameters.

In the female animals of mid-high dose group, the mean percentage of basophil granulocytes, mean hemoglobin concentration, hematocrit value and mean activated thromboplastin time (APTT) was below these of the control group. The mean percentage of reticulocytes (RET) slightly exceeded the control value in female animals.

450 mg/kg bw/day

All examined hematological parameters were comparable to the control in male animals of the high dose group.

Statistical significance was detected at the higher mean percentage of reticulocytes in female animals at 450 mg/kg bw/day when compared to control.

These slight but statistically significant differences with respect to control were not considered toxicologically relevant as these were at a low magnitude and were only observed in the lower dose groups (BASO, HGB, HCT or APTT) or were not related to doses (RET). Moreover all values remained well within the historical control ranges.

 

Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 50, 100, 300 or 450 mg/kg bw/day. Slight elevation of alanine amino transferase activity in female animals at 450 mg/kg bw/day was probably related to the test item effect on liver function (in accordance with liver weight changes).

50 mg/kg bw/day

In the male animals, statistical significance was observed at the slightly lower mean total bilirubin concentration (TBIL) with respect to the control.

In the female animals of low dose group, there were no statistically significances with respect to their control at the examined clinical chemistry parameters.

100 mg/kg bw/day

The mean concentration of bilirubin and sodium (Na+) and albumin/ globulin ratio (A/G) were slightly below the relevant control in male animals.

In the female animals, slightly lower mean activity alkaline phosphatase (ALP) was detected when compared to the control.

300 mg/kg bw/day

The mean concentration of bilirubin was below and the mean concentration of total protein (TPROT) was above the control value in male animals.

In the female animals, the albumin/ globulin ratio was slightly below the relevant control.

450 mg/kg bw/day

Slight but statistically significant differences were observed between the control and treated male animals at the lower mean concentration of total bilirubin, at the higher mean concentration of total protein and at the lower mean albumin: globulin ratio.

In the female animals of the high dose group, the mean activity of alanine amino transferase (ALT) was slightly elevated while the mean activity of alkaline phosphatase (ALP) was lower with respect to the control.


The total protein concentrations exceeded the control value and the mean albumin: globulin ratio was below the control value.

The slight elevation ofalanine amino transferaseactivity(female animals at 450 mg/kg) might be indicative of the test item influence on hepatic function.

Although the differences between the control and test item treated groups were statistically significant, there was no

dose relevance for most of the above mentioned parameters (TBIL, Na+, TPROT and A/ G in male animals; ALP and A/G in female animals) and values of these parameters remained well within the historical control ranges. Therefore these changes were considered to be toxicologically irrelevant.

Specific macroscopic alterations indicative of adverse test item effect were not observed in the organs or tissues at any

dose levels – 50, 100, 300or 450 mg/kg bw/dayat the necropsy. Dark color of the liver might be related to the test item

influence in accordance with liver weight changes in male animals at 450 mg/kg bw/day.

Control group

There were no macroscopic changes in male animals (5/5).

Slight hydrometra was noted for single control female animal (1/5).

50 mg/kg bw/day

Thymic hemorrhages were observed in on male animal (1/5) and slight or moderate hydrometra in female animals

(3/5) administered with the low dose.

100 mg/kg bw/day

In the male animals, pyelectasia (2/5) and one side smaller than normal seminal vesicle (1/5) were detected.

There were no macroscopic changes in female animals (5/5).

300 mg/kg bw/day

Hemorrhages were seen in the thymus in one male animal (1/5) and slight hydrometra in one female animal (1/5).

450 mg/kg bw/day

In the male animals, thymic hemorrhages (1/5), pyelectasia (1/5) and darker than normal color of the liver (2/5) were detected at the necropsy.

There were no macroscopic changes in female animals (5/5).

Dilatation of renal pelvis (pyelectasia) and smaller than normal seminal vesicles are common individual changes in experimental rats. These were observed independently from doses and were not related to the test item.

Thymic hemorrhage is frequently observed in experimental rats and is considered related to theexsanguination procedure i.e. it was consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.

Hydrometra, related to the female sexual cycle, is also a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs these were judged to be toxicologically not relevant.

 

Slightly but statistically significantly higher mean weights of liver(absolute or relative to body or brain weights)were

indicative of a test item influence both in male and female animalsat 50, 100, 300 or 450 mg/kg bw/day and probably were coinciding with changes in clinical chemistry parameters (female) and necropsy observations (male) at 450 mg/kg bw/day). Changes at 50 mg/kg bw/day were with negligible degree.

50 mg/kg bw/day

Statistically significant difference with respect to the control was detected at the higher mean liver weight relative to body weight in male and female animals and at the lower mean weight of adrenal glands (absolute and relative to body and brain weights) in male animals.

100 mg/kg bw/day

The mean liver weights (absolute and relative to body and brain weights) were higher than in the control group in male animals.

In the female animals, the liver weights relative to body weight was slightly above the control value. Statistical

significance was also detected at the higher mean brain weight relative to body weight and lower mean body weight relative to brain weight with respect to the control in female animals.

300 mg/kg bw/day

The mean liver weights (absolute and relative to body and brain weights) exceeded the control value both in male and female animals.

450 mg/kg bw/day

Statistically significant difference with respect to the control was observed at the higher mean liver weights (absolute and relative to body and brain weights) both in male and female animals and at the higher mean brain weight relative to body weight as well as the lower mean body weight relative to brain weight in male animals.

Additionally, statistically significant differences with respect to the control were noted for the lower mean thymus weights (absolute and relative to body and brain weights), for the higher mean brain weight relative to body weight and adrenal weights (relative to body and brain weights), and for the lower mean body weight relative to brain weight in female animals.

 

Statistically significant reduction of thymus weights (absolute and relative to body and brain weights) with respect to

controls in female animals dosed with 450 mg/kg bw/day was probably due to an increased physiologic involution in the lack of any systemic changes referring to immunotoxic relevance.

The changes in weight of adrenal glands were with minor degree and not considered to have toxicological significance in male animals at 50 mg/kg bw/day and in female animals at 450 mg/kg bw/day.

Statistical significances in brain weight relative to body weight as well as in body weight relative to brain weight were

originated from the slightly lower mean fasted body weight in female animals at 100 mg/kg bw/day and in male and female animals at 450 mg/kg bw/day.

(Appendices 7.1 and 7.2)

Conclusions:
Reaction Mass of N-Buthylphthalimide and N-Propylphthalimide and N-sec-Butylphthalimide did not induce adverse effects in male or female
Hsd.Han:
Wistar rats after the consecutive 14-day oral (by gavage) administration at 50, 100, 300 or 450 mg/kg bw/day.

450 mg/kg bw/day caused clinical signs with short duration (male and female), a slightly reduced body weight gain (male and female), elevated AST
activity (female), macroscopic changes on liver (dark color in male animals) and elevation in liver weights (male and female). All these changes were
related to the test item however were not considered to be adverse.

At 300 mg/kg bw/day, slight clinical signs, slightly depressed body weight gain and elevation in liver weights (male and female, each) were observed in
minimal degree.

At 100 mg/kg bw/day, slight clinical signs, slightly depressed body weight gain (female) and elevation in liver weights (male and female) were foud.

There were no toxicologically significant changes in the examined parameters at 50 mg/kg bw/day.

Based on the observations made in this toxicity study, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study) were determined as follows:

Group 1 Vehicle control
Group 2 50 mg/kg bw/day
Group 3 100 mg/kg bw/day
Group 4 300 mg/kg bw/day

Executive summary:

The objective of this study was to obtain first information on the toxic potential of Reaction mass of N-buthylphthalimide and n-propylphthalimide and N-sec-butylphthalimide in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study).The dose setting with 0, 50, 100, 300 and 450 mg/kg bw/day is based on the results of an acute oral toxicity study of Reaction mass of N-buthylphthalimide and n-propylphthalimide and N-sec- butylphthalimide in rats and in agreement with the Sponsor. The LD50value of the test item was between 300 and 2000 mg/kg bw with an estimated cut off value of 500 mg/kg bw.Five groups ofHsd.Han: of Wistar ratsconsisting of five animals per group and sexwere administered orally (by gavage) once daily at 0 (vehicle only), 50, 100, 300 and 450 mg/kg body weight/day (mg/kg bw/day) doses in nominal concentrations of 0, 10, 20, 60 and 90 mg/mL corresponding to a 5 mL/kg bw dosing volume. A groupof vehicle (sunflower oil) treated animals (n= 5/sex) served as a control.The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.The Reaction mass of N-buthylphthalimide and N-propylphthalimide and N-sec-butylphthalimide concentrations in the samples used for administration of animals varied within the range from92 %to107 %in comparison to the nominal values, thereby confirming proper dosing.

Detailed clinical observationswere performed daily after the treatment and before the necropsy.Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted on all animals one day after the last treatment (on Day 14). Selected organs were weighed. 

The results of this study were summarized as follows:

Mortality:There was no mortality during the course of the study.

 

Clinical observations:Reaction Mass of N-Buthylphthalimide and N-Propylphthalimide and N-sec-Butylphthalimide caused clinical signs at 100, 300 or 450 mg/kg bw/dayin male or female animals during the two weeks treatment period. All signs were with minimal degree and short duration (approximately 15 minutes for salivation, nuzzling up or chewing the bedding material; 2 hours for activity decrease) and animals fully recovered thereafter.

 

Body weight and body weight gain: The body weight gain was slightly reduced at 100 mg/kg bw/day in female animals and at 300 and 450 mg/kg bw/day doses in male and female animals on week 1 and for study overall but this did not result in significant body weight change compared to control group.


Food consumption:The mean daily food consumption was slightly and transiently less with respect to their control (male and female) at 300 and 450 mg/kg bw/day on week 1 in accordance with the body weight changes at these doses.

 

Hematology and blood coagulation:Hematological evaluation did not reveal test item related significant changes in the examined parameters at 50, 100, 300 or 450 mg/kg bw/day (male and female).

 

Clinical chemistry:Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters at 50, 100, 300 or 450 mg/kg bw/day (male and female). Slight elevation inALT activity in female animals dosed with 450 mg/kg bw/day might be indicative a test item effect on hepatic function and histological examination could reveal the nature of this change.

 

Gross pathology: Test item related pathologic findings were not detected in male or female animals at 50, 100, 300 or 450 mg/kg bw/dayat the necropsy. Dark color of the liver in two male animals (2/5) probably was related to the test item effect and histological examination could reveal the nature of this change.

 

Organ weight: The liver weights (absolute and relative to body and brain weights) were significantly elevated in male animals at 100 mg/kg bw/day and in male and female animals at 300 and 450 mg/kg bw/day with respect to controls.

Statistically significant reduction of thymus weights (absolute and relative to body and brain weights) with respect to controls in female animals dosed with 450 mg/kg bw/day was probably due to an increased physiologic involution in the lack of any systemic changes referring to immunotoxic relevance. Histological examination could reveal the nature of this change.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Justification for classification or non-classification