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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of the present study, p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) administered at 60, 200 or 600 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.

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

NOAEL for systemic toxicity of male/female rats: 600 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats:600 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19, September, 2017-24 May, 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)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
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:
2000 July
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 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 or test system and environmental conditions:
Animals

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: 92 – 97 days
the treatment: Female animals: 92 – 97 days
Body weights at start of 346 – 392 g for male animals
the study: 209 – 247 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
Acclimatization time: 27 days


Animal husbandry

Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder
Animal room no.: 21/B

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;
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.
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. Final (permanent) animal numbers were given
according to allocation into the treatment groups after randomization, as follows:

Identity number of parental animals
GROUPS DOSE
(mg/kg bw/day) MALES FEMALES
Group 1 0 101 – 112 121 – 132
Group 2 60 201 – 212 221 – 232
Group 3 200 301 – 312 321 – 332
Group 4 600 401 – 412 421 – 432
The boxes were marked by identity cards, with information about study number, strain, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Color of cards also referred to doses. Boxes were arranged in such a way that possible effects due to cage placement were minimized.

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.

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was formulated in the vehicle (distilled water) in concentrations of 12, 40 and 120 mg/mL.
The test item was administered orally via gavage. 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.
A constant treatment volume of 5 mL/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.
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. For one female animal in each of the 60 and 200 and 600 mg/kg bw/day groups (no.232, 324, 424, respectively), male pair was replaced by a proven male (no.211, 303, 403, respectively).
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 (12, 40 and 120 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 04, 2017 and November 13, 2017
Date of analysis: October 05, 2017 and November 14, 2017
Concentration of the test item in the dosing formulations varied between the range of 91 % and 99 % in comparison to the nominal values.
Results and details of analysis are attached to the Report
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
Recovery of p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) from distilled water formulations was within the acceptance criteria
(relative to nominal concentrations: 104 % at ca. 1 mg/mL and 104 % at ca. 200 mg/mL).
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation
in a separate analytical study (Toxi-Coop Study no. 805-100-2254). p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) proved to be stable at
room temperature for three days (recovery was 98 % of starting concentration at ~1 mg/mL and 100 % at ~200 mg/mL).
Duration of treatment / exposure:
The experimental period involved 27 days of acclimatization (including 14 days for examination of estrous cycle) and 50-65 days treatment/observation period (depending on the effectiveness of mating) and necropsy days. One female animal at 60 mg/kg bw/day was administered for 42 days and subjected to necropsy on Day 42 due to its early death.
The day of first treatment is considered as day 0 of examination.
Frequency of treatment:
once a day
Details on study schedule:
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 51 days). Females were additionally exposed through the gestation period and up to lactation days
13 - 16, i.e. up to the day before necropsy (altogether for 50-65 days; one female animal at 60 mg/kg bw/day was administered for 42 days due to its early
death). 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 treatment for 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.
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 possible 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 at termination on post-partum/ post-natal day 13 and from all parent male animals at
termination.
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 whole of 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 dam
at 60 mg/kg bw/day. In addition, uterus of one female at 60 mg/kg bw/day, and the spleen in male animals at 60, 200 or 600 mg/kg bw/day were also
processed and evaluated histologically on the basis of macroscopic observations at the necropsy.
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
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:
Males
A PM M ¿
Acclimatization Pre-mating period Mating period Post mating period FOB
Blood sampling
Necropsy
Organ weighing††
on Day 51


Females
A PM M G ¿ PP/PN ¿
Acclimatization period Pre-mating period Mating period Gestation period Delivery Lactation period FOB
27 days† 14 days 1-17 days 21-24 days 13 – 16 days Blood sampling
Necropsy
Organ weighing
Day 54 ††,
Day 42 †††
Remark: † Pre-treatment examination of estrous cycle included
†† = For animals selected for toxicity examinations
††† = For one female at 60 mg/kg bw/day

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 51 days (14 days pre-mating and 1-17 days mating, plus 20 – 36 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 – 17 days mating period and throughout pregnancy and at least up to and including day 13
post-partum or the day before sacrifice. 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.
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 44. 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 days 7, 13, and post-mating days 20, 27, 34, 41, 48 and 50 for male animals), pre-mating 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 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

Blood samples for determination of serum levels of thyroid hormones (T4) were drawn as described at 4.3.9.3 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
¿ from all parent male animals at termination on Days 51
Details of sampling – number of pups per litters sampled on postnatal days 4 and 13; parental male and female animals
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.
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, 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.
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 weight was 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).

Necropsy

Gross necropsy was performed on each animal.
One female animal (no. 225) at 60 mg/kg bw/day died and was subjected to necropsy immediately after the death on Day 42.
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 Days 51.
¿ Dams on post-partum day 14 or shortly thereafter (Days 51, 54, 56, 58, 62, 64, 65).
¿ Non-pregnant females on Day 50.
¿ 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 five male and five female
animals randomly selected from each group:

List of organs to be preserved
Adrenal glands
Aorta
Bone with bone marrow and joint (femur)
Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
Eyes (lachrymal gland with Harderian glands)
Female mammary gland
Gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina)
Heart
Kidneys
Large intestines (caecum, colon, rectum, including Peyer’s patches),
Liver
Lungs (with main stem bronchi; inflation with fixative and then immersion;)
Lymph nodes (submandibular, mesenteric)
Muscle (quadriceps)
Esophagus
Pancreas
Pituitary
Prostate
Salivary glands (submandibular)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin
Small intestines (representative regions: duodenum, ileum, jejunum)
Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
Spleen
Sternum
Stomach
Thymus
Thyroid + parathyroid
Trachea
Urinary bladder

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.
The thyroid weight will be determined – if relevant – after fixation.
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 randomly selected animals in the control and high dose
(600 mg/kg bw/day).
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.
Postmortem examinations (offspring):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
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.
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
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.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality at 60, 200 or 600 mg/kg bw/day groups during the course of study (male and female).
One female animal at 60 mg/kg bw/day died on gestation day 24 (Day 42) probably due to fetal deaths. Histopathological examination revealed hepatic necrosis in connection with the dead fetuses.
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 60, 200 or 600 mg/kg bw/day during the entire
treatment period. A slightly depressed body weight gain was observed at 600 mg/kg bw/day (male and female) resulting in only slight body weight difference
with respect to their control (<10 %) therefore these slight changes in bodyweight gain were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was not adversely affected at 60, 200 or 600 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). In compliance with the slight body weight changes, minor and transient differences with respect to the control were observed at 600 mg/kg bw/day, which however were judged to be toxicologically not relevant.
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 60, 200 and 600 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The bile acid concentration was elevated in male and female animals at 200 and 600 mg/kg bw/day with respect to their control indicative some disturbances in the enterohepatic circulation or the hepatobiliary system.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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, 60, 200 or 60 mg/kg bw/day groups, on Day 44).
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.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
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 (60, 200 and 600 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):
There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups
(60, 200 and 600 mg/kg bw/day). A slightly lower percentage of implantations, higher percentage of pre-implantation loss and total intrauterine mortality
at 600 mg/kg bw/day were judged to be of little or no toxicological significance as the mean values were comparable with the historical control.
The examined parameters of reproductive ability were comparable in the control and in 60, 200 and 600 mg/kg bw/day groups (male and female).
Mortality

There was no test item related mortality at 60, 200 or 600 mg/kg bw/day groups during the course of study (male and female).
One female animal (no.225) at 60 mg/kg bw/day was in moribund condition on Day 42 (gestation day 24) with the following clinical signs: paleness, prone
position, piloerection, decreased body tone, vaginal bleeding. The animal died before the euthanasia. Necropsy observation revealed pale skin, eye, mucous membranes and visceral organs and 6 matured but died pups in the uterus. Histopathological examination revealed hepatic necrosis in connection with the
dead fetuses, as the probable cause of moribund condition and death.

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 parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities
in the control and at 60, 200 or 600 mg/kg bw/day.
Two knots (~1 cm in diameter) in the right armpit was noted for one female (1/12, animal no.129) in the control group during the daily clinical observations from gestation day 20 until the end of the study. Similarly, this observation was also present during the weekly detailed clinical observations on gestation day
21, lactation days 0, 4 and 13.
These findings were considered to be individual ones and not related to the test item.

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, 60, 200 or 60 mg/kg bw/day groups, on Day 44).
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.

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 60, 200 or 600 mg/kg bw/day during the entire treatment period. A slightly depressed body weight gain was observed at 600 mg/kg bw/day (male and female) resulting in only slight body weight difference with respect to their control (<10 %) therefore these slight changes in bodyweight gain were considered to be toxicologically not relevant.

The mean body weight and body weight gain was similar in male animals in the control and 60 mg/kg bw/day groups.
Statistical significance with respect to the control was detected in male animals at 200 mg/kg bw/day at the slightly lower mean body weight gain between
Days 0 and 7.
In the male animals at 600 mg/kg bw/day, the mean body weight was slightly lower than in the control from Day 7 up to the end of the treatment period as the mean body weight gain was significantly reduced between Days 0 and 7, between Days 34 and 41 and if summarized, i.e. between Days 0 and 50.
The mean body weight and body weight gain was similar in female animals in the control and 60 and 200 mg/kg bw/day groups during the course of
premating, gestation and lactation periods.
There were no significant differences between the control and 600 mg/kg bw/day treated female animals in the mean body weight although the mean
body weight gain was lower than in the control group during the pre-mating period (between Days 0 and 7 and between Days 0 and 13). The mean body weight was slightly below the control in high dose treated female animals during the entire gestation period reaching statistical significance on gestation days 0, 7 and 21. The mean body weight and body weight gain was comparable with that of control in dams at 600 mg/kg bw/day during the lactation period.

Food consumption

Compared to the control animals, toxicologically significant differences were not detected in the mean daily food consumption at 60, 200 or 600 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).
In compliance with the slight body weight changes, minor and transient differences with respect to the control were observed at 600 mg/kg bw/day,
which however were judged to be toxicologically not relevant.
The mean food consumption was similar in male animals in the control and 60 mg/kg bw/day groups.
Statistical significance was noted in male animals for the slightly lower mean daily food consumption at the 600 mg/kg bw/day group between Days 0 and
7, for the slightly higher mean daily food consumption at the 200 and 600 mg/kg bw/day group between days 41 and 48.
There were no significant differences in the mean daily food consumption of female animals at 60 or 200 mg/kg bw/day with respect to their control during
the pre-mating, gestation or lactation periods.
In female animals at 600 mg/kg bw/day slightly reduced food consumption was observed during the entire study, however reaching statistical significance
only between Days 7 and 13 in the pre-mating period and between gestation days 0 and 7. These differences with respect to their control were with minor
degree and were judged to be toxicologically not relevant.


Estrous cycle

A test item influence on the estrous cycle was not detected at any dose level (60, 200 or 600 mg/kg bw/day).
There were no significant differences between the control and 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

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups
(60, 200 and 600 mg/kg bw/day). The slight but statistically significant differences compared to their control at 600 mg/kg bw/day – at the lower
percentage of implantations, at the higher percentage of pre-implantation loss and total intrauterine mortality – were judged to be of little or no toxicological
significance as the mean values were comparable with the historical control.
The delivery data of dams were comparable with the control at 60 and 200 mg/kg bw/day.
Slight but statistically significant difference with respect to the control was noted for the lower percentage of implantations, for the higher percentage of pre-implantation loss and total intrauterine mortality at 600 mg/kg bw/day.
The number of pregnant females and dams delivered, the mean number of corpora lutea, the number of post-implantation loss, the duration of pregnancy, and pups births (total, live, still birth, viable) and live birth index were comparable in all groups (60, 200 and 600 mg/kg bw/day).


Reproductive ability

The examined parameters of reproductive ability were comparable in the control and in 60, 200 and 600 mg/kg bw/day groups (male and female).
Statistical significance was observed at the slightly lower copulatory index of male animals at 60, 200 and 600 mg/kg bw/day because
one male animal (1/12) in each of the test item treated groups failed to mate within 14 days. The fertility index of male animals at 60 mg/kg bw/day was higher than in the control group as two control male animals (2/12) did not fertilize their female partners.
In the female animals at 60 mg/kg bw/day, the fertility index was higher than in the control group as two sperm positive control females (2/12) was not
pregnant. The gestation index at 60 mg/kg bw/day was lower than in the control as one pregnant female died on its gestation day 24 before delivery.
These minor changes in the copulatory, fertility or gestation indices (male or female) were considered to be not related to the test item because similar
incidences also occur in non-treated experimental rats of this strain moreover there was no dose relevance.

The percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals.

Hematology and blood coagulation

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 60, 200 or 600 mg/kg bw/day.
At 60 mg/kg bw/day, the examined hematological and blood coagulation parameters were comparable with the control in male animals.
In the female animals of the low dose group, statistically significant differences with respect to their controls were noted for the slightly longer mean activated partial thromboplastin time (APTT).
There were no significant differences between the control and 200 mg/kg bw/day dose treated male animals.
In the female animals of the mid dose group, the mean corpuscular hemoglobin concentration (MCHC) was slightly lower, the mean activated partial
thromboplastin time was slightly longer than in the control group.
At 600 mg/kg bw/day, slight but statistically significant differences with respect to their controls were detected in the male animals at higher mean
corpuscular (erythrocyte) volume (MCV), at the lower mean corpuscular hemoglobin concentration and at the higher mean percentage of reticulocytes
(RET).
In the female animals of high dose group, statistically significant difference with respect to their control was detected at the higher mean activated partial
thromboplastin time (APTT).
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 (MCV, MCHC, RET, APTT). The mean and individual values of reticulocytes in male animals at 600 mg/kg bw/day exceeded also the historical control range however there were no related findings (signs of anemia, hemolysis or hemorrhage, etc.) therefore this change was considered to be non-adverse. Similarly, the slight changes in MCHC in female animals and in the mean activated partial thromboplastin time in female animals were independent
from doses therefore were judged to be indicative of biological variation and not related to the test item effect.

Clinical chemistry

The bile acid concentration was elevated in male and female animals at 200 and 600 mg/kg bw/day with respect to their control indicative some
disturbances in the enterohepatic circulation or the hepatobiliary system.
In the male animals at 60 mg/kg bw/day slight statistical significance was noted for the slightly lower mean concentration of total bilirubin (TBIL)
when compared to the control.
In the female animals of the low dose group, all examined clinical chemistry parameters were comparable to their control.

At 200 mg/kg bw/day, higher mean concentrations of urea and bile acids (BAC) and lower mean concentration of cholesterol (CHOL) were
observed in comparison with the control group.
In the female animals of the mid dose group, there were no statistically significant differences with respect to their control in the investigated parameters.
However the mean concentration of bile acids was higher than in the control especially in animal no. 328.
At 600 mg/kg bw/day, statistically significant difference with respect to the control was observed at the higher mean alanine aminotransferase activity (ALT), at the higher mean concentrations of total bilirubin, urea and bile acids as well as at the lower mean concentration of glucose.
In the female animals at 600 mg/kg bw/day, the mean bile acids concentration was significantly higher than in the control group and in the historical control.
Statistically significant differences with respect to their control in male and female animals at 60, 200 or 600 mg/kg bw/day (ALT, TBIL, UREA, GLUC and
CHOL) were considered to be toxicologically not relevant as mean values were well within historical 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 slightly lower than in the control group in parental male animals at 600 mg/kg bw/day.
Statistically significant difference with respect to the control was noted for the slightly lower thyroid hormone (free T4) level in parental male animal at 200 and 600 mg/kg bw/day. The values at 200 mg/kg bw/day were within or marginal to historical control ranges (2.72±0.39 ng/dL; n=96; min: 1.91 ng/dL; max = 3.86 ng/dL) therefore this minor difference was considered to be toxicologically not relevant.
Although there were no histological changes in the organs of hypothalamic-pituitary-thyroid axis of examined animals at 600 mg/kg bw/day a test item
influence on thyroid function was assumed.


Necropsy

Specific macroscopic alterations indicative of test item effect were not observed in the organs or tissues at any dose levels 60, 200 or 600 mg/kg bw/day
at the necropsy.
In dead female animal (1/12 at 60 mg/kg bw/day) – which was in moribund condition on Day 42 (gestation day 24) and died immediately before the
euthanasia – necropsy observation revealed pale skin, eyes, mucous membrane and visceral organs and 6 matured pups in the uterus.
Thymic hemorrhage was observed in one control male animal (1/12).
Pepper-size ovarian cyst (1/10) and hydrometra (1/10) were noted for control dams. There were no macroscopic findings in non-pregnant control
female animals (2/2).
At 60 mg/kg bw/day, in one male animal (1/12), smaller than normal testes and epididymides were observed.

Slight hydrometra was noted for one dam (1/11) of low dose group.
At 200 mg/kg bw/day, dark, lobe-like, solid formation in the abdominal fatty tissue (close to the liver) was observed in one male animal (1/12).
There were no macroscopic findings in dams (10/10) or non-pregnant female animals (2/2) administered with 200 mg/kg bw/day.
At 600 mg/kg bw/day, dark, lobe-like, solid formation in the abdominal fatty tissue (close to the liver; 1/12), enlarged (4/12) or dark colored spleen (8/12)
and thymic hemorrhage (3/12) were observed male animals.
In the female animals of the high dose group, hydrometra (1/10 dam), pale kidneys (1/10 dams) and enlarged, dark colored spleen (1/10 dam) were detected.
In one of the non-pregnant females of the high dose group, hard, yellowish brown, lentil-size formation was seen in the right ovary (1/2).
Enlarged or dark colored spleen noted for several animals at 600 mg/kg bw/day were not accompanied by adverse histopathological changes therefore was
judged to be independent from the administration of the test item.
Tissue formations in fatty tissue or ovary and ovarian cyst were considered to be individual changes. These are common in experimental rats of this strain.
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 on the examined organ weights in male or female animals at 60, 200 or 600 mg/kg bw/day.
At 60 mg/kg bw/day, the mean testes weight relative to the brain weight was slightly below that of the control in male animals.
In the female animals of the low dose group, the weight of the examined organs was comparable to the control.
There were no significant differences in the examined organ weights when compared to their control in male at 200 mg/kg bw/day.
In the female animals at 200 mg/kg bw/day, statistical significance was detected at the slightly lower mean thymus weight and at the slightly higher mean
spleen weight relative to body weight.
At 600 mg/kg bw/day, the mean fasted body weight was slightly below the control in male animals therefore the mean brain weight relative to body weight
was higher and mean body weight relative to brain weight was lower than in the control. The mean weights of the spleen (absolute, relative to body and
brain weights) were statistically significantly higher than in the control in male animals at 600 mg/kg bw/day. The mean liver weight relative to body
weight was
slightly higher than in the control group in male animals administered with the high dose.

In the female animals at 600 mg/kg bw/day, statistical significance was detected at the slightly lower mean thymus weight and at the slightly higher mean spleen weight relative to body weight.
The slight changes in the mean spleen weights were in accordance with macroscopic observations in male animals. Histological examinations revealed
congestion in the spleen without other pathological changes therefore it was judged to be a postmortem alteration without toxicological significance.
The slight differences in weights of some organs (testes, thymus, liver) with respect to their control were judged to be toxicologically not relevant but
indicative of individual variations.

Histopathology

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 600 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 (600 mg/kg/bw/day) or in the spleen at 60, 200 or 600 mg/kg bw/day.
In dead female animal at 60 mg/kg bw/day (1/1), histological investigation revealed hepatic necrosis in connection with the fetal death, as the probable cause of moribund condition and death.
In the male animals belonging to the test item treated and control groups (12/12 control; 1/1 at 60 mg/kg bw/day; 2/2 at 200 mg/kg bw/day; 12/12 at 600 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 (12/12 control; 2/2 at 200 mg/kg bw/day; 12/12 at 600 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 (1/10 control dam; 1/1 at 60 mg/kg bw/day, 1/10 dam at 600 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.
Congestion was observed in the endometrium without inflammatory lesions and in connection with the pregnancy in dead female animal at 60 mg/kg bw/day
(1/1).

In accordance with macroscopic observation on spleen (enlarged, dark colored), histological investigation revealed congestion, without other pathological
findings (for example hyperplasia, increased extramedullary hemopoiesis, inflammation, tumor cells etc.) in the spleen – 4/12 male at 600 mg/kg bw/day.
The congestion in the splenic red pulp could be in connection with a possible incomplete exsanguination of the organ, and could be considered as a
postmortem phenomenon without toxicological significance.
In animals selected for full histological examinations, minimal alveolar emphysema in the lungs (1/5 control female, 1/5 male and 1/5 female at
600 mg/kg bw/day) and acute hemorrhage in the thymus (2/5 control male, 3/5 male at 600 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 (3/5 male and 2/5 female) and 600 mg/kg bw/day (1/5 male) groups.
Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Mesothelioma in two male animals (1/1 at 200 mg/kg bw/day and 1/1 at 600 mg/kg bw/day) and ovarian cyst in one control female animal (1/10) were
considered to be spontaneous individual disorder occurring also in not treated experimental rats.
Histological examination did not reveal abnormal finding to correlate with necropsy observation: pale kidneys (1/10 dam) and yellowish-brown lentil-size hard formation in the ovary (1/2 non-pregnant) at 600 mg/kg bw/day, both.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines,
the liver (except hematoma), 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.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity of male/female rats
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for reproductive performance of male/ female rats
Key result
Critical effects observed:
no
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 (cold and/or not suckled, and one pup had bite marks) was the highest in the mid dose group (200 mg/kg bw/day).
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 mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no differences between the control and test item treated (60, 200 or 600 mg/kg bw/day) groups in the survival indices. There were no significant differences between the control and test item treated groups (60, 200 or 600 mg/kg bw/day) in the mean number of dead offspring (including missing pups) per litter.
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 (60, 200 and 600 mg/kg bw/day) on postnatal days 0, 4 and 13.
Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the higher male pup weights at 60 mg/kg bw/day for the lower female pup weights at 600 mg/kg bw/day and on postnatal day 4. The difference with respect to the control was minor therefore it was considered to be toxicologically not relevant.
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:
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
The anogenital distance (male and female) or nipple retention (male) were not affected.
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.
Four pups were subjected to necropsy on postnatal day 0. Two male pups in the control group and one male pup in the 200 mg/kg bw/day had no macroscopic changes in the organs or tissues and the lung flotation test was negative referring to intrauterine death of these offspring. In the fourth pups at 60 mg/kg bw/day, autolyzed visceral organs were detected on post-natal day 0. Therefore determination of stillborn or liveborn was not feasible for this pup.

There were no macroscopic changes in the organs or tissues in one female dead offspring at 600 mg/kg bw/day subjected to necropsy on postnatal day 1.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
Mortality

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were
no differences between the control and test item treated (60, 200 or 600 mg/kg bw/day) groups in the survival indices. There were no significant
differences between the control and test item treated groups (60, 200 or 600 mg/kg bw/day) in the mean number of dead offspring (including missing pups)
per litter.

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 (cold and/or not suckled, and one pup had bite marks) was the highest in the mid dose group
(200 mg/kg bw/day).


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 60, 200 and 600 mg/kg bw/day).
The anogenital distance, both absolute and normalized, in male and female rats of all the treated groups were consistent with the control.
Nipples/areoles were not visible in any of the examined male offspring in the control or 60, 200 or 600 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 (60, 200 and 600 mg/kg bw/day) on postnatal days 0, 4 and 13.
Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for
the higher male pup weights at 60 mg/kg bw/day for the lower female pup weights at 600 mg/kg bw/day and on postnatal day 4. The difference with
respect to the control was minor therefore it was considered to be toxicologically not relevant.

Necropsy

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Four pups were subjected to necropsy on postnatal day 0. Two male pups in the control group and one male pup in the 200 mg/kg bw/day had no
macroscopic changes in the organs or tissues and the lung flotation test was negative referring to intrauterine death of these offspring. In the fourth pups
at 60 mg/kg bw/day, autolyzed visceral organs were detected on post-natal day 0. Therefore determination of stillborn or liveborn was not feasible for this
pup.

There were no macroscopic changes in the organs or tissues in one female dead offspring at 600 mg/kg bw/day subjected to necropsy on postnatal day 1.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for F1 Offspring
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present study, p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) administered at 60, 200 or 600 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: 600 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats:600 mg/kg bw/day

NOAEL for F1 Offspring: 600 mg/kg bw
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 60, 200 and 600 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.

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) wasadministered orally (by gavage) once daily at 0 (vehicle only),60, 200 and 600 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 of12, 40 and 120 mg/mL calculated by the active ingredient content andcorresponding to a 5 mL/kg bw dosing volume.A group of vehicle (distilled water) 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.

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was stable indistilled waterin concentrations of ~1 mg/mL and ~200 mg/mL at room temperature for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) concentrations in the dosing formulations varied within the range between91 % and 99 %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 51 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 16, i.e. up to the day before necropsy (altogether for 50-65 days; one female animal at 60 mg/kg bw/day was administered for 42 days due to its early death). 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.

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 possible 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 at termination on post-partum/ post-natal day 13 and from all parent male animals at termination.

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 dam at 60 mg/kg bw/day. In addition, uterus of one female at 60 mg/kg bw/day, and the spleen in male animals at60, 200 or 600 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 60, 200 or 600 mg/kg bw/day groups during the course of study (male and female).

One female animal at 60 mg/kg bw/day died on gestation day 24 (Day 42) probably due to fetal deaths. Histopathological examination revealed hepatic necrosis in connection with the dead fetuses.

 

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 (60, 200 or 600 mg/kg bw/day) during the entire treatment period.

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.

 

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 60, 200 or 600 mg/kg bw/day during the entire treatment period. A slightly depressed body weight gain was observed at 600 mg/kg bw/day (male and female) resulting in only slight body weight difference with respect to their control (<10 %) therefore these slight changes in bodyweight gain were considered to be toxicologically not relevant.


Food consumption

The mean daily food consumption was not adversely affected at 60, 200 or 600 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). In compliance with the slight body weight changes, minor and transient differences with respect to the control were observed at 600 mg/kg bw/day, which however were judged to be toxicologically not relevant.

 

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (60, 200 and 600 mg/kg bw/day).

 

Reproductive performance

There were no toxicologically relevant differences in the evaluated delivery parameters of dams between the control and test item treated groups (60, 200 and 600 mg/kg bw/day). A slightly lower percentage of implantations, higher percentage of pre-implantation loss and total intrauterine mortality at 600 mg/kg bw/day were judged to be of little or no toxicological significance as the mean values were comparable with the historical control.

The examined parameters of reproductive ability were comparable in the control and in 60, 200 and 600 mg/kg bw/day groups (male and female).

 

Hematology

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 60, 200 and 600 mg/kg bw/day.

 

Clinical chemistry

The bile acid concentration was elevated in male and female animals at 200 and 600 mg/kg bw/day with respect to their control indicative some disturbances in the enterohepatic circulation or the hepatobiliary system.

 

Necropsy

Macroscopic findings related to the effect of the test item were not found in male and female animals at 60, 200 and 600 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 and60, 200 and 600 mg/kg bw/dayP-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) groups at the end of the treatment period.

 

Histopathology

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 600 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 600 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, p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) administered at 60, 200 or 600 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:                 600 mg/kg bw/day

 

NOAEL for reproductive performance of male/ female rats: 600 mg/kg bw/day

 

NOAEL for F1 Offspring:                                                          600 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Under the conditions of the present study, p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) administered at 60, 200 or 600 mg/kg bw/day by oral gavage did not impair the development of the F1 offspring from birth to post-natal day 13 at any dose level after repeated oral administration of dams (Hsd.Han: Wistar rats).

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

NOAEL for F1 Offspring:       600 mg/kg bw

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the OECD 422 study, the substance p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) is considered not toxic for reproduction according to the CLP regulation 1272/2008/EC.

Additional information