Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine

EC Number:

241-924-6

EC Name:

Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine

Cas Number:

18016-43-8

Molecular formula:

C38H69N3O – C58H111N3O3

IUPAC Name:

(9Z)-octadec-9-enoic acid; bis(2-aminoethyl)amine

Details on test material:

- Name of test material (as cited in study report): Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine
- Physical state: liquid
- Analytical purity: Reaction product of Oleic acid and N-(2-aminoethyl)ethane-1,2- diamine: Elemental-analysis yielded 100.4 g/100g
"Triamide"-content : 5.2 g/100g determined by HPLC-analysis (for details see analytical report No. 11L00415)
- Lot/batch No.: 11000829U0
- Stability under test conditions: The stability of the test item under storage conditions over the test period will be guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Ambient (RT)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A.
- Age at study initiation: 10-11 weeks (7-8 weeks at delivery)
- Weight at study initiation: 219 - 226 g for males and 163 - 170 g for females (upon delivery)
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polycarbonate cages measuring 42.5x26.6x18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
After mating the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages measuring 42.5x26.6x18 cm for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet: ad libitum
- Water :ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 2°C
- Humidity (%): 55% +- 15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each concentration (concentrations of 20, 62.5 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was a suitable vehicle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Week 1 and last Week of treatment were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.

Duration of treatment / exposure:

Males:
Animals were dosed once a day, 7 days a week for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Day 35 of study). Males were treated for a total of 34 days.
Females:
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 80, 250, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in consultation with the Sponsor based on the results of a 14-day range finding study in Wistar rats, in which dose levels of 0, 300, or 1000 mg/kg body weight/day were administered orally. Males dosed at 1000 mg/kg bw/day had a distinctly decreased body weight gain compared to controls and males dosed with 300 mg/kg bw/day (body weight gains from day 0 to 13 were 18.1, 19.8, and 1.1 g at ascending dose levels). Additionally, males at 1000 mg/kg bw/day showed body weight loss between day 10 and 13 (2.4 g). Therefore the dose of 1000 mg/kg bw/day was considered to be too high as top dose for the present study.

- Rationale for animal assignment: random

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/ DETAILED CLINICAL OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.


BODY WEIGHT: Yes
Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Body weight data of females during pairing phase were not reported in this report, but will be archived with all raw data.

FOOD CONSUMPTION:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY INVESTIGATIONS
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation.

The blood samples collected were divided into tubes as follows:

EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests
No anticoagulant for hormone assay


HAEMATOLOGY: Yes
Haematocrit
Haemoglobin
Red blood cell count
Red blood cell distribution width
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation test
Prothrombin time
Activated partial thromboplastin time

Blood sample for animal no. 93790046 was not analysed since it was coagulated.

CLINICAL CHEMISTRY: Yes
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Inorganic phosphorus
Total bilirubin
Total cholesterol
Bile acids
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Values of gamma-glutamyltransferase below the limit of quantification are reported in the appendix as NT (Not Taken).

Hormone determinations

Prior to necropsy, blood samples (approximately 1.2 mL) were taken from the abdominal vena cava of the same animals and under the same conditions mentioned in section 4.4 and from 5 male and 5 female spare untreated rats from the same batch.
Blood samples were collected into tubes without anticoagulant, centrifuged and the serum obtained was divided in several aliquots and frozen at -80°C for possible hormone determinations (T3, T4). These measurements were not performed, since no compound-related effects on the thyroids were observed.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observation Battery Tests

Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination.
Each animal was removed from the home cage and observed in an open arena.
The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.
Animals were examined in an open arena for a minimum of three minutes. Observed parameters, described by an evaluation scale, are indicated below:

Removal (from cage): Easy, Difficult, Very difficult
Handling reactivity: Normal, Slow, Moderate, Marked
Lachrymation: Absent, Slight, Marked
Palpebral closure: Absent, Slight, Moderate, Marked
Salivation: Absent, Slight, Marked
Piloerection: Absent, Present
Rearing: Absent, Intervals of number of times (i.e. 1-3, 4-7, 8-10)
Spasms: Absent, Tonic spasms, Clonic spasms, Tonic-clonic spasms
Myoclonia: Absent, Present
Mobility impairment: Absent, Slight, Moderate, Marked
Arousal (animal activity): Very slow, Slow, Normal, Moderate, Marked
Vocalisation: Absent, Present
Stereotypies: Absent, Present
Unusual respiratory pattern: Absent, Present
Bizarre behaviour: Absent, Present
Urination: Absent, Intervals of number of times (i.e. 1-3, 4-6)
Defecation: Absent, Intervals of number of times (i.e. 1-3, 4-6)
Tremors: Absent, Present
Gait (one of the following options): Normal
Ataxia (Slight, Moderate, Marked)
Hunched posture (Slight, Moderate, Severe)
Pronation
Forelimbs drag (Slight, Moderate, Marked)
Hindlimbs drag (Slight, Moderate, Marked)

All observed parameters are reported in a group incidence table. Individual data are not included in this report. Data are reported until Week 6 of study (all females).

Grip strength and sensory reactivity to stimuli

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. For males the tests were performed on Day 31 of the study and for females on Day 3 post partum.

Motor activity assessment (MA)

Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for 60 minutes) by an automated activity recording device. Measurement was performed using a computer generated random order. For males the test was performed on Days 29 and 30 of study and for females on Day 3 post partum,

Sacrifice and pathology:

Necropsy

Parental animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.

Parental Males

The males were killed after the mating of all females, after a total of 34 days of treatment.

Parental Females

The females with live pups were killed on Day 4 post partum.
The females which did not give birth 25 days after positive identification of mating were killed shortly after (Day 26 post coitum).


The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding animal found dead: 93790062) and the required tissue samples preserved in fixative and processed for histopathological examination.

Females
All females were examined also for the following:

a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Pups
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities, sex confirmation was performed by gonadal inspection.

Tissues fixed and preserved

Samples of all the tissues (all parental animals) listed in the table (ü) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in the table (ü). After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides of all males in the control and high dose groups were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

The examination was restricted as detailed below:

a) Reproductive organs: cervix, clitoral gland, ovaries, uterus and vagina from all parental females
b) Reproductive organs: coagulating glands, epididymides, preputial gland, prostate gland, seminal vesicles and testes from all parental males
c) Tissues specified in the table from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose groups killed at term.
d) Tissues specified in the table from the animals (no. 62) dying during the treatment period.
e) All abnormalities in all groups.

On the basis of the histopathological changes observed between control and high dose groups (5 males/group), the examination was extended to the thymus of the remaining males of the control and high dose groups and in all males of Groups 2 and 3.

Other examinations:

Organ weights

From all animals completing the scheduled test period, the organs indicated in the table (ü) were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test when ‘n’ was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
One male (no. 62) receiving 800 mg/kg bw/day was found dead on Day 6 of study.
Salivation was the main clinical sign observed on Day 3 while no clinical signs were observed the day before death.
Macroscopically, changes consisted of: dark red lungs, dark thymus and cervical lymph nodes.
Microscopically, the main changes consisted of presence of: exudate in the lumen of the trachea, associated with necrosis of the mucosa and subchronic inflammation in the submucosa; presence of exudate in the lumen of the pulmonary bronchi. Other observed lesions included: atrophy of the lymphoid tissue in the thymus, cervical and mesenteric lymph nodes, atrophy of the spleen and congestion of the adrenals.
The cause of death of this animal is suggested to be related to complications associated with mis-dosing.

Two females were found not pregnant: one control and one receiving 800 mg/kg bw/day.
The number of females with live pups on Day 4 post partum was 9 in the control, 10 in the low dose (80 mg/kg bw/day), 10 in the mid-dose (250 mg/kg bw/day) and 9 in the high dose group (800 mg/kg bw/day).

At the daily clinical examination, the following observations were recorded:
- Treated males receiving the dose levels ≥ 250 mg/kg bw/day showed salivation throughout the study. In particular, this sign involved all treated males receiving 800 mg/kg bw/day where hair loss was also noted. In addition, rales were also occasionally noted in a few treated males of the highest dose levels. This clinical sign was not considered to be compound-related due to its transient appearance.
The incidence and the nature of the other clinical signs recorded (staining on the body surface and scabs) were not considered toxicologically significant.
- Before the mating period, treated females (7/10) receiving 800 mg/kg bw/day showed salivation. Occasionally one female receiving 80 mg/kg bw/day showed the same sign.
Hairloss was recorded in 6 out of 10 treated females receiving 800 mg/kg bw/day.
Rales were also noted in two females receiving 250 (no. 47) and 800 (no. 61) mg/kg bw/day for one day.
- During the post coitum period, salivation and hair loss were still noted in most females receiving 800 mg/kg bw/day.
Rales were again noted for one day in the female (no. 47) receiving 250 mg/kg bw/day.
- During the post partum period, the incidence of females affected by salivation and hairloss decreased compared to the other periods of the study. In the female (no. 47) receiving 250 mg/kg bw/day rales persisted for two days.

BODY WEIGHT AND WEIGHT GAIN
Means of body weight and body weight gain were comparable between control and treated males throughout the study.

Slightly lower body weights were noted before pairing (Days 8 and 15 of study) and during the post coitum period (Days 7 and 14) in females receiving 250 mg/kg bw/day. These changes did not exceed -6%. During post partum period, the body weight of treated females was comparable to the control group.

Body weight gain of treated females did not show any differences between controls, during both post coitum and post partum periods.

Terminal body weight was unaffected by treatment in both sexes.

FOOD CONSUMPTION
Food consumption was unaffected by treatment in both sexes during the study.

HAEMATOLOGY
Slightly lower lymphocyte and monocyte counts were recorded in males dosed with 800 mg/kg bw/day. Changes were approximately 33% when compared with controls.
Lymphocyte counts were also lower in males treated with 250 mg/kg/day (26%). Relative neutrophil counts were statistically significantly higher (approximately 50 to 60%) and relative lymphocyte counts were statistically significantly lower (approximately 10 to 15%) in males at 800 and 250 mg/kg bw/day, without apparent dose-dependency. Since only one sex was affected and the values were within the historical control range of the rat strain used, this differences were considered not to be toxicologically relevant.
Females did not show any relevant changes. Red blood cell counts and haematocrit values were statistically significantly higher at 250 mg/kg bw/day, without dose-dependency.

Coagulation

A slightly lower activated partial thromboplastin time was recorded in animals treated with 800 mg/kg bw/day (9% in males and 16% in females; statistically significant in males only). The values were within the historical control range of the rat strain used. Therefore, and due to the low magnitude of the difference this finding was considered of no toxicological importance.

CLINICAL CHEMISTRY
The most relevant finding observed was the lower creatinine concentration in animals receiving 800 mg/kg bw/day (35% in males and 22% in females). This finding was considered not to be toxicologically relevant, since lower, and not higher values were recorded.
In addition, glucose was lower in males dosed with 250 and 800 mg/kg bw/day, with no dose-relation (approximately 23%). Since only one sex was affected and the values were within the historical control range of the rat strain used, this differences were considered not to be toxicologically relevant.
Sodium levels were statistically significantly higher in treated males. The difference was of minimal magnitude (1%), therefore considered of no toxicological significance.

NEUROBEHAVIOUR
Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Motor activity and sensory reaction to stimuli measurements recorded at the end of treatment were comparable between control and treated groups in animals of both sexes.
The statistically significant differences noted in land foot splay in treated females receiving 80 and 250 mg/kg bw/day and in grip strength in females at 250 mg/kg bw/day were considered incidental, since they were observed in one sex only and without any correlation with the dose.

ORGAN WEIGHTS
Some statistically significant differences were noted in the absolute or relative organ weight, such as:
- Lower absolute brain and prostate weights in males receiving 800 mg/kg bw/day (-5%, and -16%, respectively).
- Higher relative adrenals weight in females receiving 800 mg/kg bw/day (+16%).

All the above differences were of low magnitude and not accompanied by histological findings. Therefore, they were considered not treatment-related.

GROSS PATHOLOGY
No treatment-related changes were noted.
A range of lesions were seen, mostly having a comparable incidence in control and treated animals and therefore are not considered as related to treatment.

HISTOPATHOLOGY
The only changes noted in the thymus of the males treated with the high dose consisted of mild atrophy of the cortex. This change was seen in 4/9 terminally killed animals from the high dose, compared to 1/10 case seen in the concurrent control group. This change is suggested to reflect a stress-related effect.
A range of lesions were seen, mostly having a comparable incidence in control and treated animals and therefore are not considered as related to treatment. Examples of incidental lesions included nephropathy, inflammatory cell foci in the liver and atrophy of the thymus in the female groups.

Spermatogenic cycle
No alterations were noted.

A detailed qualitative examination of the testes was performed on 10 animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subjected Russell, 1990; Creasy, 1997; Creasy, 2002.
The PAS-stained sections were used to identify the spermatogenic stages.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant systemic effects were observed

2

Dose descriptor:

NOEL

Effect level:

80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: non adverse local effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The purpose of this study was to generate information on toxic effects on rats after repeated oral dosing with Oleic acid, compound with N-(2-aminoethyl)ethane-1,2-diamine (80, 250 and 800 mg/kg bw/day), as well as on effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring.

 

Males

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 34 days.

During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated.

Clinical pathology investigations (haematology and clinical chemistry) were also evaluated.

At necropsy a detailed external and internal examination was performed.

The histopathological examination was carried out in five males of control and high dose groups randomly, selected. The identification of the stages of the spermatogenic cycle was performed in all males of the control and high dose groups.

In addition, reproductive organs such as coagulating glands, epididymides, preputial gland, prostate gland, seminal vesicles and testes were examined histopathologically on all parental males.

Since histopathological changes were observed in control and high dose males, the examination was then extended to the thymus of the remaining males of the control and high dose groups and in all males of Groups 2 and 3.

 

One male receiving 800 mg/kg bw/day was found dead on Day 6 of study. The cause of death of this animal is suggested to be related to complications associated with mis-dosing.

Hair loss and salivation were the main clinical signs noted throughout the study in males receiving the dose levels ≥ 250 mg/kg bw/day.

No differences in body weights and food consumption were observed in treated males compared to the control group.

No adverse findings were recorded in clinical pathology investigations (haematology and clinical chemistry).

Fertility index and copulatory index were unaffected by treatment.

No relevant differences were recorded in the absolute and relative organ weights of treated animals.

No treatment-related changes were noted at macroscopic and microscopic observations.

 

Females

Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum.

During the in-life phase, body weight, body weight gain, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption and mating performance were evaluated.

Clinical pathology investigations (haematology and clinical chemistry) were also evaluated.

 

The histopathological examination was carried out in five females of control and high dose groups randomly selected. In addition, reproductive organs such as cervix, clitoral gland, ovaries, uterus and vagina were examined histopathologically on all parental females.

Clinical signs of pups as well as necropsy examination of pups sacrificed at termination or unscheduled deaths were recorded.

At necropsy a detailed external and internal examination was performed. Litter data, sex ratios and gestation length were recorded.

 

Salivation and hair loss were observed in treated females receiving 800 mg/kg bw/day.

Body weight and food consumption did not show relevant changes during the in-life phase.

Fertility index and copulatory index were unaffected by treatment and litter data parameters and sex ratio did not show differences.

No changes of toxicological significance were recorded at haematological and clinical chemistry evaluations.

Absolute and relative organ weights were unaffected by treatment.

No treatment-related changes were noted at macroscopic and microscopic observations.

 

Evaluation

 

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for reproductive and developmental toxicity was considered to be 800 mg/kg bw/day for males and females.

 

Based on the occurrence of salivation, which might be a local reaction to irritancy or taste of the test compound, the NOEL (No Observed Effect Level) was considered to be 80 mg/kg bw/day.