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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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:

- 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 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

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurs or 14 days had elapsed.

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

Details on study schedule:

N/A

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

Parental animals: Observations and examinations:

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.

PARTURITION AND GESTATION LENGTH

A parturition check was performed from Day 20 to Day 25 post coitum.
Females which did not gave birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0 post partum.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation is made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

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.

Litter observations:

As soon as possible, after parturition is considered complete (Days 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.

Postmortem examinations (parental animals):

Necropsy

Parental animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.

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.

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.

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.

Postmortem examinations (offspring):

SACRIFICE
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.

GROSS NECROPSY
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.

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.

Reproductive indices:

Copulatory Index, Fertility Index, Pre-coital interval, Pre- implantation loss, Pre-birth loss

Offspring viability indices:

Pup loss at birth, Cumulative pup loss on Day 4 post partum, Sex ratios were calculated at birth and on Day 4 post partum

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
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.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Gestation periods were similar in control and treated groups. All dams with the exception of two not pregnant females (one in control and one high dose groups) gave birth between Days 21 and 22 post coitum (mean value).
Corpora lutea, implantations, pre-implantation loss data, total litter size and pre-birth loss (percentage) were, in general, similar in control and treated groups. In particular, pre-birth loss data was higher in females receiving 80 mg/kg bw/day when compared to the control group. However, this change was not considered of toxicological significance since this it was attributable to two low-dose females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

systemic, reproduction and development

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant effects were observed

Results: F1 generation

Details on results (F1)

VIABILITY / BODY WEIGHT (OFFSPRING)
No differences between control and treated groups were recorded in litter data parameters and sex ratios, at birth, on Day 1 and Day 4 post parum.

CLINICAL SIGNS (OFFSPRING)
Clinical signs noted in pups throughout the study were comparable across groups and considered unrelated to treatment.

GROSS PATHOLOGY (OFFSPRING)
No abnormalities were recorded in the decedent pups.
Malrotated left forepaw noted in one pup of the mid-dose group (250 mg/kg bw/day) was considered incidental and not treatment related.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant effects were observed

Overall reproductive toxicity

Reproductive 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.