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Toxicological information

Toxicity to reproduction

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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:
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

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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: 221 - 234 g for males and 160 - 182 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: 13 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 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched was suspended in the vehicle (corn oil) and brought to the final volume appropriate for each concentration (concentrations of 10, 31.25 and 100 mg/mL).
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) with one exception. One control male (no. 93810016) died during the pre-pairing period. Therefore, the assigned female (no. 93810015) was mated with the proven male (no. 93810018) of the same treatment group. For this female, the mating phase started on Day 16 of the study instead of Day 15 of the other animals. In addition, also one high dose female (no. 93810069) died during the pre-pairing period. Therefore, the assigned male (no. 93810070) was not assigned to mating with any 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 occurred.
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 10 to 100 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.

Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method, in the range from 5 to 200 mg/mL. The software used for this activity was Analyst 1.5.2 (Ab Sciex).
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.

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, 40, 125, 400 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: Dose levels had been selected in consultation with the Sponsor and were based on the results of a 14-day oral range-finding study with groups of four male and female rats, in which all animals had to be sacrificed in moribund condition on day 3 of the study at a daily dose of 1000 mg/kg bw/day. The dose of 300 mg/kg bw/day was tolerated. Based on these results, the dose of 400 mg/kg bw/day was selected as the high dose for the present study.

- Rationale for animal assignment (if not random): 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.
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.

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 give 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 were first detected in the cage was considered Day 0 post partum.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was 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 evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, 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 subject Russell, 1990; Creasy, 1997; Creasy, 2002.
The PAS-H 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.
Litter observations:
As soon as possible, after parturition was 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 dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
Euthanasia

Parental animals were killed by exsanguination under isofluorane anaesthesia.
Pups were euthanised (Day 4 post partum) by intraperitoneal injection of Sodium Thiopenthal.

Parental Males

The males were killed after the mating of all females (after 32-33 days of treatment).

Parental Females

The females with live pups were killed on Day 4 post partum.
One female with total litter loss was killed on the day of the occurrence of total litter loss (Day 2 post partum).
The females which did not give birth 25 days after positive identification of mating were killed shortly after.

Necropsy

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 animals sacrificed for humane reasons or found dead) 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 or uteri of females with no visible implantations were immersed in a 10-20% 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 were examined for external abnormalities and sex confirmation 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, Harderian glands, 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.

In the first instance the examination was restricted as detailed below:

a) Reproductive organs: cervix, clitoral gland, ovaries, uterus and vagina from all parental females
b) Reproductive organs: coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes from all parental males
c) Tissues specified in the table (ü) from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
d) Tissues specified in the table (ü) from all animals dying during the treatment period
e) All abnormalities in all groups

The examination was extended to include the lungs, including mainsten bronchi (both sexes), thymus and jejunum (females only) from the remaining animals of the control and high dose groups. The examination was also extended to the thymus (females only) from all other dose groups.
Postmortem examinations (offspring):
SACRIFICE
Pups were euthanised (Day 4 post partum) 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 were examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was 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 animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Two cases of premature death occurred during the study. One male of the control group was found dead on Day 2 of treatment but the cause of death could not be determined. One female of the high dose group was found dead on Day 3 of treatment and the cause of death was considered as a consequence of mis-dosing.

Two females were found not pregnant: one control and one mid-dose.
One high dose female was sacrificed for total litter loss on Day 2 post partum.
The number of females with live pups on Day 4 post partum was: 9 in each of the control and mid-dose groups (125 mg/kg bw/day), 10 in the low dose group (40 mg/kg bw/day) and 8 in the high dose group (400 mg/kg bw/day).

Salivation and decreased muscle tone were the main clinical sign observed in all high dose males throughout the study and in all high dose females during post coitum and post partum periods. Salivation was considered most probably related to the taste of the compound and therefore considered not to be adverse. Decreased muscle tone occurred as an isolated finding, since no alterations were observed in the functional observation battery tests, especially in the grip strength and foot splay tests, and was therefore not considered to be toxicologically relevant. In addition, rales were also occasionally noted in 5 out of 10 treated males and females of the high dose group. This clinical sign was not considered to be compound-related due to its transient appearance. Due to the low incidence, other clinical signs recorded were considered of no toxicological importance

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males of the high dose group showed a slight decrease in body weight during the first week of administration (approximately 13 g). Body weights were slightly, but statistically significant reduced (approximately 8%) in the high dose males at the end of Weeks 3 and 4 of treatment, when compared to controls. However, these changes were mainly attributable to one male (no. 93810066), which had clinical signs, particularly lower body weights, and histopthological changes suggestive for mis-dosing.
No difference in body weight was detected in treated females during the whole study, when compared to controls.
Body weight gain of treated animals did not show differences of toxicological importance, when compared to controls.
Terminal body weight was slightly decreased in treated males with statistical significance in the high dose group (10%). No differences were observed in females.
Food consumption was unaffected by treatment in both sexes during the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
All females mated both in the control and treated groups.
Oestrous cycle, copulatory index and fertility index were similar in treated and control groups. Pre-coital interval was slightly higher in the high dose group (4.1 days versus 2.4 days in controls), and the number of copulation plugs was slightly lower (1.6 versus 3.1 in controls). However, these parameters were within the physiological range of the rat strain used. Therefore these differences are considered not to be toxicologically revelant.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No alterations were noted
A detailed qualitative evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, 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 subject Russell, 1990; Creasy, 1997; Creasy, 2002.
The PAS-H 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)
No significant differences were found in the number of corpora lutea, implantations, gestation length and total litter size between control and treated groups.
All dams, with the exception of two not pregnant females, gave birth between Days 21 and 23 post coitum.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Some statistically significant differences were noted in the absolute and/or relative organ weight of both sexes.

In males:
- Lower absolute seminal vesicles weight in high dose males (22%).
- Higher relative adrenals (27%), brain (15%), kidneys (15%) and testes (13%) weights in high dose males.

In females:
- Lower absolute and relative ovaries weights in low, mid- and high dose females (20%, 19% and 26% respectively for the absolute weight and 21%, 21% and 25% respectively for the relative weight).
- Lower absolute and relative thymus weight in high dose females (28% and 27% respectively).

The increased relative organ weights and the lower absolute seminal vesicle weights in males were considered to be due to the reduced terminal body weight. None of the above differences was accompanied by histological findings. Therefore, they were considered of no toxicological importance.

GROSS PATHOLOGY/HISTOPATHOLOGY (PARENTAL ANIMALS)
Detailed macroscopic observations were reported for individual animals in the study.
Microscopic evaluation was performed on 5 randomly selected animals from each of Groups 1 and 4 (males and females). In addition, the reproductive organs from all male and female animals from all groups, as well as all macroscopic abnormalities from all groups were examined histologically.
Potential treatment-related lesions were detected in the lungs, including bronchi, of both sexes and in the jejunum and thymus of the females only. The lungs were suggested to be checked in the rest of the animals of both sexes in Groups 1 and 4. Moreover, the thymus and jejunum were suggested to be checked in the rest of the females in Groups 1 and 4, while in all female animals from Groups 2 and 3, only thymus was examined.
For ease and clarity of reporting, only tissues with diagnoses have been reported in the incidence tables.

Unscheduled death

Two cases of premature death occurred during the study.
The list of the findings observed in the individual animals is as follows:
Animal no. 93810016 (male control group):
Macroscopically, the following changes were noted: caecum: distended; lungs: incomplete collapse and dark color; thymus: red color; prostate and seminal vesicle: small size.
Microscopically, the changes were as follows: liver: inflammatory cell foci; thymus and lungs: congestion. The cause of death could not be determined.

Animal no. 93810069 (female high dose group):
Macroscopically, the following changes were noted: muzzle: red staining; stomach and jejunum: red mucosa; liver: a single pale raised area; thymus: red color; trachea: a single abnormal area; lungs: incomplete collapse and dark read areas; and pancreas: pallor.
Microscopically, the changes were as follows: stomach: multifocal erosions in the glandular mucosa; liver: congestion; thymus: mild atrophy; jejunum: autholysis; trachea: mild mucosal necrosis associated with mild submucosal subchronic inflammation; lungs: bronchi - mild necrosis, associated with mild submucosal subchronic inflammation and presence of inflammatory exudate in lumen. The changes in the trachea and lungs are suggested to be related to the mis-dosing of the test item. Other changes noted in the lungs consisted of oedema and congestion and are considered as agonal changes.
The cause of death is considered as related to mis-dosing of the test item.

Terminal kill

Macroscopic observations

No treatment-related changes were noted.
Even though there was an increased incidence of reduced size of thymus when comparing the incidence in the high dose females (n=6) to the control group (n=1), histopathological evaluation did not confirm a treatment effect.

Microscopic observations

No treatment-related findings were noted.

In a single male rat (no. 93810066) from the high dose, the bronchi and the alveoli in the centriacinar areas contained mucus-like fluid. This change was associated with goblet-cell metaplasia of the epithelium lining the bronchi, peribronchial fibrosis, presence of polymorphonuclear cells and histiocytes within the mucus.
The lung changes are suggested to be related to mis-dosing of the test item.
All other observed changes are considered as incidental findings, unrelated to treatment with the test compound.
The incidence of thymic atrophy in the female groups was as follows: mild grade: 9/10; 10/10; 9/10 and 8/10, respectively in the control, low, mid and high dose. A single case of moderate degree of thymic atrophy was noted in the female high dose group. Due to almost comparable incidence of thymic atrophy seen in the control and treated groups, this lesion is not considered as related to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
systemic, reproduction and development
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects were seen up to the highest dose level tested
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY/BODY WEIGHT (OFFSPRING)
No significant differences were observed on litter data parameters and sex ratios at birth, on Day 1 and Day 4 post partum.
Litter weight was slightly lower in the high dose group, due to eight litters versus nine in controls, but mean pup weight was comparable in all groups. One female of the high dose group had total litter loss on Day 2 post partum, which occurs also spontaneously in control animals and was considered incidental, since only one animal was affected.

CLINICAL SIGNS (OFFSPRING)
No compound-related effects were observed. Apparently no food intake (milk) and small appearance were noted in pups of the control and treated groups. Pallor was also observed in a few pups of treated groups.

GROSS PATHOLOGY (OFFSPRING)
Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects were seen up to the highest dose level tested
Remarks on result:
other: Generation: P and F1 (migrated information)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The toxic effects on rats of both sexes after repeated dosing with 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated.

The dosages used were 0 (control), 40, 125 and 400 mg/kg/day.

One control male and one high dose females died during the study, the deaths being not compound-related.

One control female and one mid-dose femalewere found not pregnant at necropsy.

In addition, one high dose female was sacrificed for total litter loss on Day 2 post partum.

The number of females with live pups on Day 4 post partum was: 9 in each of the control and mid-dose groups, 10 in the low dose group and 8 in the high dose group.

Decreased muscle tone was observed in males and females of the high dose group.This finding was considered not to be toxicologically relevant, since no effects were seen in the functional observation battery tests, especially in the grip strength and foot splay tests.Males of the high dose showed a slight body weight loss during the first week of the study, and body weights remained slightly decreased compared to the other groups throughout the study. However, these changes were mainly attributable to one male, which was probably mis-dosed. No findings of toxicological significance were noted during the in-vivo phase, both in male and female animals.

No toxicologically relevant differences were found in terms of mating performance and reproductive parameters.

No significant differences were found on litter data parameters and sex ratio.

No toxicologically significant changes were observed in clinical pathological investigations.

No treatment-related changes were observed both in male and female animals at post mortem macroscopica nd microscopic examinations and organ weight analysis.

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both systemic toxicity and reproduction/developmental toxicity was considered to be 400 mg/kg/day for males and females

Applicant's summary and conclusion