Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity of the test substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimnetal Phase: 07 December 2017 to XXXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
There was a deviation to the study plan for thyroid hormone assessment relating to sampling; however, the deviation was no considered to have affected the scientific integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST strain rats

- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.

- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males approximately 11 weeks old, females approximately 12 weeks old
- Weight at study initiation: Males 278 to 362g, females 192 to 231g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over-trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum (Supplied from polycarbonate bottles attached to the cage containing mains drinking water supply)
- Acclimation period: 19 days
DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis were available for the diet. All diet and water used on the study was considered to be of acceptable quality and not to have interfered with the outcome of the study.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the test facility maintained rodent facility.
- Temperature (°C): 19 - 25ºC
- Humidity (%): 30 -70%
- Air changes (per hr): Fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.
- Environmental Enrichment: Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

IN-LIFE DATES: February 2018 (first day of treatment) to 01 April 2018 (final day of necropsy).

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on two occasions and analysed for concentration of the test substance by HPLC. The results indicated that the prepared formulations were within 89-106 % of the nominal concentration.

The test substance in Arachis Ol BP at concentrations of 2.5 mg/L and and 250 mg/L was confirmed to be stable for up to 28-days when stored refrigerated.
Duration of treatment / exposure:
Approximately six weeks for males and up to eight weeks for females.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control Group
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
12 male and 12 female
Control animals:
yes, concurrent vehicle
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase, females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination (Day 43 for males and Day 13 post partum for females).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: five males amd five females


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination (Day 43 for males and Day 13 post partum for females).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: five males and five females

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Battery of functions tested: sensory activity / forelimb and hindlimb grip strength / motor activity

IMMUNOLOGY: No

OTHER: Blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analysed for Thyroxine (T4).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, and then an additional internal examination was performed.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin:


Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Cecum
Colon
Cowpers Glands
Duodenum
Esophagus
Eyes
Glans Penis
Gross lesions
Heart Ileum (including peyer’s patches)
Jejunum
Kidneys
LABC (levator ani-bulbocavernous) muscle
Liver
Lungs (with bronchi)
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating Epididymides gland)
Skin Spinal cord (cervical, mid-thoracic and lumbar)
Spleen
Stomach
Testes
Thyroid/Parathyroid
Trachea
Thymus
Urinary bladder
Uterus & Cervix (with oviducts)
Vagina







Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

Data were analysed using the decision tree from the Provantis ™ Tables and Statistics Module.

Data not analysed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment.

Eight males treated with 1000 mg/kg bw/day displayed increased salivation throughout the study and six females treated with 1000 mg/kg bw/day also showed increased salivation throughout the study, albeit at a much lower frequency than males. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered to represent the distaste of the test item rather than an adverse effect of treatment.

Two males treated with 300 mg/kg bw/day showed an isolated incident of noisy respiration. At this low frequency and in the absence of a similar effect at 1000 mg/kg bw/day, this was considered to indicate difficulty in dosing these particular animals on these occasions.

Incidental findings, unrelated to treatment included one control male with an open wound and scabbing between Days 22 and 29, one control male with a scab between Days 22 and 29, one female treated with 75 mg/kg bw/day with generalised fur loss from Day 36 to termination, one female treated with 300 mg/kg bw/day with a scab and generalised fur loss on Days 5 and 6, and one female treated with 300 mg/kg bw/day with staining around the right eye from Day 46 to termination.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 300 mg/kg bw/day was found dead on Day 12. This female did not show any clinical signs prior to being found dead and no macroscopic or microscopic abnormalities were observed to determine the cause of death. In isolation, this was considered unrelated to treatment.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reticulocytes for males treated with 300 and 1000 mg/kg bw/day were statistically significantly higher (p<0.01) compared to controls. However, in the absence of any supporting histopathological correlates in male animals, this was considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males treated with 75 mg/kg bw/day showed a statistically significant increase (p<0.01) in cholesterol compared to controls. All individual values were within the historical control range; whereas one individual control value was lower than the historical control range. In the absence of a similar effect at 300 or 1000 mg/kg bw/day or any associated histopathological correlates, the intergroup difference was considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Ovary weights, both absolute and relative to terminal body weight were statistically significantly higher (p<0.01) for females treated with 300 and 1000 mg/kg bw/day when compared to controls. In the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were considered of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings which were considered unrelated to treatment, included one control female and one male treated with 75 mg/kg bw/day with a pale area on the liver, one female treated with 75 mg/kg bw/day with a small thymus, one male treated with 300 mg/kg bw/day with a discoloured and pale liver and a dark spleen, one male treated with 1000 mg/kg bw/day with a discolored and pale liver, and one male treated with 1000 mg/kg bw/day with a dark spleen.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the spleen increased hematopoiesis was present in all treated female groups when compared to controls. The changes in the spleen were of low grade severity. In the absence of organ weight change, obvious toxicity or changes in the bone marrow the change in the spleen of females treated with the test item at all doses is considered to be incidental. No such effects were detected in treated males.
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects observed hence value is NOEL.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
immune system
Conclusions:
The oral administration of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester to rats by gavage, at dose levels of 75, 300 and 1000 mg/kg bw/day, did not result in any adverse treatment-related effects.

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males. A No Observed Adverse Effect Level (NOAEL) for females was considered to be 1000 mg/kg bw/day due to microscopic spleen changes evident in treated females which were considered to be incidental in nature.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test substance, following repeated dosing.  The assessment of repeated dose toxicity was made as part of a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test which also assessed potential adverse effects of the test item on reproduction. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

 

Method

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 75, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same period.

 

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum.

 

Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals for thyroid hormone analysis and samples from the adult males were analysed for Thyroxine (T4).

 

All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

 

Results

 

(Adult Responses)

 

Mortality

There were no treatment-related deaths.

 

One female treated with 300 mg/kg bw/day was found dead on Day 12. This female did not show any clinical signs prior to being found dead and no macroscopic or microscopic abnormalities were observed to determine the cause of death. In isolation, this was considered unrelated to treatment.

 

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 75, 300 and 1000 mg/kg bw/day.

 

Behavioural Assessment

Behavioural Assessments did not indicate any effect of treatment for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 75, 300 or 1000 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 75, 300 or 1000 mg/kg bw/day.

 

Body Weight

There was no effect of treatment on bodyweight or body weight gains for males at 75, 300 or 1000 mg/kg bw/day throughout the study. No effect in body weight development was evident in females during maturation, gestation or lactation at 75, 300 or 1000 mg/kg bw/day.

 

Food Consumption

There was no effect of treatment on food consumption for males at 75, 300 or 1000 mg/kg bw/day throughout the study. No effect on food consumption was evident in females during maturation, gestation or lactation at 75, 300 or 1000 mg/kg bw/day.

 

Water Consumption

There was no effect of treatment on water consumption for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Laboratory Investigations

 

Haematology

Assessment of haematology parameters did not indicate any obvious effect of treatment for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Blood Chemistry

Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Pathology

 

Necropsy

Neither the incidence nor the distribution of macroscopic necropsy findings for adults indicated any obvious effect of treatment at 75, 300 or 1000 mg/kg bw/day.

 

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males did not identify any obvious effect of treatment or indication of endocrine disruption at 75, 300 or 1000 mg/kg bw/day.

 

Organ Weights

Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 75, 300 or 1000 mg/kg bw/day.

 

Histopathology

The following treatment-related microscopic abnormality was detected:

 

Spleen: Increased haematopoiesis was present in all treated female groups when compared to controls.

 

Conclusion

The oral administration of 2-Propenoic acid, 2-[[(octadecylamino)carbonyl]oxy]ethyl ester to rats by gavage, at dose levels of 75, 300 and 1000 mg/kg bw/day, did not result in any adverse treatment-related effects.

 

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day for males. A No Observed Adverse Effect Level (NOAEL) for females was considered to be 1000 mg/kg bw/day due to microscopic spleen changes evident in treated females which were considered to be incidental in nature.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification