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Toxicological information

Repeated dose toxicity: oral

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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: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl sebacate
EC Number:
203-431-4
EC Name:
Dimethyl sebacate
Cas Number:
106-79-6
Molecular formula:
C12H22O4
IUPAC Name:
1,10-dimethyl decanedioate
Test material form:
other: solid to liquid (melting point 26°C)
Details on test material:

Appearance Colourless solid to colourless liquid (melting point 26ºC)

Correction factor No
Storage conditions Sealed container 15 to 25°C In the dark
Additional storage conditions as detailed in Sponsor supplied documentation.
GMO status Non-GMO
Chemical class : Not provided
Archive sample :Responsibility of Covance (nominal 500 mg). Stored at 15 to 25°C. See section 8.7
Other information :A certificate of analysis will be supplied by the
Sponsor for each batch of test article.

Additional batch numbers may be used and detailed in the study records.

Surplus test article will be returned to the Sponsor prior to or on issue of the final report (unless required for additional studies).

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal information
Species, strain, supplier Rat: HsdHan™:WIST from Harlan, Bicester, UK.
Specification At start of dosing: 10 to 12 weeks old.
Justification A readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics.

Environment
Housing : Cages conforming to the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989).
Housing density: Groups of up to four (pre-pairing and post-pairing), one female with one male (pairing) or individually (mated females).

Relevant animals were housed individually for approximately 24 hours# prior to FOB assessment.
# During the mating period, any male or female thatdid not show evidence of mating on the day prior to their FOB assessment, were continued to be paired for mating. On the day of testing, the animal was removed from the mating cage and housed singly for approximately 1 hour before testing. After testing, animals were placed back into mating cages as required.
Rooms exclusive to study Yes
Target temperature range 20 to 24°C.
Target humidity range 45 to 65%.
Air changes 15 -20 air changes/hour.
Photo-period 12 hours nominal.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Administration volume: 5 mL/kg. Individual dose volumes will be based on individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test article Dimethyl Sebacate, was formulated in corn oil for administration to the study animals.
Formulations prepared at 10 and 200 mg/mL were analysed to determine homogeneity and stability. The formulations were to be considered be homogeneous if the coefficient of variation (CV) of the results was £ 6.0% and the homogeneity results were within ± 10% of the mean. The formulations were to be considered stable if the mean of the results at each time point were within ±10% of the mean at 0 hour.
Formulations prepared for use in Weeks 1, 3 and 6 of the study were analysed to determine homogeneity and achieved concentration. The formulations were to be considered be homogeneous if the coefficient of variation (CV) of the results was £ 6.0% and the homogeneity results were within ± 10% of the mean. The target range for the preparation of the formulations was 90 to 110% of nominal.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to the scheduled necropsy. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily, 7 d/w
The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- until sacrifice (at least 5 weeks in total),

in the females:
- 2 weeks before pairing,
- during the pairing period (3 weeks),
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for females with no delivery.

Day 1 corresponds to the first day of the treatment period.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per group.
Control animals:
yes, concurrent vehicle
Details on study design:
The males and females were administered the test item at 100, 300 or 1000 mg/kg/day The rationale for dose-level selection was based on a weeks range finding study. In this latter study, daily oral (gavage) administration of Dimethyl Sebacate at 100, 300 and
1000 mg/kg/day in a 14 Day oral (gavage) range-finding study (Covance Study Number 8273520) was generally well tolerated, with no unscheduled deaths or remarkable clinical signs. There were no dose-limiting post-dosing observations. Mouth rubbing was noted immediately after dosing, throughout the majority of the dosing period in all male animals (including controls); this lasted for up to 2 hour post-dose on one occasion at 1000 mg/kg/day. Mouth rubbing was also seen immediately after dosing in the majority of females (including controls), but on fewer occasions than males. Salivation and paddling was also observed post-dosing in all groups, for up to 2 hours in males given Dimethyl Sebacate. There were no adverse effects of dosing on body weight, body weight gains or food consumption in males or females. At terminal necropsy, there were no adverse macroscopic findings or effects on organ weights. Therefore, based on these findings, the same dose levels are considered suitable for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
Positive control:
none.

Examinations

Observations and examinations performed and frequency:
HEALTH MONITORING
Observe animal in cage to monitor health status.

Any animal which shows marked signs of ill health or overt toxicity may be isolated and may be killed and subject to the relevant terminal procedures.
Animal cohort Frequency of observation
All animals Twice daily: beginning and end (nominal) of the working day.


CLINICAL EXAMINATIONS
Individual animal record maintained on days of body weight recording.
Animal cohort Frequency of observation
All animals Once weekly.


POST-DOSING OBSERVATIONS
Observations related to time of dosing.

Character and timing of reactions to treatment were recorded.

Animal cohort Frequency of observation
All animals Daily: upon return to the home cage and at 0.5, 1, 2 and 4 hours post dose.


BODY WEIGHT
Individual body weight
Animal cohort Frequency of observation
Males Day -7 (randomisation body weight check). Weekly.
Day of (prior to) necropsy.
Females Day -7 (randomisation body weight check).

Weekly prior to pairing and until confirmation of mating.
Days 0, 7, 14 and 20 of gestation. Days 1 and 4 post partum.


FOOD CONSUMPTION
Recorded in g; calculated as g/animal/day.
Animal cohort Frequency of observation
Males Over the same intervals as the body weights prior to pairing and post-pairing.
Females Pre-pairing:
Over the same intervals as the body weights prior to pairing.

FUNCTIONAL OPERATION BATTERY (FOB)
At the time of testing, the observer were unaware of each animals dose level.
Where possible the observations were performed at the same time on each occasion.

OBSERVATIONS
Before removal from the home cage, the animal were observed and evaluated for the following:
Posture, convulsion, activity, excessive vocalisation, gait, arousal upon opening cage, tremor.

Each animal were removed from its cage, and observed for the following: Ease of removal, lacrimation, ease of handling, lacrimation type, excessive vocalisation, salivation, tremor, respiration, convulsion, piloerection, palpebral closure, appearance of fur, exophthalmus, any other abnormalities.

FUNCTIONAL OBSERVATION BATTERY: The first five males and the first five females were evaluated once at the end of the treatement period. The FOB included touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity.
LABORATORY INVESTIGATIONS: Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology, urinalysis and blood biochemistry parameters.
Sacrifice and pathology:
Animals were killed with an intraperitoneal injection of sodium pentobarbitone. Death was confirmed by cervical dislocation.

- TERMINAL PROCEDURES
- Necropsy
All adult animals including decedents were subjected to necropsy.
The adult scheduled necropsies were performed after an overnight period without food. Where possible the scheduled necropsies were carried out in group order for all adult animals. Each adult was given isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, the animal was exsanguinated by the severing of major blood vessels. A full macroscopic examination was performed under the general supervision of a pathologist and all lesions were recorded.
the animal was exsanguinated by the severing of major blood vessels.
The uterus of each littering female and any mated not pregnant female was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.

The following tissues from each animal were preserved in 10% neutral buffered formalin unless otherwise indicated.

W = weighed; E = processed and examined microscopically

adrenals W E Peyer’s patch E
animal identification# pituitary E
aorta E popliteal lymph nodes E
bone marrow smear (femur) (1) (2) E prostate# W E
brain W E rectum E
caecum E sciatic nerves E
colon E seminal vesicles# W E
duodenum E spinal cord cervical E
femur with bone marrow and stifle joint E spinal cord lumbar E
gross lesions (variable)# E spinal cord thoracic E
heart W E spleen W E
ileum E sternum with bone marrow E
jejunum E stomach E
kidney W E testes and epididymides# (3) (4) W E
larynx E thymus W E
liver W E thyroids + parathyroids W E
lungs with mainstem bronchi and bronchioles E tongue E
mandibular lymph nodes W E trachea E
mesenteric lymph nodes E trachea bifurcation E
oesophagus E ureters E
ovaries# W E urinary bladder# E
oviducts E uterus including cervix# W E
pancreas E vagina# E
1 – see clinical pathology section (Section 3.7.3)
2 – tissue taken into bovine albumin; smear prepared; air dried, then fixed in methanol
3 - tissue taken into Bouin’s fixative and processed at least to block stage
4 – sections of testes were stained with Periodic Acid Schiff-Haematoxylin (PASH) for qualitative assessment of
spermatogenic stages
# - all adult animals
Bone designated for histopathological examination was decalcified using Kristenson’s fluid.

Organ weights
Adult animals were weighed before necropsy.
The organs of the five highest numbered main study animals/sex/group denoted by ‘W’ in the tissue list above were dissected free from fat and other contiguous tissue and weighed before fixation (the testes and epididymides were weighed for all animals). Left and right organs were weighed together.
Histopathology
All tissues denoted by ‘E’ in the tissue list above from the five highest numbered animals/sex in Groups 1 and 4 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, stained with haematoxylin and eosin (slide stage) and examined microscopically by the study pathologist.
Statistics:
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3 and 4) were compared. All variables were analysed with a two-sided risk except where stated below.
Body weight gains, terminal body weights, food intake, locomotor activity data, function observational battery data, haematology, clinical chemistry and urine analysis variables were analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>=0.01), pairwise comparisons with control were made using Dunnett's test. A linear contrast was performed to determine whether there was a relationship between increasing dose and response. A significant trend (P<0.05) was only reported where none of the pairwise comparisons was significant.
No analysis was performed for clinical pathology parameters with values above or below the limit of the assay.
Organ weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett's test, for each sex separately, using the necropsy body weight as covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Levene's test for equality of variances across the groups was also performed for all organs and, in all cases, this showed no evidence of heterogeneity (P>=0.01).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
for the males, most of the samples were clotted for the females
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
The target range for the preparation of the formulations was 90 to 110% of nominal. Results were within the range 97 to 106%, with a CV of 0.30 to 1.43%, and therefore acceptable for dosing. Test article was not detected in the Group 1 control samples.

There was no mortality associated with administration of dimethyl Sebacate that were attributed to the treatment with the test item.

Clinical observations
There were no treatment-related adverse clinical observations or necropsy findings during the study.

Summary of post-dosing observations

For males, minor post-dosing observations such as mouth-rubbing, paddling and salivation were seen immediately post-dose or occasionally for longer from Day 9 or 12 of dosing throughout the dosing period on many occasions in the majority of animals receiving 1000 mg/kg/day, few animals receiving 300 mg/kg/day, rarely in animals receiving 100 mg/kg/day,. For females administered 1000 mg/kg/day, there was an isolated incident of mouth rubbing and salivation immediately post-dosing only on Day 12 and Day 13 of dosing in the pre-pairing period. During gestation for females that received 1000 mg/kg/day mouth rubbing was observed each day after dosing and was frequently associated with paddling and occasionally associated with salivation. Mouth rubbing and paddling were also seen for females that received 100 or 300 mg/kg/daybut the frequency and number of animals involved was lower.
These observations were considered to be indicative of taste aversion rather than systemic toxicity.

Body weight
The variations observed at the high dose-level were slight and not statistically significant. Therefore the slight decrease was not considered to be of biological significance.

Food consumption
There was no effect of Dimethyl Sebacate administration on mean food intake either before pairing in males and females or in females during gestation and lactation.

Functional observation battery

Locomotor activity data was highly variable, and there was considered to be no effect of treatment with Dimethyl Sebacate. For males that received 300 or 1000 mg/kg/day, there was a single occasion at 12-14 minutes when the total activity count was marginally lower activity than the controls achieving statistical significance (p<0.05). However this was not apparent for the total mobile count and was not seen for the females and was therefore considered to be incidental.

Locomotor activity
Locomotor activity data was generally highly variable, with no clear adverse effect of treatment seen.

Haematology

For males there were marginally fewer reticulocytes than controls in all the treated groups. However, the mean values fell within the background historical control range for this parameter ( 141.5 to 275.7 109/L ) The platelet count was slightly higher in all treated male groups, but the values were within the historical control range of 760 to 1173 109/L . Therefore these observations were considered to be within normal variation and not related to treatment with the test article. Other parameters were variable with no clear differences from the controls.
For females, almost all the sampling tubes were clotted, therefore no analysis was possible.

Clinical chemistry

Tthere was considered to be no effect of treatment with Dimethyl Sebacate on clinical chemistry parameters. Clinical chemistry parameters were generally variable with slight differences showing statistical significance on occasion, however these were often not dose-proportional and considered not to be biologically significant.

Urine analysis
Urine analysis showed no significant difference between the groups following Dimethyl Sebacate administration.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: 1000 mg/kg/day is the limit dose.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily oral admistration of Dimethyl Sebacate in Wistar Han rats at 100, 300 and 1000 mg/kg/day were performed according to OECD 422 guideline. Males and females were given the test item for at least 6 weeks. No changes that could be related to the treatment were observed on clinical sings, body weight, food consumption, motor activity, Hematology and biochemistry parameters. No treatment related changes were observed at necropsy and histopathology exmaination. The NOAEL for general toxicity of males and females was therefore 1000 mg/kg/day.
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Wistar Han Rat were performed by oral gavage with Dimethylsebacate (Rhodes, 2013). This study was performed according to GLP and OECD guideline 422.

Males were dosed once daily for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. The males were treated for at least six weeks.

Females were dosed once daily for two weeks prior to pairing, during the pairing period and until Day 4post-partuminclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum. Dosing was deferred or omitted if the animal was in or near parturition. In total males and females were dosed for approximately 6 to 8 weeks.

The selected dose-levels were 100, 300 and 1000 mg/kg/day based on the results of the dose-range finding study.

Body weights, clinical signs, food consumption and functional observation battery testing were recorded during the treatment period. Blood samples were taken for Haematology and clinical chemistry and urine samples were collected and analysed.

Analysis trials performed on concentrations from 10 to 200 mg/mL showed that the formulated test material was stable and homogenous. Prepared dosing formulations were found to be in the target range for achieved concentration, and homogenous.

There were no treatment-related deaths, adverse clinical observations, or necropsy findings during the study. Minor post-dosing observations associated with taste-aversion were seen in many treated animals during the study.

There was no clear effect of treatment on mean food intake and mean body weight either before pairing in males and females or in females during gestation. The Functional Observation Battery and Locomotor activity data showed no clear effect of treatment. Haematology, Clinical chemistry parameters and urine analysis showed no adverse effect of treatment. The few organ weights and histopathological changes were not considered to be treatment related. In conclusion, 1000 mg/kg/day was considered to be the NOAEL (No Observed Adverse Effect Level) for the males and females general toxicity.