Dimethyl sebacate

Endpoint summary

Description of key information

There is an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rat (Rhodes, 2013). This study was performed according to GLP and OECD 422 guideline.No systemic toxicity was observed at the limit dose of 1000 mg/kg/day.

There is also a 13 weeks toxicity key study by the oral in rats performed according to OECD 408. No effect was observed at the top dose-level of 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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.

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).

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.

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.

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

chronic toxicity: oral

Remarks:

43038 TCR

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2015 - 23 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories France, l’Arbresle, France- Age at study initiation: at the beginning of the treatment period, the animals were 6 weeks old- Weight at study initiation: the males had a mean body weight of 150 g (range: 132 g to 172 g) and the females had a mean body weight of 147 g (range: 135 g to 170 g)- Fasting period before study: no- Housing: the animals were housed in twos of the same sex and in the same group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust- Diet (e.g. ad libitum): all animals had free access to SSNIFF R/M-H pelleted maintenance diet- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)- Acclimation period: the animals were acclimated to the study conditions for a period of 13 days before the beginning of the treatment period.ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2°C- Humidity (%): 50 ± 20%,- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air- Photoperiod (hrs dark / hrs light): 12h/12hIN-LIFE DATES: From 23 November 2015 to 23 February 2016.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: VEHICLE - Justification for use and choice of vehicle (if other than water): suitable formulation in corn oil - Concentration in vehicle: 20, 60 and 200 mg/mL - Amount of vehicle (if gavage): 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High Performance Liquid Chromatography with UV detection (HPLC-UV).Once in Weeks 1, 4, 8 and 13.A sample is taken from control and test item dose formulations and analyzed using the validated method.

Duration of treatment / exposure:

At least 13 weeks (i.e. 91 or 92 days).

Frequency of treatment:

Once a day, in the morning.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex and per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, based on the results of an OECD 422 study where three groups of Wistar Han rats received daily by gavage the test item at 100, 300 or 1000 mg/kg/day. Ptyalism and mouth rubbing were the only test item treatment-related findings in the study. The NOAEL was established at 1000 mg/kg/day for parental, fertility and developmental toxicity.Therefore, the same dose-levels were chosen in the present study.

Observations and examinations performed and frequency:

MORTALITY / MORBIDITY: Yes- Time schedule: Each animal was checked for mortality and morbidity once a day during the acclimation period and twice a day during the treatment period.CLINICAL OBSERVATIONS: Yes- Time schedule: Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.BODY WEIGHT: Yes- Time schedule for examinations:The body weight of each animal was recorded:- once before the beginning of the treatment period,- on the first day of treatment,- at least once a week until the end of the study.FOOD CONSUMPTION: Yes- Time schedule: The quantity of food consumed by the animals in each cage was recorded once a week, over a 7 day period, during the study.OPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations:Ophthalmological examinations were performed:- on all animals, before the beginning of the treatment period,- on all animals, on one occasion at the end of the treatment period.HAEMATOLOGY: Yes- Time schedule: for all animals at the end of the treatment period.CLINICAL CHEMISTRY: Yes- Time schedule: for all animals at the end of the treatment period.URINALYSIS: Yes- Time schedule: for all animals at the end of the treatment period.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: All animals were evaluated once in Week 12 (before the daily treatment). - Battery of functions tested: touch response, forelimb grip strength, pupillary reflex, visual stimulus, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature (at the end of the observation period) and motor activity.SEMINOLOGY: Yes- Time schedule for examinations: Before sacrifice at the end of the treatment period in all males.OTHER: Monitoring of estrous cycle The estrous cycle stage was determined for each female from a fresh daily vaginal lavage (stained with methylene blue), for 21 consecutive days before the end of the treatment period.Organ weightsThe body weight of each animal was recorded before sacrifice at the end of the treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY: YesSacrificeOn completion of the treatment period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital.Macroscopic post-mortem examinationA complete macroscopic post-mortem examination was performed on all animals.Preservation of tissuesFor all study animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes, Harderian glands, right testis and the right epididymis which were fixed in Modified Davidson's Fixative).Preparation of histological slidesAll tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).As no indication of hyaline droplets accumulation was observed, no immunostaining was performed.HISTOPATHOLOGY: YesA microscopic examination was performed on:- the control- and high-dose animals (groups 1 and 4),- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3).Testicular staging was performed for control- and high-dose males. A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item treatment-related clinical signs were limited to ptyalism observed in all animals given 1000 mg/kg/day (generally from Week 2 until the end of the study). This sign, commonly observed when a test item is administered by gavage, was considered not to represent an adverse effect. Ptyalism was also noted in some animals given 100 or 300 mg/kg/day (generally from Week 4 until Week 10 at the latest) but at a similar incidence than in the control group and could be related to the vehicle (corn oil).Reflux at dosing noted in 1/10 males and 1/10 females at 100 mg/kg/day on one occasion and thin appearance noted in 1/10 females given the same dose-level from Day 87 (Week 13) were considered to be of no toxicological importance as they did not correlate with any other signs, were isolated and not dose related.The other clinical signs recorded during the study, i.e. alopecia, scabs, thinning of hair, and/or chromorhynorrhea were observed both in control and/or test item-treated animals with no dose relationship; they were considered to be incidental and therefore unrelated to the test item treatment.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight and mean body weight change were unaffected by the test item treatment in both sexes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Higher mean food intake was observed in females given 300 mg/kg/day between Weeks 2 and 8 (+9 to +13% vs. controls; p < 0.05 in Weeks 3, 4 and 6) and in females given 1000 mg/kg/day from Week 2 (+11 to +20% vs. controls; p < 0.05 or p < 0.01 between Weeks 2 and 10). These differences were considered to be test item-related but of minor toxicological importance.Mean food consumption of males was unaffected by the test item treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were observed at the end of the treatment period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item treatment effects were observed on the hematology parameters at the end of the treatment period in both sexes when compared to control group.Statistically significant differences between control and test item-treated animals, namely in hemoglobin concentration (males given 100 mg/kg/day) and mean neutrophil counts (males given 300 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude and noted with no dose relationship.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item treatment effects were observed on the blood biochemistry parameters at the end of the treatment period in both sexes when compared to control group.Statistically significant differences observed between control and test item-treated animals, namely in glucose level (males given 100 mg/kg/day) and in triglyceride levels (females given 100 or 300 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude and/or noted with no dose-relationship.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The only statistically significant differences from controls consisted of slightly lower mean pH value in males given 1000 mg/kg/day (6.3 vs. 7.0 in controls; p < 0.05). As this difference was of minimal amplitude and not associated with any other findings, this was considered to be of minor toxicological importance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, a higher mean number of horizontal movements were noted in females (+27% vs. controls). In view of the slight severity, and as there was no correlating higher mean number of rearing movements, this finding was considered to be of no toxicological importance.Differences from controls in defecation and urination were noted in isolated animals at all dose-levels. In view of the very slight severity and incidence, and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the test item treatment. Other changes consisted of higher or lower mean landing foot splay values in males and females given 1000 mg/kg/day, respectively (males: 105 mm vs. 92 mm in controls; females: 78 mm vs. 93 mm in controls) and of lower rectal temperature in males given 1000 mg/kg/day (37.9°C vs. 38.3°C in controls). These findings were considered to be of no toxicological importance as they were minimal, of opposite trend and/or did not correlate with any other findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related organ weight changes were noted in adrenal glands in males and kidneys in both genders at 1000 mg/kg/day (see table 1).Increased adrenal glands weight changes were observed in male rats treated at 1000 mg/kg/day (relative weight statistically significant) and correlated with tan discoloration observed in 3/10 males at necropsy. As it did not correlate with any microscopic finding, it was considered to be treatment-related but of minor toxicological significance.Increased kidneys weights were noted in male and female rats treated at 1000 mg/kg/day, p < 0.01 for relative weights in both sexes and for absolute weights in females. As it did not correlate with any microscopic finding, it was considered to be treatment-related but of minor toxicological significance.Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Diffuse tan discoloration of the adrenals was observed in 1/10 males treated at 300 mg/kg/day and 3/10 males treated at 1000 mg/kg/day. This correlated with increased adrenals weights in males treated at 1000 mg/kg/day but did not correlate with any microscopic finding.The few other macroscopic findings noted at the end of the treatment period were of those commonly recorded in the species and none were considered to be related to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic changes were noted in test item-treated animals.A careful qualitative histopathological examination of the testes was performed. There were no effects on the germ cells or interstitial (Leydig) cells detected.All microscopic findings noted in treated animals were considered to be incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the species.

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle: no effects observed.Mean estrous cycle length and mean numbers of cycles were not affected by the test item treatment.Seminology: effects observed, non-treatment-related.No test item-related effects were noted on the epididymal sperm motility or morphology. Lower mean values for sperm parameters recorded in males given 300 mg/kg/day were due to the contribution of one male which showed small epididymis together with little translucent seminal liquid. As this was isolated without dose-relationship, these differences were considered to be incidental.

Key result

Dose descriptor:

LOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: kidney

Remarks:

The No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 1000 mg/kg/day.

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Table 1:

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	100	300	1000	100	300	1000
Exam. animals / Num. of animals	10/10	10/10	10/10	10/10	10/10	10/10
- Final body weight	-4	-4	-5	+4	+5	+2
- Adrenal glands
. absolute	+8	+3	+17	-5	-4	+3
. relative	+11	+6	+24*	-10	-9	+1
- Kidneys
. absolute	-3	+2	+6	+2	+4	+16**
. relative	+1	+7	+12**	-2	-1	+14**

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Conclusions:

The toxicity of the test item was evaluated after daily administration (gavage) to Wistar rats at dose-levels of 100, 300 or 1000 mg/kg/day for 13 weeks.Only non-adverse treatment-related changes were observed at the high dose-level of 1000 mg/kg/day. Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

This GLP study was carried out according to OECD test guideline No. 408 (21 September 1998).

 

Methods

Three groups of ten male and ten female Wistar rats received the test item by daily oral administration for 13 weeks. The test item was administered at 100, 300 and 1000 mg/kg/day in the vehicle (corn oil) under a constant dosage-volume of 5 mL/kg/day. A control group of ten animals per sex received the vehicle alone under the same experimental conditions.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using a High Performance Liquid Chromatography with UV detection analytical method.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12.

Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly.

Ophthalmological examinations were performed on all animals before the beginning of the study and on control and test item-treated animals at the end of the treatment period (Week 13).

Hematology, blood biochemistry and urinalysis investigations were performed at the end of the treatment period (Week 13).

The estrous cycle was determined for all females over 21 consecutive days at the end of the treatment period. Seminology investigations (count, motility and morphology) were performed on all males at sacrifice at the end of the treatment period.

On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period.

 

Results

Actual concentrations of the test item in the dosage forms administered to the animals during the study remained within an acceptable range (-1.3% to +6.8%) compared to the nominal concentrations.

 

No unscheduled deaths occurred during the study.

Ptyalism was observed in all high-dose animalsand was considered not to be an adverse effect.

The Functional Observation Battery was unaffected by the test item treatment.

Body weight and body weight change were not affected by the test item treatment but episodes of higher food consumption were noted in females given 300 (in Weeks 3, 4 and 6) or 1000 mg/kg/day (between Weeks 2 and 10).

No ophthalmology findings were observed at the end of the treatment period.

Estrous cycle, the epididymal sperm motility and morphology, and the spermatozoa count were unaffected by the test item treatment.

At hematology and blood biochemistry investigations, no test item-related changes were noted.

At urinary investigations, lower pH value, considered to be of minor toxicological importance due to the minimal amplitude and isolated nature, was noted in males given 1000 mg/kg/day.

 

At pathology investigations, increased adrenals weights in males and increased kidneys weights in both genders were noted at 1000 mg/kg/day and were considered to be related to treatment with the test item.

No treatment-related microscopic changes were noted at the end of the treatment period in this study.

Conclusion

The toxicity of the test item was evaluated after daily administration (gavage) to Wistar rats at dose-levels of 100, 300 or 1000 mg/kg/day for 13 weeks.

 

Only non-adverse treatment-related changes were observed at the high dose-level of 1000 mg/kg/day.

 

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to OECD 408 and GLP.

Additional information

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Wistar Han Rat was 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.

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted according to REGULATION (EC) No 1272-2008 and Annex VI of Commission Directive 2001/59/EC.