Diisodecyl sebacate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/04/2019 - 22/04/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

See Principles of Methods

Principles of method if other than guideline:

Deviations

Due to a miscalculation during the preparations of the formulas, for one day (30 May 2019, Day 22), Top dose animals received only 60% of the nominal concentration. Given that for the rest of the 28-days long treatment period, the animals received the correct dosage, this deviation is considered to have no impact on the outcome of the study and interpretation of the results.

Due to technical reasons, the actual relative humidity range was 29– 60% instead of 3070 % as it was indicated in the Study Plan. This deviation is considered to have no impact on the outcome of the study and interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Diisodecyl sebacate

EC Number:

249-047-0

EC Name:

Diisodecyl sebacate

Cas Number:

28473-19-0

Molecular formula:

C26H50O4

IUPAC Name:

1,10-bis(2-methylnonyl) decanedioate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Standard strain for repeated-dose studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals approximately 10 weeks old at time of treatment. Acclimation period was 13 days.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dosing formulations were administered daily starting from Day 0 for 28 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw was administered to all animals in all groups. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Vehicle:

propylene glycol

Details on oral exposure:

The dosing formulations were administered daily starting from Day 0 for 28 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw was administered to all animals in all groups. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Control animals were treated concurrently with the vehicle only (1% Tween 80 in propylene glycol). Animals were not treated on the day of necropsy.

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of Charles River Laboratories Hungary Kft. using a validated HPLC-UV method (Charles River Laboratories Hungary Kft. study code 18/296-316AN [5]).

Top, middle and bottom duplicate samples were taken from test item formulations once during the study, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the middle of the vehicle control solution for concentration measurement.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

General clinical observations were made at least twice daily, at the beginning and towards the end of the working day as practical, with the exception of Day 28, when only a detailed observation was performed. From Day 16, some non-adverse clinical changes had been seen in the animals, hence an additional post-dose clinical observation was added to the daily routine.

Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.

Body weight measurement

Body weight was recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 27 (last treatment day) and prior to necropsy (fasted) on Day 28.

Food consumption measurement

The determination of food consumption was performed for all groups once a week. The remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Weekly food consumption was calculated.

Sacrifice and pathology:

On Day 28, blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia.

After an overnight period of food deprivation of animals, two blood samples were collected; one for haematology (in tubes with K3-EDTA, 1.6 mg/mL blood) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Terminal procedures and Macroscopic evaluation

Gross necropsy was performed on all animals at the end of treatment period on Day 28. The animals were euthanized by exsanguination under pentobarbital anaesthesia.

After exsanguination, the external appearance was examined, the cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

On completion of the macroscopic observation and organ weight measurements, the carcasses were discarded. No tissue preservation was performed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant body weight changes in the 1000 mg/kg bw/day dose group (males/females) when compared to the control rats at any occasion.

Lower body weight gains were observed from Day 7 up to Day 14, and from Day 21 up to Day 27 in the males of the 1000 mg/kg bw/day dose group, reaching statistical significance in the Day 7- Day 14 period (p < 0.01). The overall body gain difference between the control and males receiving 1000 mg/kg bw/day were slightly less than 10 percent (-9.8%) throughout the study. Significantly higher body weight changes were observed in the females receiving 1000 mg/kg bw/day between Day 21 and Day 27 when compared to the control.

These findings correlate well with the similar food consumption findings (decreased food consumption at the onset of treatment in males receiving 1000 mg/kg bw/day, increased food consumption in the last week of treatment in females receiving 1000 mg/kg bw/day).

However, the growth curve for both sexes correlates well with the control group and historical control data, therefore these changes, while the correlation with the treatment could not be excluded, are not considered to be an adverse effect.

In summary, there were no adverse effects on body weight or on body weight gain that were ascribed to the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first week, lower food consumption was measured by 8-8.6% in the males and females receiving 1000 mg/kg bw/day. The animals showed adaptation to the test item, but in the males, a much smaller (-3.6%) difference was still observed during the second week. These changes were considered to be test item related.

Higher food consumption (20.7%) was observed in the females receiving 1000 mg/kg bw/day; this change was considered to be incidental, and most probably due to the low number of animals in the study.

Haematological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment noted in the haematology parameters.

Minor variations were observed, predominantly in the exposed females; HGB, MCHC, relative basophil cells, RDW, and LUC values, attaining statistical significance. The changes were considered to be incidental and related to individual variation. All data is within the normal range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment noted in the clinical chemistry parameters.

Statistically significant values were recorded only for ALT/GPT values of the females receiving 1000 mg/kg bw/day. When compared to the control group animals, the difference was only 40%, which is consistent with the organ weight and histopathological changes of the livers, but not indicating any adversity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed on the liver and kidney weight for both sexes.

The absolute and relative liver weights in the 1000 mg/kg bw/day dose group were significantly (p<0.01) increased in both sexes, by about 17% in males and by about 54% in females.

Kidney weights were increased in the 1000 mg/kg bw/day dose group in both sexes by about 13%, reaching statistical significance in the males only (p<0.05).

Furthermore, adrenal glands were significantly higher in the females of the 1000 mg/kg bw/day dose group.

Based on the relative change in the organ weights and the histopathological results, the changes in the liver were considered to be adaptive responses to the treatment. Without histopathological examination, the adversity of the kidney weight changes could not be determined.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed.

Bilateral small size in the adrenal in 1/5 male animals and small right adrenal in 1/5 male animals was recorded for the 1000 mg/kg bw/day dose group The toxicological significance of these findings is unclear in the absence of histopathological examination, and in the absence of similar changes in the females receiving 1000 mg/kg bw/day.

All other observed changes were considered incidental or within the normal historical background.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the histopathological examination of the livers, minimal to mild centrilobular hypertrophy was observed in the males and females of the 1000 mg/kg bw/day dose group, indicating an adaptive change most probably due to the increased xenobiotic metabolism activity of the liver. The kidneys were not examined.

Effect levels

Key result

Dose descriptor:

dose level:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

not measured/tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Following administration of a 1000 mg/kg bw/day of the test material, effects were observed that were attributed to the test item. These included:

- Lower food consumption in male animals
- Increase in liver and kidney weights
- Increase in ALT/GPT associated with the increase in organ weights

Executive summary:

CEREPLAS DIDS administered by oral gavage to Wistar rats for 28 consecutive days at the dose level of 1000 mg/kg body weight/day in 1% Tween 80 in propylene glycol at a dose volume of 5 mL/kg body weight, caused the following effects:

Increase of the liver and kidney weights were recorded in the animals receiving 1000 mg/kg bw/day, with no correlating adverse histopathology findings. The liver weight increase in females, which was about 54%, might limit the practicability of

testing at higher dose levels; the liver weight increase in the males was approximately 16%, which is expected to be compatible with longer term administration.

In selecting dose levels for the following OECD 408 study, the highest dose is proposed to be 1000 mg/kg bw/day; lower dose levels should be set to ensure that a NOAEL can be determined.