Dodecanoic acid, 2,2-dimethyl-3-oxopropyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-21 and 2016-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

468-880-2

EC Name:

-

Cas Number:

102985-93-3

Molecular formula:

C17H32O3

IUPAC Name:

2,2-dimethyl-3-oxopropyl dodecanoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: 43 – 50 days old (male/female)
- Weight at study initiation: males: 148 - 182 g; females: 96 - 150 g
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil (Helianthii annui oleum raffinatum)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil in a frequency based on the stability features of the test item in the vehicle (stable at room temperature at least for one day, at 5 +/- 3°C for 7 days)

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle: 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored at room temperature until the analysis. Formulation samples were diluted with hexane and then analysed by a GC-FID method.
Measured concentrations varied between 92 and 98 % of the nominal concentrations and all formulations were considered to be homogeneous.

GC conditions:
Column: Column: Elite-5MS, 30 m x 0.32 mm x 0.25 μm, No: 926895
Temperature program: 1 min hold, 165°C to 200°C at 10°C/min, 1 min hold
Injector: 250°C
Detector: FID, 250°C
Carrier gas: nitrogen, 2 mL/min, constant flow
Split ratio: 20
Injection volume: 1 μL

Duration of treatment / exposure:

90 or 91 days (depending on the day of necropsy)

Frequency of treatment:

7 days/week basis, every day at a similar time (+/- 2 hours)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex in the control and dose groups; 5 animals/ sex in the control and high dose groups for recovery observations

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 1000, 300 and 100 mg/kg bw/day is based on findings obtained in previous repeated dose toxicity studies with the test item in the rat (Reproduction/Developmental Toxicity Screening Test, Study no. 666.421.2959; 28-Day Oral Toxicity Study, LAB Study no. 04/892-100P).
- Rationale for selecting satellite groups: five animals per sex in the control and in the high dose group (according to guideline)
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighing was performed on day 0, then weekly. Fasted body weight was measured on day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The food consumption was determined on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period
- Dose groups that were examined: all control and high dose test animals prior to test termination (day 89)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90 and day 91) and on recovery animals at the end of recovery period (day 118)
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90 and day 91) and on recovery animals at the end of recovery period (day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (day 84)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: Yes
- Time schedule for examinations: During the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy)
- Dose groups that were examined: all female animals
- Examinations: A vaginal smear was prepared from each female. The type of cycle (regular or irregular), number of days in pro-estrous, estrous and diestrous, number of cycle during the two weeks, number of animals with prolonged diestrous, number of animals with prolonged estrous were evaluated.

SPERM EXAMINATION
- Time for examinations: at Necropsy
- Dose groups that were examined: 10 animals of the control and 10 animals at high dose group
- Quantitative examinations: Testes and epididymides were frozen at the necropsy and enumeration was performed later. The total number of homogenization of one side testis was enumerated.
- Qualitative examinations: Sperm motility was determined from sample of ductus deferens at the necropsy. For the evaluation of the sperm motility the mean percentage of motile and immotile sperms was determined. The total sperm count and number of immotile sperms were recorded. A morphological evaluation of ductus deferens sperms sample was performed from the same animals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, including organ weights (see table No. 4); All animals were necropsied one day after the last treatment (main groups) or after four weeks recovery period (recovery groups).
HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups. In addition, the kidneys were also processed histologically in single male animal of 300 mg/kg bw/day dose group due to macroscopic observations at the necropsy.

Statistics:

Statistical analysis was done for the following data: body weight, food consumption, estrous cycle, hematology, blood coagulation, clinical chemistry, organ weight data and sperm parameters.
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations.

Mortality:

no mortality observed

Description (incidence):

No animals died during the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of male and female animals was not affected in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 mg/kg bw/day).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day).

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology examinations did not reveal clear test item related adverse changes in the evaluated parameters (1000, 300 and 100 mg/kg bw/day). Slight changes in the percentage of neutrophil granulocytes and lymphocytes in male animals as well as in the white blood cell count in female animals at 1000 mg/kg bw/day were observed. However, these changes remained well within the historical control ranges and were therefore considered to be a sign of biological variation and not test item related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Adverse findings were not detected in any of the clinical chemistry parameters in male or female animals at 1000, 300 and 100 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the behaviour or in reactions to different type of stimuli at the end of the treatment period (male and female, 1000, 300 or 100 mg/kg bw/day).
The behaviour and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A test item related increase in splenic weight parameters was observed at 1000 mg/kg bw/day in male and more pronounced in female animals. These spleen weight parameter increases were not reversible in females at the end of the recovery period. In general, these organ weight changes corresponded with macroscopic and histopathological findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged spleen was observed in one male and in 5/10 female animals at 1000 mg/kg bw/day at the terminal necropsy but not in any animals per sex after recovery.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed hyperplasia in the spleen in male and in a higher incidence in female animals at 1000 mg/kg bw/day. These findings consisted of erythroid and myeloid cells as well as lymphoid cells. The hyperplasia was observed only in 3/10 male and 6/10 female animals at the end of the dosing period and was not detected in the recovery groups. Thus, it was considered to be reversible.

Histopathological findings: neoplastic:

not examined

Details on results:

SPERM ANALYSIS
Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 1000 mg/kg bw/day.

ESTROUS CYCLE
A test item related influence on the estrous cycle was not detected (1000, 300 and 100 mg/kg bw/day).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) was determined to be 300 mg/kg bw/day for male and female Wistar rats.

Executive summary:

The objective of this study was to obtain information on the possible health hazards after repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in the high dose and control dose animals in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day doses corresponding to concentrations of 0, 500, 150 and 50 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. Each five animals/sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle (sunflower oil) for the test item and its sufficient stability in the vehicle was analytically verified up front. Recovery of the test item was 99 % of nominal concentrations at 50 and 500 mg/mL in sunflower oil, respectively. Thus, it was proved to be stable at room temperature of at least 7 days. Concentrations of the test item in the dosing formulations varied between ranges of 92 % and 98 % of nominal concentrations at each analytical occasion confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups.

The results of study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. The body weight development of male and female animals was not affected in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (1000, 300 and 100 mg/kg bw/day). Hematology examinations did not reveal clear test item related adverse changes in the evaluated parameters (1000, 300 and 100 mg/kg bw/day). Slight changes in the percentage of neutrophil granulocytes and lymphocytes in male animals as well as in the white blood cell count in female animals at 1000 mg/kg bw/day were observed. However, these changes remained well within the historical control ranges and were therefore considered to be a sign of biological variation and not test item related. Adverse findings were not detected in any of the clinical chemistry parameters in male or female animals at 1000, 300 and 100 mg/kg bw/day. Enlarged spleen was observed in one male and in 5/10 female animals at 1000 mg/kg bw/day at the terminal necropsy but not in any animals per sex after recovery. A test item related increase in splenic weight parameters was observed at 1000 mg/kg bw/day in male and more pronounced in female animals. These spleen weight parameter increases were not reversible in females at the end of the recovery period. In general, these organ weight changes corresponded with macroscopic and histopathological findings. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 1000 mg/kg bw/day. Histopathological examinations revealed hyperplasia in the spleen in male and in a higher incidence in female animals at 1000 mg/kg bw/day. These findings consisted of erythroid and myeloid cells as well as lymphoid cells. The hyperplasia was observed only in 3/10 male and 6/10 female animals at the end of the dosing period and was not detected in the recovery groups. Thus, it was considered to be reversible.

Under the conditions of the present study, 1000 mg/kg bw/day dose of the test item induced alterations in the spleen in the form of macroscopically enlargement associated with increases in spleen weight and accompanied by splenic hyperplasia (erythroid, myeloid and lymphoid) in male and female animals after consecutive 90-day oral (gavage) administration to Hsd.Han:Wistar rats. Splenic alterations were fully reversible in male and female animals except the increase in spleen weight in female animals.

There were no toxicologically relevant changes in the examined parameters in male or female animals at 300 mg/kg bw/day or 100 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 300 mg/kg bw/day for male and female Hsd.Han:Wistar rats.