Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 April 2019 to 03 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP conditions

Justification for type of information:

Study already available for a registration outside EU, and owned by the company having sold the REACH LoA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid

EC Number:

946-682-1

Cas Number:

2166089-27-4

Molecular formula:

Non applicable (UVCB)

IUPAC Name:

Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid

Test material form:

liquid

Details on test material:

- Appearance: Limpid liquid
- Storage condition of test material: Keep container tightly closed. Preferably store in the original packaging. Store at room temperature, protect from humidity.

Specific details on test material used for the study:

Storage conditions: Keep at ambient temperature(15-30℃).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 209-251 g for males and 161-201 g for females.
- Fasting period before study: not reported
- Housing: On arrival, rats were reared in a plastic cage with a volume of 420 mm x 270 mm x 200 mm, and the cage was covered with sterilized shavings pad. Male and female animals were reared in different cages, and no more than 5 animals per cage.
Each cage was clearly labeled with a color-coded cage card indicating study number, group number, cage number, dosage level, animal number(s), and sex.
- Diet: Breeding Rodent Diet., ad libitum
- Water: City drinking water, ad libitum
- Acclimation period: According to the health screening instructions provided by the veterinarian during the quarantine period, the experimental animals used in this test were in good health. During the adaptation period, there were no obvious abnormalities in the cage observation, body weight, food intake of the animals, indicating that the batch of animals can be used for experiments.

The animals care and use in this study were also in compliance with the Guide for the Care and Use of Laboratory Animals (2011) issued by The National Academies Press. The facility had passed the accreditation of Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
All procedures in this protocol were in compliance with the 3R principle (Reduction, Replacement and Refinement), the study did not unnecessarily duplicate any previous study. The animal use of this study had been reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of the facility. The IACUC Number of this study was IACUC-19-067.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2°C to 25.8°C
- Humidity (%): 40.8% to 69.5%.
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle, 8:00 a.m. light and 8:00 p.m. dark.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance formulations and vehicle were administered by gavage needle.

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item (5.0, 15.0 or 70 g) was weighed in a beaker. Approximately 50% of the final volume of vehicle was added and magnetically stirred until it was uniformly mixed. It was then made up to the required volume with vehicle and mixed with a magnetic stirrer until homogenous.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item. Formulations were prepared weekly.

VEHICLE
- Type: Propylene glycol
- Concentration in vehicle: 20, 60 and 200 mg active ingredient/mL
- Dose volume administered (for vehicle and treatment groups): 5 mL/kg bw/day

STORAGE
Test formulations were stored after preparation under refrigerated conditions, and kept at ambient temperature temporarily before exposure.
Propylene Glycol were kept at ambient temperature.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The animals were treated with the test substance daily for a period of 90 days. Animals in a satellite group (vehicle control group and high dose group) scheduled for follow-up observations were kept for extra 28 days after exposure without treatment.
The first day of exposure was designated as D1, the day after the last day of exposure (D90) was designated as rD1 which was the first day of recovery.

Frequency of treatment:

The animals were dosed with the test substance daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

No. of animals per sex per dose:

15 animals per sex in Vehicle Control and High Dose group, 10 animals per sex in Low Dose group and Mid Dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level.
In previous study, no toxic effect related to the exposure of the test item was observed when SD rats were administered with the test substanceat 922 mg/kg daily for 14 days.
Based on the data available from the 28 day toxicity study and the suggestion from Sponsor, three dose levels were established in this study: 100, 300 and 1000 mg/kg. And the vehicle Propylene Glycol group was set as the control group.

The high dose level for the current OECD408 study was therefore set at 1000 mg/kg/day with intermediate and low dose levels of 300 and 100 mg/kg/day chosen to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.

- Rationale for animal assignment: On the day of grouping (D0), animal weight was measured and ophthalmic examination was performed. Based on body weights and sex, 100 animals were randomized and assigned into four groups, which were Vehicle Control, Low Dose group (100 mg/kg), Mid Dose group (300 mg/kg), High Dose group (1000 mg/kg),

- Post-exposure recovery period: An additional satellite group of ten animals (five per sex) in the control and in the top dose group was used for observation of reversibility, persistence, or delayed occurrence of toxic effects. These animals were treated for a period of 90 days, followed by a 28-day period without treatment.

Examinations

Observations and examinations performed and frequency:

The following parameters and end points were evaluated in this study: mortality and clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology parameters (haematology, coagulation, clinical chemistry, urinalysis and bone marrow smears), gross necropsy findings, organ weights, and histopathologic examinations.


CLINICAL OBSERVATIONS: Yes
- General clinical observations were made at least once a day, and preferably at the same time(s) each day. The health condition and toxic effects of the animals were recorded.
- Detailed clinical observations were made in all animals once before the first exposure and D8, D15, D22, D29, D36, D43, D50, D57, D64, D71, D78, and D85 of the exposure periods and rD1, rD8, rD15, and rD22 of the recovered periods. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activities (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded.
- Sensory reactivity to stimuli of different types (e.g. auditory, visual stimuli and proprioceptive stimuli), assessment of grip strength and motor activity assessment were conducted on D78.

MORTALITY: Yes
- During the observation, the number of dead animal and the time of death were recorded. Animals which died during the study were dissected duly to figure out the causes of death. During the study, all animals were observed twice a day for mortality

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of animal were determined on D1, D8, D15, D22, D29, D36, D43, D50, D57, D64, D71, D78, and D85 of the exposure periods and rD1, rD8, rD15, and rD22 of the recovered periods.
- A fasted body weight which used by calculating Organ-to-body weight ratios were collected prior to terminal sacrifice.

FOOD CONSUMPTION: Yes
- Measurements of food consumption for 48 hours were made once a week.
Feed was added quantitatively about 400 g/ cages, and the daily intake (g/day) of each rat was calculated according to the amount of feed remaining.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Veterinarians used ophthalmoscopes to examine the eyelids, corneas, iris, conjunctiva, pupils, lenses, vitreous bodies, and fundus of animals in the Vehicle Control and high dose groups at D90 and rD28.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected by Vacuum Blood Collection Needle via abdominal aorta and added to marked scale (approximately 1.5 ml) in Vacuum Collection Tubes which containing EDTA-K2 on D91 and rD29.
- Anaesthetic used for blood collection: 3% sodium pentobarbital solution (45 mg/kg) injected intraperitoneally.
- Animals fasted: Not reported
- Parameters checked (haematology): White Blood Cell Count (WBC), Red Blood Cell Count (RBC), Hemoglobin (Hb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Percentage of Neutrophil (NEUT), Percentage of Lymphocyte (LYMP), Percentage of Monocyte (MONO), Percentage of Eosinophil (EOS) and Percentage of Basophil (BASO).

HEMOAGGLUTINATION: Yes
- Time schedule for collection of blood: Blood samples were collected by Vacuum Blood Collection Needle via abdominal aorta and added to marked scale (approximately 2.0 ml) in Vacuum Collection Tubes which containing Sodium citrate on D91 and rD29. After blood collection, the supernatant was taken after centrifugation at 1500 g for 10 min.
- Anaesthetic used for blood collection: 3% sodium pentobarbital solution (45 mg/kg) injected intraperitoneally.
- Animals fasted: Not reported
- Parameters checked (hemoagglutination): Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected by Vacuum Blood Collection Needle via abdominal aorta and added to marked scale (approximately 3.0 ml) in Vacuum Collection Tubes which containing coagulant on D91 and rD29. After blood collection, the supernatant was centrifuged at 1500 g for 10 min after 30-60 min at room temperature.
- Anaesthetic used for blood collection: 3% sodium pentobarbital solution (45 mg/kg) injected intraperitoneally.
- Animals fasted: Not reported
- Parameters checked (clinical chemistry): Aspartate Amino Transferase (AST), Alanine Amino Transferase (ALT), Urea Nitrogen (BUN), Creatinine (Cr), Total protein (TP), Albumin (Alb), Glucose (Glu), Total Cholesterol (TC), Sodium (Na+), Potassium (K+) and Chloride (Cl-).

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis were performed by metabolic cage method in animals which prior to scheduled sacrifice on D87 and rD25.
- Parameters checked: Appearance (color and clarity), Specific Gravity (SG), pH Value, Glucose (GLU), Urobilirubin (BIL), Ketone Body (KET), Blood Cells (BLD), White Blood Cell (WBC), Protein (PRO), Nitrite (NIT) and Urobilinogen (URO).

BONE MARROW SMEARS: Yes
- Time schedule for collection of urine: After euthanasia, the femur (left) of each animal was taken and bone marrow smears were carried out on D29 and rD15. The smears were fixed with formalin buffer solution and stained with Wright Giemsa, dried and preserved. The Bone Marrow Morphology test was not made.

Sacrifice and pathology:

GROSS NECROSPY
After anesthesia, the surviving animals were quickly bled and euthanized after blood collection.
The remaining animals were euthanized by CO2 inhalation.
After euthanasia, animals were subjected to full, detailed gross necropsy which included careful examination of external surface of the body, and the cranial, thoracic, thoracic, and abdominal cavities and their contents on the day at the end of exposure phase and recovery phase, which was D91, D92 and rD29.

ORGAN WEIGHTS
The tissues and organs of the necropsied animals in schedule were weighed as below: brain, heart, liver, kidney, adrenal gland, thymus, spleen, testis, epididymis, ovary, uterus.
Organ-to-body weight ratios were calculated as percentages.

The following tissues and organs were preserved in the corresponding fixation medium: All gross lesions, brain (representative regions including cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, thyroid, parathyroid, thymus, esophagus, salivary gland, stomach, small and large intestine (including Peyer's patches), liver, pancreas, kidney, adrenal gland, spleen, heart, trachea and lung, aorta, gonads and accessory sex organs, uterus, mammary gland, prostate, bladder, lymph nodes, peripheral nerves (sciatic nerve) adjacent to muscles, skin.
It was recommended that testes and epididymides be fixed by immersion in modified Davidson’s fixative. The other tissues or organs were fixed in neutral buffered formalin.

HISTOPATHOLOGY
Full histopathological examination was carried out on organs and tissues of all animals in the high-dose group and the vehicle control group, and the results showed no relevant toxic pathological changes. Therefore further pathological examination was not performed on organs and tissues of all animals in other dosage groups and recovery phase.

Optional endpoint(s):

None

Other examinations:

None

Statistics:

DATA COLLECTION
The data of the test and observation were recorded in appropriate tabular forms or collected by output of instrument computers. The measured data did not exceed the precision of the measuring instrument.

STATISTICS
All data of index detection were processed by SPSS software according to the group and gender, and are indicated as Mean ± SD. Statistic results of dose groups were compared with those of control group at significance level of 0.05, 001 and 0.001 in final report.
For descriptive data, such as symptoms of clinical observations, urine appearance and histopathological findings, were collected in a descriptive manner based on sex.
For quantitative data, such as body weights, food consumptions, hematology index, biochemistry index and organ weights and other indicators to analyze, homogeneity test of variance were selected between dose groups and control group.
For ranked data, such as urine index (except urine appearance), nonparametric tests (Kruskal-Wallis, H test, K-W, H test) were used between dose groups and control group.
Statistical analysis were not carried out, if the number of samples is less than or equal to 2 (N=2).
The clinicopathological data of unscheduled autopsied animals were not statistically analyzed and reserved as raw data only.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No obvious abnormality was observed in all animals during this study and in the weekly detailed clinical observation.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During this study, there was no significant difference between the body weight of each dose group and the vehicle control group (P≥0.05).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During this study, the amount of food consumption in each group was equivalent to that in the control group. Although the food consumption of some dose group were statistically significant in week 1, week 2, week 6, week 13, but the changes in these index were small and also there was no dose dependent relationship,, therefore the changes of food consumption were not related to the exposure of the test substance.

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 abnormal sign of eye lesions were noted in both vehicle control group and High-dose group at the termination of the study.

Haematological findings:

no effects observed

Description (incidence and severity):

No obvious abnormality was observed in hematological index both at the end of the exposure phase and recovery period.
No obvious abnormality was observed in Coagulation index both at the end of the exposure phase and recovery period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of exposure phase, compared with the vehicle control group, although some indexes (ALT) were statistically significant, the changes in these index were small, and just in one dosage, therefore the changes of biochemistry index above were not related to the exposure of the test substance. Also, at the end of recovery phase, biochemistry index in high group was equivalent to that in the control group.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of exposure phase, compared with the vehicle control group, although some indexes (KET, SG, PH, PRO) were statistically significant, the changes of the index were small, there was no obvious dose dependent relationship. Therefore the changes of urinalysis index above were not related to the exposure of the test substance.
No obvious abnormality was observed in urinalysis indexs at the end of the recovery period.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No obvious abnormality was observed in all animals during the functional observation on week 11 of exposure phase.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No obvious abnormality was observed in organ weight and coefficient both at the end of the exposure phase and recovery period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

According to the gross anatomy, some animals found abnormal of kidney, lung, thymus, spleen, adrenal gland, pituitary gland, stomach, etc., because some of them had no histopathological changes, or the incidence of pathological changes was low and the degree was light, or only in one side, it was speculated that there was no correlation with exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were some pathological changes in the control and high dose group, such as the infiltration of focal inflammatory cells in the heart, the infiltration of tiny granuloma in the liver, the infiltration of interstitial inflammatory cells in the prostate, etc. because of the low incidence, the slight degree, and they were common spontaneous lesions, it was considered that they were not related to the exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

No related Clinical observations (general clinical observations, detailed clinical observations and functional observations), body weight, food consumption, clinical pathology (hematology, blood coagulation, biochemistry and urine), gross pathology and histopathological examination in all animals showed no related changes of the test substance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Target system / organ toxicity

Key result

Critical effects observed:

no

System:

other:

Applicant's summary and conclusion

Conclusions:

Under the present experimental conditions, SD rats were administered with the test substance Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid (CAS #2166089-27-4) daily for 90 days at the dose level of 100, 300 and 1000 mg/kg bw/day. No changes related to the exposure of the test substance were noted. The NOAEL of the test substance was 1000 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test substance when administered via oral gavage to Sprague Dawley rats daily for a period of 90 days. The derived data allowed for the characterization of the test substance toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect Level (NOAEL), so relevant information can be obtained to assess the possible hazards and evaluate the safe dose.

 

100 SPF healthy SD rats (50 males 50 females) were employed. Based on body weights and sex, the rats were randomly divided into four groups including Vehicle Control (15 animals per sex), Low Dose group (100 mg/kg, 10 animals per sex), Mid Dose group (300 mg/kg, 10 animals per sex), High Dose group (1000 mg/kg, 15 animals per sex). Each rat was administered with Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid (CAS #2166089-27-4) of corresponding concentrations or vehicle daily for 90 days with dose volume of 5 ml/kg. 10 Animals in Control group and High Dose group continued to observe for 28 days. During the study, all animals were observed once daily for mortality and moribund status. Detailed clinical observations, the Body weight of each animal and food consumption were recorded once a week. Ophthalmic examinations were taken on the day prior to exposure and at the termination of the study. All animals in this study were subjected to Clinical Pathology examinations (Hematology, Blood Coagulation, Biochemistry and Urinalysis), Gross necropsy and Histopathology examinations at the end of exposure phase or recovery phase.

 

No mortality and moribund Status was observed in the study and no clinical signs of toxicity were noted in any of the animals through clinical observations. There was no statistically significant change of body weight and food consumption in any dosage group. There was no change of ophthalmic in High Dose group. At the end of exposure phase and recovery phase, changes related to exposure were not found in the indexes of hematology, blood coagulation, biochemistry and urine. All animals had no obvious gross pathological changes at the end of exposure phase and recovery phase. Changes of organ weight related to the exposure of the test substance were not found. Animals in high dose group and control group had no obvious pathological change related to the exposure at the end of exposure phase.

 

Under the present experimental conditions, SD rats were administered with the test substance Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid daily for 90 days at the dose level of 100, 300 and 1000 mg/kg bw/day. No changes related to the exposure of the test substance were noted. The NOAEL of Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid was 1000 mg/kg bw/day.