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

Endpoint summary

Description of key information

Oral route

- subacute toxicity study in rat (OECD 407, GLP, rel. 1) : NOAEL = 1000 mg/kg bw/day in Sprague-Dawley rats.

- subchronic toxicity study in rat (OECD 408, GLP, rel.1): NOAEL = 1000 mg/kg bw/day in S-D rats.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

Specific details on test material used for the study:

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

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.

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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

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.

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.

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.

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

Key result

Critical effects observed:

no

System:

other:

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February - 06 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline 407 without deviations impacting the integrity or validity of the study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Revised 3 october 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

31 May 2008 (L142/210-L142/215)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 02 April 2019

Limit test:

no

Specific details on test material used for the study:

Storage conditions: At ambient temperature (15 to 25°C) in the dark

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, UK
- Age at study initiation: 48 to 54 days.
- Weight at study initiation: Males: 228-289 g; females: 152-210 g
- Housing: Animals were housed in groups up to four by sex in polycarbonate or polypropylene body cages with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: Pelleted diet (Teklad 2014C), ad libitum (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, changed at appropriate intervals. Ad libitum (except during urine collection).
- Acclimation period: 13 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours light : 12 hours dark.

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item was weighed into a suitable container. 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.

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

STABILITY:
- Before commencement of treatment, the suitability of the proposed mixing procedures was determined and homogeneity and stability was confirmed for the test item iin propylene glycol formulations at nominal concentrations of 1 mg/mL and 200 mg/mL, during magnetic stirring for up to 2 hours, at ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 15 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analyzed for achieved concentration of the test item.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response,
repeatability, method accuracy and precision.
The mean concentrations of the test item in formulations analyzed for the study were within set limits of +10% and -15% of nominal concentrations. The difference from mean remained within 16%. The wide precision of individual values and procedural recovery values observed reflect the complexity of test item (a multi-constituent), the formulation (an emulsion) and the analytical method (HPLC-ELSD).

Duration of treatment / exposure:

28 days followed by a 14 day recovery period

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Expressed in terms of material as supplied

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of a 7-day preliminary study in rats (Covance No.BL47QJ). In this study test material was administered at dose levels of 250, 500 and 1000 mg active ingredient/kg bw/day. No premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.
The high dose level for the current OECD407 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: Animals were randomly allocated to groups of five by sex and the treatment groups allocated to each cage using random letter tables. The group mean bodyweights were then determined to ensure similarity between the dose groups.

- Post-exposure recovery period: five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes (Sensory Reactivity and Grip Strength: Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, Grip strenght)
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as
appropriate. Animals were observed immediately before dosing, after dosing, one to two hours after completion of all dosing and as late as possible in the working day.

MORTALITY: Yes
- Time schedule: A viability check was performed near the start and end of each working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Day 1), on Days 1, 8, 15, 22, 28 and 29 of the treatment phase, Days 1, 8, 14 and 15 of the recovery phase and before necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION:
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein on the Day 29 for all main study animals.
- Anaesthetic used for blood collection: Yes, under light general anesthesia induced by isoflurane
- Animals fasted: Yes, after overnight withdrawal of food
- Parameters checked (haematology): Hemoglobin, haematocrit, erythrocyte count, total and differential leucocyte count, absolute reticulocyte count, mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), mean cell volume (MCV), platelet count and red cell distribution width (RDW). Additional blood samples were taken to check the Prothrombin time (PT) and the Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein on the Day 29 for all main study animals and on the recovery Day 15 for all recovery phase animals.
- Anaesthetic used for blood collection: Yes, under light general anesthesia induced by isoflurane
- Animals fasted: Yes, after overnight withdrawal of food
- Parameters checked (clinical chemistry):Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea (during recovery phase), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos) and Total protein (Total Prot) used for calculation of albumin to globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight on the Day 29 for all main study animals and on the recovery Day 15 for all recovery phase females in an individual metabolism cage, without food or water.
- Parameters checked: Clarity and Color (App), Volume (Vol), pH (for recovery phase males only) and Specific gravity (SG) using manual methods.
Ketones (Keto), Bile pigments (Bili) and Blood pigments (UBld) using Multistix reagent strips interpreted using the Clinitek®500 instrumen.
Protein (Prot and T-Prot), Glucose (U-Gluc and T-Gluc) and Creatinine (U-Creat and T-Creat) using a Cobas 6000 Analyzer.

BIOMARKERS AND PROTEIN ELECTROPHORESIS: Yes
- Time schedule for collection of blood: Blood samples (1mL) were withdrawn from the sublingual vein on the Day 29 for all main study animals and on the recovery Day 15 for all recovery phase animals, kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for ten minutes at 4°C) and then stored on deep frozen (approximately -60°C to -90ºC).
- Anaesthetic used for blood collection: Yes, under general anesthesia induced by isoflurane
- Animals fasted: Yes, after overnight deprivation of food and water
- Parameters checked: Albumin (Alb), alpha-1 globulin (a1), alpha-2 globulin (a2), beta-1 globulin (b1), beta-2 globulin (b2) and gamme-globulin (Gamma). Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration.

Sacrifice and pathology:

- GROSS PATHOLOGY (see Table 1 below): On completion of the dosing period, main study animals were killed following four weeks of treatment (on Day 29) and recovery animals were killed following four weeks of treatment and two weeks of recovery (on Day R15), by Carbon dioxide asphyxiation followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

- ORGAN WEIGHTS (see Table 1 below) :For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled termination.
- FIXATION: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of Testes (in modified Davidson’s fluid) and Eyes (in Davidson’s fluid).

- HISTOPATHOLOGY (see Table 1 below): Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Other examinations:

None

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
- A parametric analysis, as Williams' test or Dunnett's test, was performed if Bartlett's test for variance homogeneity was not significant at the 1% level and a nonparametric analysis, as Shirley's test or Steel's test, was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations for grip strength, motor activity, body weight, organ weight and clinical pathology data.
For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, Fisher’s exact tests were performed.

-For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.
- Probability values were calculated as: p < 0.01 **, p < 0.05 * and P ≥ 0.05 (not significant)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs observed in relation to dose administration and no test item-related changes in clinical condition and grip strength assessment during Week 4 of treatment for males and females at any dose investigated.
There was some inter-group variation, with hind limb grip strength values in some treated groups being marginally lower than Control, however, there was no evidence of a doserelationship in either sex.
There was considered to be no effect of treatment on motor activity.
The total group mean high beam scores for males that received 300 or 1000 mg/kg/day were slightly lower than Control, however, a review of the individual data revealed that one control
male and one male that received 100 mg/kg/day had atypically high total high beam scores which increased the overall group mean total scores for the Control and 100 mg/kg/day groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female that received 300 mg/kg/day was found dead on Day 17 of treatment.
There were no test item-related changes in clinical condition and no signs observed in relation to dose administration for this animal. Macroscopic examination revealed a perforated oesophagus, abnormal clear fluid in the thoracic cavity and abnormal, pale, frothy fluid in the trachea. These findings indicated that the female was mis-dosed and this death was therefore not associated with treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight gains for all groups of treated males and females were considered to be unaffected by treatment.
Males given 100 or 300 mg/kg/day showed slightly low mean body weight gain during three and two weeks of the dosing period, respectively, and females in these groups showed slightly low mean body weight gain during Days 8-15 of treatment. In the absence of any effects on body weight gain at 1000 mg/kg/day, these slight differences in the low and intermediate dose groups were considered incidental.
Body weight gain throughout the recovery period was slightly higher than Control for males that previously received 1000 mg/kg/day and slightly lower than Control for females that previously received 1000 mg/kg/day; these differences were considered incidental.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of treatment on the food intake of males or females at any dose level investigated.
The food intake of all groups of treated males was slightly lower than Control during the treatment period, however, no dose response was apparent and these minor differences were considered fortuitous.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

A visual assessment of water intake did not reveal any test-item related effects.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haematological investigation performed after four weeks of treatment revealed, when compared to Controls, a statistically significant slight increase mean cell haemoglobin concentration and a statistically significant slight decrease in mean cell volume in males at 1000 mg/kg/day; similar differences were not observed in females in this dose group.
Total white cell concentrations were slightly lower than Control, in a non dose-dependent manner in females given 300 or 1000 mg/kg/day, with statistical significance attained in the 1000 mg/kg/day group. The differences in total white cell concentrations were attributable to decreases in lymphocyte, basophil and large unstained cell concentrations in both groups of females, with statistical significance attained for the majority of the differences from Control. Similar differences were not evident in males.
Platelet counts were statistically significantly lower than Control in females given 1000 mg/kg/day, and prothrombin time was statistically significantly shorter than Control in males at 1000 mg/kg/day. All other differences from Control were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal
biological variation.
These changes were considered not to be of toxicological significance as they were minor, lacked consistency between sexes and were not associated with any micropathological correlates.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed after four weeks of treatment revealed, urea levels were slightly (but not statistically significantly) lower than Control in males and females that received 1000 mg/kg/day. Following a two-week recovery period urea levels remained slightly lower than Control for males that previously received 1000 mg/kg day but were marginally higher than Control for females that previously received 1000 mg/kg/day, indicating complete or partial recovery. Triglyceride concentration was higher than Control in all groups of treated males with statistical significance attained in males that received 1000 mg/kg/day, however, no dose response was apparent and no similar effect was observed in the females.
An increase in sodium concentration was apparent in females given 1000 mg/kg/day, and phosphorus and potassium concentrations were statistically significantly lower than Control for these females.
In addition, there were no micropathological correlates for either this change or the other minor changes seen after four weeks of treatment; statistically high triglyceride concentration in males and statistically high sodium and low potassium and phosphorus concentrations in females, therefore, these biochemical changes were not considered to be adverse.
All other differences from Control were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation. In addition, there was no effect of treatment on the biochemical examination of the blood serum for protein electrophoresis and albumin/globulin parameters performed after four weeks of treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of the urine after four weeks of treatment revealed that pH was statistically significantly lower than Control for males that received 1000 mg/kg/day, however, no similar
effect was observed in the females and following a two-week recovery period pH for males that previously received 1000 mg/kg/day was similar to Control.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organ weights after four weeks of treatment revealed, a dose-related decrease in body weight adjusted thymus and spleen weights was apparent for all groups of treated females with statistical significance attained at 1000 mg/kg/day for thymus weights, however, no similar effect was observed in males. Following a two-week recovery period, spleen weights were essentially similar to Control, however, thymus weights remained lower than Control for females that previously received 1000 mg/kg/day.
Group mean body weight adjusted liver weights were statistically significantly higher than Control in males that received 100 mg/kg/day, however, there was no such difference in males that received 300 or 1000 mg/kg/day or in females at any dose level, therefore, this slight difference was considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after four weeks of treatment and after two weeks of recovery did not reveal any treatment-related findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A finding related to treatment was observed in the kidneys of males: Minimal or slight hyaline droplet accumulation was observed in the kidneys in the majority of males administered 1000 mg/kg/day. The incidence and distribution of all other findings were considered to be unrelated to treatment.
The microscopic examination performed after 2 weeks of recovery revealed no test item-related findings. Full recovery of hyaline droplet accumulation was observed following examination of the kidneys from Control males and males previously administered 1000 mg/kg/day. No test item-related findings were observed in other tissues with macroscopic abnormalities which were presented for microscopic examination.

Histopathological findings: neoplastic:

not examined

Details on results:

HISTOPATHOLOGY
Hyaline droplet accumulation was apparent in the kidneys of the majority of males administered 1000 mg/kg/day, however this microscopic abnormality showed full recovery.
The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of alpha-2μ-globulin, a common finding in male laboratory rats (Khan et al, 2002). Hyaline droplet accumulation is specific to the male rat and is not generally considered to be significant in man. The presence of hyaline droplets in the kidneys may be associated with the change in urinary pH apparent in the high dose males at the end of the dosing period, which also showed complete recovery after two weeks off-dose. In the absence of other degenerative changes, this finding was considered to be non-adverse. Since these findings are male-rat-specific and have no relevance to human health they can be excluded when consideration of the No-Observed-Adverse-Effect-Level is made.

ORGAN WEIGHT FINDING
The dosage-related decrease in body weight adjusted thymus and spleen weights for all groups of treated females were not associated with any macroscopic or microscopic changes.
Following the two-week recovery period, body weight adjusted spleen weights were slightly higher than Control, however, thymus weights remained lower than Control for females that previously received 1000 mg/kg/day. As these organ weight changes were not associated with any other findings, these changes were not considered to be adverse.

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 effects observed

Critical effects observed:

not specified

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of Dodecanoic acid, mixed diesters with dipropylene glycol and nonanoic acid (CAS #2166089-27-4) was concluded to be 1000 mg/kg bw/day in CD rats.

Executive summary:

In a 28-days repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, three groups, each comprising five male and five female Crl:CD(SD) rats, received the test item, by gavage. Dose levels were selected on the basis of the results of a 7-day preliminary study in rats.In this study test material was administered at dose levels of 250, 500 and 1000 mg active ingredient/kg bw/day. No premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.The high dose level for the current OECD407 study was therefore set at 1000 mg/kg/day with intermediate and low dose levels of 300 and 100 mg/kg/day. A similarly constituted control group received the vehicle, propylene glycol, at the same volume dose as the treated groups. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover.

 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

 

Treatment with the test item at doses up to and including 1000 mg/kg/day was well tolerated; there were no treatment-related mortalities and no test item-related clinical signs or effects on body weight gain or food and water consumption. Neurobehavioural assessments conducted during Week 4 of treatment did not reveal any adverse effects on sensory reactivity, grip strength or motor activity at any dose level investigated.

The haematological investigation performed after four weeks of treatment revealed, when compared to Controls, a statistically significant slight increase mean cell haemoglobin concentration and a statistically significant slight decrease in mean cell volume in males at 1000 mg/kg/day; similar differences were not observed in females in this dose group. Total white cell concentrations were slightly lower than Control, in a non dose-dependent manner in females given 300 or 1000 mg/kg/day, with statistical significance attained in the 1000 mg/kg/day group. The differences in total white cell concentrations were attributable to decreases in lymphocyte, basophil and large unstained cell concentrations in both groups of females, with statistical significance attained for the majority of the differences from Control. Similar differences were not evident in males. Platelet counts were statistically significantly lower than Control in females given 1000 mg/kg/day, and prothrombin time was statistically significantly shorter than Control in males at 1000 mg/kg/day.

Biochemical examination of the blood plasma performed after four weeks of treatment revealed, urea levels were slightly lower than Control in males and females that received 1000 mg/kg/day. Following a two-week recovery period urea levels remained slightly lower than Control for males that previously received 1000 mg/kg day but were marginally higher than Control for females that previously received 1000 mg/kg/day. At the end of the treatment period, triglyceride concentration was statistically significantly higher than Control in all groups of treated males and increased sodium concentration and decreased phosphorus and potassium concentrations were apparent for females that received 1000 mg/kg/day.

Analysis of the urine after four weeks of treatment revealed that pH was statistically significantly lower than Control for males that received 1000 mg/kg/day, however, no similar effect was observed in the females and following a two-week recovery period pH for males that previously received 1000 mg/kg/day was comparable to Control.

Analysis of organ weights after four weeks of treatment revealed, when compared to Control, a dose-related decrease in body weight adjusted thymus and spleen weights was apparent for all groups of treated females with statistical significance attained at 1000 mg/kg/day for thymus weights, however, no similar effect was observed in males. Following a two-week recovery period, spleen weights were slightly higher than Control, however, thymus weights remained lower than Control for females that previously received 1000 mg/kg/day.

Macroscopic examination performed after four weeks of treatment and after two weeks of recovery did not reveal any treatment-related findings.

The histopathological change of hyaline droplet accumulation was observed in the kidneys of the majority of males that received 1000 mg/kg/day, however, full recovery was observed.

 

Test item-related hyaline droplet accumulation was evident in the kidneys of the majority of males administered 1000 mg/kg/day, however, this is a male rat-specific phenomenon and full recovery was observed. In the absence of any effects which were deemed to be adverse, within the context of this study, the No Observed Adverse Effect Level (NOAEL) was concluded to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study complete and sufficient to fulfill the endpoint requirements (Klimisch 1 and GLP)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute oral toxicity study (OECD 407, GLP, rel. 1)

In a 28-days repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, three groups, each comprising five male and five female Crl:CD(SD) rats, received the test item, by gavage. Dose levels were selected on the basis of the results of a 7-day preliminary study in rats. In this study test material was administered at dose levels of 250, 500 and 1000 mg active ingredient/kg bw/day. No premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.The high dose level for the current OECD407 study was therefore set at 1000 mg/kg/day with intermediate and low dose levels of 300 and 100 mg/kg/day. A similarly constituted control group received the vehicle, propylene glycol, at the same volume dose as the treated groups. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover.

 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

 

Treatment with the test item at doses up to and including 1000 mg/kg/day was well tolerated; there were no treatment-related mortalities and no test item-related clinical signs or effects on body weight gain or food and water consumption. Neurobehavioural assessments conducted during Week 4 of treatment did not reveal any adverse effects on sensory reactivity, grip strength or motor activity at any dose level investigated.

The haematological investigation performed after four weeks of treatment revealed, when compared to Controls, a statistically significant slight increase mean cell haemoglobin concentration and a statistically significant slight decrease in mean cell volume in males at 1000 mg/kg/day; similar differences were not observed in females in this dose group. Total white cell concentrations were slightly lower than Control, in a non dose-dependent manner in females given 300 or 1000 mg/kg/day, with statistical significance attained in the 1000 mg/kg/day group. The differences in total white cell concentrations were attributable to decreases in lymphocyte, basophil and large unstained cell concentrations in both groups of females, with statistical significance attained for the majority of the differences from Control. Similar differences were not evident in males. Platelet counts were statistically significantly lower than Control in females given 1000 mg/kg/day, and prothrombin time was statistically significantly shorter than Control in males at 1000 mg/kg/day.

Biochemical examination of the blood plasma performed after four weeks of treatment revealed, urea levels were slightly lower than Control in males and females that received 1000 mg/kg/day. Following a two-week recovery period urea levels remained slightly lower than Control for males that previously received 1000 mg/kg day but were marginally higher than Control for females that previously received 1000 mg/kg/day. At the end of the treatment period, triglyceride concentration was statistically significantly higher than Control in all groups of treated males and increased sodium concentration and decreased phosphorus and potassium concentrations were apparent for females that received 1000 mg/kg/day.

Analysis of the urine after four weeks of treatment revealed that pH was statistically significantly lower than Control for males that received 1000 mg/kg/day, however, no similar effect was observed in the females and following a two-week recovery period pH for males that previously received 1000 mg/kg/day was comparable to Control.

Analysis of organ weights after four weeks of treatment revealed, when compared to Control, a dose-related decrease in body weight adjusted thymus and spleen weights was apparent for all groups of treated females with statistical significance attained at 1000 mg/kg/day for thymus weights, however, no similar effect was observed in males. Following a two-week recovery period, spleen weights were slightly higher than Control, however, thymus weights remained lower than Control for females that previously received 1000 mg/kg/day.

Macroscopic examination performed after four weeks of treatment and after two weeks of recovery did not reveal any treatment-related findings.

The histopathological change of hyaline droplet accumulation was observed in the kidneys of the majority of males that received 1000 mg/kg/day, however, full recovery was observed.

 

Test item-related hyaline droplet accumulation was evident in the kidneys of the majority of males administered 1000 mg/kg/day, however, this is a male rat-specific phenomenon and full recovery was observed. In the absence of any effects which were deemed to be adverse, within the context of this study, the No Observed Adverse Effect Level (NOAEL) was concluded to be 1000 mg/kg/day.

 

Subchronic oral toxicity study (OECD 408, GLP, rel. 1)

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.

Justification for classification or non-classification

Harmonized classification:

The substance has no toxicological classification for human health hazards according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available information, no additional self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No information is available regarding the dermal and inhalation route.