Octanoic acid, 2-phenoxyethyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Oct - 02 Dec 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in Oct 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents

Version / remarks:

adopted in 2000

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-phenoxyethyl octanoate

EC Number:

607-240-0

Cas Number:

23511-73-1

Molecular formula:

C16H24O3

IUPAC Name:

2-phenoxyethyl octanoate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality)

Details on species / strain selection:

This rat strain was used as it is recognized by international guidelines as the recommended test species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Ranges: 155 - 191 g (males); 122 - 147 g (females)
- Housing: 5 animals per sex were housed in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage enrichment (Enviro-dri, Wm. Lilico & Son, Surrey, UK).
- Diet: Pelleted rodent diet (SM R/M-Z from SNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY: Analysis of diet and water did not reveal any findings that were considered to have affected the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 - 21.9
- Humidity (%): 44 - 66
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and vehicle.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at the testing facility and on information from the sponsor.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (project 489876 of the testing facility). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

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:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 5-day dose range finding study at the same testing facility (project 497713, study period: 13 - 17 Oct 2011). Three females per group were orally exposed to 500 and 1000 mg/kg bw/day for 5 days at the same testing facility, respectively. No mortality occurred in both dose groups. In the high-dose group hunched posture was seen in all females on Day 5, and piloerection was observed in 1/3 females on Day 4. 1/3 females of the low-dose group showed salivation on Day 4. No weight gain was observed in one female of the low-dose group had, however the body weight of the other two low-dosed females and all females of the high-dose group were normal. At every dose group no abnormalities were noted during the macroscopic examination and the liver and kidney weights were reported to be normal. Based on the results of this range finding study, dose levels suggested for the main study (28-day toxicity study) were 100, 300 and 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals were observed for mortality and viability.
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, observations were made outside the cage in a standard arena.
- Time schedule: Immediately after dosing, once prior to start of treatment and at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes, the food consumption for each animal was determined weekly and mean calculated as g food/kg body weight/day.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation performed as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight for a maximum of 20 h.
- How many animals: All animals in every group
- Parameters checked: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of necropsy
- Animals fasted: Yes, animals were deprived of food overnight for a maximum of 20 h.
- How many animals: All animals in every group
- Parameters checked: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos.)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: All animals in every group
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The weight of the following organs from all animals was recorded: liver, kidneys, adrenal glands, testes, epididymides, prostate, seminal vesicles including coagulating glands, thymus, spleen, brain, heart, ovaries, uterus including cervix and thyroid including parathyroid.

HISTOPATHOLOGY: Yes. All organ and tissue samples were processed, embedded in paraffin wax, sliced and stained with haematoxylin and eosin. The samples of all tissues (except the tissues/organs listed below) collected at the scheduled sacrifice from animals in the control and high-dose group and all gross lesions were examined by a pathologist.
- The following organs and tissues were collected and fixed in a 10% buffered formalin: ovaries, adrenal glands, Peyer's patches (jejunum, ileum), brain (cerebellum, mid-brain, cortex), caecum, cervix, prostate gland, rectum, colon, duodenum, sciatic nerve, epididymides, seminal vesicles including coagulating gland, eyes including optic nerve and harderian gland, skeletal muscle, spinal cord (cervical, midthoracic, lumbar), femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, kidneys, thymus, thyroid including parathyroid, liver, trachea, lung (infused with formalin), urinary bladder, lymph nodes (mandibular, mesenteric), uterus, vagina and all gross lessions.
Following tissues/organs were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination: pancreas, aorta, pituitary gland, preputial gland, clitoral gland, salivary glands (mandibular, sublingual), skin, female mammary gland area, larynx, oesophagus, lacrimal gland (exorbital), tongue, nasopharynx.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Control: Scabs were observed in 1/5 males on Days 12 - 28 (left shoulder) and on Days 21 - 28 (right shoulder).
100 mg/kg bw/day: Missing upper incisors were noted in 1/5 males on Days 14 - 28.
1000 mg/kg bw/day: Exophthalmos was seen in 1/5 males on Days 22 - 28.

According to the study director, these findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were considered to have no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: A significantly lower body weight gain of males over Days 1 - 8 (88%) and 15 - 22 (81%) was observed compared with the control group.

This effect occured in the absence of a dose-related trend and was slight in nature, therefore this effect has no toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: A significantly lower mean of total movements was noted in males.

This effect was considered to have no toxicological relevance since the change was slight in nature (mean remained well within the range considered normal for rats of this age and strain), and the clinical appearance of these animals was normal. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: A significantly lower absolute (35%) and relative (33%) thyroid weight was observed for males compared to control animals.
300 mg/kg bw/day: The absolute (45%) and relative (41%) thyroid weight and the absolute (32%) and relative (26%) seminal vesicle weight was reduced in males compared with the control group.

These effects occurred in the absence of a dose-related trend, since the organ weights of the 1000 mg/kg bw/day group were in the same range as the organ weights of the control group. Moreover, histopathological examination of these organs revealed no abnormalities in the 100 and 300 mg/kg bw/day group. Therefore, these effects were not considered to be toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Scab formation of the skin was observed in 1/5 control males and pelvic dilation in kidneys occurred in 1/5 control males. A missing upper incisor and enlarged mandilubular lymph nodes were noted in one male of the 100 mg/kg bw/day group. In the 300 mg/kg bw/day group enlarged mandilubular lymph nodes and an accessory liver of the left median lobe were observed in 1/5 males, respectively. Several reddish foci in thymus, isolated reddish foci in stomach and exophthalmus were noted in 1/5 males of the 1000 mg/kg bw/day group, respectively.
FEMALES:
An enlarged mandilubular lymph node and isolated foci in clitorial glands were seen in 1/5 control animals. An uterus that contained fluid was noted in 2/5 females of the control group and in 1/5 females of the 100 mg/kg bw/day group. Pelvic dilation in kidneys was observed in 2/5 females of the 300 mg/kg bw/day group.

The macroscopic findings among control and treated animals were considered to be indicental without a dose-related incidence trend. These necropsy findings were therefore regarded to be of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Heart:
- Myofiber degeneration: 1/5 control females (grade 1)
- Inflammation lymphocytic: 1/5 females of the 1000 mg/kg bw/day group (grade 1)

Lung:
- Mineralization, vascular: 1/5 males of the 1000 mg/kg bw/day group (grade 2), 2/5 males of the 1000 mg/kg bw/day group (grade 1), 2/5 females of the 1000 mg/kg bw/day group (grade 1)
- Alveolar macrophages: 3/5 control males (1/5: grade 2; 2/5: grade 1) and 1/5 males of the 1000 mg/kg bw/day group (grade 1), 1/5 males of the 1000 mg/kg bw/day group (grade 1)
- Inflammation infiltrate: 2/5 control males (grade 1), 2/5 control females (grade 1), 3/5 males of the 1000 mg/kg bw/day group (grade 1), 1/5 females of the 1000 mg/kg bw/day group (grade 1)
- Alveolar inflammation: 1/5 males of the 1000 mg/kg bw/day group (grade 1), 1/5 females of the 1000 mg/kg bw/day group (grade 1)

Liver:
- Hemopoietic cells: 1/5 control males (grade 1), 1/5 control females (grade 1), 1/5 males of the 1000 mg/kg bw/day group (grade 1)
- Inflammation cell foci: 3/5 control males (grade 1), 3/5 control females (2/5: grade 1, 1/5: grade 2), 3/5 females of the 1000 mg/kg bw/day group (grade 1), 4/5 males of the 1000 mg/kg bw/day group (3/5:grade 1, 1/5: grade 2)
- Vacuolation, hepatocytes: 1/5 control females (grade 1)
- Macro noted: 1/5 males of the 100 mg/kg bw/day group (severity not scored)

Kidneys:
- Mineralization, tubular: 1/5 control males (grade 1), 1/5 control females (grade 1), 1/5 males of the 1000 mg/kg bw/day group (grade 1)
- Hyaline droplets: 1/5 control males (grade 2)
- Hyaline cast(s): 1/5 females of the 300 mg/kg bw/day group (grade 1)
- Inflammation, interstit.: 2/5 control males (grade 1), 2/5 control females (grade 1), 1/5 females of the 300 mg/kg bw/day group (grade 1), 1/5 males of the 1000 mg/kg bw/day group (grade 1), 1/5 females of the 1000 mg/kg bw/day group (grade 1)
- Inflammation, pelvic: 1/5 control males (grade 1)
- Basophilia, tubular: 3/5 control males (grade 1), 1/5 control females (grade 1), 1/5 females of the 300 mg/kg bw/day group (grade 1), 4/5 males of the 1000 mg/kg bw/day group (grade 1), 1/5 females of the 1000 mg/kg bw/day group (grade 1)
- Pelvic dilation: 2/5 females of the 300 mg/kg bw/day group (severity not scored)

Uterus:
- Proestrus epithelium: 1/5 females of the 100 mg/kg bw/day group (severity not scored)

Epididymides:
- Sperm granuloma: 1/5 males of the 1000 mg/kg bw/day group (grade 2)
- Inflammation, lymphocyti: 1/5 males of the 1000 mg/kg bw/day group (grade 1)

Thyroid gland:
- Hyperplasia/hypertrophy: 2/5 males of the 1000 mg/kg bw/day group (grade 1)

Thymus: 1/5 males of the 1000 mg/kg bw/day group (severity not scored)
- Lymphocytolysis, increased: 1/5 males of the 1000 mg/kg bw/day group (grade 1)

Eyes:
- Atrophy, retinal: 1/5 males of the 1000 mg/kg bw/day group (grade 2)

These microscopic findings were considered to be incidental effects and not related to the treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

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 toxicologically significant changes were noted in any of the parameters investigated in this study up to and including the highest dose level.

Target system / organ toxicity

Key result

Critical effects observed:

no