9-Octadecen-1-ol, (Z)-, phosphate

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:

2013-02-07 to 2013-03-07

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 3 October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, Council Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, EPA Health Effects Test Guidelines, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

UVCB: not applicable

IUPAC Name:

UVCB: not applicable

Constituent 2

Reference substance name:

UVCP: not applicable

IUPAC Name:

UVCP: not applicable

Constituent 3

Reference substance name:

9-Octadecen-1-ol, (Z)-, phosphate

EC Number:

253-455-4

EC Name:

9-Octadecen-1-ol, (Z)-, phosphate

Cas Number:

37310-83-1

Molecular formula:

C18H37O4P

IUPAC Name:

[(9Z)-octadec-9-en-1-yloxy]phosphonic acid

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Rat, Hsd.Brl.Han: of Wistar origin
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H- 1103 Budapest, Hungary
- Age at initiation: Male animals: 37 - 42 days old; Female animals: 37 - 42 days old
- Weight at study initiation: Male animals: 146 - 172 g; Female animals: 113 - 132 g
- Housing: 2 or 3 animals of the sex/ cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance"
- Water: tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70 %
- Air changes: 8 - 12 exchanges / hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (Helianthii annui oleum raffinatum)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than for three days before application.
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places (10 mL, each) from each concentration (Groups 2, 3 and 4) and measured on 2 occasions:
Date of samplings: February 07 and 28, 2013
Date of measurement: February 08 and March 01, 2013.
The samples were stored at 5 ± 3 °C until the analysis.
Similarly, five samples were taken from the control solution (group 1) and analyzed.
Formulations of reaction product of oleyl alcohol with polyphosphoric acid in sunflower oil proved to be homogeneous. Measured concentrations varied between 92 and 105 % of the nominal concentrations.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability in the chosen vehicle was verified over the range of relevant concentrations. Recovery of Reaction product of oleyl alcohol with polyphosphoric acid from sunflower oil formulations was 103 and 96 % at ~10 and ~200 mg/mL concentrations, respectively.
The test item proved to be stable in sunflower oil formulations at ~10 and ~200 mg/mL concentration levels at least for 3 days in the refrigerator
(5 ± 3°C) and for 24 hours at room temperature. A separate analytical report has provided this data.


VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 200, 60 and 20 mg/mL, applied in a dose volume of 5 mL/kg bw for 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places (10 mL, each) from each concentration (Groups 2, 3 and 4) and measured on 2 occasions:
Date of samplings: February 07 and 28, 2013
Date of measurement: February 08 and March 01, 2013.
The samples were stored at 5 ± 3 °C until the analysis.
Similarly, five samples were taken from the control solution (group 1) and analyzed.
Formulations of reaction product of oleyl alcohol with polyphosphoric acid in sunflower oil proved to be homogeneous. Measured concentrations varied between 92 and 105 % of the nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
20, 60, 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals/sex in the control and dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting based on findings obtained in a previous 14-Day oral gavage dose range finding study with Reaction product of oleyl alcohol with polyphosphoric acid in rats (Toxi-Coop study no. 559.400.3890) and was in agreement with the Sponsor. Doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment: The rat is regarded as suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in toxicity studies.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, after treatment at approximately the same time. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed with an accuracy of 1 g on days 0, 7, 14, 21 and 27. Individual body weight changes were calculated. Fasted body weight was measured on day of necropsy (day 28).

FOOD EFFICIENCY:
The food consumption was determined with an accuracy of 1 g on days 7, 14, 21 and 27 by reweighing the non-consumed diet.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment.
- Anesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table:
WBC, White Blood Cell (leukocyte) count
RBC, Red Blood Cell (erythrocyte) count
HGB, Hemoglobin concentration
HCT, Hematocrit (relative volume of erythrocytes)
MCV, Mean Corpuscular (erythrocyte) Volume
MCH, Mean Corpuscular (erythrocyte) Hemoglobin
MCHC, Mean Corpuscular (erythrocyte) Hemoglobin Concentration
RET, Reticulocytes
PLT, Platelet (thrombocyte) count
Differential white blood cell count*
* NEUT: Neutrophil (%); LYMPH: Lymphocyte (%); EOS: Eosinophil; (%);
MONO: Monocyte (%); BASO: Basophil (%),


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment.
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table:
ALT, Alanine Aminotransferase activity
AST, Aspartate Aminotransferase activity
GGT, Gamma Glutamyltransferase activity
ALP, Alkaline Phosphatase activity
TBIL, Total Bilirubin concentration
CREA, Creatinine concentration
UREA, Urea concentration
GLUC, Glucose concentration
CHOL, Cholesterol concentration
BAC, Bile acids
Pi, Inorganic phosphate concentration
Ca++, Calcium concentration
Na+, Sodium concentration
K+, Potassium concentration
Cl-, Chloride concentration
ALB, Albumin concentration
TPROT, Total Protein concentration
A/G, Albumin/ globulin ratio


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: General clinical observations were made once a day, after treatment at approximately the same time.
- Dose groups that were examined: All
- Battery of functions tested:
Sensory activity: Sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were assessed in all animals during the last exposure week. General physical condition and behavior of animals were tested. A modified Irwin test was performed.
(SOP no. ATK 001; Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioural and Physiologic State of the Mouse, Psychopharmacologia (Berlin) 13 222-257 1968).

OTHER:
Prior to necropsy, the estrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of estrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal one day after the last treatment. Animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® (details are presented in "Details of Other Materials").
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. The following organs/tissues were preserved in 4 % buffered formaldehyde solution except testes and epididymides, which were fixed in modified Davidson solution.
Table 5: List of organs preserved
adrenal glands
aorta
bone marrow (femur)
brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
eyes (lachrymal gland and Harderian glands)
female mammary gland
gonads (testes with epididymides, ovaries, uterus with cervix and vagina)
gross lesions
heart
kidneys
large intestines (cecum, colon, rectum, including Peyer’s patches)
liver
lungs (with main stem bronchi; inflation with fixative and then immersion)
lymph nodes (submandibular and mesenteric)
muscle (quadriceps)
esophagus
pancreas
pituitary
prostate
salivary glands (submandibular)
sciatic nerve
seminal vesicle with coagulating gland
skin
small intestines (representative regions: duodenum, ileum, jejunum)
spinal cord (at three levels: cervical, mid-thoracic and lumbar)
spleen
sternum
stomach
thymus
thyroid + parathyroid
trachea
urinary bladder


ORGAN WEIGHTS: The following organ weights were determined and recorded prior to preservation (wet weight):
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands as a whole;
With precision of 0.001g: adrenals, ovaries
Paired organs were measured together.
Organ weights of one female animal (1/5) in 300 mg/kg bw/day group were not determined due to the early death of animals shortly after the blood sampling (i.e. exsanguination was not feasible).

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (group 1) and high dose (group 4) groups and in female animal of 300 mg/kg bw/day, which died due to over anesthesia. The kidneys of one male animal of 100 mg/kg bw/day group were also processed histologically as one pyelectasia was detected at the necropsy.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- clinical pathology (hematology, blood coagulation, clinical chemistry)
- organ weight
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality:
No mortality occurred in the control, 1000, 300 or 100 mg/kg bw/day dose groups during the course of the treatment period
One female animal of 300 mg/kg bw/day group was found dead after the terminal blood sampling due to over-anesthesia on Day 28.

Clinical Observations:
Daily clinical observations
There were no toxic signs related to the test item effect at the daily clinical observations. The behavior and physical condition of animals were considered to be normal at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire observation period.
A test item related salivation (which is not toxicologically relevant) was observed at 1000 mg/kg bw/day (5/5 male and 5/5 female), at 300 mg/kg bw/day (2/5 male and 1/5 female) and at 100 mg/kg bw/day (2/5 female) with a dose related onset.

Weekly detailed clinical observations
The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period.

Functional observation battery
Functional observation battery did not demonstrate treatment-related changes in the behavior or in reactions to different type of stimuli at the end of the treatment period (Day 27; male and female, 1000, 300 and 100 mg/kg bw/day).

Weight Assessment
The bodyweight development was undisturbed in the test item treated animals (male and female, 1000, 300 and 100 mg/kg bw/day).
There were no statistically or biologically significant differences in male animals between the control and test item treated groups in the mean body weight and body weight gain during the entire observation period.
In the female animals of 100 mg/kg bw/day, the body weight gain was statistically significantly higher with respect to control between Days 7 and 14 and between Days 0 and 27 . These slight but statistically significant differences with respect to control were considered to be toxicologically not relevant.

Food Consumption
The daily mean food consumption was similar in the control and test item treated groups (male and female, 1000, 300 and 100 mg/kg bw/day) during the entire treatment period.

Hematology
Hematological investigations did not reveal any test item related changes in the examined parameters (male and female, 1000, 300 or 100 mg/kg bw/day).
In the male animals, small but statistically significant differences were observed in the higher mean percentage of lymphocytes (LYMPH) and basophil granulocytes (BASO) at 300 and 100 mg/kg bw/day and in the less mean percentage of neutrophil granulocytes (NEU) at 100 mg/kg bw/day with respect to the relevant control.
In the female animals, statistical significance was indicated for a slightly less mean platelet count (PLT) at 1000 mg/kg bw/day and higher mean percentage of reticulocytes (RET) at 1000 and 100 mg/kg bw/day with respect to control. The prothrombin time (PT) was slightly shorter than in the control at 300 mg/kg bw/day.
Alterations in the parameters listed above (LYMPH, BASO, NEU, PLT, RET and PT) were independent from administered doses and values remained within the historical control ranges. Therefore, the changes noted in haematology parameters were not considered to be toxicologically relevant.

Clinical Chemistry
No pathologic test item effects were detected at the evaluation of the clinical chemistry parameters (male and female, 1000, 300 or 100 mg/kg bw/day).
Statistically significant, slight differences were observed between the control and 1000 mg/kg bw/day treated male animals regarding the activity of alanine aminotransferase (ALT) and concentrations of total bilirubin (TBIL). Although the mean activity of ALT slightly exceeded the upper limit of the historical control range, there were no related effects on other clinical pathology parameters and it was also not correlated with any histological findings. The mean bile acid concentration (BAC) of male animals of 100 mg/kg bw/day group was slightly less than in the control group. However the mean concentration of total bilirubin and bile acid remained well within the historical control ranges. Therefore these findings (ALT, BAC and TBIL) were not considered to be of toxicological relevance.
In the female animals, there were no significant differences between the control and test item treated groups (1000, 300 or 100 mg/kg bw/day) in the examined parameters.

Necropsy
Necropsy observations did not reveal any test item related macroscopic findings in male or female animals, at any dose level tested.
One side pyelectasia was observed in one (1/5) male animal at 100 mg/kg bw/day). Moderate or marked hydrometra was noted for female animals of 300 mg/kg bw/day (1/5) and 100 mg/kg bw/day (2/5). Renal pyelectasia is a common observation of this strain of experimental rats and it was only present in one animal of the low dose group. Hydrometra, related to the female sexual cycle, occurred only in the low dose groups, is a frequent observation in experimental rats. Both of these findings were considered to be without any toxicological relevance in this study.

Organ Weight
There were no test item related changes in the examined organ weights in male or female animals at any dose level (1000, 300 or 100 mg/kg bw/day).
Slight, but statistically significant differences with respect to controls were observed in the less mean adrenal weights relative to body and brain weights of male animals at 100 mg/kg bw/day.
In the female animals, the absolute mean weight of liver, thymus and adrenal glands exceeded the relevant control value at 100 mg/kg bw/day. Similarly, the mean thymus weight relative to body weight (300 mg/kg bw/day) and the mean thymus weight relative to brain weight (100 mg/kg bw/day) were also higher than the control value.
All these statistically significant differences in the mean weights of adrenal glands, liver and thymus with respect to the appropriate control value were indicative of biological variation and no test item influence was supposed in the lack of similar changes in the high dose group or related histopathological lesions.

Effect levels

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 effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, Reaction Product of Oleyl Alcohol with Polyphosphoric Acid caused no signs of toxicity in male or female Hsd.Brl.Han: Wistar rats after 28-days oral (gavage) administration at 1000, 300 or 100 mg/kg bw/day.

Based on the observations made in the study the No Observed Adverse Effect Level (NOAEL) was determined as follows.

NOAEL: 1000 mg/kg bw/day for male animals
NOAEL: 1000 mg/kg bw/day for female animals

Executive summary:

The aim of this 28-Day toxicity study was to obtain first information on the toxic potential of Reaction Product of Oleyl Alcohol with Polyphosphoric

Acid in rats at three dose levels following 28-day oral administration.

The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5/sex/group) once a day at 0, 1000, 300and 100 mg/kg bw/day doses corresponding to concentrations of 0, 200, 60and 20 mg/mL, applied in a dose volume of 5 mL/kg bw for 28 days.

The suitability of the chosen vehicle, sunflower oil, for the test item at the intended concentrations was analytically verified up front. Reaction Product of Oleyl Alcohol with Polyphosphoric Acid was stable for 24 hours at room temperature and for three days in the refrigerator.

Concentration of the test item in the dosing formulations varied from 92 % to 105 % of nominal concentrations at both analytical occasions, thereby confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of the treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including hematology and clinical chemistry were conducted one day after the last treatment. Gross pathology was performed on animals on the day following the last treatment. The absolute and relative weights of selected organs were determined. A full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups.

The results of the study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle (sunflower oil) only.

No mortality occurred during the observation period. One female animal of the 300 mg/kg bw/day group was found dead shortly after the terminal blood sampling due to over-anesthesia on Day 28.

Toxic signs related to the test item were not found at any dose level at the daily and detailed weekly clinical observations and in the course of functional observation battery.

Salivation was observed during the daily clinical observations, shortly after test item administration, in male and female animals at 1000 and 300 mg/kg bw/day and in female animals of the 100 mg/kg bw/day group. This finding was considered to be toxicologically not relevant.

The body weight development was undisturbed in male and female animals at each dose level (1000, 300 and 100 mg/kg bw/day) in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level.

The food consumption was not influenced by the test item. The mean daily food consumption was similar in the control and in all test item treated groups.

Clinical pathology examinations did not reveal any pathologic changes in the examined hematology, blood coagulation or clinical chemistry parameters. A slightly higher mean activity of ALT (alanine aminotransferase)in male animals of 1000 mg/kg bw/day group was determined to be toxicologically not relevant as there were no changes in other clinical pathology parameters and there was no correlation with any histological findings.

Specific macroscopic alterations related to the test item were not found during the necropsy.

There were no test item effects on the examined organ weights at any dose level.

Histological examination did not detect any toxic or test item related lesions in the examined organs.

Under the conditions of the present study, Reaction Product of Oleyl Alcohol with Polyphosphoric Acid caused no signs of toxicity in male or female Hsd.Brl.Han: Wistar rats after 28-days oral (gavage) administration at 1000, 300 or 100 mg/kg bw/day.

 

Based on the observations made in the study the No Observed Adverse Effect Level (NOAEL) was determined as follows.

 

NOAEL:         1000 mg/kg bw/day for male animals

NOAEL:         1000 mg/kg bw/day for female animals