[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-25 to 2010-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

, adopted 1996-03-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

700-380-4

EC Number:

700-380-4

Molecular formula:

A: C25H50O2 B: C27H54O2

IUPAC Name:

700-380-4

Details on test material:

- Name of test material (as cited in study report): SymMollient S (Cetearyl nonanoate)
- Chemical name: A: Hexadecyl nonanoate; B: Octadecyl nonanoate
- Physical state: colourless liquid to solid
- Storage condition of test material: room temperature (20 +/- 5 °C), dark, dry and in original container
- Water solubility: a) 1.1 x 10^-6 mg/L (QSAR, WSKOW v1.41); b) 1.1 x 10^-8 mg/L (QSAR, WSKOW v.1.41)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst/Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: males: 292 to 339 g; females: 185 to 218 g
- Housing: individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding ('Lignocel' J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg/Germany). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (ad libitum): pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland)
- Water (ad libitum): community tap-water from Füllinsdorf
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/- 3 °C
- Relative humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (music was played during the light period)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied. SymMollient S was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Insoluble particles observed in the dose formulation for the high dose group were discarded from the solution by decanting of the dose formulation. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Based upon the results of stability analyses performed within a non-GLP Harlan Laboratories study (C76932), dose formulations were stable for at least one week.

Dose volume: 4 mL/kg bw.

VEHICLE:
Batch no.: 369109168 (Carl Roth GmbH & Co. KG, 76185 Karlsruhe)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day, samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were stored at -20 ± 5 °C until analysis.

The samples were analyzed by GC-FID. The test item was used as the analytical standard.

Results:
The SymMollient S peaks were assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms, no peak appeared at the retention time of SymMollient S and, therefore, the absence of the test item in the vehicle control samples (peanut oil) was confirmed.

The formulations investigated during the study were found to comprise SymMollient S in the range of 91.6% to 109.1% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of SymMollient S in the preparations was approved because single results found did not deviate more than 3.1% (<15%) from the corresponding mean.
In addition, the test item was found to be stable in formulations when kept 7 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item SymMollient S and peanut oil as vehicle during this study. Formulations were found to be homogeneously prepared and sufficient formulation stability at room temperature was approved.

For the results of the analysis please see also Section 8 Analytical methods (Entry: s_Morgenthal_2011).

Duration of treatment / exposure:

Main groups:
- Males: 14 days pre-pairing, throughout pairing (14 days maximum) and until the day before sacrifice (necropsy: after treatment of at least 28 days).
- Females: 14 days pre-pairing, throughout pairing (14 days maximum) and gestation periods (approx. 21 days) and until day 4 post partum (day 0 post partum: day when dam delivered all her pups7) for a total of approx. 7 weeks of treatment.
- Recovery groups (not mated animals): 40 days of treatment followed by a 14-day recovery period without treatment.

Frequency of treatment:

once daily, at approximately 24 hour intervals

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg /day
Basis:
other: actual administered

No. of animals per sex per dose:

Main groups:
0 mg/kg/day: 10 males / 10 females
100 mg/kg/day: 10 males / 10 females
300 mg/kg/day: 10 males / 10 females
1000 mg/kg/day: 10 males / 10 females

Recovery groups (not mated animals):
0 mg/kg/day: 10 males / 10 females
1000 mg/kg/day: 10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on a dose range finding toxicity study in Han Wistar rats. Four groups of 3 males and 3 females were treated by gavage with SymMollient S once daily over a 14-day treatment period. The following dose levels were used: 0, 100, 300 and 1000 mg/kg/day. Control animals were dosed with the vehicle alone (peanut oil).
Results:
- all animals survived until the scheduled necropsy.
- no clinical signs or observations were noted at any dose level in males and in females.
- no test item-related effects on food consumption, body weights or body weight gain were observed in males or females at any dose level.
- no test item-related macroscopical findings wre noted during the necropsy in males or in females at any dose level.
Based on these data, dose levels of 0, 100, 300, and 1000 mg/kg bw/day were selected for the subsequent combined repeated dose toxicity study with the reproduction/development toxicity screening test in the rat.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (main and recovery groups)
- Time schedule: viability/mortality were recorded twice daily. Also, cage-side clinical observations were conducted once daily (during acclimatization and up to day of necropsy). Additionally females of main groups were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes (main and recovery groups)
- Time schedule: once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage in all animals of main and recovery groups. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (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 or bizarre behaviour were also reported.

BODY WEIGHT: Yes (main and recovery groups)
- Time schedule for examinations: main groups: recorded daily from treatment start to day of necropsy; recovery groups: recorded daily during the treatment period and weekly the during recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main and recovery groups):
- Males: weekly during pre-pairing and after pairing periods.
- Females: pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.
- No food consumption was recorded during the pairing period.
- Recovery group males and females: treatment period days 1 - 7, 7 - 14, 14 - 21 and 21 - 28, 28 - 35 and 35 - 39; weekly during recovery period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (main and recovery groups)
- Time schedule for collection of blood: blood samples were obtained on the day of the scheduled necropsy from 5 males and 5 females from each main group and 5 males and 5 females from each recovery group (5 animals which were sacrificed at first). The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: blood samples were drawn sublingually from all animals under light isoflurane anesthesia.
- Animals fasted: the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: complete blood cell count (erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, total leukocyte count, differential leukocyte count, and platelet count) and coagulation (prothrombin time (= thromboplastin time) and activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes (main and recovery groups)
- Time schedule for collection of blood: blood samples were obtained on the day of the scheduled necropsy from 5 males and 5 females from each main group and 5 males and 5 females from each recovery group (5 animals which were sacrificed at first). The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: the animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, bile acids, globulin and albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (main and recovery groups)
- Time schedule for examinations: functional observation battery (FOB) parameters were performed in the main groups at one time during the study (males shortly before the scheduled sacrifice; females on day 3 post partum) and in the recovery groups toward the end of the recovery period. This FOB assessment was conducted following the daily dose administration.
- Dose groups examined: five males and five females from each main and recovery group.
- Battery of functions tested: animals were observed for the following:
1. Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
2. Hand-held observations: muscle tone, constituation, skin, pupil size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
3. Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behaviour, hair coat, respiration, quantity of faeces-balls and urine.
4. Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
5. Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
6. Locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Main groups: dams were sacrificed on day 5 post partum (day 0 post partum: delivery of pups). If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum (day 0 post coitum: day of mating). Males were sacrificed after treatment of at least 28 days, when no longer needed for assessment of reproductive effects.
- Recovery groups (not mated animals): males and females were sacrificed 14 days after the last day of test item administration.

All animals of main and recovery groups sacrificed were subjected to a detailed macroscopic examination.

Organ weights (parental animals):
- Testes and epididymides of all parental males of main groups and all males of recovery groups were weighed separately.
- From 5 males and 5 females selected from each main and recovery group, the following organs were trimmed from any adherent tissue and their wFt weight taken: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), liver, thymus and spleen.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all parental animals of main and recovery groups were sacrificed by an injection of sodium pentobarbital and exsanguinated. All parent animals of main groups and all animals of recovery groups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals of main groups, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites [see Salewski (1964)].

HISTOPATHOLOGY: Yes
Main groups and recovery groups: the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- males: prostate, seminal vesicles with coagulating gland, testes (in Bouin’s fixative) and epididymides (in Bouin’s fixative)
- females: ovaries
- from the five males and five females per group selected for organ weights, the following tissues were reserved in neutral phosphate buffered 4% formaldehyde solution: gross lesions, brain, spinal chord, small and large intestines (incl. Peyer’s patches), stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), uterus (with vagina), urinary bladder, lymph nodes (mesenterial, mandibular), peripheral nerve (sciatic) and bone marrow

-All organ and tissue samples to be examined were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion.
- Slides of all organs and tissues collected at terminal sacrifice from the animals in the control and high-dose main groups were examined . The same applied to all occurring gross lesions.
- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
- Histological examination of ovaries was carried out on any females that did not give birth.
- In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Statistics:

The following statistical methods were used to analyze functional observational battery, food consumption, body weights, clinical laboratory investigations and organ weight and ratios:
- Means and standard deviations of various data were calculated.
- The Dunnett-test [see Dunnett (1955)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test [see Miller (1981)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test [see Fisher (1950)] was applied if the variables could be dichotomized without loss of information.

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:

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):

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 specified

Details on results:

NOTE: THE FOLLOWING DATA IS THE DATA FOR THE MAIN GROUPS

CLINICAL SIGNS (daily) AND MORTALITY
- All males and females survived until the scheduled necropsy.
- No clinical signs were noted in males at any dose level.
- Treatment with the test item at the dose level of 1000 mg/kg bw/day caused salivation in two females during the gestation period and in two females during the lactation period. No further clinical signs which were considered to be test item-related were noted during daily observations in females at any dose level. The finding was considered a test-item related sign of discomfort and not an adverse effect.
- In control group hair loss was observed in two females.
- During the detailed weekly clinical observation, salivation was confirmed in two females at the dose level of 1000 mg/kg bw/day. No further observations were noted at any dose level in males and females.

BODY WEIGHT AND WEIGHT GAIN
Males (pre-pairing, pairing and after pairing periods):
- no effects on body weights or body weight gain were observed in males at any dose level.
- no effects on body weights or body weight gain were observed in females at any dose level.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males (pre-pairing and after pairing periods):
- no effects on food consumption were observed in males at any dose level.
Females (pre-pairing, gestation and lactation periods):
- no test item-related effects on food consumption were observed in females at any dose level.

HAEMATOLOGY
Males/Females.
- no test item-related effects on hematology parameters were observed in males at any dose level.

CLINICAL CHEMISTRY
Males/Females:
- no test item-related effects on biochemical parameters of the blood serum were observed in males at any dose level.

NEUROBEHAVIOUR
During the functional observational battery testing:
- salivation was observed in one female at the dose level of 1000 mg/kg bw/day. The finding was considered a test-item related sign of discomfort and not an adverse effect.
- no further test item-related findings were observed in males or females at any dose level.

The following findings were made on locomotor activity:
- no test item-related effects on locomotor activity of males or females were osberved at any dose level.

ORGAN WEIGHTS
Males/Females:
- no test item-related effects on organ, organ/body and organ/brain ratios were observed at any dose level.

GROSS PATHOLOGY
Males/Females:
- no test item-related findings were noted during necropsy of males at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males/Females:
- no test item-related findings were found during histopathological examination of males at any dose level.

RECOVERY GROUPS:
No effects were observed on any parameters in recovery animals of the control and high dose group during the recovery period.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since no test item related effects were noted during the study, a general NOAEL for systemic toxicity was established at the highest dose level.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

During the study, no test item-related effects were observed in male and female rats treated with different dose levels up to 1000 mg/kg bw/d. Based on these results, a general NOAEL for systemic toxicity was established at 1000 mg/kg body weight/day.