Alcohols, C18-22, distn. residues

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:

January to February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study according to OECD Guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

One control animal received a lower dosing volume on a single occasion. The relative humidity was recorded outside the acceptable range on one occasion. These deviations were considered not to have affected the study outcome.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C18-22, distn. residues

EC Number:

641-136-6

Cas Number:

1160164-88-4

IUPAC Name:

Alcohols, C18-22, distn. residues

Details on test material:

- Name of test material (as cited in study report): Alcohols, C18-22, Distn. Residues
- Substance type: product
- Physical state: waxy solid with yellowish colour
- Stability under test conditions: stable and homogeneous for 8 days at ambient dark in formulation
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

other: Han-Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 221 - 259 g (males); 165 - 191 g (females)
- Fasting period before study: no
- Housing: in groups of 3 per cage and per dose group in suspended polypropylene cages with stainless steel grid tops and solid bottom, wood shaving were used as bedding
- Diet: Rat and Mouse (modified) No. 1 Expanded SQC diet (supplied by Special Diets services limited, Essex, UK) ad libitum
- Water: water from domestics mains ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2010-01-20 To: 2010-02-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% carboxymethylcellulose (medium viscosity) with 0.2% Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: A weighed amount of the test substance was suspended in an appropriate amount of vehicle and mixed using a Silverson Mixer until a visibly homogeneous supsension was obtained. The formulations were magentically stirred for at least 60 min prior to and during sampling and splitting. All formulations were magnetically stirred for at least 60 min prior to dosing (Exception: In week 1 of treatment, dose groups 1, 2, 3 (resp. 0, 100, 300 mg/kg/d) were stirred for at least 30 min prior to dosing).

VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate (2 mL) samples of formulation from all groups were taken for analysis with regard to accuracy and concentration intended for Day 1 and Week 4 of dosing. The samples were stored at ambient laboratory temperature in the dark prior to analysis. The samples were analysed at the testing laboratory using a validated method (Charles River Study No. 425902). All samples analysed demonstrated satisfactory accuracy and concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected by the Sponsor after examination of preliminary studies carried out by the Sponsor

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality / Moribundity check: twice daily (in the morning and as late as practical each day)
Additional clinical observations: examination for reaction to treatment at regular hourly intervals throughout the day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucus membranes, respiration and excreta

BODY WEIGHT: Yes
- Time schedule for examinations: recorded twice during prerial, then daily during the dosing period, reported twice weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: quantity of food consumed by each cage measured and recorded twice each week, starting one week before the start of the treatment period up until the end of the dosing period

WATER CONSUMPTION: Yes
- Time schedule for examinations: visually monitored on a weekly basis

OPHTHALMOSCOPIC EXAMINATION: Yes
once during the pretrial period on all animals using an indirect ophthalmoscope after application of a mydriatic agent (1% Tropicamide) and during week 4 on control and group 4 (highest dose level) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to terminal kill
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: No data
- How many animals: all
- Parameters examined: haemoglobin, red blood cell count, haematocrit, mean cell haemoglobin, mean cell volume, mean cell haemoglobin concentration, red cell distribution width, reticulocytes, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unclassified cells, platelet count, prothrombin time, activated partial thromboplastin time.
Other: A blood smear was prepared from each haematology specimen. The smears were labelled, stained, stored and archived.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to terminal kill
- Animals fasted: No data
- How many animals: all
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, creatine phophokinase, urea, glucose, total bilirubin, cholesterol, total protein, albumin, globulin, albumin globulin ratio, sodium, potassium, chloride, phosphate, calcium, creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4, over a 4 h period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: animals deprived of food and water during the 4 h period in the metabolism cages
- Parameters examined: colour, specific gravity, volume, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood pigments, leukocytes, white blood cells, organisms, casts, other abnormalities, turbidity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during week 4
- Dose groups that were examined: all
- Battery of functions tested: cage side observations (posture/condition on first approach, after removal from cage: body temperature, condition of eyes, condition of the coat, presense of salivation, overall ease of handling) / observations in a standarized area (2 min observations) / funcional tests (grip strength, pain perception, landing foot splay, motor activity) / other physical or functional abnormalities not already recorded.

OTHER: none

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, post mortem examination consisting of a complete external and internal examination to include body orifices (ears, nostrils, mouth, anus, and vulva) and cranial, thoracic and abdominal organs and tissues; determination of organ weights (see table 1) from all animals before sampling and preservation, paired organs were weighed separately and the sum of the individual organs used for reporting purposes. Representative samples of the tissues were taken from all animals and fixed in 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes (see table 2), tissues were processed to paraffin wax blocks from all group 1 and 4 animals (control and highest dose level) and evaluated by the study pathologist (an internal peer review was undertaken by an appropriate qualified and experienced veterinary pathologist by independently assessing the microscope slides).

Other examinations:

Bone marrow smears were taken at necrospy, stained for groups 1 and 4 (control and highest dose level) using May-Gruenwald-Giemsa and evaluated microscopically by a pathologist.

Statistics:

All statistical tests were two-sided and performed at the 5% significance level. Males and females were analysed separately. Pairwise comparison were only performed against control group.
Body weight, food consumption, haematology, clinical chemistry and selected urinanalysis, motor activity and quantitative FOB measurement data were analysed for homogeneity of variance using the F-Max test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher's protected LSD method via Student's test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used. If the variances remained heterogeneous, then a Kruskal-Wallis non parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the nonparametric equivalent of Student's test). Where it was not possible to perform the F-Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weight as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis.
If the variances in the ANCOVA remained heterogeneous following log or square root transformation, the data was subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (ie less than 3 animals in any group), the untransformed parametric ANCOVA results were reported.
In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance: histological incindence data were analsed using Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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

Details on results:

CLINICAL SIGNS AND MORTALITY
One Group 2 female (100 mg/kg/day) died during the orbital sinus sampling, prior to termination on Day 29. The death of this animal was considered not to be related to treatment with the test substance.

BODY WEIGHT AND WEIGHT GAIN
Body weight was slightly lower in females receiving 300 or 1000 mg/kg/day than controls throughout the pretrial and dosing period. This was considered not to be a treatment-related effect because this difference was consistent from the beginning of pretrial period until the end of dosing period and the body weight gain in these groups was similar to or superios to controls.

FOOD CONSUMPTION
Food consumption was unaffected by treatment with the test substance.
Food intake of females receiving 1000 mg/kg/day was slightly lower than controls throughout the pretrial and dosing period. The changes attained statistical significance on Day 7. However, this was considered not to be a treatment-related effect because this difference was consistent from the beginning of pretrial period until the end of dosing period.

OPHTHALMOSCOPIC EXAMINATION
All findings and incidences were considered to be representative of the normal background findings observed at the testing laboratory.

HAEMATOLOGY
Slightly low monocyte counts were observed in all females receiving the test item. There was also a tendency for lower monocyte counts in males, but the changes did no attain statistical significance. The aeriology of the slight disturbances in monocyte counts in animals receiving the test item is unclear, but the changes showed no clear evidence of a dose response relationship and were considered to be of insufficient magnitude to be of toxicological significance. Furthermore, all monocyte counts in treated animals were within the historical control background range.
A number of other inter-group differences occurred when compared with controls, some of which attained statistical significance. These differences were minor, confined to one sex or lacked dosage-relationship. Consequently, these differences were attributable to biological variation.

CLINICAL CHEMISTRY
High creatine phophokinase activity was observed in males receiving 1000 mg/kg/day, however, this was considered to be the result of an individual group 4 male for which an unusually high value was recorded, thereby producing an artificially high group mean value for comparison.
Other slight variances from control values were noted for several parameters across male and female treated groups. However, due to a lack of a dose-related response, statistical significane or any further supportive evidence, these variances were considered not to be related to treatment.

NEUROBEHAVIOUR
Slight variances in motor activity from control values were noted throughout the timepoints. However, due to a lack of statistical significance, consistency between sexes, evidence of a dose-related response or further corroborating data, it was considered that these differences were not treatment related.

ORGAN WEIGHTS
Slightly high testes weights were observed in males receiving 100 or 1000 mg/kg/day. The changes attained statistical significance following absolute and covariate analysis, but not for body weight-relative organ weights. As this finding did not demonstrate a dose-related reponse and as no histopathological changes correlated, therefore, the statistical differences were considered not to reflect a toxic effect.

GROSS PATHOLOGY
All necropsy fingings noted were those commmonly seen in rats of this age and strain at the testing laboratory.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings recorded were typical backround findings for rats of this age and strain at the tesing laboratory.

OTHER FINDINGS
none

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It was concluded that repeat oral administration of Alcohols, C18-22, distn. residues to Han Wistar rats for 28 days at dose levels up to 1000 mg/kg/day was considered to be well tolerated with no obvious evidence of toxicity being noted. Therfore, the dose of 1000 mg/kg/day was considered to be a No-Observed-Effect-Level (NOEL) in this study.

Executive summary:

The objective of this study was to assess the potential toxicity of Alcohols, C18 -22, distn. residues following oral (gavage) administration for a period of at leasst 28 days. The study was designed to be compliant with OECD (Guideline No. 407), Directive 96/54/E.C.B.7 and EPA OPPTS guideline 870.3050. This study was conducted in accordance with the OECD Principles of Good Laboratory Practice.

Three goups of 5 male and 5 female Han Wistar rats were administered Alcohols, C18 -22, distn. residues orally at dose levels of 100, 300 or 1000 mg/kg/day for a period of 28 days. A similarly constituted control group received the vehicle, 1% carboxymethylcellulose (medium viscosity) with 0.2% Tween 80 at the same dose volume of 10 mL/kg body weight as the treated animals.

Animals were monitored regularly for viability and for signs of ill health or reaction to treatment. The following activities were measured and recorded at pre-determined intervals from pretrial until the completion of the study: body weights, food consumption, observations (clinical, detailed functional, standardised arena and cage), functional tests, ophthalmic examinations, haematology, coagulation, clinical chemistry, urinanalysis, gross necropsy, organ weights and histopathology.

There were no treatment-related clinical signs.

No treatment-related effects were observed during the detailed functional observation investigations.

Body weights and food consumption were unaffected by treatment.

There were no treatment-related haematology, coagulation or clinical chemistry changes.

There were no treatmennt-related ocular findings.

There were no necropsy or histology findings attributed to treatment.

In conclusion, repeat oral administration of Alcohols, C18 -22, distn. residues to Han Wistar rats for 28 days at dose levels up to 1000 mg/kg/day was considered to b e well tolerated with no obvious evidence of toxicity being noed. Therefore, the dose of 1000 mg/kg /day was considered to be a No-Observed-Effect-Level (NOEL) in this study.