Polyamide wax

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:

From 2000-04-07 to 2000-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

other: CD® (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river (UK) Limited, Margate, Kent, England
- Age at study initiation: 39 to 43 days
- Weight at study initiation: 184 to 214g for males and 145 to 182g for female
- Fasting period before study: no
- Housing: Five of one sex per cage. The cages were made of a stainless steel body with a stainless steel mesh lid and floor.
- Diet (e.g. ad libitum): ad libitum except overnight before routine blood sampling. Rat and Mouse n°1 Maintenance Diet (Special Diets Services Limited, Witham, Essex, England)
- Water (e.g. ad libitum): ad libitum, via polycarbonates bottles fitted with sipper tubes.
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycle


IN-LIFE DATES: No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v methylcellulose in purified water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared for administration as a series of graded suspensions in 1.0% w/v methylcellulose in purified water. The required amount of test material was weighed and transferred to a suitably sized mortar and ground using a pestle. A small amount of 1.0% methylcellulose was added to the mortar and test material mixed to a smooth paste. The remaining amount of 1.0% methylcellulose was added to give the require concentration and the suspension was mixed using a high-shear homogeniser to produce a homogenous suspension.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous suspensions were obtained with 1.0% w/v methylcellulose in purified water as vehicle
- Concentration in vehicle: 1.5, 15 and 100 mg/ml
- Amount of vehicle (if gavage): 10 mL/kg bw/day
- Lot/batch no. (if required): nodata
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the homogeneity assessment showed that at 1.5 and 100 mg/mL, E96096 was homogeneously distributed throughout the 1.0% methylcellulose formulations.
The mean concentrations of E96096 in formulations for dosing on Day 1 of treatment, ranged from 96.0 - 97.7 % of nominal concentrations and were considered satisfactory.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/ week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previously conducted seven-day repeated dose oral toxicity study, CD rats were dosed with 250, 500 or 1000 mg/kg bw /day of the test substance. There were no deaths. The appareance and behaviour of the animals were unaffected by treatment. Bodyweight gain, food consumption and food conversion efficiency were unaffected by treatment. There were no organ weight changes attributable to treatment with the test material and no macroscopic findings at necropsy. Dosages of 15, 150 and 1000 mg/kg were considered suitable for the subsequent 4-week study.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
Animals were inspected at least wice daily for evidence of reaction to treatment or ill-health. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.


BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed during the acclimatisation period, on the day that treatment commenced, weekly throughout the treatment period and before necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded for each week throughout the treatment period. From these records, the mean consumption per animal was calculated for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food
- How many animals: 40
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29
- Animals fasted: Yes, overnight withdrawal of food
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase activity (gGT), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment and weekly during the study. the FOB was performed prior to dosing during the treatment period and at a similar time of day on each occasion.
- Dose groups that were examined: All groups
- Battery of functions tested:
=> in the hand and standard arena observations performed before commencement and during each week of treatment: ease of removal from cage, salivation, lacrimation, exophthalmos, piloerection, fur condition, vocalisation on handling, reactivity to handling, arousal, gait, grooming, palpebral closure, posture, activity counts, rearing count, tremors, twitches, convulsion, defecation, urination.
=> Reflexes and responses were recorded before commencement and during week 4 of treatment: approach response, touch response, auditory qtartle reflex, tail pinch response, righting reflex, body temperature, landing footsplay, grip strength, pupil closure reflex.
=> Motor activity was measured before commencement and during week 4 of treatment. Activity measurements were recorded for ten six-minutes periods (a total of one hour).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (1000 mg/kg bw /day) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with - longitudinal section including bone marrow
joint
Heart - included auricular and ventricular regions
Ileum - included Peyers patch , where possible
Kidneys - included cortex, medulla and papilla regions
Liver - section from each of the five major lobes
Lungs - section from two major lobes, including bronchi
Spinal cord - transverse and longitudinal section at the cervical level
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section

Statistics:

For organ weights and bodyweight changes, homogeneity of variance was tested using Barlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwisecomparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.
For clinical pathology data, if the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values diffrent from the mode was analysed (Fisher, 1950) followed by a test for a trend in proportion (Mantel, 1963). Otherwise, a test for heterogeneity of variance between treatments was applied (Barlett, 1937). If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found) and more than two groups were being compared, group means were compared using William's test for a dose-related response (Williams, 1971-72), or if there was evidence for a non-monotonic response, Dunnett's test (Dunnett, 1955, 1964). For separate two-group comparisons, a Student's t test was used. If significant heterogeneity of variance was present (and could not be removed by a logarithmic transformation), groups were compared using Shirley's non-parametric test for a dose-related response (Shirley, 1977), or if there was evidence for a non-monotonic response, Dunn's test (Dunn, 1964). For separate two-group comparisons, a Wilcoxon rank sum test (Wilcoxon 1945) was used.
Unless stated, group mean values or incidences for the treated groups were not significantly different from those of the Controls (p>0.05).

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):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths and no clinical signs related to the treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was considered to be unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was considered to be unaffected by treatment.

FOOD EFFICIENCY
Food conversion efficiency was considered to be unaffected by treatment.

HAEMATOLOGY
There were no haematologival changes which were considered to be related to treatment.
A few inter-group differences attained statistical significance but there were minor, seen in one sex only and were considered to have arisen by chance.

CLINICAL CHEMISTRY
No clinical chemistry changes which were considered to be related with treatment.

NEUROBEHAVIOUR
There were no inter-group differences in arena observations that were considered to be associated with treatment.
Landing footsplay and grip strength measurements showed some inter-group variation during week 4 of treatment and some differences achieved statistical significance. There were, however, no consistent trends and many of the differences were apparent before commencement of treatment, an association with treatment was therefore discounted.
Motor activity was unaffected by treatment.

ORGAN WEIGHTS
Organ weights were unaffected by treatment.

GROSS PATHOLOGY
Macroscopic examination of animals killed on completion of the treatment period did not reveal any treatment-related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings which were attributable to treatment with the test material. All microscopic findings were considered to be incidental and of no toxicological importance.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The daily administration to CD rats of the test material, at dosages of 15, 150 and 1000 mg/kg/day for four weeks was not associated with any evidence of toxicity. The No-Observe-Effect Level (NOEL) is considered to be 1000 mg/kg bw/day .

Executive summary:

In a sub-acute study performed according to OECD 407 guideline and in compliance with GLP, test material diluted in 1.0% methylcellulose in purified water was administered by oral gavage to three groups of rats (5/sex) at 15, 150 and 1000 mg/kg bw/day for four weeks. A control group received vehicle alone at the same volume-dosage (10 mL/kg bw/day).

There were no deaths and no clinical signs related to treatment, no evidence of neurotoxicity during the weekly functional battery test. Bodyweight, food consumption and food conversion efficiency were not affected by treatment. There were no haematological or blood chemistry changes related to treatment. Organ weights were unaffected by treatment and there were no macroscopic or microscopic findings which were attributable to the treatment with the test material.

The No-Observed-Effect Level (NOEL) was considered to be 1000 mg/kg bw/day.