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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2002 to 31 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with international guidelines in a GLP facility.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Details on test material:
Identification of test item (as cited in study report): NALCO 01WC026/PSO
Batch number: XC1M0642

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality. Source: Charles River Deutschland, Sulzfeld, Germany.
Age at start of treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nuliparous and non-pregnant)
Randomisation: Prior to commencement of treatment, by computer generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.
Identification: Earmark and tattoo.

Animal husbandry
Conditions: A controlled environment was maintained in the roon with optimal conditions considered as being approximately 15 air chanes per hour, a temperature of 21 ± 3 degrees celsius, a relative humidity of 30-70 % and 12 hours artificial fluorescent light and 12 hours dark per day.
Temporary deviations from the light/dark cycle (with a maximum of 1 hour; main study) occurred due to performance of functional observations in the room. Also, deviations from the maximum level of relative humidity (with a maximum of 20 %) occurred which might have been caused by cleaning procedures in the room. Based on laboratory historical data, these deviations were considered not to have affected study integrity.

Accommodation
Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding. Results of bedding analyses for contaminants are examined and archived.

Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants on a regular basis. Results are examined and archived.

Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.

Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Test Item Preparation
Vehicle: Water (Milli-U)
Rationale for vehicle: Based on trial formulations performed at NOTOX
Stability of test item in vehicle: At least 4 hours (determined at NOTOX)
Method of Formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the substance.
Storage conditions: At ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analysed after the in-life phase to check homogeneity (highest and lowest concentrations) and accuracy of preparation (all concentrations). Stability in vehicle over 4 hours was also determined (highest and lowest concentrations). The analytical method used was based on the results of the development and validation of the analytical method (see Section 8 of this technical dossier).
Duration of treatment / exposure:
Oral treatment of rats was condcted by oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Frequency of treatment:
Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dosing group (20 of each sex in total).
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Observations
Mortality/viability: Twice daily
Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatement and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Functional observations: During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights: on days 1, 8, 15, 22 and 28.
Food consumption: weekly
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical Laboratory Investigations
Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7:30 and 9:30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats and collected into tubes prepared with EDTA for haemotological parameters (0.25 mL), with citrate for clotting tests (1.0 mL) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 mL).
The following parameters were determined:
Haemotology: Erythrocytes count, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular heamoglobin concentration, Platelets count, Red cell distribution width, Total leucocytes count, Differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes).
Clotting Potential: Prothrombin time, Partial thromboplastin time.
Clinical Biochemistry: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Bilrubin-total, Chloride, Cholesterol-total, Creatinine, Glucose, Phosphorous, Protein-total, Protein-albumin, Urea, Calcium, Potassium, Sodium, Haemolysis.
Sacrifice and pathology:
Pathology

Necropsy
All animals surviving to the end of the observation period were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all aninmals at necropsy and fixed in a 4 % formaldehyde solution:
Adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, duodenum, epididymies, eyes with optic nerve and Harderian gland, female mammary gland area, femur including joint, heart, lieum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung (infused with formalin), lymph nodes-mandibular/mesenteric, nasopharynx, oesophagus, ovaries, pancreas, peyer's patched (jejunum, lieum) if detectable, pituitary gland, preputial gland, prostate gland, rectum, salivary gland-mandibular/sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord-cervical/midthoracic/lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross legions.

Organ Weights
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus.

Histotechnology
All organ tissue samples, as defined under histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

Histopathology
The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group
- all gross legions of all animals
Based on the treatment related morphologic changes, kidenys were also examined from all rats of the intermediate dose groups. All abnormalities were described and included in the report.
Statistics:
The following statistical methods were used to analyse the data:
- if the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) 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

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. No statistical analysis was performed on motor activity data. 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 differnet test statistics values.

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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
OBSERVATIONS

Mortality
No mortality occurred during the study period.

Clinical Signs
There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered related to treatment.
Incidental findings that were noted included alopecia, scabs, a wound in the neck region, and brown staining of the fur. These findings are often noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidences observed, these were considered signs of no toxicological significance. Clinical signs were absent among control animals and group 3 animals and high dose females.

Functional Observations
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with NALCO 01WC026/PSO, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.

Body Weights
Body weights and body weight gain of treated animals were considered to have been unaffected by treatment with the test item.

Food Consumption
There were no differences in food consumption before or after allowance for body weight between treated and control animals that were considered to be related to treatment with the test item.
Relative food intake of high dose females appeared lower than control females during treatment. However, food intake of control females was higher than expected from similar studies. No reasonable explanation could be given for that observation. Also, body weights of these females were normal. Therefore, this change in food intake was considered to be of no toxicological significance.

CLINICAL LABORATORY INVESTIGATIONS

Haemotology
Haemotological parameters of treated rats were considered not to have been affected by treatment.
Increased white blood cell counts were noted in males dosed at 1000 mg/kg/day, the mean of which was within the normal range. Also, no (structural) lesions were noted. The higher partial thromboplastin time in females treated at 150 mg/kg/day occurred in the absence of a dose-related response. These changes were therefore to be of no toxicological sognificance.

Clinical Biochemistry
There were no differences noted between control and treated rats that were considered to be related to treatment.
At 1000 mg/kg/day, a lower albumin plasma concentration was noted in males and total protein was decreased in females. These changes were slight, occurred in opposite sexes and were within the range expected for these types of studies. In addition, morphological or biochemical support for these changes was absent. Other findings that attained statistical significance included higher glucose concentration (males, 50 mg/kg/day), lower creatinine (females, 50 mg/kg/day) and inorganic phosphate levels (females, 150 mg/kg/day). Since these changes were absent in higher dose levels, they were not considered toxicologically relevant.

PATHOLOLGY

Macroscopic Examination
There were no necropsy findings that were considered to be related to treatment with the test item.
Pelvic dilation was seen in three males at 1000 mg/kg/day, and in one male of the control group and 50 mg/kg/day group, respectively. Albeit this necropsy finding occurred at a slightly higher incidence in the high dose than would be expected, it is more often seen in untreated rats of this age and strain. Also, the pelvic dilation was not accompanied by supporting microscopic lesions. Therefore, pelvic dilation was considered to be of a spontaneous nature and not related to treatment. Incidental findings among control/treated animals included presence of an accessory liver lobe (grown together with diaphragmatic surface), red discolouration of the thymus, lungs or mandibular lymph node, red scabs, fluid in uterus, enlarged adrenals, and red(-brown) foci on adrenals or thymus. These findings are occasionally seen among rats used in these types of studies. In the absence of treatment-related incidence they were considered changes of no toxicological significance.

Organ Weights
Organ weights and orga:body weight ratios of treated animals were considered to be similar to those of control animals.
The statistically significant lower absolute brain weight of femalesdosed at 50 mg/kg/day was considered not to be a sign of toxicity in the absence of a dose-related response.

Microscopic Examination (see also attached background material for raw data)
There were no microscopic findings recorded which could be attributed to treatment with the test item.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysis of Dose Preparations

Analyses were based on the presence of three peaks in the chromatogram. Test substance formulations in water (Milli-U) were noted as stable for at least 4 hours and formed a homogenous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 93 % to 102 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type (see attached data and tables document in background information).

Applicant's summary and conclusion

Conclusions:
Wistar rats were treated with NALCO 01WC026/PSO for 28 consecutive days by oral gavage at dose levels up to 1000 mg/kg/day.
There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination that were considered to be an effect of treatment.
From the results presented a No Observed Adverse Effect Level (NOAEL) for NALCO 01WC026/PSO of 1000 mg/kg/day was established.