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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-06 to 2018-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25/09/2017-07/08/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
The 14-day dose range finder study was designed to provide information for further repeated dose toxicity studies.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 7 weeks
- Weight at study initiation: males weighed 177 to 202g, the females weighed 137 to 159g,
- Fasting period before study: no
- Housing: housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: pelleted diet, mains drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0-50-100-200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
Analytical verification of doses or concentrations:
no
Remarks:
The test item was administered within approximately two hours of it being formulated . It is assumed that the formulation was stable for this duration.
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing.
Additional observations were also made four hours following dosing (not at weekends).

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION :
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to
15. Food conversion efficiency was calculated retrospectively.


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): Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male treated with 1000 mg/kg bw/day was observed to have increased salivation postdosing (Day 5 only). Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation this finding is considered not to represent an adverse effect of treatment.
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
Gross pathological findings:
no effects observed
Conclusions:
The administration of test item , by oral gavage for a period of fourteen consecutive days to
rats, at dose levels of 250, 500 and 1000 mg/kg bw/day resulted in no toxicologically
significant effects being detected, as such, dose levels of up to 1000 mg/kg bw/day would be
suitable for use in future studies.
Executive summary:

The study was designed to provide information for further repeated dose toxicity studies. The test item was administered by oral gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for fourteen consecutive days, at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Distilled Water) over the same period. There was no adverse effect observed on mortality, clinical signs, body weight change, dietary intake and water consumption during the study. No macroscopic abnormalities were detected at necropsy.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
EC Number:
272-712-1
EC Name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Cas Number:
68909-77-3
Molecular formula:
Not applicable (UVCB)
IUPAC Name:
2-(2-hydroxyethoxy)ethan-1-ol; 2-[1-(morpholin-4-yl)ethoxy]ethan-1-amine; 2-{2-[bis(2-hydroxyethyl)amino]ethoxy}ethan-1-ol; 4-{2-[2-(morpholin-4-yl)ethoxy]ethyl}morpholine; morpholin-3-one
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: LL150693
- Expiration date of the lot/batch: 30 December 2018
- No correction for purity was made

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: Initially formulations were found to be stable for at least ten days and therefore formulations were prepared weekly and stored at approximately 4 °C in the dark. Further stability at twenty-one days was confirmed during the later stage of the study.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan(TM):WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: Males 182-226 g, Females 133 -168 g
- Fasting period before study: no data
- Housing: housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding
- Diet (e.g. ad libitum): ad libitum, Rodent 2014C Teklad Global Certified Pelleted Diet
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: nine days

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Once daily, by gavage, using a stainless steel dosing cannula attached to a disposable plastic syringe for ninety consecutive days. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Vehicle:
water
Remarks:
distilled water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services.
Initially formulations were found to be stable for at least ten days and therefore formulations were prepared weekly and stored at approximately 4 °C in the dark. Further stability at twenty-one days was confirmed during the later stage of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analyzed on two occasions for concentration of the test substance at Envigo Research Limited, Shardlow, UK, Analytical Services.
The homogeneity and stability, determined with respect to the concentration of Test Item in Water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL. The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak. The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accurracy and precision. The homogeneity and stability was performed for Test Item in Distilled Water formulations at nomincal concentrations of 2 mg/mL and 200 mg/mL when stored refrigerated for 21 days and at room temperature in the light for 24 hours. The mean concentrations of Test Item in test formulations analyzed for the study were within ± 10% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (vehicle only - distilled water)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available toxicity data including a14 day repeated dose oral (gavage) range-finding toxicity study in rats (Wood, 2019). The administration of the test item , by oral gavage for a period of fourteen consecutive days to rats, at dose levels of 250, 500 and 1000 mg/kg bw/day resulted in no toxicologically significant effects being detected, as such, it was concluded that dose levels of up to 1000 mg/kg bw/day would be suitable for the 90-day repeated dose toxicity study.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing.
- Parameters: overt signs of toxicity, ill-health or behavioral change

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Individual body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all Groups 1 to 4 animals were examined pre-treatment. During Week 12, the eyes of all control and high dose animals (Groups 1 and 4, respectively) were examined.
Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

HAEMATOLOGY: Yes
Hematological investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
- Parameters checked: The following parameters were measured on plasma from blood collected into tubes containing potassium EDTA anti-coagulant: hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, volume and hemoglobin concentration), total leukocyte count, differential leukocyte count (neutrophyls, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count (methylene blue stained slides were prepared but reticulocytes were not assessed), prothrombin time was assessed by 'Innovin' and activated partial thromboplastin time was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on day 91.
- Animals fasted: No
- How many animals: all animals
- Parameters checked: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids

FUNCTIONAL OBSERVATIONS
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Behavioral assessment: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory reactivity Tests.
- Functional performance tests:
- motor activity : 20 purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least 2 hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, reiter and Macphail, 1979).
- forelimb/hindlimb grip strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
- Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed: gasp response, vocalization, toe pinch, tail pinch, fingers approach, touch escape, pupil reflex, blink reflex, startle reflex
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint) - retained only and not processed, bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides - preserved in Modified Davidson's fluid, esophagus, eyes - fixed in Davidson's fluid, gross lesions, heart, ileum (including Peyer's patches), jejunum, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes - preserved in Modified Davidson's fluid, thymus, kidneys, liver, lungs (with bronchi) - lungs were inflated to approximately normal inspiratory volume with buffered 10% formaline before immersion in fixative, lymph nodes (mandibular and mesenteric), mammary glands, muscle (skeletal), thyroid/parathyroid, tongue - retained only and not processed, trachea, urinary bladder, uterus (with cervix), vagina
All tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Other examinations:
No data
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test(parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day from Day 19 (males) or Day 21 (females) throughout the majority of the treatment period. Observations such as increased post-dose salivation are often observed when animals are dosed via the oral gavage route and indicates unpalatability or slight irritancy of the dosing formulations, rather than evidence of true systemic toxicity.
Isolated instances of noisy respiration were noted in two males from each treatment group and one female each treated with 100 or 1000 mg/kg bw/day. At this low frequency, this is likely to indicate difficulty in dosing these animals on isolated occasions, rather than evidence of true systemic toxicity.
Incidental observations considered to be unrelated to treatment included one animal of either sex treated with 10 mg/kg bw/day exhibiting isolated occurrences of generalized fur loss, and two males and one female treated with 10 mg/kg bw/day showing staining of the fur/head for short periods of time.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths on the study.
One female treated with 10 mg/kg bw/day was killed in extremis on Day 81 due to the severity of observations noted, these included ptosis, pilo-erection, lethargy, hypothermia, hunched posture, pallor of the extremities, dehydration, red fluid from the snout and staining around the snout. Necropsy showed a pale brain, dark colored contents throughout the gastro-intestinal tract, a raised limiting ridge in the stomach and a pale uterus. There were no histopathological changes to account for the early death.
One male treated with 10 mg/kg bw/day was found dead on Day 90 during the blood sampling procedure; no clinical signs were observed prior to death, and no abnormalities were seen at necropsy. There was tracheal necrosis at histopathology, suggesting possible dosing trauma or reflux.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight:
There was no adverse effect detected for body weight development at 10, 100 or 1000 mg/kg bw/day.

Body weight gain:
Males from all treatment groups showed a statistically significant reduction (p<0.05) in body weight gain during the first week of treatment. Improvement was evident thereafter, however, body weight gain was again statistically significantly reduced (p<0.01) during Week 11 for males treated with 1000 and 100 mg/kg bw/day. During Week 12 for these males, body weight gain exceeded controls and during Week 13, body weight gain was comparable to controls. As a consequence of the slight reductions in body weight gain at 1000 mg/kg bw/day, overall body weight gain was slightly lower than controls. Overall, the fluctuations in body weight gain were not considered to represent an adverse effect based on improvement evident throughout the majority of the treatment period. No such effects were detected in any treated female animals. For more details, please refer to Table 1 in the section 'Additional information on results'.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on food consumption for either sex at 10, 100 or 1000 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on food conversion efficiency for either sex at 10, 100 or 1000 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects on the hematological parameters measured for either sex at 10, 100 or 1000 mg/kg bw/day.
* Platelet count: Males treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.01) in platelet count in relation to controls. All of the individual values were within the background control ranges (Table 2b), and in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance.
* Neutrophils: Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in neutrophil count in relation to controls. All of the individual values were within the background control ranges, and 5/10 individual control values actually exceeded the background control range. In isolation, and in the absence of any histopathological correlates, these findings were considered to be the result of normal biological variation.
For more details, please refer to ection 'Additional information on results'.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 10, 100 or 1000 mg/kg bw/day.
* Aspartate aminotransferase: Males treated with 100 or 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in aspartate aminotransferase (ASAT); with the exception of one animal per dose group, all of the individual values were within the background control range. However, it was noted that the majority of the control values were at the higher end of the background control range. A reduction in ASAT is generally considered not an adverse effect, and therefore of no toxicological significance.
* Urea: Males treated with 1000 mg/kg bw/day also had a statistically significant reduction (p<0.05) in urea compared to the controls. The majority of the individual values were within the background control range, and without any histopathological correlates, this finding was considered to be incidental and of no toxicological significance.
* Calcium: Females from all treatment groups exhibited statistically significant increases in calcium (p<0.05); however a true dose relationship was not apparent for either parameter. The individual values for all treated females were within background control ranges, and this finding is likely due to normal biological variation.
* Bilirubin: Females from all treatment groups exhibited statistically significant increases in bilirubin (p<0.05 or p<0.01); however a true dose relationship was not apparent for either parameter. The individual values for all treated females were within background control ranges, and this finding is likely due to normal biological variation.
* Chloride: Females treated with 1000 mg/kg bw/day exhibited a statistically significant reduction (p<0.05) in chloride concentration, with 5/10 values below the background control ranges. In the absence of any corresponding histopathological changes, this reduction is considered to be a result of normal biological variation and of no toxicological significance.
For more details, please refer to Table 3a and 3b in section 'Additional information on results'.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral assessments: There were no treatment-related changes in behavioral parameters observed
Functional performance tests: There were no treatment-related changes in functional performance. The final 20% of activity was statistically significantly lower (p<0.05) than controls for all treated males. A true dose related response was not evident, there was no effect on overall activity or any corresponding effect seen in females, and no clinical signs of neurotoxicity were evident. The intergroup differences were therefore considered not to be of toxicological significance.
Sensory reactivity assessments: There were no treatment-related changes in sensory reactivity.
For more details, please refer to Table 4 in section 'additional information on results'
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Liver weight: Both males and females treated with 100 or 1000 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in liver weight; both absolute and relative to terminal body weight, in a dose related manner. The majority of the individual values were within the background control ranges, however, the increased weights correlate with the histopathological changes of increase rarefaction in the liver.
Kidney weight: Females treated with 100 or 1000 mg/kg bw/day showed a statistically significant increase (p<0.05 or p<0.01, respectively) in kidney weights; both absolute and relative to terminal body weight in a dose related manner. The majority of the individual absolute values were within the background control ranges; with the exception of two absolute values at 1000 mg/kg bw/day which were above these ranges. The majority of relative to body weight values were within the background control ranges, with the exception of two and four values above the background control ranges (100 and 1000 mg/kg bw/day, respectively). As there were no histopathological correlates the intergroup differences were considered to be of no toxicological significance.
For more details, please refer to Table 5a and 5b in section 'Additional information on results'.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Premature deaths: The male treated with 10 mg/kg bw/day that was found dead on Day 90 and did not show any macroscopic abnormalities. One female treated with 10 mg/kg bw/day that was killed in extremis on Day 81 exhibited a pale brain, dark colored contents throughout the gastro-intestinal tract, a raised limiting ridge in the stomach and a pale uterus.
Terminal kill: There were no toxicologically significant abnormalities detected in animals of either sex treated up to 1000 mg/kg bw/day.
The following macroscopic abnormalities detected were without treatment-related histopathogical correlates or showed a true dose-related response and were considered not to be of toxicological significance.
* One male treated with 1000 mg/kg bw/day exhibited an enlarged liver and reddened lungs.
* One further male treated with 1000 mg/kg bw/day had a raised limiting ridge in the stomach.
* One male treated with 100 mg/kg bw/day had a raised limiting ridge in the stomach.
* One female treated with 100 mg/kg bw/day exhibited a pale and mottled liver.
* A further female treated with 100 mg/kg bw/day had reddened lungs.
* One male treated with 10 mg/kg bw/day had small epididymides, prostate, seminal vesicles and testes.
* One female treated with 10 mg/kg bw/day had reddened lungs.
No macroscopic abnormalities were detected in female animals treated with 1000 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Premature Decedents:
There were two premature decedents during the study, both deaths were considered to be unrelated to test item administration.
* Male 30 receiving the test item at 10 mg/kg bw/day was found dead during the bleeding procedure on Day 90. At histopathology there was tracheal necrosis suggesting a possible dosing trauma or reflux.
* Female 32 receiving the test item at 10 mg/kg bw/day was killed in extremis on Day 81. There was dark colored contents in the gastrointestinal tract and several pale tissues at necropsy. No changes were noted at histopathology to account for the early death.

Terminal Kill:
Liver: Increased rarefaction was present in both males and females at 1000 mg/kg bw/day. This was considered to be associated with minor metabolic changes, and correlated with the increased liver weights at necropsy. This finding is generally considered to be non-adverse.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Key result
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 adverse treatment related effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Body weight gain

Groups

(sex) 

 From Day

To Day

1

8

8

15 

15

22 

22

29

29

36

36

43

43

50

50

57

57

64 

64

71 

71

78

78

85 

85

91

Abs

Gain

1

91 

 %

Gain

1

91

control

(M)

 Mean

SD

38.3

4.9

29.9

4.6

26.0

4.9

22.7

4.6

14.8

5.5

12.4

4.7

14.0

4.2

10.0

3.4 

10.5

4.8

9.2

2.4

10.9

3.0

5.8

4.5 

5.7

3.9

210.2

27.8 

99.9

11.7

10 mg/kg bw

(M)

Mean

SD

34.4*

5.5 

28.5

4.5 

23.8

3.5 

21.3

3.3 

16.2

7.2 

12.4

2.9 

14.7

3.3 

11.7

5.9

11.1

3.8 

9.6

4.6 

9.1

3.9

8.8

3.8

3.4

3.4

203.1

30.1

97.8

12.7

100 mg/kg bw

(M)

Mean

SD

34.5*

4.4

29.3

5.2

24.2

5.6 

22.1

4.9

14.1

2.1

11.2

2.5 

15.4

3.2 

10.3

27.5 

10.0

30.3

10.8

2.3 

7.4**

2.6

7.8

2.8

3.9

3.7

201.0

29.0

95.0

11.2

1000 mg/kg bw

(M)

Mean

SD

33.4*

4.7

27.3

5.4

23.9

5.3

19.6

3.2

14.9

6.0 

10.9

3.2 

14.5

5.0

8.9

3.8

8.3

4.2 

10.1

3.4

6.2**

1.6

7.7

3.0

5.3

4.2 

191.0

34.6

93.2

13.2

control

(F)

Mean

SD

13.1

3.0

13.4

4.1

9.8

4.4

11.1

6.2 

5.5

3.9

6.6

3.9

2.9

4.9

5.8

4.0

3.9

2.3

1.6

2.6

3.7

4.5 

5.4

3.2 

-1.2

3.6

81.6

7.0

52.5

3.1 

10 mg/kg bw

(F)

Mean

SD

13.1

6.0 

13.8

5.1 

8.5

4.7 

11.1

7.1 

2.8

3.7

10.3

4.1

0.5

3.5 

3.4

4.3

3.3

3.4 

4.2

4.2

1.0

4.7 

3.8

4.5 

3.9

4.0

80.4

16.5 

52.4

8.6

100 mg/kg bw

(F) 

Mean

SD

14.2

7.8 

10.2

4.7

10.6

3.8

12.4

5.9 

4.4

4.6 

5.9

3.4

5.0

4.7 

4.0

4.2

3.6

3.5

3.1

3.4 

2.2

4.3

7.3

5.0 

0.1

5.1 

 83.0

16.7

54.0

9.5

1000 mg/kg bw

(F) 

Mean

SD

15.1

5.0

10.8

2.9 

9.5

5.6

11.2

4.3 

4.9

5.4

5.8

5.5 

2.7

5.6

5.4

3.8 

3.8

3.7 

1.3

3.7

2.2

6.3 

5.3

4.1 

-0.6

5.0

77.4

11.3

50.7

7

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

abbreviations: M males; F females; SD standard deviation

Table 2a: Hematological values (platelet count and neutrophils)

Groups

Sex

control

M

10 mg/kg bw

M

100 mg/kg bw

M

1000 mg/kg bw

M

control

F

10 mg/kg bw

F

100 mg/kg bw

F

1000 mg/kg bw

F

 Neut (10^9/l) (mean +/-SD)

 2.0929

+/-0.509

 1.876

+/- 0.428

 1.488

+/-0.299

 1.728

+/-0.605

 1.180

+/-0.302

 1.452

+/-0.350

1.230

+/-0.360 

 0.742**

+/-0.295

 PLT  (10^9/l) (mean +/-SD)

 507.4

+/-118.2

 567.1

+/-126.0

 580.5

+/-96.0

 659.8**

+/-89.4

541.9

+/-104.7 

 579.8

+/-142.7

 513.1

+/-179.0

 498.2

+/-213.5

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

Abbreviations: Neut: neutrophils, PLT: platelet count

Table 2b: Normal Ranges for Hematological parameters in the Wistar Han™:RccHan™:WIST Strain Rat (appropriate strain and age)

 Hematology Male ranges*    No of animals  Female ranges*   No of animals 
 Neut (10^9/l)

 0.20 - 2.05

(1.12) [0.46]

 173

 0.11 -1.18

(0.65) [0.27]

 173
 PLT (10^9/l)

 473 - 732

(603) [65]

 171

 446 - 785

(615) [85]

 172

* Range = mean ± 2 standard deviations values in brackets indicate (group mean) and [standard deviation]  

Table 3a: Group Mean Blood Chemical Values

Group

(sex)

control

(M)

10 mg/kg bw

(M)

100 mg/kg bw

(M)

1000 mg/kg bw

(M)

control

(F)

10 mg/kg bw

(F)

100 mg/kg bw

(F)

1000 mg/kg bw

(F)

 Urea (mg/dl)

 44.2

+/-4.7

 45.8

+/-8.7

41.3

+/-15.5 

 32.3*

+/-11.6

 43.8

+/-7.4

 44.2

+/-4.2

54.6

+/-24.5 

41.5

+/-7.9 

 Cl- (mmol/l)

 101.5

+/-6.9

104.2

+/-7.7

102.9

+/-7.7

104.5

+/-5.3

 101.4

+/-2.1

 100.7

+/-4.0

100.4

+/-4.4 

97.3*

+/-4.9 

 Ca++ (mmol/l)

2.698

+/-0.119

2.357

+/-0.893 

2.710

+/-0.086 

2.424

+/-0.858 

 2.536

+/-0.224

 2.761*

+/-0.118

2.718*

+/-0.199 

 2.702*

+/-0.185

 ASAT (IU/l)

110 .7

+/-28.5

 99.0

+/-62.8

 85.4*

+/-21.7

 82.6*

+/-28.6

 103.3

+/-42.3

 90.4

+/-38.0

105.2

+/-28.9 

82.9

+/-19.1 

 Bili (mg/dl)

 0.847

+/-2.433

 0.130

+/-0.030

0.105

+/-0.044 

0.117

+/-0.049 

 0.063

+/-0.052

 0.114*

+/-0.025

0.082*

+/-0.054 

0.118**

0.034 

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

Table 3b Normal Ranges for Blood Chemical Values in the Wistar Han™:RccHan™:WIST Strain Rat

 Blood chemistry  Male  No of animals   Female  No of animals
 Urea (mg/dl)

 30 - 55

(43) [6]

176

 30 - 62

(46) [8]

 172

 Cl- (mmol/l)

 99 - 107

(103) [2]

176 

 99 - 108

(104) [2]

 171
 Ca++ (mmol/l)

 1.24 - 3.12

(2.18) [0.47]

176

 1.22 - 3.17

(2.20) [0.49]

 177
 ASAT (IU/l)

 45 - 122

(83) [19]

 174

 21 - 173

(97) [38]

178 
 Bili (mg/dl)

0.05 - 0.16

(0.11) [0.03] 

 173

 0.04 - 0.13

(0.09) [0.02]

158

* Range = mean ± 2 standard deviations values in brackets indicate (group mean) and [standard deviation]  

Table 4: Group Mean Functional Test Values and standard Deviations (Mean +/-SD)

Group

(Sex)

control

(M)

10 mg/kg bw

(M)

100 mg/kg bw

(M)  

1000 mg/kg bw

(M)

control

(F) 

10 mg/kg bw

(F)

100 mg/kg bw

(F) 

1000 mg/kg bw

(F) 

 last 20% activity

 89.8

+/-54.3

36.8*

+/-71.5 

 34.9*

+/-48.5

38.4*

+/-44.5 

 60.1

+/-57.8

61.6

+/-49.2 

 100.7

+/-39.2

44.6

+/-47.6 

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

Table 5a: Group Mean (+/- SD) Organ Weights with Corresponding Relative (% of Body Weight)

 Group

(sex)

control

(M)

10 mg/kg bw 

(M)

100 mg/kg bw 

(M)  

1000 mg/kg bw 

(M)

control

(F) 

10 mg/kg bw 

(F) 

100 mg/kg bw 

(F)   

1000 mg/kg bw 

(F) 

Kidneys (g)

 2.30695

+/-0.28602

 2.34176

+/-0.29851

2.31172

+/-0.22578 

 2.35556

+/-0.32921

1.45316

+/-0.10376 

1.45564

+/-0.11038

1.53995*

+/-0.14362

 1.60262**

+/-0.15012

Kidneys (%)

0.549

+/-0.056

0.571

+/-0.064

0.562

+/-0.045 

0.595

+/-0.037

0.614

+/-0.030 

0.626

+/-0.048

0.654*

+/-0.048 

 0.697**

+/-0.051

 Liver (g)

 13.6783

+/-1.97583

13.0220

+/-2.22944 

 14.2141*

+/-1.73269

14.4790**

+/-1.86953 

7.85257

+/-0.82498 

8.11848

+/-1.23007 

8.41874*

+/-1.17860

 8.48672*

+/-0.93354

 Liver (%)

3.245

+/-0.311

3.156

+/-0.327 

 3.444*

+/-0.228

 3.660**

+/-0.204

3.314

+/-0.302 

3.474

+/-0.313

3.559*

+/-0.296

 3.688*

+/-0.331

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

Table 5b Normal Ranges for Absolute and Body Weight-Relative Organ Weight Values in the Wistar Han™:RccHan™: WIST Strain Rat

 Organ weight  Male  No of animals  Female  No of animals
 Kidney absolute weight (g)

 1.7255 - 2.8352

(2.2804) [0.2774]

 162

1.2110 - 1.8014

(1.5062) [0.1476] 

 164
 Kidney relative weight (%)

 0.4589 - 0.6654

(0.5622) [0.0516]

 165

 0.5233 - 0.7051

(0.6142) [0.0454]

 160
 Liver absolute weight (g)

 10.0828 - 16.2974

(13.1901) [1.5537]

 160

 6.5535 - 10.3234

(8.4384) [0.9425]

 162
 Liver relative weight (%)

 2.7896 - 3.7163

(3.2529) [0.2317]

161 

 2.8487 - 4.0584

(3.4536) [0.3024]

162 

* Range = mean ± 2 standard deviations values in brackets indicate (group mean) and [standard deviation]

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to rats by gavage, at dose levels of 10, 100 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects for body weight development, food intake, clinical observations, hematological or blood chemistry parameters, or necropsy findings. The histopathological changes of increased rarefaction in the liver at 1000 mg/kg bw/day was considered to be non adverse, therefore the No Observed Adverse Effect Level (NOAEL) for either sex was considered to be 1000 mg/kg bw/day. The substance is therefore not to be classified as STOT RE according to criteria laid down in the CLP regulation.