Registration Dossier

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:
2018-01-17 to 2018-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TE767F05G
- Analytical purity: 99.7%. N ocorrectoin fo rpurity was made
- Expiration date of the lot/batch: 05 June 2019

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: The stability and homogeneity of the test item formulations determinations showed the formulations to be stable for four hours. Formulations were therefore prepared daily and dosed within four hours of preparation.

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 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 6 weeks old
- Weight at study initiation: Males 186-214 g, Females 134-165 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 softwood flake bedding
- Diet (e.g. ad libitum): ad libitum, Rodent 2014C Teklad Global Certified Diet
- Water (e.g. ad libitum): ad libitum, no data
- Acclimation period: six days

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water are considered not to contain any contaminants at a level that might have affected 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:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for four hours.
- Formulations were therefore prepared daily and dosed within four hours of preparation.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 0, 2.5, 7.5 and 25 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on four occasions and analyzed for concentration of test item at Covance CRS Research Limited. The results indicate that the prepared formulations were within 90% and 105% of the nominal concentration.
The homogeneity and stability, determined with respect to the concentration of Test Item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 100 mg/mL. The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a achromatographic 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 (recovery) and precision, limit of quantification (LOQ). The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed at nominal concentrations of 2.5 mg/mL and 100 mg/mL during ambient storage.The mean concentrations of Test Item in test formulations analyzed for the study were within applied limits ± 10%, 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 - Arachis oil BP)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
100 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 a 14 day range finder toxicity study in the rat
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: 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: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: 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 Lewis). All tissues from control and 100 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):
The observed clinical effects were considered not to be treatment-related.
Increased salivation was evident in all males treated with 100 mg/kg bw/day and in nine females treated with 100 mg/kg bw/day between Days 1 and 88 (males) and Days 12 and 89 (females). Noisy respiration was evident in all males treated with 100 mg/kg bw/day from Day 1 onwards and in all females from this treatment group between Days 3 and 87. These observations were mainly sporadic in frequency. Decreased respiratory rate, hunched posture, lethargy and labored respiration were also noted in one or two animals treated with 100 mg/kg bw/day on isolated occasions. On one occasion only, one male treated with 30 mg/kg bw/day and two females treated with 10 mg/kg bw/day showed noisy respiration. Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and represent difficulties in dosing particular animals rather than evidence of true systemic toxicity.

The following observations were considered to be incidental and unrelated to treatment. Red/brown staining around the right eye in one male treated with 100 mg/kg bw/day and generalised fur loss in two females treated with 10 mg/kg bw/day and in one control female.

For more details, please refer to Table 1a and 1b in section 'Additional information on results'.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three unscheduled deaths occured during the study.
One female treated with 100 mg/kg bw/day was found dead on Day 80. One male and one female treated with 100 mg/kg bw/day were euthanized in extremis on Days 56 and 78 (respectively).

The female that was found dead on Day 80 showed noisy/gasping respiration, a decreased respiratory rate, pilo-erection and hunched posture on Day 80 and had previously shown increased salivation.

The male treated with 100 mg/kg bw/day that were euthanized in extremis on Day 56 showed noisy/gasping/labored respiration, a decreased respiratory rate, hunched posture, pilo-erection and lethargy and had previously shown increased salivation. The female treated with 100 mg/kg bw/day that was euthanized in extremis on Day 78 showed noisy/gasping/labored respiration, a decreased respiratory rate, hunched posture, pilo-erection, pallor of the extremities and increased salivation.

There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in body weight or body weight gain.
Males treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during Week 2. An increase in body weight gain is considered not to represent an adverse effect of treatment.
For more details, please refer to Table 2 in section 'Additional information on results'.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption.
There was no adverse effect of treatment on food consumption for either sex at 10, 30 or 100 mg/kg bw/day.
For more details, please refer to Table 3 in section 'Additional information on results'.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food conversion efficiencies.
There was no adverse effect of treatment on food conversion efficiency for either sex at 10, 30 or 100 mg/kg bw/day.
For more details, please refer to Table 4 in section 'Additional information on results'.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in water consumption.
Dialy visual inspection of water bottles did not reveal any inter-group differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed during ophthalmoscopic examination of animals of both sexes from the control group and surviving 100 mg/kg bw/day during Week 12 of the treatment period.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in hematology.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The observed clinical effects were considered not to be treatment-related.
Males from all treatment groups showed a statistically significant reduction (p<0.05) in alanine aminotransferase. A true dose related response was not evident, all of the individual values were within historical control range and no associated histopathological correlates were evident, therefore, the intergroup differences were considered not to be of toxicological importance. Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in glucose. The majority of individual values were within historical control range and there were no associated histopathological correlates, therefore the intergroup difference was considered not to be of toxicological importance.
For more details, please refer to Table 1a and 1b 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 assessments.
Instances of noisy respiration were evident in a total of nine males and seven females treated with 100 mg/kg bw/day between Weeks 1 and 13 (males) and during Weeks 2, 5-7, 9-13 (females). Hunched posture was evident in one of these males during Week 1 only and labored respiration and a decreased respiratory rate was evident in another of these males during Week 2 only. Noisy respiration was also evident in two males treated with 30 mg/kg bw/day during either Week 1 or Week 11 and in one control male during Week 7. These correlated with the daily clinical observations seen in these treatment groups. Pilo-erection and hunched posture was evident in one male treated with 10 mg/kg bw/day during Weeks 8 and 9 only.
No such effects were evident in females treated with 30 or 10 mg/kg bw/day.
Functional performance tests:There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.
Sensor reactivity assessments: There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects detected in organ weights measured.
Males from all treatment groups showed a statistically significant reduction (p<0.05) in kidney weight both absolute and relative to terminal body weight. With the exception of one individual relative value at 30 and 100 mg/kg bw/day, all remaining individual values were within historical control ranges. In the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight and a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. Although the majority of individual brain and spleen weights for treated animals were outside of the historical control ranges, the majority of control values were also outside of the historical control ranges. In the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities observed at terminal necropsy. The female treated with 100 mg/kg bw/day that was found dead on Day 80 had gaseous distension in the stomach, red contents in the ileum and jejunum and dark lungs. The male treated with 100 mg/kg bw/day that was euthanized in extremis on Day 56 had gaseous distension in the cecum, colon and ileum and red lungs. The female treated with 100 mg/kg bw/day that was euthanized in extremis on Day 78 had gaseous distension in the cecum, duodenum, ileum, jejunum and stomach, red lungs and enlarged adrenals. Reddened lungs may have been the result of possible aspiration of the test item, with both of these animals showing significant respiratory observations prior to being terminated.
One control male, one control female, one male and two females treated with 10 mg/kg bw/day, one male and one female treated with 30 mg/kg bw/day and two males and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examinations revealed agonal congestion in the lungs for a number of these animals and in view of the finding being present in a control animal, these observations were considered unrelated to treatment with the test item. One female treated with 100 mg/kg bw/day had a fluid filled left horn of the uterus and a mass on the cervix. In the absence of any associated
treatment-related histopathological correlates these findings were considered not to be of toxicological significance. One male treated with 10 mg/kg bw/day had pale kidneys. In the absence of a similar effect at the higher dosages the intergroup difference was considered not
to be of toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following microscopic abnormalities were detected:
- The male euthanized in extremis on Day 56 had ulceration with necrosis/inflammation in the trachea (moderate) and agonal congestion in the lungs. Examination of the nasopharynx showed inflammatory/necrotic exudate as well as atrophy.
The female that was found dead on Day 80 had focal, minimal hyperplasia in the nonglandular stomach. Examination of the nasopharynx proved inconclusive.
- The female euthanized in extremis on Day 78 had ulceration with necrosis/inflammation in the trachea (marked) and mild inflammatory change in the lungs. The tracheal change is considered to be the cause of the poor clinical condition.

Terminal Necropsy
- Stomach: Hyperplasia of the non-glandular epithelium, minimal or mild was present in five males and three females treated with 100 mg/kg bw/day. Minimal, focal hyperplasia of the non-glandular region was present in one male treated with 30 mg/kg bw/day but in the
absence of any other changes in this group at this low incidence and severity this is considered incidental.
- Trachea: Ulceration with necrosis/inflammation was present in one male treated with 100 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 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 1a: Summary incidence of daily clinical observations (male)

   Group 1
0 (control)
 Group 2
10 mg/kg bw/day
Group 3
30 mg/kg bw/day 
 Group 4
100 mg/kg bw/day
 Killed 'in extremis'  -  -  -

 1 animal

Day 56

 Scheduled kill

 10 animals

Day 91

 10 animals

Day 91

 10 animals

Day 91

 9 animals

Day 91

 Staining around the eyes  -  -  -

 1 animal

Day 9

 Increased salivation  -

 1 animal

Day 88

 -  10 animals
From day 1 to day 88
 Noisy respiration  -  -

 1 animal

Day 75

 10 animals
From day 1 to day 90
 Laboured respiration  -  -  -  1 animal
Day 56
 Gasping respiration  -  -  -  1 animal
Day 56
 Decreased respiratory rate  -  -  -  2 animals
From day 6 to day 68
 Pilo-erection  -  -  -  1 animal
Day 56
 Lethargy  -  -  -  1 animal
Day 56
 Hunched posture  -  -  -  3 animals
From day 6 to day 88

Day numbers relative to start date

Tabel 1b: Summary incidence of daily clinical observations (female)

   Group 1
0 (control)
 Group 2
10 mg/kg bw/day
 Group 3
30 mg/kg bw/day
 Group 4
100 mg/kg bw/day
 Killed 'in extremis'  -  -  -  1 animal
Day 78
 Scheduled kill 10 animals
Day 91
 10 animals
Day 91
10 animals
Day 91 
8 animals
Day 91
 Found dead  -  -  - 1 animal
Day 80
 Increased salivation  -  -  -  9 animals
From day 12 to day 89
 Noisy respiration  -  2 animals
From day 63 to day 84
 -  10 animals
From day 3 to day 87
 Laboured respiration  -  -  -  2 animals
From day 61 to day 78
 Gasping respiration  -  -  -  2 animals
From day 78 to day 80
 Decreased respiratory rate  -  -  -  4 animals
From day 6 to day 80
 Pilo-erection  -  -  -  2 animals
From day 78 to day 80
 Lethargy  -  -  -  1 animal
From day 6 to day 7
 Hunched posture  -  -  -  4 animals
From day 6 to day 80
 Generalised fur loss  1 animal
From day 57 to day 63
 2 animals
From day 37 to day 90
 -  -
 Pallor of the extremities  -  -  -  1 animal
Day 78

Day numbers relative to start date

Table 2: Group Mean Body Weight Gains

Increase in Bodyweight (g) - Day numbers relative to start date

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
N

31.5
6.5

10

 24.8

4.9

10

 24.8

3.8

10

22.7

3.9

10

16.8

4.1

10 

12.7

5.2

10 

11.6

2.2

10 

15.0

7.1

10 

11.3

7.2

10 

10.0

3.4

10 

9.7

3.2

10 

8.5

4.3

10 

5.4

2.2

10 

204.8

25.2

10 

103.3
11.4

10 

10 mg/kg bw

(M)

 Mean

SD

N

 34.2

4.5

10

26.9

5.7

10 

25.4

5.4

10 

21.9

4.8

10 

15.3

4.5

10 

13.4

4.4

10

14.2

5.6

10 

15.0

6.4

10 

13.3

3.1

10 

14.1

4.6

10 

9.2

2.9

10 

10.7

4.4

10 

6.0

3.8

10 

219.6

22.5

10 

111.0

10.9

10 

30 mg/kg bw

(M)

 Mean

SD

N

35.9

6.0

10

25.8

4.1

10

23.3

5.5

10 

22.4

5.0

10 

14.7

5.6

10 

13.2

7.3

10 

12.4

5.9

10 

14.7

4.4

10

10.8

4.2

10

12.4

6.3

10 

7.1

5.1

10 

9.2

4.0

10 

6.3

4.4

10 

208.2

34.9

10 

105.0

18.9

10 

100 mg/kg bw

(M)

 Mean

SD

N

29.1

10.1

10 

30.1*

3.2

10 

26.7

2.8

10 

21.7

4.3

10 

18.3

7.6

10 

16.5

5.1

10 

10.3

5.4

10

16.8

4.0

10.7

2.6

10.7

4.2

9.6

4.0

7.4

3.0

4.6

8.9

216.4

34.1

108.5

19.4

control
(F)

 Mean

SD

N

13.1

3.3

10

11.2

7.1

10 

10.3

5.8

10 

9.3

6.1

10 

4.0

4.6

10 

7.1

6.0

10 

3.2

6.1

10 

4.6

4.4

10 

0.0

3.4

10 

4.9

4.9

10 

1.7

4.9

10 

3.7

5.0

10 

0.1

4.4

10 

73.2

18.6

10 

46.9

10.9

10 

10 mg/kg bw

(F)

 Mean

SD

N

 16.4

3.8

10

11.4

4.6

10 

14.4

5.5

10 

8.3

2.9

10 

5.3

5.2

10 

5.0

5.4

10 

5.3

4.2

10 

4.6

2.3

10 

2.1

2.4

10 

2.8

4.7

10 

5.0

3.7

10 

3.6

4.6

10 

-1.8

5.4

10 

82.4

14.1

10 

53.2

9.4

10 

30 mg/kg bw

(F)

 Mean

SD

N

15.0

5.3

10

13.2

6.6

10 

11.9

4.4

10 

8.1

3.5

10 

2.5

4.2

10 

7.9

6.2

10 

6.5

3.4

10 

1.7

2.6

10 

3.1

4.0

10 

4.8

4.7

10 

3.8

3.3

10 

1.7

4.1

10 

-0.8

3.7

10 

79.4

17.2

10 

52.0

9.6

10 

 100 mg/kg bw

(F)

 Mean

SD

N

15.9

9.3

10 

13.0

5.0

10 

7.9

4.0

10 

13.3

4.4

10 

7.6

6.7

10 

5.3

4.6

10 

4.4

5.0

10 

3.6

5.8

10 

3.1

6.7

10 

1.5

4.2

10 

4.6

5.2

10 

0.3

3.6

0.4

2.8

81.5

11.6

52.2

8.6

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

Increase in Bodyweight (g)

Day numbers relative to start date

Table 3: Group Mean Food consumptions       

Group

(Sex)

From

To 

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 

 control

(M)

 Mean

N

19.3

10

 20.4

10

20.6

10 

21.1

10 

20.3

10 

20.3

10 

20.0

10 

20.0

10 

20.1

10 

19.8

10 

19.7

10 

19.3

10 

19.4

10 

10 mg/kg bw

(M)

Mean

SD

19.8

10 

21.1

10 

21.0

10 

21.1

10 

20.4

10 

20.7

10 

19.9

10 

20.2

10 

20.7

10 

20.7

10 

20.5

10 

20.5

10 

20.0

10 

30 mg/kg bw

(M) 

 Mean

SD

21.5

10

22.0

10 

21.8

10 

22.1

10 

21.0

10 

21.4

10 

21.1

10 

21.3

10 

21.5

10 

21.7

10 

20.5

10 

20.5

10 

19.8

10 

100 mg/kg bw

(M) 

 Mean

SD

 18.3

10

 21.9

10

 21.3

10

20.8

10 

19.8

10 

21.1

10 

20.1

10 

20.2

10 

22.2

20.2

20.2

20.2

19.8

control

(F)

Mean

SD 

14.8

10 

15.8

10 

15.8

10 

16.0

10 

15.4

10 

15.6

10 

15.4

10 

14.7

10 

14.7

10 

15.1

10 

15.1

10 

15.0

10 

13.7

10 

10 mg/kg bw

(F) 

 Mean

SD

15.0

10 

16.3

10 

16.7

10 

16.5

10 

16.0

10 

16.1

10 

15.4

10 

15.1

10 

15.4

10 

15.2

10 

15.7

10 

15.3

10 

14.3

10 

30 mg/kg bw

(F) 

 Mean

SD

14.4

10 

14.6

10 

14.9

10 

14.6

10 

14.7

10 

14.6

10 

14.4

10 

13.6

10 

14.2

10 

14.5

10 

14.1

10 

14.1

10 

12.5

10 

100 mg/kg bw

(F) 

 Mean

SD

13.9

10

15.5

10 

15.5

10 

15.5

10 

15.7

10 

15.0

10 

14.7

10 

14.4

10 

14.0

10 

14.1

10 

14.5

10 

14.3

13.2

Day numbers relative to start date

Table 4: Food efficiency

Group

(sex)

From

To 

1

8

15 

15

22 

22

29 

29

36 

36

43 

43

50 

50

57 

57

64 

64

71 

71

78 

78

85 

85

91 

control

(M)

 Mean

N

23.2

10

17.4

10 

17.2

10 

15.4

10 

11.9

10 

8.9

10 

8.2

10 

10.8

10 

8.0

10 

7.2

10 

7.0

10 

6.3

10 

4.7

10 

10 mg/kg bw

(M) 

Mean

SD 

24.6

10 

18.1

10 

17.4

10 

14.9

10 

10.7

10 

9.2

10 

10.0

10 

10.6

10 

9.2

10 

9.7

10 

6.4

10 

7.6

10 

5.0

10 

30 mg/kg bw

(M)

Mean

SD 

23.8

10 

16.8

10 

15.2

10 

14.5

10 

9.9

10 

8.8

10 

8.5

10 

9.9

10 

7.2

10 

8.0

10 

5.0

10 

6.5

10 

5.1

10 

100 mg/kg bw

(M) 

Mean

SD 

22.4

10 

19.6

10 

17.9

10 

14.8

10 

13.1

10 

11.1

10 

7.4

10 

11.8

6.9

7.5

6.7

5.3

3.7

control

(F) 

Mean

SD 

12.6

10 

10.2

10 

9.2

10 

8.4

10 

3.8

10 

6.7

10 

2.8

10 

4.5

10 

-0.1

10 

4.8

10 

1.5

10 

3.5

10 

0.3

10 

10 mg/kg bw

(F) 

Mean

SD 

15.6

10 

10.0

10 

12.3

10 

7.2

10 

4.8

10 

4.4

10 

4.8

10 

4.4

10 

2.0

10 

2.6

10 

4.5

10 

3.4

10 

-2.0

10 

30 mg/kg bw

(F) 

Mean

SD 

15.1

10 

12.9

10 

11.3

10 

8.0

10 

2.5

10 

7.6

10 

6.3

10 

1.9

10 

3.1

10 

4.8

10 

3.8

10 

1.8

10 

-1.0

10 

100 mg/kg bw

(F) 

Mean

SD 

16.3

10 

11.9

10 

7.2

10 

12.3

10 

7.0

10 

5.0

10 

4.3

10 

3.5

10 

3.1

10 

1.5

10 

4.5

10 

2.3

-1.9

Days numbers relative to start date

Table 5: Group Mean Hematological Values

 Group

(sex)

 

 Hb

g/dl

 RBC

10^12/l

 Hct

%

 MCH

pg

 MCV

fl

 MCHC

g/dl

 WBC

10^9/l

 Neut

10^9/l

 Lymph

10^9/l

 Mono

10^9/l

 Eos

10^9/l

 Bas

10^9/l

 C

Seconds

 PLT

10^9/l

 APTT

Seconds

control

(M)

Mean

SD

N

16.47

1.56

10

8.807

0.644

10 

47.26

3.8

10 

18.68

0.88

10 

53.64

1.91

10 

34.79

0.59

10 

7.85

1.56

10 

2.842

0.688

10 

4.959

1.278

10 

0.000n

0.000

10 

0.051

0.062

10 

0.000n

0.000

10 

9.39

0.56

10 

542.5

101.5

10 

14.70

1.71

10 

10 mg/kg bw

(M) 

Mean

SD

16.02

1.70

10

8.462

0.747

10 

45.26

3.65

10 

18.83

1.18

53.62

1.70

35.12

1.96

7.90

2.05

10 

3.122

0.984

10 

4.745

1.107

10 

0.000n

0.000

10 

0.035

0.049

10 

0.000n

0.000

10 

9.55

0.48

10 

593.3

129.7

10 

14.48

1.96

10 

30 mg/kg bw

(M)

Mean

SD

15.93

1.22

10 

8.481

0.590

10 

45.5

3.21

10 

18.87

0.63

53.94

1.28

10 

34.83

1.15

7.67

2.62

10 

3.090

1.251

10 

4.538

1.583

10 

0.010n

0.032

10 

0.033

0.061

10 

0.000n

0.000

10 

9.26

0.84

10 

510.8

139.2

10 

14.73

1.96

10 

100 mg/kg bw

(F) 

Mean

SD

 16.28

1.65

9

 8.617

0.789

9

47.58

4.65

18.87

0.82

55.20

1.74

34.19

0.75

8.92

3.24

3.618

1364

5.286

1.923

0.012n

0.037

0.010

0.023

0.000n

0.000

9.54

0.72

600.8

118.7

15.29

1.50

control

(F)

Mean

SD

N

15.39

0.55

10

8.151

0.347

10 

44.49

1.65

10 

18.91

0.58

10 

54.59

1.59

10 

34.62

0.49

10 

6.45

2.46

10 

1.608

0.435

10 

4.776

2.021

10 

0.000n

0.000

10 

0.068

0.073

10 

0.000n

0.000

10 

8.63

1.06

10 

631.5

108.3

10 

14.52

1.41

10 

10 mg/kg bw

(F)

Mean

SD

N

15.24

0.34

10 

8.091

0.300

10 

44.27

0.93

10 

18.83

0.54

10 

54.73

1.39

10 

34.39

0.35

10 

8.10

2.29

10 

2.008

0.346

10 

6.033

2.072

10 

0.000n

0.000

10 

0.060

0.070

10 

0.000n

0.000

10 

9.18

0.85

10 

650.1

85.7

10 

15.98

1.92

10 

30 mg/kg bw

(F) 

Mean

SD

14.85

1.44

10 

7.647

0.820

10 

43.06

4.31

10 

19.41

0.88

10 

56.41

2.94

10 

34.48

0.33

10 

8.41

2.67

10 

2.054

0.668

10 

6.293

2.093

10 

0.008n

0.025

10 

0.057

0.061

10 

0.000n

0.000

10 

8.97

1.03

10 

613.3

49.6

10 

16.33

2.02

10 

100 mg/kg bw

(F) 

Mean

SD

15.33

1.26

8.110

0.674

44.74

3.27

18.90

0.24

55.23

1.05

34.23

0.47

8.10

2.70

2.079

0.772

5.945

1.984

0.000n

0.000

0.078

0.128

0.000n

0.000

8.98

0.79

577.3

228.2

13.76

1.37

Abbreviations: Hb - hemoglobin, GBC - Erytrhocyte count, Hct - hematocrit, MCH - mean copuscular hemoglobin, MCV - mean corpuscular volume, MCHC - mean corpuscular hemoglobin concentration, WBC - total leukocyte count, Neut - neutrophils, Lymph - lymphocytes, Mono - monocytes, Eos - eosinophils, Bas - basophils, C - prothrombin time, PLT - platelet count, APTT - activated partial thromboplastin time

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of the test substance for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 30 or 100 mg/kg bw/day resulted in the unscheduled death of one male and two females treated with 100 mg/kg bw/day.
These sporadic deaths were not associated with target organ histopathological changes which could have suggested that mortality or morbidity was associated with, or a consequence of, systemic toxicity. The evidence of degenerative and inflammatory changes in the upper respiratory tract of these animals supports the conclusion that these deaths were associated with the process of gavage dose administration of an irritant material and the pathology was due to gastric reflux. The microscopic stomach changes identified in surviving animals of either sex at 100 mg/kg bw/day were also considered to be the result of gastric irritancy rather than attributable to true systemic toxicity. Therefore, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day.