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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:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: Body responsible for the test
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report):unoxolTM3 ,4 Dialdehyde
- Physical state:liquid
- Impurities (identity and concentrations): Cyclohexanecarboxaldehyde 0.28%, Hydroxyaldehyde 0.23%, Unknowns 0.51%
- Lot/batch No.:200502771 -6M
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Housing: Group housing of 5 animals per sex in Macrolon plastic cages with sterilised sawdust as bedding
material and paper as cage-enrichment
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Altromin
(code VRF I)L,ag e, Germany
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: Acclimatisation
period was at least 5 days before start of treatment under laboratory
conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 23.4OC
- Humidity (%): 43 - 74%
- Air changes (per hr): approximately 15 air changes per
hour
- Photoperiod (hrs dark / hrs light): 12 hours
artificial fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: 21 February 2006 To: 21 March 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Method of administration:
Gavage
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable; the test substance was dosed undiluted in the main study.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
No. of animals per sex per dose:
Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 450 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 450 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day
Details on study design:
The test substance was administered daily for 28 days by oral gavage to SPF-,bred Wistar rats.
One control group and three treated groups were tested, each consisting of 5 males and 5
females.
The following parameters were evaluated: clinical signs daily; functional observation tests; body
weight and food consumption weekly; water consumption between days 21-25; clinical
pathology and macroscopy at termination; organ weights and histopathology on a selection of
tissues.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort,
which was considered not transient in nature or was likely to become
more severe, were sacrificed for humane reasons based on OECD
guidance document on humane endpoints (ENVIJMIMONOI 200017).
The time of death was recorded as precisely as possible.

Clinical signs: At least once daily, detailed clinical observations were made in all
animals. Once prior to start of treatment 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 (abbreviations mentioned in the respective tables indicated
between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL LIR), static
righting reflex (STATIC R) and grip strength (GRIP) (Score 0 =
normallpresent, score 1 = abnormallabsent).
- motor activity test (recording period: 12 hours during overnight for
individual animals, using a computerised monitoring system,
Pearson Technical Services, Debenham, Stowmarket, England).
Sacrifice and pathology:
All animals surviving to the end of the observation period and all moribund animals 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 animals at necropsy and fixed in a neutral phosphate buffered
4% formaldehyde solution:
Identification marks: not processed
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex]
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides
(Eyes with optic nerve [if detectable] and
Harderian gland)
(Female mammary gland area)
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
(Salivary glands - 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 [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions
Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ
involvement.
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. No statistical analysis was performed on histopathology findings. Group means
were calculated for continuous data and medians were calculated for discrete data (scores) in
the summary tables. 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 different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Mortality:
mortality observed, treatment-related
Description (incidence):
both sexes affected
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males affected
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
males affected
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
FOB findings present in females; Motor activity decreased in males
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
both sexes affected
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
both sexes affected
Details on results:
Clinical observations:
One male at 1000 mg/kg/day was sacrificed in week 3.


One female at 150 mg/kg/day was found dead in week 3. A
cause of death could not be established based on
histopathological assessment.


Hunched posture was shown by all animals at 1000 mg/kg/day
and by all females at 450 mg/kg/day in week 1, and
reappeared at 1000 mg/kg/day from week 3 onwards along with
abdominal swelling and/or piloerection in all high dose
males. Laboured respiration was incidentally shown by a few
males at 1000 mg/kg/day.
Lethargy, general tremors, piloerection, hunched posture, uncoordinated movements, laboured
respiration and ptosis shown by high dose male no. 19 prior to sacrifice were considered to
reflect the moribund status of this animal.
Salivation shown by all animals at 150 mg/kg/day and higher was considered to be a
physiological response related to irritant properties and/or taste of the test substance rather than
a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its
time of occurrence (i.e. after dosing).
Incidental findings included chromodacryorrhoea and scabs. These findings are commonly
noted in rats of this age and strain which are housed and treated under the conditions in this
study. At the incidence observed, these were considered signs of no toxicological significance.
No clinical signs were shown by control animals.



Body weights and body weight gain of males at 1000 mg/kg/day
were reduced, achieving a level of statistical significance
in all cases. One high dose male lost weight between days
22 and 28. Body weight gain of females at 1000 mg/kg/day was
also slightly reduced with statistical significance in week
1, but recovered to levels similar to controls as treatment
progressed. Body weights and body weight gain of other
treated animals remained in the same range as controls over
the 4-week study period.


Food consumption of males at 1000 mg/kg/day was reduced
throughout treatment. Food consumption after correction for
body weight was also reduced in high dose males in the first
week, but was similar to control levels towards the end of
treatment.


Food consumption (before or after correction for body
weight) of females at 1000 mg/kg/day as well as of other
dose groups was considered to have been unaffected by
treatment with the test substance.

Laboratory findings:
The following statistically significant changes
distinguished animals at 1000 mg/kg/day from control
animals:

- Increased white blood cell counts in males and females;

- Reduced red blood cell counts in females;


- Reduced haemoglobin levels in females.

- Increased platelets in males



- Reduced alkaline phosphatase activity was seen in males
at 150 mg/kg/day and higher and in females at 450 and 1000
mg/kg/day;

Effects in organs:
Treatment resulted in a marked irritant effect on the
stomach down to the lowest administered dose of 150
mg/kg/day. The primary and most severe finding was
forestomach ulceration with submucosal inflammation in the
stomach which was recorded in a dose related fashion in all
dose groups, reaching very severe grade at 1000 mg/kg/day.
These findings correlated to necropsy findings.

The following necropsy findings distinguished treated from
control animals:

- Thickening of the limiting ridge of the stomach and/or of
the forestomach (all males at 150 mg/kg/day and higher
(three high dose males also showed thickening of the
glandular mucosa of the stomach or of the entire stomach),
2/5 females at 150 mg/kg/day, 5/5 females at 450 mg/kg/day
and 4/5 females at 1000 mg/kg/day;

- Stomach grown together with the abdominal wall (1/5 males
at 1000 mg/kg/day);

- Irregular surface of the forestomach (4/5 males and
females at 150 mg/kg/day, 5/5 males and females at 450
mg/kg/day (one of these males showing a general irregular
surface of the stomach) and 4/5 males and 5/5 females at
1000 mg/kg/day;

- Red foci on the glandular mucosa of the stomach (1/5
females at 450 mg/kg/day);

- Red discolouration of the mesenteric lymph nodes (1/5
males and females at 450 mg/kg/day, and 4/5 males and 3/5
females at 1000 mg/kg/day)




- Reduced heart weight in males at 1000 mg/kg/day

- Increased liver weight was seen in females at 450 and
1000 mg/kg/day and increased liver to body weight ratio in
males at 1000 mg/kg/day and females at 450 and 1000
mg/kg/day

- Reduced thymus weight and thymus to body weight ratio in males and females at ,1000
mg/kg/day (only statistically significant for absolute thymus weights of males)

- Reduced kidney weights (not statistically significant) and increased kidney to body weight
ratio in males at 1000 mg/kg/day

- lncreased adrenal weights and adrenal to body weight ratio in males and females at 1000
mg/kg/day

- Reduced spleen weight (males at 1000 mg/kg/day; not statistically significant)




Stomach -
- forestomach diffuse hyperkeratosis at a slight to very severe degree was
noted in all treated animals except for one group 2 female

- forestomach inflammation at a minimal to moderate degree was recorded in
three group 2 and five group 3 animals

- glandular inflammation at a minimal or slight degree was seen in two group
2, three group 3 and one graup 4 rats

- forestomach squamous hyperplasia at moderate or severe degree was seen
in all treated animals except for one group 2 female

- forestomach ulceration with submucosal inflammation at slight to very severe
degree was recorded in three group 2, five group 3 and all ten group 4
animals

Liver -
- diffuse midzonal/centrilobular hypertrophy at a minimal degree was noted in
six group 4 animals.

Adrenal glands -
- multifocal vacuolation in zona fasiculata at a slight degree in one group 4
male

- diffuse cortical hypertrophy at slight degree in four group 4 males

Mesenteric lymph node -
- yellow-brown pigment at a minimal to slight degree was recorded in one
group 3 and nine group 4 rats

- congestion/erythrophagocytosis at minimal to severe degree was recorded in
six group 3 and all group 4 rats

- histiocytosis at a minimal or slight degree was seen in four group 4 animals
lymphoid atrophy at a slight degree was noted in two group 4 males

Effect levels

Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
< 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Key target organ was the stomach/gastrointestinal tract. Observations associated with significant local irritation at all dose levels.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

TABLE 1. BODY WEIGHTS (GRAM) SUMMARY

 MALES
     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 TREATMENT          
 DAY 1  MEAN  187  184  187  188
 WEEK 1  ST. DEV  6.2 14.7   7.9  13.6
   N  5  5  5
           
 DAY 8  MEAN  239  237  236  202**
 WEEK 2  ST. DEV  7.4  12.6  6.5  14.2
   N  5  5  5  5
           
 DAY 15  MEAN  285  274  276  237**
 WEEK 3  ST. DEV  11.7  8.8  9.8  8.7
   N  5  5  5  5
           
 DAY 22  MEAN  321  313  313  261**
 WEEK 4  ST. DEV  18.2  14.6  13.0  16.2
   N  5  5  5  4
           
 DAY 28  MEAN  344  339  333  259**
 WEEK 4  ST. DEV  24.5  17.3  14.5  17.5
   N  5  5  5  4

TABLE 2. BODY WEIGHT GAIN (%) SUMMARY

 MALES
     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 TREATMENT          
 DAY 8  MEAN  28  29  27  8**
 WEEK 2  ST. DEV  2.1  4.3  3.2  7.7
   N  5  5  5  5
           
 DAY 15  MEAN  53  49  48  27**
 WEEK 3  ST. DEV  6.1  8.8  5.9  8.5
   N  5  5  5  5
           
 DAY 22  MEAN  71  71  68  42**
 WEEK 4  ST. DEV  10.1  9.1  3.4  8.1
   N  5  5  5  4
           
 DAY 28  MEAN  84  85  79  41**
 WEEK 4  ST. DEV  12.8  12.2  5.7  17.2
   N  5  5  5  4

TABLE 3. HAEMATOLOGY SUMMARY

MALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 WBC  MEAN  9.6  10.1  13.2  16.3*
 10E9/l  ST. DEV  2.6  2.6  2.1  4.4
   N  5  4  5  4
           
 Neutrophils  MEAN  14.8  21.3  20.0  22.8
 %WBC  ST. DEV  4.2  8.6  9.4  6.4
   N  5  4  5  4
           
 Lymphocytes  MEAN  84.6  78.5  79.4  75.0+
 %WBC  ST. DEV 3.5   8.4  8.7  7.4
   N  5  4  5  4
           
 Monocytes  MEAN  0.4  0.3  0.6  2.3
 %WBC  ST. DEV  0.9  0.5  0.9  1.7
   N  5  4  5  4
           
 Eosinophils  MEAN  0.2  0.0  0.0  0.0
 %WBC  ST. DEV  0.4  0.0  0.0  0.0
   N  5  4  5  4
           
 Basophils  MEAN  0.0  0.0  0.0  0.0
 %WBC  ST. DEV  0.0  0.0  0.0  0.0
   N  5  4  5  4
           
 Red Blood Cells  MEAN  7.67  7.80  7.61  7.73
 10E12/l  ST. DEV  0.63  0.26  0.22  0.76
   N  5  4  5  4
           
 Reticulocytes  MEAN  2.6  3.1  2.6  4.1
 %RBC  ST. DEV  0.3  0.3  0.2  1.2
   N  5  4  5  4
           
 RDW  MEAN  12.1  12.9  12.4  13.8**
 %  ST. DEV  0.5  0.3  0.4  0.4
   N  5  4  5  4
           
 Haemoglobin  MEAN  9.5  9.2  9.3  9.4
 mmol/l  ST. DEV  0.5  0.2  0.2  0.9
   N  5  4  5  4
           
 Haematocrit  MEAN  0.454  0.453  0.447  0.469
 l/l  ST. DEV  0.025  0.009  0.008  0.046
   N  5  4  5  4
           
 MCV  MEAN  59.4  58.1  58.8  60.7
 fl  ST. DEV  2.4  1.2  1.1  0.8
   N  5  4  5  4
           
 MCH  MEAN  1.24  1.18  1.22  1.22
 fmol  ST. DEV  0.05  0.03  0.03  0.02
   N  5  4  5  4
           
 MCHC  MEAN  20.90  20.35*  20.69  20.06**
 mmol/l  ST. DEV  0.21  0.14  0.35  0.17
   N  5  4  5  4
           
 Platelets  MEAN  850  824  1162**  1190**
 10E9/l  ST. DEV  67  122  141  11 6
 N  5  4  5  4
           
 PT  MEAN  17.2  16.2  15.3**  18.8*
 s  ST. DEV  0.8  1.0  0.7  0.6
   N  5  4  5  4
           
 APTT  MEAN  15.2  13.9  13.4  15.7
 s  ST. DEV  3.1  2.2  1.2  1.7
   N  5  4  5  4

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1 % (**) level

 FEMALES
     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 WBC  MEAN  7.7  6.6  7.9  12.3**
 10E9/l  ST. DEV  1.8  0.7  1.1  1.2
   N  5  4  5  5
           
 Neutrophils  MEAN  11.4  14.5 12.4 21.2 
 %WBC  ST. DEV  3.9  6.8  4.4  10.4
   N  5  4  5  5
           
 Lymphocytes  MEAN  86.2  84.8  87.0  76.8
 %WBC  ST. DEV  4.8  7.1  4.0  10.8
   N  5  4  5  5
           
 Monocytes  MEAN  0.4  0.8  0.6  2.0
 %WBC  ST. DEV  0.5  1.0  0.9  1.2
   N  5  4  5  5
           
 Eosinophils  MEAN  0.0  0.0  0.0  0.0
 %WBC  ST. DEV  0.0  0.0  0.0  0.0
   N  5  4  5  5
           
 Basophils  MEAN  0.0  0.0  0.0  0.0
 %WBC  ST. DEV  0.0  0.0  0.0  0.0
   N  5  4  5  5
           
 Red Blood Cells  MEAN  7.65  7.22  7.28  7.17*
 10E12/l  ST. DEV  0.30  0.32  0.21  0.30
   N  5  4  5  5
           
 Reticulocytes  MEAN  2.3  2.2  2.1 3.6
 %RBC  ST. DEV  0.7  0.1  0.4  1.3
   N  5  4  5  5
           
 RDW  MEAN  11.2  11.9  11.6  13.4**
 %  ST. DEV  0.6  0.5  0.4  0.6
   N  5  4  5  5
           
 Haemoglobin  MEAN  9.0  8.7  8.8  8.4*
 mmol/l  ST. DEV  0.3  0.2  0.2  0.5
   N  5  4  5  5
           
 Haematocrit  MEAN  0.429  0.413  0.416  0.411
 l/l  ST. DEV  0.013  0.012  0.009  0.020
   N  5  4  5  5
           
 MCV  MEAN  56.1  57.2  57.2  57.3
 fl  ST. DEV  1.5  2.0  1.6  1.7
   N  5  4  5  5
           
 MCH  MEAN  1.18  1.20  1.22  1.17
 fmol  ST. DEV 0.05  0.04  0.03  0.04
   N  5  4  5  5
           
 MCHC  MEAN 20.94  21.04  21.21  20.45
 mmol/l  ST. DEV  0.38  0.39  0.35  0.23
   N  5  4  5  5
           
 Platelets  MEAN  1019  1020  1068  1073
 10E9/l  ST. DEV  218  46  155  372
   N  5  4  5  5
           
 PT  MEAN  17.8  18.5  15.8  18.5
 s  ST. DEV  1.0  4.0  0.6  0.6
   N  5  4  5  5
           
 APTT  MEAN  16.3  16.0  14.2  14.7
 s  ST. DEV  2.3  2.8  2.1  2.9
   N  5  4  5  5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1 % (**) level

TABLE 4. CLINICAL BIOCHEMISTRY SUMMARY

MALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 ALAT  MEAN  32.4  26.9  31.2  41.2
 U/l  ST. DEV  3.8  26.9  7.1  11.0
  N  5  5  5  4
           
 ASAT  MEAN  84.6  78.2  77.0  91.2
 U/l  ST. DEV  4.1  5.8  6.7  36.2
   N  5  5  5  4
           
 ALP  MEAN  144  102**  99**  89**
 U/l  ST. DEV  32  9  14  7
   N  5  5  5  4
           
 Total protein  MEAN  60.4  58.9  57.6  56.6
 g/l  ST. DEV  1.6  1.4  2.7  5.1
   N  5  5  5  4
           
 Albumin  MEAN  30.8  29.8  28.8  27.5**
 g/l  ST. DEV  0.8  1.0  0.5  2.2
   N  5  5  5  4
           
 Total bilirubin  MEAN  2.0  1.9  1.8  2.0
 umol/l  ST. DEV  0.1 0.3  0.2  0.3
   N  5  5  5  4
           
 Urea  MEAN  4.7  5.0  4.5  6.6
 mmol/l  ST. DEV  0.5  0.4  0.2  3.2
   N  5  5  5  4
           
 Creatinine MEAN  37.8  35.8  35.6  38.1
 umol/l  ST. DEV  1.8  3.2  0.6  3.5
   N  5  5  5  4
           
 Glucose  MEAN  6.58  5.80  6.24  7.11
 mmol/l  ST. DEV  0.63  0.41  1.24  3.13
   N  5  5  5  4
           
 Cholesterol  MEAN  1.70  1.63  2.05  2.40*
 mmol/l  ST. DWV  0.44  0.43  0.35  0.29
   N  5  5  5  4
           
 Sodium  MEAN 136.3  138.0  136.9  137.9
 mmol/l  ST. DEV  0.9  1.4  0.8  1.5
   N  5  5  5  4
           
 Potassium  MEAN  3.83  3.79  4.24  4.07
 mmol/l  ST. DEV  0.22  0.06  0.67  .045
   N  5  5  5  4
           
 Chloride  MEAN  99  101  99  98
 mmol/l  ST. DEV  1  2  1  2
   N  5  5  5  4
           
 Calcium  MEAN  2.70  2.66  2.60  2.61
 mmol/l  ST. DEV  0.14  0.06  0.21  0.14
   N  5  5  5  4
           
 Inorg. Phos  MEAN  2.65  2.51  2.86  2.79
 mmol/l  ST. DEV  0.29  0.09  0.029  0.07
   N  5  5  5  4
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level                                                                       

FEMALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 ALAT  MEAN  24.4  25.4  24.4  34.7**
 U/l  ST. DEV  2.7  4.8  1.7  7.2
   N  5  4  5  5
           
 ASAT  MEAN  74.3  80.6  76.7  77.4
 U/l  ST. DEV 6.1  2.9  6.1  6.9
   N  5  4  5  5
           
 ALP  MEAN  74  71  52*  58
 U/l  ST. DEV  12  12  8  12
   N  5  4  5  5
           
 Total Protein  MEAN  60.5  58.6  59.1  57.2*
 g/l  ST. DEV  1.2  1.5  1.8  2.7
   N  5  4  5  5
           
 Albumin  MEAN  31.1  30.3  30.2  27.7**
 g/l  ST. DEV  0.8  0.3  1.3  1.0
   N  5  4  5  5
           
 Total bilirubin  MEAN  2.3  2.2  2.2  1.8*
 umol/l  ST. DEV  0.1  0.3  0.3  0.1
   N  5  4  5  5
           
 Urea  MEAN  6.5  6 .1  5.3  4.8*
 mmol/l  ST. DEV  0.9  1.2  0.4  0.7
   N  5  4  5  5
           
 Creatinine  MEAN  39.8  42.0  40.0  42.3
 umol/l  ST. DEV  1.3  5.0  3.2  2.9
   N  5  4  5  5
           
 Glucose  MEAN  6.42  6.50  6.23  5.84
 mmol/l  ST. DEV  0.40  0.31  0.74  0.58
   N  5  4  5  5
           
 Cholesterol  MEAN  1.88  1.63  2.04  1.97
 mmol/l  ST. DEV  0.39  0.26  0.45  0.36
   N  5  4  5  5
           
 Sodium  MEAN  135.3  138.0*  136.2  133.6
 mmol/l  ST. DEV  1.4  0.2  1.6  1.6
   N  5  4  5  5
           
 Potassium  MEAN  3.68  3.70  3.79  4.00
 mmol/l  ST. DEV  0.21  0.13  0.31  0.22
   N  5  4  5  5
           
 Chloride  MEAN  99  102  99  100
 mmol/l  ST. DEV  1 1  1  4
   N  5  4  5  5
           
 Calcium  MEAN  2.68  2.68  2.70  2.68
 mmol/l  St. DEV  0.05  0.04  0.06  0.09
   N  5  4  5  5
           
 Inorg Phos  MEAN  2.33  2.27  2.24  2.33
 mmol/l  ST. DEV  0.0.8  0.22  0.19  0.17
   N  5  4  5  5

*/** Dnnnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 5. URINALYSIS SUMMARY

MALES              

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 Volume  MEAN  29  23  42  30
 ml  ST. DEV  11  8  3  18
   N  5  5  5  4
           
 Specific Gravity  MEAN  1.011  1.015  1.006  1.021
   ST. DEV  0.006  0.008  0.002  0.026
   N  5  5  5  4
           
 Clarity  MEDIAN  1  2  1  1
 score 1/3  N  5  5  5  4
           
 Colour  MEDIAN  1  1  1  1
 score 1/5  N  5  5  5  4
           
 pH  MEAN  6.6  6.6  6.7  6.3
   ST. DEV  0.7  0.2  0.4  0.3
   N  5  5  5  4
           
 Blood  MEDIAN  0  0  1  1
 score 0/3  N  5  5  5  4
           
 WBC  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Bilirubin  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Urobilinogen  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Protein  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Ketones  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Glucose  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 WBC - sed  MEDIAN  0  0  0  1
 score 0/3  N  5  5  5  4
           
 RBC - sed  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Casts  MEDIAN  0  0  0  0
 score 0/3  N  5  5  5  4
           
 Epithelial Cells  MEDIAN  1  1  1  1
 score 0/3  N  5  5  5  4
           
 Crystals  MEDIAN  1  1  0  1
 score 0/3  N  5  5  5  4
           
 Bacteria  MEDIAN  3  3  3  2
 score 0/3  N  5  5  5  4
           
 Other  MEDIAN  2  2  2  2
 score 0/3  N  5  5  5  4
           
 Sodium  MEAN  17.3  21.7  19.6  22.1
 mmol/l  ST. DEV  4.1  6.5  9.6  14.9
   N  4  5  5  4
           
 Sodium  MEAN  0.47  0.47  0.80  0.50
 mmol/TPV  ST. DEV  0.15  0.05  0.36  0.27
   N  4  5  5  4
           
 Potassium  MEAN  51.0  69.2  18.8  48.3
 mmol/l  ST. DEV  29.5  40.6  8.0  64.4
   N  5  5  5  4
           
 Potassium  MEAN  1.25  1.38  0.77  0.64+
 mmol/TPV  ST. DEV  0.40  0.44  0.29  0.16
   N  5  5  5  4
           
 Calcium  MEAN  0.50  0.42  0.28  0.48
 mmol/l  ST. DEV  0.55  0.15  ---  0.35
   N  3  4  1  4
           
 Calcium  MEAN  0.010  0.008  0.011  0.010
 mmol/TPV  ST. DEV  0.009  0.002  ---  0.003
   N  3  4  1  4

+/++ Steel-test significant at 5% (+) or 1 % (++) level

FEMALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000MG/KG
 Volume  MEAN  14  12  25  25
 ml  ST. DEV  5  10  9  8
   N  5  4  5  5
           
 Specific gravity  MEAN  1.019  1.016  1.011  1.013
   ST. DEV  0.007  0.006  0.007  0.006
   N  5  3  5  5
           
 Clarity  MEDIAN  2  1  1  1
 score 1/3  N  5  3  5  5
           
 Colour  MEDIAN  1  1  1  1
 score 1/5  N  5  3  5  5
           
 pH  MEAN  6.4  6.5  6.1  5.8**
   ST. DEV  0.2  0.0  0.2  0.3
   N  5  3  5  5
           
 Blood  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 WBC  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Bilirubin  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Urobilinogen  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Protein  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Ketones  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Glucose  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 WBC-sed  MEDIAN  1  1  0+  0
 score 0/3  N  5  3  5  5
           
 RBC-sed  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Casts  MEDIAN  0  0  0  0
 score 0/3  N  5  3  5  5
           
 Epithelial cells  MEDIAN  1  1  1  1
 score 0/3  N  5  3  5  5
           
 Crystals  MEDIAN  1  1  1  0
 score 0/3  N  5  3  5  5
           
 Bacteria  MEDIAN  3  2  2  2
 score 0/3  N  5  3  5  5
           
 Other  MEDIAN  1  1  1  1
 score 0/3  N  5  3  5  5
           
 Sodium  MEAN  27.1  29.1  21.3  15.3
 mmol/l  ST. DEV  9.3  4.2  6.1 0.9
   N  5  3  5  3
           
 Sodium  MEAN  0.34  0.46  0.48  0.34
 mmol/TPV  ST. DEV  0.07  0.15  0.09  0.13
   N  5  3  5  3
           
 Potassium  MEAN  84.9  68.1  41.7  30.5*
 mmol/l  ST. DEV  36.3  20.1  27.5  15.9
   N  5  3  5  5
           
 Potassium  MEAN  1.01  1.02  0.84  0.67+
 mmol/TPV  ST. DEV  0.10  0.25  0.17  0.16
   N  5  3  5  5
           
 Calcium  MEAN  2.66  1.81  1.88  4.06
 mmol/l  ST. DEV  2.01  1.09  2.38  2.51
   N  5  3  5  5
           
 Calcium  MEAN  0.034  0.028  0.031  0.089
 mmol/TPV  ST. DEV  0.027  0.017  0.027  0.032
   N  5  3  5  5

+/++ Steel-test significant at 5% (+) or 1 % (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1 % (**) level

TABLE 6. FUNCTIONAL OBSERVATIONS SUMMARY

FEMALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 AT WEEK 4          
 HEARING  MEDIAN 0  0  0  0
 SCORE 0/1  N  5  4  5  5
 PUPIL L  MEDIAN  0  0  0  1+
 SCORE 0/1  N  5  4  5  5
 PUPIL R  MEDIAN  0  0  0  1+
 SCORE 0/1  N  5  4  5  5
 STATIC R  MEDIAN  0  0  0  0
 SCORE 0/1  N  5  4  5  5
 GRIP  MEDIAN  0  0  0  0
 SCORE 0/1  N  5  4  5  5

+/++ Steel-test significant at 5% (+) or 1% (++) level

TABLE 7. MOTOR ACTIVITY MEASUREMENTS SUMMARY

MALES       

WEEK 4       

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 total high  MEAN  2727  2272  2379  1049*
 sensor count  ST. DEV  895  887  874  329
   N  5  5  5  4
           
 total low  MEAN  6113  6334  6154  4390
 sensor count  ST. DEV  777  1590  1148  968
   N  5  5  5  4

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 8. ORGAN WEIGHTS (GRAM) SUMMARY

MALES       

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 BODY W.  MEAN  313  308  301  234**
 (GRAM)  ST. DEV  22  16  12  14
   N  5  5  5  4
           
 BRAIN MEAN  1.96  1.99 2.00  1.86
 (GRAM)  ST. DEV  0.10  0.05  0.05  0.04
   N  5  5  5  4
           
 HEART  MEAN  1.082  1.120  1.079  0.910*
 (GRAM)  ST. DEV  0.080  0.110  0.095  0.084
   N  5  5  5  4
           
 LIVER  MEAN  9.47  9.06  9.51  8.82
 (GRAM)  ST. DEV  1.24  0.50  0.71  0.27
   N  5  5  5  4
           
 THYMUS  MEAN  0.387  0.441  0.397  0.210*
 (GRAM)  ST. DEV  0.092  0.111  0.113  0.077
   N  5  5  5  4
           
 KIDNEYS  MEAN  2.56  2.62  2.56  2.25
 (GRAM)  ST. DEV  0.17  0.27  0.10  0.18
   N  5  5  5  4
           
 ADRENALS  MEAN  0.062  0.070  0.061  0.078**
 (GRAM)  ST. DEV  0.003  0.009  0.003  0.009
   N  5  5  5  4
           
 SPLEEN  MEAN  0.738  0.917*  0.748  0.601
 (GRAM) ST. DEV  0.087  0.062  0.082  0.173
   N  5  5  5  4
           
 TESTES  MEAN  3.33  3.56  3.47  3.26
 (GRAM)  ST. DEV  0.25  0.36  0.22  0.21
   N  5  5  5  4
           
 EPIDIDYMIDES  MEAN  0.898  0.976  0.986  0.817
 (GRAM)  ST. DEV  0.073  0.181  0.092  0.072
   N  5  5  5  4

*/** Dunnett-test based an pooled variance significant at 5% (*) or 1 % (**) level

FEMALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 BODY W.  MEAN  197  203  204  190
 (GRAM)  ST. DEV  4  24  13  6
   N  5  4  5  5
           
 BRAIN  MEAN  1.82  1.81  1.87  1.84
 (GRAM)  ST. DEV  0.09  0.04  0.07  0.05
   N  5  4  5  5
           
 HEART  MEAN  0.741  0.830  0.830  0.763
 (GRAM)  ST. DEV  0.045  0.074  0.098  0.048
   N  5  4  5  5
           
 LIVER  MEAN  6.21  6.71  7.14*  7.43**
 (GRAM)  ST. DEV  0.27  0.62  0.47  0.35
   N  5  4  5  5
           
 THYMUS  MEAN  0.314  0.355  0.375  0.216
 (GRAM)  ST. DEV  0.065  0.104  0.063  0.044
   N  5  4  5  5
           
 KIDNEYS  MEAN  1.68  1.65  1.70  1.67
 (GRAM)  ST. DEV  0.10  0.11  0.13  0.05
   N  5  4  5  5
           
 ADRENALS  MEAN  0.067  0.075  0.076  0.084**
 (GRAM)  ST. DEV  0.008  0.004  0.009  0.008
   N  5  4  5  5
           
 SPLEEN  MEAN  0.561  0.597  0.598  0.611
 (GRAM)  ST. DEV  0.065  0.108  0.050  0.046
   N  5  4  5  5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 9. ORGAN/BODY WEIGHT RATIOS (%) SUMMARY

MALES       

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 BODY W.  MEAN  313  308  301  234**
 (GRAM)  ST. DEV  22  16  12  14
   N  5  5  5  4
           
 BRAIN  MEAN  0.63  0.65  0.66  0.80**
 (%)  ST. DEV  0.04  0.03  0.02  0.04
   N  5  5  5  4
           
 HEART  MEAN  0.346  0.364  0.359  0.389
 (%)  ST. DEV  0.021  0.041  0.035  0.033
   N  5  5  5  4
           
 LIVER  MEAN  3.02  2.94  3.16  3.77**
 (%)  ST. DEV  0.23  0.16  0.12  0.19
   N  5  5  5  4
           
 THYMUS  MEAN  0.123  0.143  0.131  0.089
 (%)  ST. DEV  0.023  0.038  0.035  0.032
   N  5  5  5  4
           
 KIDNEYS  MEAN  0.82  0.85  0.85  0.96*
 (%)  ST. DEV  0.03  0.12  0.03  0.08
   N  5  5  5  4
           
 ADRENALS  MEAN  0.020  0.023  0.020  0.033**
 (%)  ST. DEV  0.002  0.002  0.001  0.004
   N  5  5  5  4
         
 SPLEEN  MEAN  0.236  0.298  0.248  0.256
 (%)  ST. DEV  0.022  0.025  0.020  0.072
   N  5  5  5  4
           
 TESTES  MEAN  1.07  1.15  1.16  1.39**
 (%)  ST. DEV  0.06  0.07  0.07  0.05
   N  5  5  5  4
           
 EPIDIDYMIDES  MEAN  0.289  0.315  0.328  0.349
 (%)  ST. DEV  0.039  0.045  0.027  0.013
   N  5  5  5  4

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES

     GROUP 1  GROUP 2  GROUP 3  GROUP 4
     0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 BODY W.  MEAN  197  203  203  190
 (%)  ST. DEV  4  24  13  6
   N  5  4  5  5
           
 BRAIN  MEAN  0.93  0.90  0.91  0.97
 (%)  ST. DEV  0.06  0.11  0.04  0.05
   N  5  4  5  5
           
 HEART  MEAN  0.377  0.413  0.406  0.401
 (%)  ST. DEV  0.029  0.055  0.044  0.019
   N  5  4  5  5
           
 LIVER  MEAN  3.15  3.32  3.49** 3.91** 
 (%)  ST. DEV  0.09  0.17  0.09  0.19
   N  5  4  5  5
           
 THYMUS  MEAN  0.159  0.173  0.183  0.114
 (%)  ST. DEV  0.031  0.039  0.026  0.024
   N  5  4  5  5
           
 KIDNEYS  MEAN  0.85  0.82  0.83  0.88
 (%)  ST. DEV 0.04  0.06  0.06  0.04
   N  5  4  5  5
           
 ADRENALS  MEAN  0.034  0.037  0.037  0.044**
 (%)  ST. DEV  0.004  0.005  0.003  0.005
   N  5  4  5  5
           
 SPLEEN  MEAN  0.285  0.299  0.293  0.322
 (%)  ST. DEV  0.034  0.067  0.032  0.023
   N  5  4  5  5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TABLE 10. MACROSCOPIC FINDINGS SUMMARY

MALES       

      GROUP 1  GROUP 2  GROUP 3  GROUP 4
      0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 INTERCURRENT DEATH           
Animals examined           1
Animals affected           1
Stomach        
  Thickened        1
  Irregular surface        1
 Liver        
  Accentuated lobular pattern        1
  Enlarged        1
  Discolouration        1
 Seminal vesicles        
  Reduced in size        1
 Thymus        
  Reduced in size        1
 Mesenteric l. node        
  Discolouration        1
 END OF TREATMENT        
 Animals examined  5  5  5  4
 Animals without findings  5  0  0  0
 Animals affected  0  5  5  4
 General observations        
  Gi-tractus distended with gas  0  0  0  1
 Stomach        
  Thickened  0  5##  5##  4##
  Grown together with:  0  0  0  1
  lrregular surface  0  4##  5##  4##
 Liver        
  Diaphragmatic hernia  0  0  1  0
  Focus/foci  0  0  0  1
 Pancreas        
  Nodule(s)  0  0  0  1
 Thymus        
  Reduced in size  0  0  0  1
Lymph nodes         
  Enlarged  0  0  0  1
 Mesenteric l. node        
  Discolouration  0  0  1  4##
 Mandibular l. node        
  Enlarged  0  1  0  0

# / ## Fisher's Exact test significant at 5% (#) or 1 % (##) level

FEMALES

   GROUP 1  GROUP 2  GROUP 3  GROUP 4
   0 MG/KG  150 MG/KG  450 MG/KG  1000 MG/KG
 INTERCURRENT DEATH        
 Animals examined    1    
 Animals affected    1    
 General observations        
  Cannihalism:organ missing    1    
 END OF TREATMENT        
 Animals examined  5  4  5  5
 Animals without findings  4  0  0  0
 Animals affected  1  4  5  5
 Stomach        
  Focus/foci  0  0  1  0
  Thickened  2  5##  4#
  Grown together with:  0  0  0  1
  Irregular surface  0  4##  5##  5##
 Liver        
  Diaphragmatic hernia  0  0  1  0
 Pancreas        
  Nodule(s)  0  0  0  1
 Uterus        
  Contains fluid  0  0  1  1
 Lymph nodes        
  Enlarged  0  0  1  2
 Mesenteric l. node        
  Discolouration  0  0  1  3
 Mandibular l. node        
  Enlarged  1  0  0  0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Applicant's summary and conclusion

Conclusions:
The test material produced significant treatment related local effects (stomach and GIT) at all dose levels. The LOAEL for local effects was therefore the lowest dose of 150 mg/kg bw/day. All other findings were considered likely to be secondary to the local effects and as such the Systemic NOAEL is potentially 1000 mg/kg.
Executive summary:

The test substance was administered daily for 28 days by oral gavage to SPFbred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; water consumption between days 21-25; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Treatment resulted in a marked irritant effect on the stomach down to the lowest administered dose of 150 mg/kg/day. The primary and most severe finding was forestomach ulceration with submucosal inflammation in the stomach which was recorded in a dose related fashion in all dose groups, reaching very severe grade at 1000 mg/kg/day. These findings correlated to necropsy findings. Ulceration of the forestomach was the cause of moribundity and subsequent sacrifice of one male at 1000 mg/kg/day. Clinical signs

and deviations in clinical pathology parameters observed at 450 and 1000 mg/kg/day as well as lower body weights and food intake at 1000 mg/kg/day were also considered to have resulted from the stomach effects noted at these dose levels. Increased adrenal weights and reduced thymus weights at 1000 mg/kg/day were considered to be a secondary non-specific stress response. Similarly, the alterations noted in the mesenteric lymph node recorded variously at 450 and 1000 mg/kg/day as well as higher white blood cell counts at 1000 mg/kg/day may be regarded as secondary reactive responses to the inflammation and necrosis in the stomach.

The stomach was identified to be the primary target organ of UNOXOL™ 3,4 DIALDEHYDE following oral administration. Based on the stomach effects observed at dose levels down to 150 mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for local toxicity of UNOXOL™ 3,4 DIALDEHYDE could not be established. All other findings were either considered to have resulted secondarily from the stomach effects or were considered not to constitute an adverse effect. Therefore, a NOAEL of 1000 mg/kg/day for systemic toxicity of UNOXOL™ 3,4 DIALDEHYDE was established.