1,3- AND 1,4-CYCLOHEXANDICARBOXYALDEHYDE

Repeated dose toxicity: oral

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 for cross-reference:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-020-3

EC Name:

-

Molecular formula:

C8H12O2

IUPAC Name:

cyclohexane-1,3-dicarbaldehyde; cyclohexane-1,4-dicarbaldehyde

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 or test system 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	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	0	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.