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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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: performed in accordance with OECD and GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
Guideline study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
EC Number:
228-768-4
EC Name:
2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
Cas Number:
6358-31-2
Molecular formula:
C18H18N4O6
IUPAC Name:
2-[(2-methoxy-4-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxobutanamide
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- Name of test material (as cited in study report): Hansa Brillantgelb 2GX70 [Pigment Yellow 74]

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: F344/DuCrl, SPF
- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Deutschland
- Age at first administration: ca. 7 weeks
- Weight at first administration: males: 165 g, females: 121 g
- Identification: By tattooing the individual animal number into the right pinna and cage tag.
- Housing: Optimal hygienic conditions. Makrolon cages Type IV (33 cm x 55 cm area, 20 cm height). The animals were caged in groups (3 or 4 animals of one group and one sex per cage). The first 4 animals of a group were housed together in one cage and the remaining ones in groups of 3 in two more cages.
- Diet: Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum.
- Water: Tap water, acidified with HCl to pH ¿3, from an automatic watering system, ad libitum.
- Nesting material: Nesting material and nibbling wood bricks (sized 10 cm x 2 cm x 2 cm), same material as the bedding material, were offered freshly once a week.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: Average of 21.9 °C.
- Humidity: Average of 49 % .
- Air changes: 12 per hour.
- Photoperiod: Artificial light from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered after being blended with (respectively dissolved in) the vehicle. Preparations of the test substance were made freshly every day shortly before the administration to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was neither soluble in nor miscible with any of the commonly used aqueous vehicles (water, water with modified cellulose, with or without detergent). Therefore corn oil was used as vehicle, which allowed the preparation of suitable suspensions.
- Concentration in vehicle: Appropriate preparations were made to allow a uniform dose volume for all groups.
- Amount of vehicle: 5 mL per kg body weight.
- Purity: nutritional grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was analysed by a validated HPLC-method. A linear calibration line was established. The variance homogeneity was checked over the working range of 10.1 to 51.8 mg/L.
Stability: Determined on Day 1.
Test conditions: Ambient temperature, exposed to light. The concentrations of preparations for the low dosed group were checked. A loss of at most 5 % within 4 h was tolerated. The stability of higher concentrated suspensions is considered better than that of lower ones.
The test substance was found to be sufficiently stable in the preparations for at least 4 h.

Homogeneity: Determined on Days 1 and 37. 3 samples of 1.0 mL each of the preparations for the three dosed groups A, B and C+CS were taken using the syringe plus probe by the technician at the beginning, in the middle and at the end of the dosing procedure for the group concerned. A deviation of the individual samples from the mean of at most ± 10 % was tolerated.
Actual concentration: Determined on Days 1 and 37. The same samples as for the determination of homogeneity were used. A deviation of the mean of the 3 samples from the target concentration of at most ± 10 % was tolerated.
The concentrations and the homogeneity of the test substance in the preparations were within the chosen limits at both sampling terms.
Duration of treatment / exposure:
Day 1 was the day of the first administration of the test substance.
The animals of all groups were treated with the test substance solutions or with the vehicle once a day on 91 (males) or 92 (females) consecutive days.
Animals of the satellite groups KS and CS were kept after cessation of dosing without a further administration for 29 (females) or 30 (males) additional days.
Frequency of treatment:
Once a day.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg: control group K and also control satellite group KS
Basis:
other: nominal, gavage
Remarks:
Doses / Concentrations:
50 mg/kg: low dose, group A
Basis:
other: nominal, gavage
Remarks:
Doses / Concentrations:
200 mg/kg: mid dose, group B
Basis:
other: nominal, gavage
Remarks:
Doses / Concentrations:
1000 mg/kg: high dose, group C and also high dose satellite group CS
Basis:
other: nominal, gavage
No. of animals per sex per dose:
10 males and 10 females in the main groups K, A, B and C.
6 males and 6 females in the satellite groups KS and CS.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses chosen were derived from and based on the results of a dose range finding study. Groups of 5 male and 5 female rats each were administered 100 mg or 316 mg or 1000 mg test substance in 5 mL corn oil per kg body weight orally once a day for 14 consecutive days. Investigations performed: Animal observations, body weights and feed consumption, necropsy.
Summarized results: All animals survived until the terminal sacrifice. There were no treatment related observations in life. Body weights and feed consumption were not notably affected. All animals were found to be grossly normal at the post mortem examination.
1000 mg per kg body weight is the highest feasible dose for a subchronic toxicity study and was thus chosen as high dose. The low dose, where no toxic effects are anticipated and the mid dose were chosen by the sponsor.
- Rationale for animal assignment (if not random): randomisation procedure.
- Rationale for selecting satellite groups: A control group KS and a high dose group CS was used to investigate the development of changes after finishing of dosing.
- Post-exposure recovery period in satellite groups: 29 days for females and 30 days for males.
- Bias control: The sequence of animals for clinical observations, blood sampling, blood and urine analyses, necropsy and histopathology was randomised.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 7, 14, 21, 28, 36, 42, 49, 56, 63, 70, 77, 85, 91: all animals of all groups. Days 98, 105, 112, 119: all animals of the satellite groups. Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights were determined:
Day 1 to 91: Once a week, all animals. Days 91 to 120: Satellite groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Determined per cage in weekly intervals in all animals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day 0: All animals of all groups. Day 86: All animals of all groups. Examination of the right eyes of all animals by adspection and by fundus examination, in case of the presence of lesions, and if considered as necessary, additionally by a slit lamp examination
An additional ophthalmoscopical examination at the end of the recovery period in the animals of groups KS and CS was omitted, as no test substance related alterations were noted at the end of the dosing period.
- Dose groups that were examined: each group.

HAEMATOLOGY: Yes
- Blood samples were taken from the retrobulbar vein plexus of the left eyes in the morning.
- Time schedule for collection of blood: Blood was taken on Day 92 from all male animals of groups K, A, B and C, on Day 93 from all female animals of groups K, A, B and C and of all animals of groups KS and CS on Day 121.
- Anaesthetic used for blood collection: Yes, slight ether anaesthesia
- Animals fasted: Yes. Feed was offered again immediately after the blood sampling.- Parameters determined (abbreviations, when commonly used):
¿ Red blood cell count (RBC)
¿ Haemoglobin concentration (HGB)
¿ Haematocrit (HCT)
¿ Mean corpuscular haemoglobin (MCH)
¿ Mean corpuscular haemoglobin concentration (MCHC)
¿ White blood cell count (WBC)
¿ Mean cell volume (MCV)
¿ Platelet count (PLT)
¿ Differential white blood cell count (% of the different cell species)
¿ Prothrombin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Blood samples were taken from the same animals and at the same time as for haematology. Plasma (with Li-heparin as anticoagulant) was obtained by centrifugation of the blood samples after incubation at 37 °C for about 1 hour.
- Parameters determined (abbreviations, when commonly used):
¿ alanin aminotransferase (ALT, GPT)
¿ albumin (ALB)
¿ alkaline phosphatase (AP)
¿ aspartate aminotransferase (AST, GOT)
¿ cholesterol (CHOL)
¿ creatinine (CREA)
¿ gamma-glutamyltransferase (GGT)
¿ glucose (GLU)
¿ potassium (K)
¿ sodium (Na)
¿ total protein (TP)
¿ urea (UREA)

URINALYSIS: Yes
- Time schedule for collection of urine:
Urine was collected overnight by placing the animals in metabolism cages and cooling the urine with ice.
Day 91/92: All males of groups K, A, B and C.
Day 92/93: All females of groups K, A, B and C.
Day 120/121: All animals of groups KS and CS.
- Parameters determined:
¿ total amount (weighed),
¿ colour, turbidity, appearance (visual),
¿ osmolarity (Osmometer)
¿ pH (pH-Meter),
¿ proteins, glucose, ketones, urobilinogen, bilirubin, erythrocytes, haemoglobin (semi-quantitative, test sticks),
¿ sediment analysis (microscopically).
- Animals fasted: Yes. Animals were given continuous access to drinking water, but no feed was offered during the urine collection time.

NEUROBEHAVIOURAL EXAMINATION: Yes.
Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) was performed outside the home cage in a standard arena.
On Day 87: all male animals of all groups.
On Day 91: all female animals of all groups.
On Day 119: all animals of groups KS and CS.
The eyelid and auricular reflexes (tactile and proprioceptive) were tested by slightly touching the cornea and the interior of a pinna with a nylon cord of approximately 0.6 mm diameter. Shaking of the head was considered as positive response.
Acoustic reactivity was tested by response to a moderate sound (clapping of the hands). Twitching of the body was considered as positive.
Visual reactivity was derived from the reaction to a dark sheet of paper, brought near to the animal without any physical contact. Turning towards the paper was considered as positive.
For the testing of the righting reflex, the animals were held between the front- and hind legs, turned on their backs and dropped from a height of 30 cm onto the bedding material of the standard arena. A landing on all four legs is considered to be a normal reaction.
The measurement of the forelimb grip strength was determined using a Digital Force Gauge DFIS 100.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes .
Animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death on
Day 92: groups K, A, B and C (males).
Day 93: groups K, A, B and C (females).
Day 121: groups KS and CS.
The following organs / tissues were taken from all animals and fixed in Bouin's solution:
gross lesions, tissue masses or tumours
adrenal glands
aorta
brain
caecum
coagulating glands
epididymides
eyes
heart
kidneys
large intestine (colon)
liver
lungs
lymph nodes (mandibular, mesenteric)
oesophagus
ovaries
pancreas
pituitary gland
prostate
rectum
salivary glands
sciatic nerve
seminal vesicles
skeletal muscle (thigh)
skin, mammary glands
small intestine (duodenum, ileum, jejunum), prepared as "Swiss Roll"
spinal cord (cervical, thoracal, lumbar)
spleen
sternum with bone marrow
stomach
testes
thymus
thyroid and parathyroid glands
trachea.
urinary bladder
uterus


ORGAN WEIGHTS
Fresh weights of the following organs were determined of all animals at necropsy:
adrenal glands (both together)
brain
epididymides (both together)
heart
liver
kidneys (both together)
ovaries (both together)
spleen
testes (both together)
thymus

HISTOPATHOLOGY: Yes.
Groups K and C: Histopathological examination was performed in all animals of all fixed organs or tissues listed above.
Groups KS, A, B and CS: No histopathological examination was performed in groups KS, A, B and CS, as there was no indication for test substance related effects found in group C.
The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:
Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:
-Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons were included in these sections.
-Spinal cord (three sections, a cervical, a thoracal and a lumbar).
-Liver (two sections).
The small intestine (duodenum, jejunum and ileum) was fixed and trimmed to form two "Swiss Rolls" (one of the cranial and one of the caudal part), according to "Moolenbeek and Ruitenberg: The Swiss Roll, a simple technique for histological studies of rodent intestine"; Lab.Animals 15 (1981), p.57-59.
The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 ¿m were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB.
The frozen liver samples were not subjected to an examination for fatty changes in frozen sections, as there was no indication for alterations noted in the H&E stained sections.
To describe the severity of lesions, the following grades were applied, if appropriate: minimal (1), mild (2), moderate (3), marked (4), severe (5).
The term "focal" together with a higher degree of severity also stands for "multifocal".
Other examinations:
None.
Statistics:
Analysis of variance followed by the Scheffé-test used for all data with means and standard deviations determined, comparison of more than two groups.
t-test used for all data with means and standard deviations determined, for comparison of two groups only.
H-test of Kruskal and Wallis followed by the test of Nemenyi used for counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled.
Chi2-test used for counted events.
Fisher's exact test used for counted events, if the Chi2-Test was not applicable.
Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used. Groups K and KS and C and CS were treated separately for statistical analysis.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Daily observation: No animal died. The faeces of all dosed animals were stained yellow. As the test substance itself is a yellow dye, the stain of the faeces is attributed to excreted test substance. One single high dosed male was found to be stained on parts of the body surface. The stain of the faeces and of the body's surface are not considered to be toxic effects.
Isolated cases of chromodakryorrhoea, alopecia and one broken incisor lack a dose relationship and are not related to the test substance.
Detailed observations: As in the daily observations, isolated cases of chromodakryorrhoea, alopecia and one broken incisor were noted, but lack a dose relationship and are not related to the test substance.

BODY WEIGHT AND WEIGHT GAIN:
There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
In the body weight gain, isolated terms with significant differences in the mid or high dosed groups, with both higher and lower body weight gains, are considered to be incidental significances.

FOOD CONSUMPTION
There were no noteworthy differences or dose related trends noted in the feed consumption of both sexes. The differences between the groups are too small the give an indication for a toxic effect.

OPHTHALMOSCOPIC EXAMINATION: No indications for ocular lesions were noted at the examinations before the first dosing and at the end of the dosing period. An additional examination at the end of the recovery period was therefore omitted.

HAEMATOLOGY
There were no relevant differences during the dosing period. Small but significant changes were observed at the end of the recovery period: RBC and haemoglobin decreased in males; MCV increased in males; MCHC decreased in females. The differences are minor in dimension, were noted only at the end of the recovery period and lack corresponding effects at the end of the dosing period. Thus they are not regarded as test substance related and may rather be incidental.

CLINICAL CHEMISTRY
Alanin aminotransferase (males) lowered in the mid dosed group and elevated in the high dosed group; cholesterol (females) increased in the mid dosed group; urea (females) decreased in the recovery group.These significant group differences lack a dose related trend. Thus none of the significant group differences is attributed to the test substance.

URINALYSIS
In the females, the low dosed group produced significantly less urine than the controls. As there was no dose relationship in the mid and the high dosed group, this is not related to the test substance.
All dosed groups of the females had a significantly lower urinary pH than the controls, but there was no dose related trend in pH. All mean urine pH data were in a range, commonly seen in control rats. Thus they are not regarded as test substance related.
In the males, there were no significant or otherwise noteworthy group differences in urinalysis results.

NEUROBEHAVIOUR: Neither test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.
There was no significant group difference in the grip strengths.

ORGAN WEIGHTS
The significantly altered thymus, brain and ovarian weights lack a dose relationship and are considered to be incidental significances.
Significantly elevated liver weights of the high dosed females may be possibly test substance related. As to the lack of any indications for hepatotoxicity, the changes in female liver weights may be part of an adaptive response. There was neither significant difference nor a corresponding trend in the males.

GROSS PATHOLOGY
In the vast majority of all dosed animals, the content of the gastrointestinal tract was found to be stained yellow. This is attributed to the presence of the test substance and not regarded as toxic effect.
Isolated cases of small testes, fatty tissue necrosis in mesenteric fat, ovarian cysts are considered to be parts of the background pathology without relation to the test substance.

HISTOPATHOLOGY:
There was no test substance related alteration noted histopathologically.
Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence.

OTHER FINDINGS: None.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

Only parameters are listed for which a significant difference between at least one treatment group and the respective control group was observed for at least one sex. All parameters not showing significant differences are omitted from the tables. A significant difference to the respective control group is indicated by a grey background.

Haematology results.

parameter (sex)

low dose

(% of the negative controls)

mid dose

(% of the negative controls)

high dose

(% of the negative controls)

high dose
after recovery
(% of the negative controls)

red blood cell count(males)

100

101

101

97

haemoglobin conc.(males)

100

100

101

97

mean cell volume(males)

100

100

100

102

mean corpuscular haemoglobin conc.(females)

105

109

115

99

Clinical biochemical results.

parameter (sex)

low dose

(% of the negative controls)

mid dose

(% of the negative controls)

high dose

(% of the negative controls)

high dose
after recovery
(% of the negative controls)

alanin aminotransferase(males)

84

83

88

102

cholesterol(females)

84

86

95

96

urea(females)

92

94

97

89

Urinalysis findings.

parameter (sex)

low dose

mid dose

high dose

high dose
after recovery

total amount(females, % of the negative controls)

58

76

85

133

pH(females; control: pH 7.1; control recovery: pH 7.3)

6.4

6.7

6.1

7.3

Organ weights.

parameter (sex)

low dose

(% of the negative controls)

mid dose

(% of the negative controls)

high dose

(% of the negative controls)

high dose
after recovery
(% of the negative controls)

thymus(males, absolute weight)

121

113

114

120

thymus(males, organ weight/body weight ratio)

121

115

114

120

thymus(males, organ weight/brain weight ratio)

120

118

115

120

brain(males, absolute weight)

100

96

99

100

liver(females, absolute weight)

105

105

110

103

liver(females, organ weight/body weight ratio)

104

103

109

99

liver(females, organ weight/brain weight ratio)

104

104

111

100

ovaries(females, absolute weight)

107

92

87

110

Applicant's summary and conclusion

Conclusions:
A 90-day toxicity study in Fischer F344 rats was performed with the test item. Groups of 10 male and 10 female rats received 0, 50, 200 and 1000 mg/kg/day by oral gavage in corn oil for 90 days. The study included additional control and high dose groups of 6 males and 6 females each analyzed after a 4-week recovery period. All animals survived until scheduled termination without any toxic signs in life. In haematology, clinical biochemistry and urinalysis some scattered significant differences, altogether without a clear dose response and all only minor in severity, were noted. Post mortem examination including histopathology did not reveal any test substance related toxic alterations. The test substance caused elevated liver weights in the high dosed females. As there were no corresponding histopathological or clinical-biochemical alterations found, the most likely interpretation is an adaptive response by enzyme induction. No parallel trend was present in the males. The effects did not persist until the end of the recovery period. No other test substance-related effect was noted. The No-observed-adverse-effect-level (NOAEL) of the test item was 1000 mg/kg bw/day in both sexes.
Executive summary:

Guidelines applied

EC-Guideline 2001/59/EC, B.26.,
OECD-Guideline 408, 21 September 1998.

Dose levels

Based on the results of a 14-day dose range finding study (which was part of the present study), the dose levels for this 90-day oral gavage study were selected to be 0, 50, 200 and 1000 mg/kg/day.

Study outline

The test substance, formulated in corn oil, was administered daily for 90 days orally by gavage to SPF-bred Fischer F344/DuCrl rats. One control group (plus a control satellite group) and 3 test substance treated groups (plus a high dose satellite group) were tested, each consisting of 10 males and 10 females (6 males and 6 females in the satellite groups).

Evaluated parameters

Chemical analyses of formulations were conducted twice in the in-life phase of the study to assess concentration and homogeneity.

The following parameters were evaluated: Clinical signs (daily plus weekly in more detail), functional observations (terminally), ophthalmoscopy (initial and terminal), body weights and feed consumption (weekly), haematology plus clinical biochemistry plus urinalysis (terminal), necropsy, organ weights and histopathology.

Results

Accuracy and homogeneity of test substance preparations in corn oil were demonstrated by analyses.

All animals survived until scheduled termination without any toxic signs in life. In haematology, clinical biochemistry and urinalysis some scattered significant differences, altogether without a clear dose response and all only minor in severity, were noted. Post mortem examination including histopathology did not reveal any test substance related toxic alterations. Liver weights of the females were elevated with a dose dependency, which may indicate an adaptive response rather than a toxic effect.

A frequently noted yellow stain of the gastrointestinal contents and of the faeces is attributed to the presence of the administered test substance itself and not regarded as a toxic effect.

Discussion and Conclusion

The test substance caused elevated liver weights in the high dosed females. As there were no corresponding histopathological or clinical-biochemical alterations found, the most likely interpretation is an adaptive response by enzyme induction. No parallel trend was present in the males.

No other test substance related effect was noted.

The effects did not persist until the end of the recovery period.

The No-observed-adverse-effect-level (NOAEL) of the test substance was at 1000 mg per kg body weight in both sexes.

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