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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:
16 January 2008 to 24 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Resin 835A
- Molecular formula: C20H28O2
- Physical state: solid
- Lot/batch No.: 02117
- Expiration date of the lot/batch: one year after manufacture
- Stability under test conditions: stable
- Storage condition of test material: ambient temperature, in the dark, may be used under light

Test animals

Species:
rat
Strain:
other: Fischer F344/DuCrl, SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: group means between 179 and 186 g (males) and between 135 and 136 g (females)
- Housing: Group caging (2 animals of one group and one sex per cage). Makrolon cages Type III (39 cm x 23 cm area, 18 cm height).
- Diet: Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany. Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood sampling.
Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).
- Water: Tap water, acidified with HCl to pH >=3, from an automatic watering system, ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).
- Bedding material: Aspen wood chips (ABEDD Dominik Mayr KEG, 8580 Köflach, Austria), autoclaved. Bedding material was changed weekly.
- Environmentla enrichment: Nesting material and nibbling wood bricks (sized 10 cm x 2 cm x 2 cm), same material and source as the bedding material, were offered freshly once week.
- Acclimation period: 4 days


ENVIRONMENTAL CONDITIONS
- Temperature: Average of 12.9 °C (continuous control and recording).
- Humidity (%): Average of 49 % (continuous control and recording).
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 24 January 2008 To: 21 May 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1 % aqueous (water pro analysis, Merck item No. 1.16754.5000) solution of Na-carboxymethyl cellulose ("CMC", high viscosity, item No. C-5013, Lot. No. 98H0328, Sigma) plus 0.01 % Tween 80 (Polyoxyethylensorbitan monooleate, item No. 822187, Merck)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a suspension in the vehicle.
Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

VEHICLE
- Justification for use and choice of vehicle: Adding both a mucilaginous agent plus a detergent was necessary to achieve a sufficiently stable and homogenous suspension.
- Concentration in vehicle: 10, 31.6 and 100 mg per mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): see above
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability: Not determined, as data on the stability of the test substance in an identical preparation were already available from a previously performed 28-Day Toxicity Study (see also endpoint study record "Key. Hruby 2007. 28-Day Oral Toxicity Rats").
Homogeneity: Determined on Days 5 and 27. 3 samples of 2.0 mL each of the preparations for each test substance group 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 5 and 27. 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.
Chemical analysis was performed by HPLC. The method is described extensively in the report.
Duration of treatment / exposure:
90 days (males) or 91 days (females). Animals of the satellite groups (control and high dose) were kept after cessation of dosing without a further administration for 28 (females) or 29 (males) additional days.
Frequency of treatment:
Once a day, on 7 days per week.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg per kg body weight
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
100 mg per kg body weight
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
316 mg per kg body weight
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
1000 mg per kg body weight
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
10 males and 10 females each for negative control, low dose, mid dose and high dose.
6 males and 6 females each for negative control satellite group and for high dose satellite group.
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 28 Day Toxicity Study (see endpoint study record "WoE. Hruby 2007. 28-Day Oral Toxicity Rats").
Doses of 100mg, 316 mg and 1000 mg per kg body weight were used in the 28-Day Study. Indications for a fatty change in the liver were noted in the high dosed females, while in the males no test substance related effects were found at any dose level. The No Observed Effect Level (NOEL) was therefore at 1000 mg/kg in the males and at 316 mg/kg in the females.
The high dose shall induce a clear toxicity, but no or at most isolated mortality. A dose of 1000 mg per kg b.w. is not exceeded as high dose. The low dose shall induce no toxic effect. The mid dose is interpolated geometrically.

- Rationale for selecting satellite groups: to check the reversibility of possible adverse effects
- Post-exposure recovery period in satellite groups: 28 days (females) or 29 days (males)
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, plus a daily check for viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Days 0, 5, 12, 19, 26, 33, 40, 47, 54, 62, 68, 75, 82 and 89 for all animals of all groups; on Days 103, 110 and 117 for 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: Day 1 to 90: once a week for all animals of all groups; on Days 99 to 118 for all animals of the satellite groups.

FOOD CONSUMPTION:
- Determined per cage in weekly intervals in all animals: Day 1 to 90: for all animals of all groups; on Days 99 to 118 for all animals of the satellite groups.

OPHTHALMOSCOPIC EXAMINATION: Yes
Days 0 and 85: 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 the satellite groups was omitted, as no test substance related alterations were noted at the end of the dosing period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 91 from all male animals and on Day 92 from all female animals of the control group and the low, mid and high dose groups; on Day 119 from all animals of the satellite groups.
- Anaesthetic used for blood collection: Yes (slight ether anaesthesia)
- Animals fasted: Yes, overnight
- How many animals: all (see above)
- Parameters examined:
--- 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
- Time schedule for collection of blood: on Day 91 from all male animals and on Day 92 from all female animals of the control group and the low, mid and high dose groups; on Day 119 from all animals of the satellite groups.
- Animals fasted: Yes, overnight
- How many animals: all (see above)
- Parameters examined:
--- 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: Day 90/91: all males of the negative control group and of the low, mid and high dose groups. Day 91/92: all females of the negative control group and of the low, mid and high dose groups.
An additional urinalysis at the end of the recovery period in the animals of the satellite groups was omitted, as no test substance related alterations were noted at the end of the dosing period.
Urine was collected overnight by placing the animals in metabolism cages and cooling the urine with ice. Animals are given continuous access to drinking water, but no feed was offered during the urine collection time.
- Parameters examined:
--- total amount (weighed),
--- colour, turbidity, appearance (visual),
--- osmolarity (Osmometer)
--- pH (pH-Meter),
--- proteins, glucose, ketones, urobilinogen, bilirubin, erythrocytes, haemoglobin (semi-quantitative, test sticks, supplied by Boehringer Mannheim),
--- sediment analysis (microscopically).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Day 89 for all male animals of all groups, on Day 90 for all female animals of all groups and on Day 117 for all animals of the satellite groups
- Dose groups that were examined: see above
- Battery of functions tested:
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.
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 from Chatillon, Greensboro, North Carolina, USA.
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 91 (males of the negative control group and the low, mid and high dose groups), Day 92 (females of the negative control group and the low, mid and high dose groups) and on Day 119 (all animals of the satellite groups).
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
- coagulation 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: Yes
Fresh weights of the following organs were determined of all animals at necropsy:
- adrenal glands (both together)
- brain
- epididymides (both together)
- heart
- kidneys (both together)
- liver
- ovaries
- spleen
- testes (both together)
- thymus
- uterus

HISTOPATHOLOGY: Yes
Negative control group, high dose group: Histopathological examination was performed in all animals of all fixed organs or tissues listed above.
Low and mid dose groups, satellite groups: A histopathological examination was performed in all livers, as there was an indication for a test substance related effect found in the high dose group.
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 µm were stained with haematoxylin and eosin. Evaluation of slides was performed using a light microscope Leica-DMRB.
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: all data with means and standard deviations determined, comparison of more than two groups.
t-test: 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 counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled.
Chi2-test: counted events.
Fisher's exact test: 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. Main and satellite groups were treated separately for statistical analysis.
Numerical data have been rounded for presentation; a manual recalculation therefore may yield slightly different results to those given in the tables.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No test substance related death occurred during the study. All animals survived until their scheduled sacrifice.
No test substance related abnormalities were noted at the daily and the detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN (see also the respective tables)
The high dosed and high dosed satellite group males had significantly reduced body weights at several terms of the first half of the administration period. This difference in body weights did not persist any more towards the end of the administration period.
In the high dosed females a significantly reduced body weight was noted at two terms and in the mid dosed females at one term of the administration period. In contrast to the males, a tendency towards lower body weights, but without gaining statistical significance, was present in the females till the end of the study.
In the body weight gain similar effects were noted, though even less clear as in the body weights. In both sexes, the body weight gain was somewhat lower in the first half of the exposure period and equal or even higher in the second half, when compared with the controls.
Reduced body weights in the high dosed groups are considered to be test substance related effects, though only poorly expressed ones.

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. No statistical evaluation of the results is useful due to group caging.

HAEMATOLOGY (see also the respective tables)
There were several statistically significant differences found.
Only the group differences in the platelet count are regarded as test substance related. All other significant group differences are minor in dimension and are thus not given toxicological relevance.

CLINICAL CHEMISTRY (see also the respective tables)
There were several statistically significant differences found.
Lowered enzyme activities (alanin aminotransferase, aspartate aminotransferase) are not considered as toxic effects, they may be rather due to an incidentally higher mean of the control group.
The elevated activity of alkaline phosphatase and a reduced total protein and reduced albumin may be due to impaired liver function.

URINALYSIS
Neither significant group differences nor any other indications for a test substance related effect was noted in urinalysis.

NEUROBEHAVIOUR (see also the respective tables)
In both sexes, dosed animals turned out to be more active during the observation in a standard arena. The findings indicating higher exploratory activities (e.g. "straightening up") were seen more often, with differences becoming statistically significant. A higher physical activity and higher exploratory activity are interpreted as biologically positive and not as toxic effects. Thus, no indication for toxicity is derived from this.
All other observations were within normal limits.
In grip strength determination, the high dosed recovery females were found to have higher grip strengths than the corresponding controls. Again, this is not interpreted as a toxic response.

ORGAN WEIGHTS (see also the respective tables)
Elevated liver weights of the females at the end of the dosing period may be caused by the presence of vacuoles, as noted histologically.
The other significantly altered organ weights at the end of the dosing period are not accompanied by any findings, which might indicate a specific toxic effect. It cannot be excluded, that these effects were treatment related.
The significant group differences in spleens and adrenal glands at the end of the recovery period are considered as incidental significances without a toxicological relevance.

GROSS PATHOLOGY
A few isolated findings, altogether interpreted as part of the background pathology without relation to the test substance were noted at necropsy.

HISTOPATHOLOGY (see also the respective tables)
The only test substance related effect was the presence of cytoplasmatic vacuoles in the centrolobular hepatocytes. This might be due to a mild fatty change or a proliferation of lysosomes.
Some isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

For details on the results see the attached file "Key_Hruby 2009_90d_Oral_Tox_Rats_Tables.pdf":

Table 1: Mean body weights

Table 2: Mean body weight gain

Table 3: Mean haematological data

Table 4: Mean clinical biochemistry data

Table 5: Survey of functional observations

Table 6: Mean grip strength

Table 7: Mean organ weights

Table 8: Survey of the histopathological findings

Applicant's summary and conclusion

Conclusions:
The test substance induced mild hepatic alterations, notable as hepatocellular vacuoles and by some clinical-chemical parameters.
A decrease in the number of platelets in the blood and some organ weight changes were noted in addition.
All test substance related findings were present only in a low grade of severity and never became life-threatening.
Changes in the high dosed group are interpreted as possibly adverse ones. In the mid and the low dosed group only the presence of hepatocellular vacuoles is attributed to the test substance, but, in the absence of clear signs of toxicity, they are interpreted rather as adaptive changes.
There was no pronounced sex difference in the response to the test substance.
None of the test substance related effects persisted until the end of the recovery period.
The No-observed-adverse-effect-level (NOAEL) of "Resin 835A" was at 316 mg per kg body weight and day in both sexes, based on possible adverse effects in the high dosed group.
According to EU-Directive 2001/59/EC, the application of "R48" is not considered as necessary for "Resin 835A", as no severe toxic effects were noted even at a dose of 1000 mg/kg.
Executive summary:

Aim of the study

This study was performed to evaluate the toxicity of “Resin 835A” after a repeated oral administration to rats, according to the EC-Guideline 2001/59/EC, method B.26., and to the OECD-Guideline 408, 21 Sept. 1998.

A 28-Day Study preceded this study (report ARC--L-2453, “WoE. Hruby 2007. 28-Day Oral Toxicity Study Rats”).

Methods

Test substance: “Resin 835A”.

Vehicle: 0.1 % aqueous solution of Na-carboxymethylcellulose plus 0.01 % Tween 80.

Test substance preparation for administration: Freshly suspended in the vehicle.

Route of test substance administration: Oral via gavage, 1/day.

Dose volume: 10 mL test substance preparation or vehicle per kg body weight.

Test system (animals): Rats, Fischer, F344/DuCrl. 10 males and 10 females in groups K, A, B and C and 6 males and 6 females in groups KS and CS.

Groups, doses:

K (negative control group): vehicle only,

KS (negative control satellite group):vehicle only,

A (low dose group): 100 mg “Resin 835A” per kg body weight,

B (mid dose group): 316 mg “Resin 835A” per kg body weight,

C (high dose group): 1000 mg “Resin 835A” per kg body weight,

CS (high dose satellite group): 1000 mg “Resin 835A” per kg body weight.

The doses chosen were derived from and based on the results of the 28 Day Toxicity Study.

Administration period:

1/day for 90 (males) or 91 (females) consecutive days (Days 1-90/91).
The animals of the satellite groups were kept for additional 28 days(females) or 29 days (males) without further dosing.

Investigations:

·       Analyses of the test substance preparations:
In selected samples. For concentration and homogeneity.

·       Animal observations:
All animals, 1/day, plus a daily check for viability.

·       Detailed clinical observations:
All animals, 1/week.

·       Functional observations:
All animals, once, in the last week of the dosing period. All animals of groups KS and CS in the last week of the recovery period.

·       Ophthalmoscopy:
All animals, prior first dosing and on Day 85.

·       Body weights:
All animals, 1/week.

·       Feed consumption:
All animals, for each week.

·       Haematology:
All male animals of groups K, A, B and C on Day 91, all female animals of groups K, A, B and C on Day 92 and all animals of groups KS and CS on Day 119.

·       Clinical biochemistry:
All male animals of groups K, A, B and C on Day 91, all female animals of groups K, A, B and C on Day 92 and all animals of groups KS and CS on Day 119.

·       Urinalysis:
All male animals of groups K, A, B and C on Day 90 and all female animals of groups K, A, B and C on Day 91.

·       Necropsy with gross pathological examination:
All male animals of groups K, A, B and C on Day 91, all female animals of groups K, A, B and C on Day 92 and all animals of groups KS and CS on Day 119.

·       Organ weight determination:
Selected organs in all animals at necropsy.

·       Histopathological examination:
Selected organs or tissues in all animals of groups K and C. Organs and tissues with suspected test substance related alterations in all groups.


Results

·       Analyses of the test substance preparations:
The preparations examined were found to be within acceptable limits of concentration and homogeneity.

·       Mortality:
There was no test substance related mortality.

·       Observations in life, clinical and functional observations, and ophthalmoscopy:
No test substance related alterations.

·       Body weights and body weight gain:
At several terms reduced body weights and body weight gain; primarily, but not exclusively, in the high dosed animals.

·       Feed consumption
No test substance related alterations.

·       Haematology:
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 this table. A significant difference to the respective control group is indicated by a black background:

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)

mean corpuscular haemoglobin (males)

101

102

102

101

mean corpuscular haemoglobin (females)

100

101

101

101

platelet count (males)

97

94

91

106

red blood cell count (females)

99

98

98

104

haemoglobin conc. (females)

99

99

99

105

haematocrit (females)

100

100

100

104

mean cell volume (females)

101

102

103

100


·       Clinical biochemistry, organ weight determination:
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 this table. A significant difference to the respective control group is indicated by a black background.

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)

102

95

89

69

alanin aminotransferase (females)

71

72

74

111

alkaline phosphatase (males)

108

105

118

94

aspartate aminotransferase (males)

97

91

91

71

urea (males)

106

102

108

111

creatinine (females)

100

102

86

105

total protein (females)

98

98

96

103

albumin (females)

100

99

97

104

testes (males, absolute weight)

98

95

94

102

spleen (males, organ weight/brain weight ratio)

101

99

96

95

adrenals (males, absolute weight)

113

94

96

79

adrenals (males, organ weight/brain weight ratio)

112

93

96

78

liver (females, absolute weight)

106

97

110

86

liver (females, organ weight/body weight ratio)

106

100

115

92

liver (females, organ weight/brain weight ratio)

105

96

108

87

uterus (females, absolute weight)

95

86

71

72

uterus (females, organ weight/body weight ratio)

95

88

75

77

uterus (females, organ weight/brain weight ratio)

94

85

70

73

·       Necropsy with gross pathological examination and histopathology:
In all dosed groups hepatocellular vacuoles were noted, being reversible within the recovery period of 4 weeks.

·       Satellite groups: No persistence of the test substance induced lesions, but in a few cases possible compensatory contrary differences.


Discussion and Conclusion

The test substance induced mild hepatic alterations, notable as hepatocellular vacuoles and by some clinical-chemical parameters.

A decrease in the number of platelets in the blood and some organ weight changes were noted in addition.

All test substance related findings were present only in a low grade of severity and never became life-threatening.

Changes in the high dosed group are interpreted as possibly adverse ones. In the mid and the low dosed group only the presence of hepatocellular vacuoles is attributed to the test substance, but, in the absence of clear signs of toxicity, they are interpreted rather as adaptive changes.

There was no pronounced sex difference in the response to the test substance.

None of the test substance related effects persisted until the end of the recovery period.

The No-observed-adverse-effect-level (NOAEL) of "Resin 835A" was at 316 mg per kg body weight and day in both sexes, based on possible adverse effects in the high dosed group.

According to EU-Directive 2001/59/EC, the application of "R48" is not considered as necessary for "Resin 835A", as no severe toxic effects were noted even at a dose of 1000 mg/kg.