Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 27th July 1995)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 3rd October 2008)
Deviations:
yes
Remarks:
no testing of the endocrine system
Qualifier:
according to
Guideline:
other: 96/54/EEC of September 30, 1996, Part B, 1996
Qualifier:
according to
Guideline:
other: Japan/MHW 1987
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Only animals free from clinical signs of disease were used for the study. The rats were identified clearly by ear tattoo.
The rats were housed singly in type DK 111 stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dust-free embedding, supplied by SSNIFF, Soest, FRG). Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, Germany (floor area about 800 cm2 ) and small amounts of absorbent material (see above). The animals were housed in a fully air-conditioned room. Central air-conditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity. The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.). The animal room was completely disinfected using a disinfector ('AUTEX', fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week. The cleansing liquid used was water containing about 0.1% lncidin perfect (supplied by Henkel, Duesseldorf, Germany). The food used was ground Kliba maintenance diet rat/mouse/ hamster, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test substance was weighed out and thoroughly mixed with a small amount of food. Then corresponding amounts of food were added to this premix in order to obtain the desired concentration, and mixing was carried out for 10 minutes in a laboratory mixer. The mixtures were prepared weekly and kept cold (+4°C). The food was changed twice a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the diet was demonstrated over a period of up to 4 days at room temperature and up to 4 days in the refrigerator. As the mixtures were stored no longer than this time period, the stability was guaranteed.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm
Remarks:
corresponding to (m/f): 142.3 / 153.3 mg/kg bw/d
Dose / conc.:
5 000 ppm
Remarks:
corresponding to (m/f): 478.5 / 529.9 mg/kg bw/d
Dose / conc.:
15 000 ppm
Remarks:
corresponding to (m/f): 1462.9 / 1552.1 mg/kg bw/d
No. of animals per sex per dose:
Control group: 10 animals per sex
1500 and 5000 ppm groups: 5 animals per sex
15000 ppm group: 10 animal per sex
Control animals:
yes, plain diet
Details on study design:
In a test study the test item was administered to groups of 3 male and 3 female Wistar rats at dose levels of 1000, 5000 and 15000 ppm for 2 weeks. Food consumption, water consumption, body weight and clinical signs were recorded. As no substance-related findings or signs of toxicity were observed, the following dose levels were selected for the present study: 15000 ppm as high concentration; this concentration results in an test substance intake of at least 1,000 mg/kg body weight/day which is the highest dose to be tested for non-toxic test substances in a 'limit test'; 5000 ppm: as mid dose and 1500 ppm: as low dose.
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Examinations

Observations and examinations performed and frequency:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further general clinical examinations were carried out daily. Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined:
1 . abnormal behavior during "handling"
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

Food consumption was determined weekly over a period of 4 days and calculated as mean food consumption in grams per animal and day.
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period and the recovery period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption. The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and food consumption.
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1 . posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open fleld observations: The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:
1 . behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes

Sensorimotor tests/reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1 .approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-Systern (Golumbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 12.45 p.m. in the main groups and at about 2.00 p.m. in the recovery groups. The number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Sacrifice and pathology:
The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weight parameters from all animals sacrificed at scheduled dates were determined:
1. anesthetized animals
2. liver
3. kidneys
4. adrenal glands
5. testes
6. epididymides
7. ovaries
8. uterus
9. spIeen
10. brain
11. heart
12. thymus

The following organs or tissues were fixed in 4% formaldehyde solution:
1. all gross lesions
2. brain
3. pituitary gland
4. thyroid glands with parathyroid glands
5. thymus
6. trachea
7. lungs
8. heart
9. liver
10. spIeen
11. kidneys
12. adrenal glands
13. testes/ovaries
14. uterus/vagina
15. epididymides, prostate gland, seminal vesicles
16. stomach (glandular and non-glandular)
17. duodenum, jejunum, ileum
18. cecum, colon, rectum
19. urinary bladder
20. Iymph nodes (mandibular and mesenteric)
21. sciatic nerve
22. bone marrow (femur)
23. eyes
24. spinal cord (cervical, thoracic and lumbar cord)

The immunorelevant organs and tissues were evaluated according to the following parameters:
Thymus:
- increased/decreased grade of cortico-medullar ratio (related only to area)
- increase of starry sky cells
- changes of cellular density in the cortex
- changes of cellular density in the medulla
Whenever the histopathologic evaluation of the thymus did not reveal a morphologic alteration of these items and/or whenever no other histologic finding was noted, the thymus was diagnosed as "no abnormalities detected".

Spleen:
- altered cellular composition of follicles
- changes of the cellularity of PALS, Iymphoid follicles, marginal zone, red pulp
- altered number of germinal centers
Whenever the histopathologic evaluation of the spieen did not reveal a morphologic alteration of these items and/or whenever no other histologic finding was noted, the spleen was diagnosed as "no abnormalities detected".

Lymph nodes (mesenteric and mandibular lymph nodes):
- changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
- altered number of germinal centers
- altered cellular composition of paracortex
- changes of the cellularity of sinus
Whenever the histopathologic evaluation of the Iymph nodes did not reveal a morphologic alteration of these items and/or whenever no other histologic finding was noted, the lymph nodes were diagnosed as "no abnormalities detected".

Peyer's patches (of the jejunum):
-changes of the cellularity of follicles (including mantle zone and germinal centers)
-changes of the cellularity of interfollicular area
Whenever the histopathologic evaluation of the Peyer's patches did not reveal a morphologic alteration of these items and/or whenever no other histologic finding was noted here or in the jejunum, the jejunum (including the Peyer's patches) was diagnosed as "no abnormalities detected".
Statistics:
Statistics of clinical pathology
Means and standard deviations of each test group were calculated for several parameters.
Further statistical analyses were performed for:
(1) CIinical pathology parameters, except differential blood count: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
(2) Urinalysis, except volume, colour, turbidity and specific gravity.

Statistics of pathology
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hyphothesis of equal medians.
For the animals of the recovery group: Pairwise comparison of each dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:
Mortality: No animal died during the study.

Clinical examinations:
Discoloration of feces (yellowish) was observed in all males and females of dose group 2 (5000 ppm) and 3 (15000 ppm). During the recovery period, discoloration returned to normal. This finding is clearly attributable to the ingestion and excretion of the test substance (dye stuff) and thus related to the physical (tinctorial) properties of the test substance, but does not represent a toxicologically relevant finding or adverse effect.
One female animal of dose group 2 showed an injury from day 21 until necropsy. Due to the single occurrences, this finding was assessed as being incidental in nature.

Food consumption: No substance-related effects were obtained.

Food efflciency: In high dose males, food efficiency was significantly decreased on day 35. Due to the isolated occurrence and the lack of a dose-response relationship, this was assessed as being incidental.

Body weight: No substance-related effects were obtained.

Functional observational battery:
Results on day 24/25 (main groups and recovery groups):
- Home cage observations: No substance-related findings were observed.
- Opon field observations: One male animal in dose group 2 and one male animal in dose group 3 showed discoloration of feces. This discoloration was caused by the tinctorial properties of the test substance (dye-stuff). Therefore, it does not represent a toxicologically relevant finding. All other deviations from "zero values" were equally distributed between treated groups and controls or occurred in single animals only. Therefore, these observations were considered to have been incidental.
- Sensonimotor tests/reflexes: No substance-related effects were observed. No statistically significant changes regarding feces, rearing, grip strength, and landing foot-splay test were observed.

Motor activity measurement:
- Regarding the overall motor activity, no statistically significant deviations were seen in treated males and females.
- Comparing the single intervals with the control groups, in females of dose group 3 an isolated statistically significant increased value was obtained. Due to the single observation this finding was assessed as being incidental.

CLINICAL PATHOLOGY
- Hematology: There are no treatment-related changes in the hematological parameters measured.
- Clinical chemistry: Compound-related differences in clinical chemistry parameters were not evident at any dose level in either males or females.
- Urinalyses: No treatment-related changes were found in the urinalyses of either sex.

PATHOLOGY
Animals of the main group (F1):
- Absolute weights: In males of the mid dose group, the mean brain weight was slightly although significantly decreased (- 5.7%). In female rats, the mean brain weight was slightly although significantly decreased in all treatment groups (-5.0%, -5.0%, -4.9% in Iow, mid and high dose group, respectively). This was regarded to be incidental as no dose-response relationship was evident. The other mean absolute weight parameters of the animais of the main group (F1) did not show significant differences when compared with the concurrent control group.
- Relative weights (related to terminal body weight): The mean liver weight of male rats was slightly although significantly decreased in the high (-6.9%) and in the mid dose groups (-3.0%). The other mean relative weight parameters (when related to terminal body weight) of the animals of the main group (F1) did not show significant differences when compared with the concurrent control group.
- Gross lesions: The only gross lesions noted were a small black erosion/ulcer in the mucosa of the glandular stomach of a mid dose male and a focal lesion in the skin of the right foreleg of a mid dose female rat. They were regarded incidental and unrelated to treatment. The other gross lesion noted were related to the physical (i. e. tinctorial) properties of the test article, that caused a yellow discoloration of the contents of the glandlular stomach of all high dose males and females. This was, hence, not regarded to represent an adverse effect.
- Histopathology: The gross lesions noted in the mucosa of the glandular stomach of a mid dose male and in the skin of a mid dose female rat were correlated with a meaningful microscopic finding. However, these microscopic findings were considered to have developed spontaneously and unrelated to treatment. The discoloration of the contents of the glandular stomach Iacked a morphologic correlate. Although they were treatment related - as a consequence of the intrinsic,physical (i.e. tinctorial) properties of the test article - they were not regarded to represent a treatment-related adverse effect.
All other microscopic findings recorded were either single observations, or they occurred in control animais only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females.
No morphologic correlate was obtained in brain of high dose female rats that may explain its slight although significantly decreased mean absolute weight. Also, there was no morphologic correlate for the significantly increased mean relative liver weight of male rats in the high dose group.
After a 4-week application period, there was no indicatiori of an affection of the organs of the central or peripheral nervous system, the reproductive systemn, and the immune system by the test article.

Animals of the recovery group:
- Absolute weights: In males of the high dose group, the mean brain weight was slightly although significantly increased (+4.1 %). In females of the high dose group, the mean thymus weight was slightly although significantly decreased (-17.4%). Both was regarded incidental. The other mean absolute weight parameters of the animals of the recovery group did not show significant differences when compared with the concurrent control group.
- Relative weights (related to terminal body weight): In females of the high dose group, the mean thymus weight was slightly although significantly decreased (-16.8%). This was regarded incidental. Ihe other mean relative weight parameters (when related to terminal body weight) of the animals of the recovery group did not show significant differences when compared with the concurrent control group.
- Gross lesions: The only gross lesions noted was a small black erosion/ulcer in the mucosa of the glandular stomach of a control male. This was, of cause, an incidental observation.
- Histopathology: The grossly noted erosion/ulcer in the glandular stomach of one control male was not correlated histopathologically. However, this gross lesions was anyway considered to have developed spontaneously and unrelated to treatment.
No morphologic correlate was obtained for the significantly increased mean absolute brain weight of treated males and for the significantly decreased mean absolute and relative weights of thymus of treated females. They were, hence regarded to have developed spontaneously and unrelated to treatment.
After a 2-week recovery period, there was no indication of a morphologic affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune systemn by the test article.

- In summary:
15,000 ppm (1462.9 mg/kg bw/d in males; 1552.1 mg/kg bw/d in females): no substance-related toxic effects were obtained in the main as well as recovery groups.
5,000 ppm (478.5 mg/kg bw/d in males; 529.9 mg/kg bw/d in females): no substance-related toxic effects.
1,500 ppm (142.3 mg/kg bw/d in males; 153.3 mg/kg bw/d in females): no substance-related toxic effects.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Corresponds to the highest dose tested. No adverse effects occured.
Remarks on result:
other: Corresponds to the highest dose tested. No adverse effects occured.
Dose descriptor:
NOAEL
Effect level:
1 462.9 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Corresponds to the highest dose tested. No adverse effects occured.
Remarks on result:
other: Corresponds to the highest dose tested. No adverse effects occured.
Dose descriptor:
NOAEL
Effect level:
1 552.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Corresponds to the highest dose tested. No adverse effects occured.
Remarks on result:
other: Corresponds to the highest dose tested. No adverse effects occured.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion