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Description of key information

In a 28-day study with rats according to OECD TG 407 (RL1) a NOAEL of >= 200 mg active ingredient/kg bw (the highest concentration tested) was derived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 SEP 2002 to 18 DEC 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
27.07.1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30.07.1996
Qualifier:
according to
Guideline:
other: U.S. EPA: OPPTS 870.3050, Repeated dose 28 days toxicity study in rordents
Version / remarks:
July 2000
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd:Sprague Dawley (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, D-33178 Borchen, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males 100.8 - 108.5 g, females 98.5 - 102.4 g
- Housing: In transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned rooms, 5 animals per cage, separated according to sex
- Diet: ssniff® RIM-H (V 1534) ad libitum, exept for the period in which the animals were kept in diuresis cage
- Water: Tap water in plastic bottles ad libitum, exept fot the period in which the animals were kept in diuresis cages
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50 ± 20 % (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route is considered to be a potential exposure route in man.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
test substance was dissolved in the stated concentrations in deionized water daily

VEHICLE
- Concentration in vehicle: 0.0, 3.0, 11.9, 47.6 mg/mL depending on the dose group
- applied volume: 10 mL /animal / day
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
5 male and female animals from the control and the high dose group respectively were observed for a recovery time of 28 days after the end of exposure.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
corresponds to 0 mg active ingredient/kg bw/day
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
corresponds to 12.5 mg active ingredient/kg bw/day
Dose / conc.:
119 mg/kg bw/day (nominal)
Remarks:
corresponds to 50 mg active ingredient/kg bw/day
Dose / conc.:
476 mg/kg bw/day (nominal)
Remarks:
corresponds to 200 mg active ingredient/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Acute oral toxicity testing og the test substance showed that the median lethal dose (LD50) lies between 714 and 4762 mg/kg body weight in female animals corresponding to a LD50 value between 300and 2000 mg/kg body weight of the active ingredient (Study report NO.: PT02-0155).
The cut-off LD50 value for the active ingredient is 500 mg/kg according to OECD guidline 423.
In a dose range finding study, 3 male and 3 female rats received the test substance at doses of 357, 714 and 1428 mg/kg body weight, corresponding to 150, 300 and 600 mg/kg body weight active ingredient per day, over a period of 14 days. Animals were necropsied on day 15.
After administration of 1428 mg/kg body weight test substance (600 mg/kg active ingredient) the animals showed beside unspecific symptons impairments of motility and respiration. Lethality occurred on day 14 of the study in one female animal. Necropsy of the decedent animals in this dose group revealed that the stomach and intestine mucous membran was detached. Additionally small and large intestine was filled with gas or light yellow mucous. The animals of this group showed disturbance of body weight gain during the study. After administration of 357 mg/kg body weight (150 mg/kg active ingredient) and 714 mg/kg body weight (300 mg/kg active ingredient) neither deaths nor symptoms occurred. Development of body weight was not impaired. The animals killed at the end of the observation period showed no macroscopically visible changes.
Based on these results, dose levels of 0, 30, 119 and 476 mg/kg body weight test substance, (corresponding to 0, 12.5, 50 and 200 mg/kg body weight active ingredient per day), were selected for the present study.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: twice/week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: twice/week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 67 + 6.7 mg/kg body weight Ketamine-Hydrochloride + Xylazine)
- Animals fasted: Not specified
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after blood sampling
- Animals fasted: Not specified
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: a few days before termination of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at termination of the study
- Dose groups that were examined: all
- Battery of functions tested: auditory, visual, proprioceptive

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
All Parameters: The assumption of a monotonic dose-response relationship for all parameters justifies the restriction of the significance level to 5 % (per parameter and sex), using the method of: HOTHORN L, LEHMACHER W.: A Simple Testing Procedure "Control versus k Treatments" for One-sided Ordered Alternatives, with Application in Toxicology, Biom. J. 33, 179-189, Akademie Verlag

Bodyweights: The changes of parameter values compared to the treatment-free baseline values are analyzed. The baseline values themselves are not statistically evaluated, since by definition no treatment effect can be present at this stage. Method used: t-Test:
HARTUNG J., ELPERT B., KLÖSENER K. H., Lehr- und Handbuch der angewandten Statistik (1989), R. Oldenbourg Verlag, München

Clinical Pathology Data: In rat studies, the absolute values are analyzed.´ Wilcoxon's Test: HOLLANDER M., WOLFE, D. A:, Nonparametric statistical methods; Wiley Series in Probability and Mathematical Statistics (1973), John Wiley & Sons Inc., New York, with the
exact distribution after STEITBERG B, RÖHMEL J.: Exakte Verteilung für Rang- und Randomisierungstests im allgemeinen Stichprobenproblem, EDV in Medizin und Biologie 18, 12-19 (1987), Verlag Eugen Ulmer GmbH & Co., Stuttgart; Gustav Fisher Verlag KG, Stuttgart

Organ weights (absolute): t-Test (HARTUNG J., ELPERT B., KLÖSENER K.H.,Lehr- und Handbuch der angewandten Statistik (1989), R. Oldenbourg Verlag, München

Organ weights (relative to bodyweight): Wilcoxon's Test HOLLANDER M., WOLFE, D.A:, Nonparametric statistical methods; Wiley Series in Probabiltiy and Mathematical Statistics (1973), John Wiley & Sons Inc., New York, with the exact distribution after STEITBERG B, RÖHMEL J.,Exakte Verteilung für Rang- und Randomisierungstests im allgemeinen Stichprobenproblem, EDV in Medizin und Biologie 18, 12-19 (1987), Verlag Eugen Ulmer gmbH & Co., Stuttgart; Gustav Fisher Verlag KG, Stuttgart
Clinical signs:
no effects observed
Description (incidence and severity):
Behavior and state of health remained uneffected by the administration of the test compound in all dose groups. Except of one female animal of the median dose group, which showed respiratory sounds from day 18 up to day 27. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gains were not influenced by the administration of the test compound in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption remained unaffected by the administration of the test compound throughout the study in all dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
All hematological parameters remained unaffected by the administration of the test compound in all groups throughout the study, i.e., after both the treatment and after the recovery period. The statistically significantly increased hemoblobin and hematocrit values for high dose recovery females were well within the physiological range of the inhouse historical control data for this rat strain and age. Hence, they were considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum clinical chemistry examination did not reveal any compound-related alterations after the treatment and after the recovery period. All statistically significant changes of serum parameters when compared to the control were generally within the physiological range of the inhouse historical control data for this rat strain and age. Moreover they were generally confined to one gender. The phosphorus value for recovery high dose females was outside the historical range but there was no corresponding change at final examination. Hence, this finding was incidental.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis including sediment was unobstrusive in all groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurotoxicological measurements including 'open field' observations, assessment, and forelimb and hindlimb grip strength was not influenced by the administration of the test compound in all groups. The statistically significantly increased number of movements for low dose males against the control was within the physiological range for this rat strain and age. There was no dose-response relationship. Therefore, this finding was incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The terminal body weight was comparable at final and recovery in all groups. Mean absolute organ weights were not influenced by the administration of the test compound in all groups.
Mean organ weights relative to terminal body weights were not affected by the administration of the test compound in any group.
Slightly lower relative kidney and adrenal weights for high dose recovery males were considered to be incidental as they were not seen after 28 days of treatment. Moreover, there was no clinical pathology correlate either at final or recovery examination with regard to target organ toxicity.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Relevant macroscopic changes could not be seen in male or female animals in all investigated groups. The gross pathology findings were as follows:
Small uterus and ovaries in a group 3 female, and one beige formation in the right epididymis, both at the terminal necropsy. Small seminal vesicles, and a unilateral depression in the kidney at the recovery.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological findings in any organs examined (including 4 sections of nasal cavity) that could be related to the administration of the test compound in any group. Incidental and/or spontaneous findings were as follows:
One female exhibited a juvenile uterus. The alteration in the epididymis of a male was characterized as a spermatocele.
Further sporadic findings in form of inflammatory foci/ cellular infiltrations were observed in heart, liver, trachea and lungs, prostate and uterus of single animals randomly distributed over all investigated groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects in highest dose tested
Key result
Dose descriptor:
NOEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects in highest dose tested
Critical effects observed:
no

Results: Changes in hematology (recovery value day 57)

 

Group, Males

Group, Females

Parameter

1

4

1

4

Hemoglobin

150

154

136

142+

Hematocrit

0.44

0.46

040

0.43+

 Statisticaily significant p<0.05

Results: Changes in clinical chemistry (final value day 29)

 

GroupMaleses

 

Group, Fernale

Parameter

1

2

3

4

1

2            3           4

Cloride

100

102+

103+

103+

103

104        104   

104

Triglycerides

0.88

0.69

0.57

0.77

0.69

0.56-     0.53-

0.51-

+/- Statistically significant p<0.05

Results: Changes in clinical chemistry (recovery value day 57)

 

    Group Males

    Group Females

 Parameter  1  4  1  4
 Sodium 142  142 141 143+ 

 Potassium 

 6.74  5.59  5.83 5.01-
  Phosphorus  2.70  2.35  2.59 1.92- 
  Urea Nitrogen   6.24  7.18+  7.16  7.26
  Albumin  26.8  27.4  26.6

 29.2+

Albumin/Globulin Ratio

 1.03  0.92  0.93  1.03+
 Globulin  26  30+  29  28

+/- Statistically significant p<0.05

Results: Changes in relative organ weights (recovery value day 57)

 

Group Males

Group Females

Parameter

1

4

1

4

Kidneys weight

7.031

6.415-

6.211

6.271

Adrenals weight

0.1413

0.1222-

0.2606

0.2512

+/- Statistically significant p<0.05

Conclusions:
In this 28-day study according to OECD guideline 407 performed under GLP, male and female Sprague-Dawley rats were exposed to 0, 30, 119 or 476 mg test item/kg bw/d in water via gavage. This corresponds to 0, 12.5, 50 and 200 mg active ingredient/kg bw/d. Cage side observations, clinical observations, food consumption, body weight development, haematology, urin analysis, macroscopic investigations, histopathology of selected organs and assays concerning the functional observational battery did not reveal any substance related toxic effects.The NOAEL in this study is >= 200 mg active ingredient/kg bw/d.
Executive summary:

The test item did not reveal substance related adverse effects under the conditions tested (according to OECD TG 407, performed under GLP).

In this 28-day study male and female Sprague-Dawley rats were exposed to 0, 30, 119 or 476 mg test item/kg bw/d in water via gavage. These doses correspond to 0, 12.5, 50 and 200 mg active ingredient/kg bw/d. Cage side observations, clinical observations, food consumption, body weight development, haematology, urin analysis, macroscopic investigations, histopathology of selected organs and assays concerning the functional observational battery did not reveal any substance related toxic effects.The NOAEL in this study is >= 200 mg active ingredient/kg bw/d, the highest dose tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable study, GLP-compliant and according to OECD guideline 407

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item did not reveal substance related adverse effects under the conditions tested (according to OECD TG 407, performed under GLP).

In this 28-day study male and female Sprague-Dawley rats were exposed to 0, 30, 119 or 476 mg test item/kg bw/d in water via gavage. This doses corresponds to 0, 12.5, 50 and 200 mg active ingredient/kg bw/d. Cage side observations, clinical observations, food consumption, body weight development, haematology, urine analysis, macroscopic investigations, histopathology of selected organs and assays concerning the functional observational battery did not reveal any substance related toxic effects. The NOAEL in this study is >= 200 mg active ingredient/kg bw/d, the highest dose tested.

The NOAEL from the supporting study (>= 120 mg active ingredient/kg bw/d) does not contradict the key value derived, since this NOAEL represents the highest dose tested in the supporting study.

Justification for classification or non-classification

With a NOAEL of >= 200 mg active ingredient/kg bw (the highest concentration tested) in a subacute oral toxicity study, no classification according to Regulation (EC) No. 1272/2008 (CLP) is required.