[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July -Sept 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 27, 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 6 weeks
- Housing: in transparant macrolon cages (tyoe IV) on soft wood granulate in an air-conditioned room, 5 animals per cages
- Diet (e.g. ad libitum): ssniff R/M-H (V1534), ad libitum, exept for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water, ad libitum, exept for the period in which the animals were kept in diuresis cages
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 22.7. To: 2.9.2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: tylose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item wassuspended in tylose in 14 days intervals. After each measurement of the body weight, the application volume was re-calculated and adapted.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): homogenity of suspension and stability of test item in vehicle
- Concentration in vehicle: 0, 12.5, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Acute oral toxicity testing of the test substance at a dose of 2000 mg/kg bw in the rat showed that the LD50 is greater than 2000 mg/kg bw in female animals. The dose of 2000 mg/kg bw was tolerated by all animals without signs of intoxication. Based on these results, the dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d were selected for the present study.
- Post-exposure recovery period in satellite groups: yes, 5 animals/sex control and high dose group

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, once daily

BODY WEIGHT: Yes, before study start and twice weekly

FOOD CONSUMPTION: contiuously per cage (two times per week)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination and after the recovery period
- Anaesthetic used for blood collection: Yes, Ketamine-hydrochloride + Xylazine
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked:
erythrocyte count, Heinz' body counts, hematocrit, hmoglobin, maen corpuscular hemoglobin, maen corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte counts, differential leukocyte counts, leukocyte counts, coagulation time, thrombocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination and after the recovery period
- Animals fasted: Not specified
- How many animals:
all animals
- Parameters checked:
gamma-glutamyltranspeptidase, alanine aminotransferase, albumin, albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, bilirubin total, calcium, chloride, cholesterol, creatinine, globuline, glucose, inorganic phosporous, potassium, sodium, total protein, triglycerides, urea, uric acid

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
Once before the first treatment and weekly thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Each animal was assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination were evaluated.
At the termination of the study sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) was evaluated including startle reflex (click response), response to approach with the finger to the nose of the animal, and righting reflex. The presence and absence of pupillary constriction was assessed using a pen flashlight directed into the eye. Assessments of motor function were performed including measurement of motor activity as well as forelimb and hindlimb grip strength. The animals were evaluated for motor activity during a 60-minute period in an 16-station automated motor activity monitoring device. Activity counts were recorded by the interruption of photocells in 3-minute-intervals to give a total of 20 intervals. Fore- and hindlimb grip strength were measured by a strain gauge device

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS:
the following organs were weighed and the organ body weight ratios calculated:
adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus

HISTOPATHOLOGY: Yes, of contol and high dose animals
adrenals, bone marrow, brain with medulla oblongata, epididymides, heart, small intestine, large intestine, kidneys, liver, lungs, lymph nodes (mandibular, iliac), nerve (sciatic nerve), ovaries, prostate, seminal vesicle, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid gland with parathyroids, trachea, unrinary bladder, uterus, all gross lesions.
Additionally, esophagus was investigated histologically in order to check if the defense reaction observed during intubation was caused by local effects.

Statistics:

Dosing and Recovery Phase data of body weight (from Day 1 of dosing, twice weekly) will be analyzed for statistical significance (p ≤ 0.05) within each sex by a 1-way ANOVA with a two sided ordinal step-down trend test (see Tukey et al., 1985). Hematological parameters, serum chemistry parameters, urine volume and organ weights are analyzed either as described above, if normally distributed, or by the Jonckheere trend test with corrections for ties (Jonckheere AR, 1954; Lin, F.O. and Haseman, J.K., 1975), if not normally distributed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals treated with the test item showed temporarily a defense reaction during gavage, starting in the first study week and lasting up to day 12 in individual animals. Only one female animal of the high dose group showed a defense reaction in the whole treatment period during intubation. The extent and duration of the defense reaction was correlated with the dose administered.
The animals of the mid and high dose group showed by compound discolored feces from day 9 (mid dose) or 4 (high dose) up to day 29 or 30 of the study.
Symptoms like stilted gait, squatting posture, hypoactivity, respiratory sounds or diarrhea were observed in individual animals of the high dose group (2 males, 6 females). Symptoms started in male animals at the end of the first study week and lasted up to day 14 or 21. In females, such symptoms were observed from day 10 onwards but disappeared in most animals up to day 14.
Only two females showed stilted gait and respiratory sounds up to day 30.
No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female animals (No. 56 and No. 60) of the high dose group died on day 8 and 9. Additionally, two male animals of the high dose group were found dead on day 8 and 10 (No. 22 and 28). Necropsy showed dark red fluid in the thoracic cavity of animal 22, 28 and 60. Histopathological investigation revealed that animals found dead exhibited red-brown material on the epicardial layer of the heart (No. 22 and 28), peripulmonal on the serosa of the lungs (No. 28), or within the blood vessels of nearly all organs (No. 56) or only the brain (No. 60). The reason of death was obviously a traumatic injury or even a perforation of the esophagus during treatment caused by heavy defense reactions during intubation.

Body weight and weight changes:

no effects observed

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

Description (incidence and severity):

No opacity of the refracting media of the eyes

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant alterations in blood serum parameter were only observed at the end of the treatment period, but not after the recovery period.
On day 29 male animals showed slightly increased sodium values (mid and high dose group), while female animals showed increases in total bilirubin, AST activity, total cholesterin, triglycerides as well as decreased calcium and chloride levels, mostly in the high dose group.
It should be noted that the high mean value in AST activity of males in dose group 4 was caused by an extraordinary high values in one animal (No. 25).
All alterated values were still within the normal range. Therefore none of the alterations was considered to represent a severe toxic effect. Remarkably, total bilirubin and triglyceride values in females of the high dose group were distinctly increased compared with the control, (while other alterations were slight to moderate) to an extent that a substance related effect with this dose can not be excluded.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant alterations in organ weigths were detected after repeated administration of the test substance in all dose groups.
Statistically significant alterations in organ weights were only detected at the end of the treatment period (final value day 29), but not at the end of the recovery period. These alterations included increased absolute testes weight, increased relative kidneys weight and decreased relative thymus weight in male animals, as well as decreased absolute weight of adrenal glands in female animals of the high dose group.
As alterations were slight, all values are within the historical control data range, no clear dosedependency occurred and no histopathological correlates were detected, these alterations are not considered as relevant test substance related findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic changes could not be seen in male or female animals of the low and mid dose group. Two male and one female animal which died early showed dark red fluid in the thoracic cavity probably caused by a perforation of the esophagus during treatment as a consequence of the severe defense reactions of the animals. One of these males showed additionally small testes, large stomach and a small right kidney, which were not considered as substance related effects as no comparable effects were noted in other animals. The fourth animal which died early showed no macroscopic findings.

Two further males of the high dose group showed an elevation in the right hepatic lobe, one of them showed additionally spleen constriction. These findings are considered to be incidental without substance relation. The other animals showed no macroscopic findings except of two animals of the recovery group which showed also findings (No. 38: elevation in the right liver lobe; No. 39: deformed spleen).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological investigation of the animals which died early revealed red-brown material on the epicardial layer of the heart (No. 22 and 28), peripulmonal on the serosa of the lungs (No. 28), or within the blood vessels of nearly all organs (No. 56) or only the brain (No. 60). These results suggested that death was obviously caused by a traumatic injury or even a perforation of the esophagus during treatment as consequence of the heavy defense reactions during intubation.
No other alterations in these and all other animals of the study were noted which could be related to administration of the test compound.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, repeated administration of the test substance at dose levels of 62.5 and 250 mg/kg body weight and day did not cause any biologically relevant substance related alterations.
Repeated administration of 1000 mg/kg body weight HPP Additiv D11 caused reversible signs of toxicity in individual animals such as impairments of motility and respiration. Additionally, slight but not yet toxicologically relevant alterations in clinical chemistry (increase in triglyceride and total bilirubin levels in females) were observed.
With regard to the present study the 'No Observed Adverse Effect Level' (NOAEL) is 250 mg/kg body weight per day.

Executive summary:

Groups of male and female rats received the test item by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for a period of 28 days. On day 29 five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly. Once before the first treatment and weekly thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes and damage to the oral mucosa. Neurotoxicological measurements including assessment of sensory function, motor activity,

forelimb and hindlimb grip strength were conducted at the end of the treatment period. Hematological examinations and clinical chemistry were carried out at the end of the treatment period and after the recovery period. Urinalysis was performed at the end of the treatment period only.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes. Body weights, hematological and clinical chemistry data, urine volume, absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analyzed with the aid of a statistical program to show differences compared to the controls.

Body weight gain and food consumption, hematological, neurotoxicological and urine values as well as organ weights were not influenced by the administration of the test compound in all dose groups.

Temporarily, a dose-dependent defense reaction during gavage was observed with all animals treated with the test compound. As the defense reaction was observed only in correlation with intubation but not during animal maintenance, and as no histopathological or clinical correlates were observed it is not considered as substance related effect affecting health, but as temporary reaction on the repeated intubation procedure.

Two female animals (No. 56 and No. 60) of the high dose group died on day 8 or 9. Additionally, two male animals of the high dose group were found dead on day 8 and 10 (No. 22 and 28).

Necropsy showed red fluid in the thoracic cavity of animal 22, 28 and 60. Histopathological investigation revealed that animals found dead exhibited red-brown material on the epicardial layer of the heart (No. 22 and 28), peripulmonal on the serosa of the lungs (No. 28), or within the blood vessels of nearly all organs (No. 56) or only the brain (No. 60). The reason of death was obviously a traumatic injury or even a perforation of the esophagus during treatment caused by heavy defense reactions during intubation.

The animals of the mid and high dose group showed feces discolored by the compound from day 4 or 9, respectively, up to day 29 or 30 of the study. Sporadically, stilted gait, squatting posture, hypoactivity, respiratory sounds or diarrhea were observed in individual animals of the high dose group (2 males, 6 females). Effects were reversible in most animals during the treatment period. Only two females showed symptoms up to the end of the treatment period. Clinical chemistry revealed some alterations at the end of the treatment period (final value). However all alterated values were still within the normal range. Therefore none of the alterations was considered to be toxicologically relevant. Total bilirubin and triglyceride values in females of

the high dose group were distinctly increased compared with the control group to an extent that a substance related effect with this dose can not be excluded, even if no morphological correlate was observed. No alterations in clinical chemistry data were observed in the recovery groups. Macroscopic changes could not be seen in male or female animals of the low and mid dose group. Two male and one female animal of the high dose group which died early showed dark red fluid in the thoracic cavity. No other relevant macroscopic findings were observed in this dose group.

Beside findings in animals died early as described above, no test substance related findings were observed in the histopathological investigation.

With regard to the present study the 'No Observed Adverse Effect Level' (NOAEL) is 250 mg/kg body weight per day.