Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

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:

March 24, 2017 - January 25, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 May 2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

3 October 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

EC Number:

943-175-7

IUPAC Name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch no.: E6.2-F04
- Purity, including information on contaminants, isomers, etc.: Total Rhamnolipid: 90.8 [wt %]; Ashes (>800°C from d.w.) 6.2 [wt %]: Water (Karl Fischer) 1.65 [wt %]

Test animals

Species:

rat

Strain:

other: CD® rats

Details on species / strain selection:

The rat was selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: Males: 53 days; Females: 60 days
- Weight at study initiation: Males: 268.6 g - 295.3 g; Females: 215.8 g - 254.3 g
- Fasting period before study:
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm
- Diet (e.g. ad libitum): A certified commercial diet; ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum


DETAILS OF FOOD AND WATER QUALITY: Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Drinking water is examined according to the 'Deutsche Trinkwasserverordnungm2001' [German Regulations on Drinking Water 2001] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year. In addition, drinking water samples taken at the CRO are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range).
- Humidity (%): 55% ± 10% (maximum range).
- Photoperiod: 12 h dark /12 h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua dest. (sterile)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle (sterile aqua dest.) to the appropriate concentrations. The administration formulation were continuously agitated by stirring throughout the entire administration procedure. Foaming of the test item formulation was avoided during the whole test item preparation and administration period. Test item formulations with concentrations of 20, 60 and 200 mg/mL were prepared. The test item
formulations were freshly prepared every day. The high dose level of 1000 mg/kg b.w. to be administered required an administration volume of 5 mL/kg b.w. Higher concentrated suspensions were too viscous to be administered.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The quantification of the test item in water was determined with HPLC-UV method

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

administration volume 5 mL/kg bw/day

Doses / concentrations 2

Dose / conc.:

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

Remarks:

administration volume 5 mL/kg bw/day

Doses / concentrations 3

Dose / conc.:

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

Remarks:

administration volume 5 mL/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for this study were selected in agreement with the Sponsor based on available toxicological data generated during a preliminary dose-range finding study. In this dose-range-finding study, repeated oral administration of 100, 300 or 1000 mg/kg bw of the test item daily for 7 days did not cause any systemic intolerance reactions, any apparent influence on the body weight, food or the drinking water consumption. At necropsy on test day 8, neither test item-related macroscopic changes in the body weight at autopsy nor changes in the relative and absolute organ weights were observed. Based on the obtained data dose levels of 100, 300 and 1000 mg/kg bw were employed in this 28-day toxicity study.
- Dose range finding studies: In this dose-range-finding study, repeated oral administration of 100, 300 or1000 mg/kg b.w. of the test substance daily for 7 days did not cause any systemic intolerance reactions, any apparent influence on the body weight, food or the drinking water consumption. At necropsy on test day 8, neither test item-related macroscopic changes in the body weight at autopsy nor changes in the relative and absolute organ weights were observed.
Based on the obtained data dose levels of 100, 300 and 1000 mg/kg of the test substance were employed in this 28-day toxicity study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed individually before and after dosing at each time of
dosing for any signs of behavioural changes, reaction to treatment or illness. Daily through the working day from 07:30 a.m. to 04:30 p.m. On Saturdays and Sunday, the animals were checked regularly from 08:00 a.m to 12:00 a.m with a final check perfomrmed at approximately 04:00 p.m.
- Cage side observations were included: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset,intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-dose on test day 1 (to allow for within-subject comparisons), post-dose on test day 1, and once weekly thereafter for all animals. In test week 4 (end of treatment), the
observations were performed prior to the laboratory investigations. The observations were made in a standard arena outside the home cage, each time at the same time of day. The observations were made within 2 hours after dosing. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to
handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: at group allocation (test day -5), on test day 1 (before first administration), daily thereafter throughout the experimental period and once a week thereafter always on the same day of the week for the animals scheduled for the recovery period.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period (treatment and recovery). The food intake per animals (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The relative food consumption (in g/kg b.w./day) was determined using the following formula: Relative food consumption (g/kg b.w./day)= (Total food given (g) - Total food left (g))/(Number of animal days# × Body weight (kg)); # The term "animal days" counts on animals day for each animal alive foe whole day; it is assument that on the day of death an animals does not eat.


WATER CONSUMPTION : Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood samples were collected from all main study and recovery animals on test day 29 (end of the treatment period),and from all recovery animals on test day 57 (end of the recovery period).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- The following parameters were examined: Haemoglobin content (HGB); Erythrocytes (RBC);
Leucocytes (WBC); Reticulocytes (reti); Platelets (PLT); Haematocrit value (HCT) (=Packed cell Volume (PCV); Differential blood count (relative)#; Differential bloos count absolute)#; Mean corpuscular volume (MCV); Mean corpuscular Haemoglobin (MCH); Mean corpuscular concentration (MCHC). # Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and
monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as for Hemaetology
- Animals fasted: Yes
- How many animals: all
- The following parameters were examined: Albumin, Globulin, Albumin /globulin ratio, Bile acids, Bilirubin (total) Cholesterol (total); Creatinine; Glucose; Protein (total), Urea (in Blood); Calcium , Chloride; Potassium; Sodium; Alanine amino-transferase (ALAT); Alkaline phosphatase (aP); Aspartate aminotransferase (ASAT); Gamma-glutamyl-transferase (gamma-GT); lactate dehydrogenase (LDH).

PLASMA/SERUM HORMONES/LIPIDS: Yes / No / Not specified
- Time of blood sample collection:
- Animals fasted: Yes / No / Not specified
- How many animals:

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the treatment period (in test week 4, before blood sampling for laboratory examinations)
- Dose groups that were examined: all dose group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Adrenal gland (2); Brain, Epididymis (2); Heart, Kidney (2), Liver ; Pvary (2); Prostate and seminal vescicles (with coagulatings glands, as a Whole), Spleen, Testicle (2); Thymus, Uterus (incl. cervix)
HISTOPATHOLOGY: Yes; Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Caecum
Epididymis (2)
Eye with optic nerve and Harderian
gland (2)
Gross lesions observed
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum,
Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver (2 lobes)
Lungs (with mainstem bronchi and
bronchioles)
Lymph node (one cervical)
Lymph node (one mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary (2)
Pancreas
Pituitary
Prostate and seminal vesicles with
coagulating glands
Salivary glands (mandibular, parotid,
sublingual)
Skin (left flank)
Spinal cord (3 sections)
Spleen
Stomach
Testicle (2)
Thymus
Thyroid (2) (incl. parathyroids)
Tissue masses or tumours (including
regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
Uterus (incl. cervix and oviducts)
Vagina

Statistics:

Toxicology and pathology data were captured, as far as possible, using the departmental computerized systems ((Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). The test item-treated groups 2 to 4 were compared with the control group 1. The following statistical methods were used: 1. Multiple t.test based on Dunnett for Body weight, Food consumption, Numerical parameters of the neurological screening; Haematology; Coagulation; Clinical chemistry; Relative and absolute organ weights
(p ≤ 0.05 and p ≤ 0.01); 2) Exact test of R.A Fisher for Histology (p<= 0.05). The following settings were used for the statistical evaluation of the parametrical
values captured by Provantis (see flow chart of decision tree on the next page):
Homogeneity of variances and normality of distribution were tested using the
BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality
of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
These statistical procedures were used for all data. Significantly different data are
indicated in the tables.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment period: None of the male and female rats treated with 100, 300 and 1000 mg/kg bw/day of the test item by oral administration for 28 days revealed any test-item related changes in behaviour, external appearence, or consistency of faeces. However, all male and 9 of 10 female animals treated with 1000 mg/kg bw /day of the test item by oral administration for 28 days revealed an increase drinking water consumption (by visual appraisal) as of test day 17. This increased usage may have been caused by animals by water spoilage to neutralise a bitter or unpleasant taste at the high dose level, the test item beiing a surface-active agent. Futhermore, 1 to 10 male and 2 of 10 female animals treated with the high dose of 1000 mg/kg bw/day revealed breathing sounds and piloerection as follows: the male animal no. 42 revealed breathing sounds and piloerection on test days 22 to 28; the females animal no. 51 revealed breathing sounds on test day 12 only; and the female animal no. 55 revealed breathing sounds on test days 16 to 28 and pilo-erection on test days 17 to 28.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment period: No test-item mortality was observed for male amd female animals treated with 100, 300 or 1000 mg/kg bw day by oral administration for 28 days. One of the 10 high dose female aniamals treated with 1000 mg/kg bw /day (animal no. 54) died after blood withdrawal for laboratory examinations of day 29 and was dissected immediately as scheduled. The death of this is due to the stress of the food withdrawal and anaesthesia and is, hence, not related to the administration of the test item. Recovery period (restricted to groups 1 and 4: None of the previously hogh-dosed anmals died or had tp be sacrficed prematurely during the recovery period.
Detailed clinical observations: Detailed clinical observations in formof an assessment of external appearance body posture, Movement and coordination capabilities, and behavior were performed for all animals pre-and post-dose on test day 1, and once weekly thereeafter for the animals scheduled onyl for the treatment period (test weeks 1 to 4). The observations were made within 2 hours after dosing. All parameters of the detailed clinical observation for all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on the day 1. All male and female control animals revealed normal values for each parameters set examined throughhout the course of the study. None of the animals treated with 100, 300 or 1000 test item kg bw /day revealed any test item-related changes in external appearence, body posture, movement and coordination capabilities and behaviour in test weeks 1 to 4 (for details see Attachments under Chapter "Overall remarks")

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4): No test-item related influence was observed on the mean body weight and the mean body weight gain of the male and female animals treated with 100, 300 or 1000 mg/kg bw/day for 28 days compared to the control animals, neither recovery period. No test item-realted differences were noted for the body weight at autopsy between the test item-.treated animals and the control animals, neither during the 4.week treatment period nor during the 28-day treatment-free recovery period. No test item-realted differences were noted for the body wight at autopsay between the test item-related animals and control animals. However, 3/20 animals of the high dose group treated with 1000/kg bw /day revealed a reduction in body weight as follows: the female animal no. 55 revelaed a considerable body weight loss up to 32.7% during the course fo the study cause by a very low food intake. Body weight at autopsy was 142.2 g compared to a group mean values of 212.3 g. The male animal no.42 revealed a low body wieght gain during the course of the study e.g, +3.9% between tes days 1 and 8 and 1% between test days 22 and 28. The male animal no. 46 revealed a low body weight gain of 0.2% between wight gain of + 0.2% +0.2% between test days 1 and 8 only, afterwards the body weight gain was within the normal range. These findings are considered possibly due to regurgitation of this surface-active test item at a fairly high required administration volume of 5 mL/kg b.w. and possibly entering the lungs. For details see (for details see Attachments under Chapter "Overall remarks").

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period and revocery period (restricted to groups 1 and 4): No test item-related influence was observed on food consumption of the male and female animals treated with 100, 300 or 1000 mg/kg bw/ day for 28 days compared to the control animals, neither during the 4-week treatment period nor during the 28-day treatment-free recovery period. However, the female animal no.55 treated with the high dose of 100 mg/kg bw/day revealed a very low food intake during the course of the study, e- g 46- 8 g/kg bw7day. this finding is considered possibly due to regurgitation of this surface-active test item at a fairly high required administration volume of 5 mL/ kg bw and possibly entering the lungs. The visual appraisal of the drinking water consumption revealed no test item-related differences between the control and the test item-treated animals.The slight but statistically significant (at p<= 0.01) increase noted for the food consumption of the high dosed male animals in the test weekes 3 and 4 may have been caused by the animals by food spoilage to neutralise a bitter or unpleasant taste at the high dose level. The statistical significant differences in relative food consumption comparted to the control animals are not considered to be test item-related but to be coincidental (for details see Attachments under Chapter "Overall remarks")

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4): No test item-related influence was observed on the haematological parameters for the male and female animals treated with 100, 300 or 1000 mg/kg b.w./day by oral administration for 28 days compared to the control animals at the end of the treatment period (test day 29) and at the end of the recovery period
(test day 57). However, the female animal no. 55 treated with the high dose of 1000 mg/kg b.w./day revealed slight changes of a few haematological parameters compared to the group mean value of the control group: increased number of erythrocytes (+37%) and neutrophilic granulocytes (+353%), increased haemoglobin content (+31%), haematocrit value (+29%) and
thromboplastin time (+72%), decreased numbers of lymphocytes (-67%) and decrease in the percentage of reticulocytes (-50%). Although the stomach tube was left in position for several seconds after administration, these findings are considered possibly due to regurgitation of this surface-active test item at a fairly high required administration volume of 5 mL/kg b.w. and possibly entering the lungs. No test item-related effects were observed for the haemoglobin content (HGB), the numbers of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the relative reticulocyte count (Reti), the haematocrit value (HCT), the relative and absolute count of neutrophilic granulocytes (Neut), lymphocytes (Lym), monocytes (Mono), eosinophilic granulocytes (Eos), large unstained cells (LUC) and basophilic granulocytes (Baso), the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) on test day 29 and on test day 57 (for details see Attachments under Chapter "Overall remarks").

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4): No test item-related influence was observed on the clinical chemistry parameters for the male and female animals treated with 100, 300 or 1000 mg/kg b.w./day by oral administration for 28 days compared to the control animals at the end of the treatment period (test day 29) and at the end of the recovery period (test day 57). However, the female animal no. 55 treated with the high dose of 1000 kg bw/day revealed slight to marked changes of several clinical chemistry parameters compared to the group mean value of the control groups: increased plasma levels of total bilirubin (+144%), urea (+471%), potassium (+24%), decreased plasma levels of albumin (-31%), globulin (-37%), cholesterol
(-85%), creatinine (-26%), glucose (-83%), total protein (-34%), calcium (-17%), chloride (-10%) and sodium (-5%), as well as increases in the plasma activities of ALAT (+453%), ASAT (+236%) and LDH (+309%). Although the stomach tube was left in position for several seconds after administration, these findings are considered possibly due to regurgitation of this surface-active test item at a fairly high required administration volume of 5 mL/kg b.w. and possibly entering the
lungs. No test item-related effects were noted on the plasma levels of albumin and
globulin and on the albumin/globulin ratio, on the plasma levels of bile acids, total
bilirubin, total cholesterol, creatinine, glucose, total protein, urea (in blood), calcium, chloride, potassium, and sodium on test day 29 and on test day 57. No test item-related influence was noted on the plasma enzyme activities of alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), and gamma glutamyl-transferase (Gamma- GT). All data are within the limits of normal biological variability. Statistically significant differences in clinical chemistry parameters compared to the control animals noted on test day 29 or on test day 57 that are not considered to be test item-related but to be coincidental are listed in the text table on the following page (for details see Attachments under Chapter "Overall remarks").

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment and recovery period (recovery restricted to groups 1 and 4): No test item related changes were noted for the relative and absolute organ weights of the animals treated with 100, 300 or 1000 mg/kg bw/day of the animals by repeated oral administration at the terminal sacrifice on test day 29 or at recovery sacrifice on the test day 57. However, the female animal no. 55 treated with the high dose of 1000 mg/kg bw/day revealed a 3 fold decrease absolute liver weight, a 4-fold decreased absoluted spleen wiegh, and an 11-fold decreased absolute thymus weight compared to the groups mean value of the control group. Although the stomach tube was left in position for several seconds after administration, these findings are considered possibly due to regurgitation of this surface-active test item at a failry high required adminstration volume of 5 mL/kg bw and possibly entering the lungs leading to a poor food intake and considerable body weight loss. Statistically significant differences in organ weights compared to the econtrol animals in the test weeks 4 or 8 that are not considered to be test item-realted are listed under under Chapter "Overall remarks".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period: The macroscopic inspection at necropsy did not reveal any test item-related
changes in the organs and tissues of the animals treated with 100, 300 or 1000 mg/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 29). The following incidental not test item-related findings were noted: One male animal treated with 1000 mg/kg b.w./day revealed a thickened mucosa of both stomach and caecum and in one other high dosed male animal both caecum and small intestines were inflated. The female animals appeared to reveal enlarged adrenals in 1 of 5 intermediate dosed animals treated with 300 mg/kg b.w./day and in 4 of 5 high dosed animals treated with 1000 mg/kg b.w./day, however, no increases in relative or absolute organ weights or any histopathological correlate were noted. One group 4 female (animal no. 55), which was emaciated at necropsy, revealed additional findings in form of dark-red discoloured adrenals, a liver reduced in size, atrophy of the musculature, a spleen reduced in size, a thymus reduced in size and a reddened stomach mucosa. These findings could be stress-related due to regurgitation of the surface-active test item. Another high dosed female animal (no. 53) revealed, besides enlarged adrenals, an enlarged spleen and a thickened stomach mucosa which were related to the administration of the test item. However, again no increases in relative or absolute organ weights or any histopathological correlate were noted. All findings are indicative of regurgitation of this surface-active test item at a fairly
high required administration volume of 5 mL/kg b.w. and possibly entering the lungs. The dilated uterus filled with clear liquid noted for 1 of 5 high dosed animals is considered to be an incidental finding.
Recovery period (restricted to groups 1 and 4): No pathologocal changes observed in the organs and tissues of the male and female rats previously treated with 1000 mg/kg bw/day for 28 days at the end of the 28-days treatment-free recovery period (test day 57). Individual finding are listed under Chapter "Overall remarks".

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The neurological screening was performed on all main study and recovery animals (group 1: n=10 per sex, groups 2 and 3: n=5 per group and sex, group 4: n= 10 per sex) at the end of treatment (in test week 4) 1 to 2 hours after dosing, and on all recovery animals (groups 1 and 4: n=5 per group and sex) at the end of the recovery period (in test week 8).
Treatment period and recovery period (restricted to groups 1 and 4): no test item-related influence was noted of any of the parameters examined during the functional observation tests, on the fore-and hind-limb grip strengh, or the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300 or 1000 mg test item/kg bw /day in test week 4 or in test week 8. A statistically significant (at p <= 0.05) increase noted the body temperature of the previously high dose male animals and for the hind leg splay of the previously high dosed female animals in comparison to the control animals at the end of the recovery period in test week 8 are considered to be coincidental effects and not to be related to the test item. This finding could be stress-related due to regurgitation of the surface-active test item (for details see Attachments under Chapter "Overall remarks")

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathological examination of variety of organs and tissues was restricted to the main study animals of control group 1 and the hih dose group 4 treated with 1000 mg//kg bw/day. The organs of males and females observed at microscopy did not reveal anychanges that could be related to the administration of the test item when dosed upto 1000 mg/kg b.w./day. Histopathology revealed only some findings in 3 of 5 female main study animals (nos. 53, 54 and 55) treated with 1000 mg/kg b.w./day, some of which were related to macroscopic findings. Although the stomach tube was left in position for several seconds after administration, these findings are considered possibly due to regurgitation of this surface-active test item at a fairly high required administration volume of 5 mL/kg b.w. and possibly entering the lungs. In female animal no. 55, which was emaciated at autopsy, microscopic observations were observed for: adrenal (congestion), liver (moderate atrophy), muscle (moderate atrophy, slight myofiber degeneration/necrosis and acute inflammatory cell infiltrate), spleen (white pulp atrophy), thymus (severe atrophy) and stomach (moderate acute inflammation, ulceration). Female animal no. 54, that died after blood withdrawal for laboratory examinations on test day 29 and was dissected immediately as scheduled, showed slight acute inflammation in the glandular submucosa of the stomach. Female animal no 53 had increased alveolar macrophage aggregates in the lung associated with multifocal acute alveolar/interstitial inflammation and localised oedema and haemorrhages and congestion of the spleen. Hence, the changes are indicative of a secondary artefact caused by the surfaceactive test item entering the lungs by regurgitation. For the detailed results of microcopic examination see Attachments under Chapter "Overall remarks".

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

TEST ITEM FORMULATION ANALYSIS

The results of the analysis of the test material concentrations in the administration formulations revealed that the test item formulations were correctly prepared by the test labor, were very homogeneous, and were stable for at least 24 hours. The actual concentrations of the test item determined in the test item formulations on test day 1 and test day 28 for groups 2 to 4 ranged from 96.2% to 101.3%. These values are considered to cover the admissible limits of 90% to 110%. 

Table 1. Test item formulation analysis

Parameter	Sampling / Handling	Range of actual test item concentration in percent of nominal concentration
Concentration	Immediately after preparation and at study termination	96.2% - 101.3%
Stability	Left at room temperature for 8h or 24h	97.2% - 101.1%
Homogeneity	At start of administration, during administration; and before administration to the last animal	96.2% - 101.1%

Applicant's summary and conclusion

Conclusions:

The test substance was administered daily as suspension in water via gavage. The aim of the present study was to obtain information on the toxicity of the test material administered daily by oral administration to rats for 28 consecutive days and to assess the reversibility of any effects at the end of a 28-day recovery period. The rats were treated with 100, 300 or 1000 mg/kg bw/day. The control animals received the vehicle (sterile aqua dest.). No test item-related deaths occured. No test item-related changes were observed for the behavior or external appearance of the animals, the detailed clinical observations, the neurological screening, the body weight, body weight gain and body weight at autopsy, the food and drinking water consumption, for any of the haematological and clinical chemical parameters, the relative and absolute organ weights, and at macroscopic inspection at necropsy at any dose level. The histopathological examination did not reveal any test item-related morphological changes. The histopathological examination did not reveal any test item-related morphological changes. No test item-related changes were noted during or at the end of the 28-day treatment-free recovery period. The experimental no-observed-effect level (NOEL) was above 1000 mg/kg bw by daily oral administration.