Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Description of key information

The target substance, Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria was tested for subacute toxicity (OECD TG 407) using male and female rats. 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 occurred. 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. 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.

 

The subchronic toxicity of the source substance jelly mushroom glycolipids from Dacryopinax spathularia (herein referred to as “AM-1”) was studied in Crl:CD(SD) rats. The test item was administered via the drinking water at concentrations of 1.5, 5.0 or 15 mg/mL for 90 days with an additional 4-week recovery period. No test articler elated deaths, clinical observations or neurological effects were noted. Decreased drinking water consumption for mid- and high-dose groups was attributable to the reduced palatability of drinking water containing higher test article concentrations. Mean body weights of high-dose males were slightly reduced beginning study week 1 due to decreased food and drinking water intake, but were not statistically significant by week 7. No test article-related adverse effects were noted for hematological or clinical chemistry, or urinalysis parameters. Statistically significant changes in select parameters were within historical control data ranges, lacked histopathological correlates, and did not occur in a consistent pattern that would suggest biological significance. Microscopic examination did not reveal any test article-related morphological changes. The no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/mL (1201 and 1423 mg AM-1/kg bw/day for male and female rats, respectively). In addition, the subchronic toxicity of AM-1 was studied in male and female Beagle dogs administered AM-1 by oral capsule at doses of 150, 500 or 1000 mg/kg/day for 90 days. AM-1 was well tolerated at all dosages and there were no test article-related effects on survival, clinical observations, neurological screening (functional observational battery) parameters, clinical pathology parameters, organ weights, macroscopic or microscopic evaluations. Test article-related changes were limited to minimal effects on food consumption and body weight changes in the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day, the highest dosage level tested. These results add to the safety database for these naturally derived jelly mushroom glycolipids with potential for use as a food ingredient.These results support the safety assessment of jelly mushroom glycolipids for potential use in food.

In a repeated dose study according to OECD guideline 407, GLP, the test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” was administered to male and female Sprague Dawley rats via gavage for 4 weeks. The following recovery period was 4 weeks.

Study design

Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley rats of both sexes after repeated dosing with the test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception,arturition and early lactation of the offspring.

The animals, 10 per sex, were assigned to four groups and administered orally, via gavage, at dose levels of 100, 300 and 1000 mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10 mL/kg.

Due to unexpected mortality and overall adverse effects during the first and the second week of treatment, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.

Therefore, it was decided to reduce the high dose level from 1000 mg/kg/day to 500 mg/kg/day, starting from Day 15 of treatment and to investigate repeated dose toxicity and recovery from any treatment-related effects, changing the current study design to a 4 week oral toxicity study followed by 4 weeks of recovery.

Mortality

A total of five treated animals were found dead during the study: one mid-dose female (300 mg/kg/day), one high dose male and three high dose females (1000 mg/kg/day). Atrophy of spleen, thymus and decreased cellularity of bone marrow were considered the factor contributory to the death of the high dose animals. These findings were considered the response to stress and related to the very high dose, or being associated with agony of these animals. In the female of the mid-dose group, the cause of death was due to a mis-dosing.

Clinical signs

Dosing Phase: Decreased activity, cyphosis, emaciated and piloerection were seen in one control male during the last week of treatment. At 100 mg/kg/day, salivation and staining on the neck were seen in single females. At 300 mg/kg/day salivation, piloerection on occasions in Weeks 1 and 2, hairloss/head in single males and females were seen during the treatment. At 1000/500 mg/kg/day, salivation, dyspnoea, rales and swollen/hard abdomen were seen in single animals, with higher incidence in females while receiving 1000 mg/kg/day. After reduction of dose level to 500 mg/kg/day, recovery from all signs was seen during the fourth week of treatment. No relevant signs were seen during the recovery period.

Neurotoxicity assessment (Functional Tests and Motor activity)

Dosing Phase: Increases in grip strength in all treated groups, statistically significant in the first trial and in the mean value of the mid-dose male and high dose female groups, were seen at the end of treatment. Measurements of motor activity and sensory reaction to stimuli were comparable between control and treated groups.

Recovery Phase: Similar trend was again seen in the grip strength at the end of the recovery period (statistical significance limited to second trial and mean value of low dose females). No relevant differences between treated and control groups were seen in the motor activity and sensory reaction to stimuli at the end of the recovery periods.

Body weight

No relevant changes were seen in treated animals, compared to controls.

Food consumption

Dosing Phase: Slight reduction in food consumption was seen in high dose females on Days 8 and 15 of the study, during treatment at 1000 mg/kg/day, compared to controls.

Recovery Phase: No relevant changes were seen in treated animals, compared to controls.

Haematology

Dosing Phase: Leucocytosis was recorded in a number of males from all treated groups, in one female dosed at 100 mg/kg/day and one female receiving 1000/500 mg/kg/day. Compared with mean control data, changes were with no dose-relation and mainly due to lymphocytosis. No changes were recorded in the coagulation parameter.

Recovery Phase: Leucocytosis was still present in a number of animals of both sexes receiving 1000/500 mg/kg/day and in one male receiving 300 mg/kg/day.

Clinical chemistry

Dosing Phase: Compared with mean control data, fluctuations of some biochemical parameters (glucose, cholesterol, creatinine, calcium) recorded in a number of animals treated at 300 and 500/1000 mg/kg/day were not considered to be suggestive of tissue/organ injury.

Recovery Phase: Changes observed were considered unrelated to treatment.

Urinalysis

No relevant changes were observed.

Terminal body weight and organ weights

No relevant changes were observed on terminal body weight, absolute and relative organ weight of treated animals, when compared to the controls.

Macroscopic observations

No remarkable differences were noted at post mortem examination in treated animals, when compared with controls, either in animals that completed the treatment or recovery period.

Microscopic observations

No treatment related changes were observed in treated animals, when compared to the

controls. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle

and to the integrity of the various cell types within the different stages; regular layering in

the germinal epithelium was noted.

Conclusion

Based on the results of the present study, the dose level of 500 mg/kg/day can be considered the NOAEL (No Observed Adverse Effect Level). This dose level could be used as high dose level for a subsequent OECD 421 study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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

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 %]

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

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

Dose / conc.:

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

Remarks:

administration volume 5 mL/kg bw/day

Dose / conc.:

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

Remarks:

administration volume 5 mL/kg bw/day

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.

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.

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".

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

Critical effects observed:

no

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%

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

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: IMD Natural Solutions, GmbH (INS) (Dortmund, Germany)
- Purity, including information on contaminants, isomers, etc.: > 92%

Species:

rat

Strain:

CD-1

Details on species / strain selection:

One hundred (100) male and 100 female CD® Crl:CD(SD) rats, ages 32e34 days, were received in good health from Charles River Germany GmbH. Animals were acclimated to the testing facility
conditions for 6e7 days prior to dose administration. Body weights at the initiation of test article administration ranged from 148.3 to 174.5 g for males and 130.2e158.5 g for females. The animals were single-housed in suitable caging in an environmentally controlledroom maintained at a temperature of 22 °C ± 3 °C (maximum range) and a relative humidity of 55% ± 15% (maximum range). The rooms were lit (ca. 150 lux at ca. 1.50 m room height) and darkened
for 12 h of each 24 h period. Granulated textured wood was used as bedding material for the cages which were changed and cleaned once a week. The basal diet (Commercial ssniff®-R/M-H V1534) and drinking water (with or without the test article) was offered to the study animals ad libitum. Periodic analysis of the bedding material, basal diet, and drinking water (without test article) are performed to verify the composition and confirm the absence of contaminants that would compromise the health of the animals or affect the results of the study. All activities related to animal care and handling were performed in accordance with the laboratory's standard operating procedures (SOPs) and based on the policies of AAALAC.

Sex:

male/female

Details on test animals or test system and environmental conditions:

One hundred (100) male and 100 female CD® Crl:CD(SD) rats, ages 32-34 days, were received in good health from Charles River Germany GmbH. Animals were acclimated to the testing facility
conditions for 6e7 days prior to dose administration. Body weights at the initiation of test article administration ranged from 148.3 to 174.5 g for males and 130.2-158.5 g for females. The animals were single-housed in suitable caging in an environmentally controlledroom maintained at a temperature of 22 °C ± 3 °C (maximum range) and a relative humidity of 55% ± 15% (maximum range). The rooms were lit (ca. 150 lux at ca. 1.50 m room height) and darkened
for 12 h of each 24 h period. Granulated textured wood was used as bedding material for the cages which were changed and cleaned once a week. The basal diet (Commercial ssniff®-R/M-H V1534) and drinking water (with or without the test article) was offered to the study animals ad libitum. Periodic analysis of the bedding material, basal diet, and drinking water (without test article) are performed to verify the composition and confirm the absence of contaminants that would compromise the health of the animals or affect the results of the study. All activities related to animal care and handling were performed in accordance with the laboratory's standard operating procedures (SOPs) and based on the policies of AAALAC.

Route of administration:

oral: drinking water

Details on route of administration:

The test article was diluted with the vehicle, drinking water (tap water) at target concentrations of 0.15%, 0.5%, and 1.5% (1.5, 5.0, and 15 g/L, respectively). Dose formulations were prepared once per week and stored refrigerated (2e8 C) until use. Prior to use for dose administration, the test article-drinking water formulations were warmed to room temperature and stirred for a minimum of 15 min. The stability and concentration of the test article in the vehicle were evaluated by analysis of samples collected at appropriate intervals throughout the study (i.e. concentration analysis was conducted on the first and last dose formulation preparations in study weeks 1 and 13; stability analysis was conducted after 7 and 14 days of room temperature storage under animal laboratory conditions).
The animals were allocated to the test groups by means of a computer generated randomization program. Main study groups were comprised of 20 animals/sex/group. An additional 10 animals/
sex for Groups 1 and 4 were designated as Recovery animals which maintained on study for a 28-day observation period following cessation of test article administration.

Vehicle:

water

Details on oral exposure:

The test article, AM-1 was orally administered to the test animals in drinking water (target concentrations of 0.15%, 0.5%, and 1.5%) for 90 days. Drinking water was selected as the vehicle to simulate beverages as the anticipated route of consumer exposure, and due to technical infeasibility of other vehicles (e.g. rodent diet) for dose formulation with this viscous test material having surfactant qualities at high concentrations. Dose levels were selected based on the results of a dose range-finding toxicity study in which no test article-related signs of systemic toxicity were noted in rats treated with dose levels of 0.1%, 0.5%, or 1.0% AM-1 in drinking water for 14 days (INS unpublished data on file). The maximum concentration in the subchronic study was increased to 1.5% in order to provide an adequate margin of safety/exposure compared
to consumer intake estimates for AM-1 at proposed use levels as a food ingredient. The test article-drinking water mixtures were freshly filled in the drinking bottles in the morning and provided ad libitum for 24 h each day (at least 60 mL/animal/day). The maximum average daily water consumption of a rat was estimated as 30-40 mL/animal/day. The control animals received fresh tap water on the same schedule/regimen as the treatment group animals.
The oral route of administration was selected as this is the anticipated route for human exposure to the test article.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

LC-MS analytical method

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

1.5 mg/L drinking water

Remarks:

132.47 ± 35.21 mg/kg bw/day for males ; 165.47 ± 22.82 mg/kg bw/day for females (group 2)

Dose / conc.:

5 mg/L drinking water

Remarks:

412.61 ± 112.94 mg/kg bw/day for males and 501.47 ± 77.96 mg/kg bw/day for females (group 3)

Dose / conc.:

15 mg/L drinking water

Remarks:

1200.52 ± 303.34 mg/kg bw/day for males; 1423.09 ± 228.10 for females (group 4)

No. of animals per sex per dose:

30 maels and female for Group 1 and 4; 20 males and females fro grops 2 and 3

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Clinical signs: Animals were observed twice daily for mortality and moribundity and at least once daily for any signs of behavioral changes, reaction to treatment or illness. Cageside observations included an examination of the skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity, and behavior patterns.
The onset, intensity, and duration of any signs observed were recorded. Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (always on the last day of the study week), detailed clinical observations were performed for all animals. These observations were made outside the home cage in a standard arena and at the same time of day each week. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, pilo-erection, pupil size, and 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 behavior (e.g., self-mutilation, walking backwards) were also recorded.
Ophthalmological examinations were performed on all animals prior to the start of administration (study week 0), at the end of the treatment period (study week 13), and at the end of the recovery -period (study week 17). All main study and recovery animals were randomly assigned to groups for neurological screening evaluation during pre-treatment and in study week 12. Recovery phase animals were also evaluated during study week 16. The screening of sensory reactivity to stimuli of different types (i.e., righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function) as well as the assessment of grip strength and motor activity assessment were conducted in all animals outside the home cage in a sound attenuated room by personnel not involved in the dosing procedure and general handling of the animals during the study duration.
Neurological symptoms: All main study and recovery animals were randomly assigned to groups for neurological screening evaluation during pre-treatment and in study week 12. Recovery phase animals were also evaluated during study week 16. The screening of sensory reactivity to stimuli of different types (i.e. righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function) as well as the assessment of grip strength and motor activity assessment were conducted in all animals outside the home cage in a sound attenuated room by personnel not involved in the dosing procedure and general handling of the animals during the study duration.
Body weight: The weight of each rat was recorded at the time of group allocation, on the first day of test article administration and once a week thereafter, and on the day of scheduled necropsy. The quantity of food provided to and left by each individual animal was recorded on a weekly basis throughout the experimental period.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week. From these data the food consumption (in g/kg bw/day) was determined for each animal.
Water consumption was recorded daily by weighing the water bottles when filled and weighing the residues on the next test day (approximately 24 h later). The residues were then discarded. A new bottle filled with the appropriate test-article formulation was made available to each animal. Possible water loss due to spilling was recorded qualitatively based on visual appraisal. The relative drinking water consumption (in mL/kg bw/day) was determined for each animal.
Clinical pathology/laboratory examinations: Blood samples for hematology, coagulation, and serum chemistry parameters were collected during study weeks 2 (Day 14), 6 (Day 42), and 13 (Day 91) for all main study and recovery animals, and during study week 17 (Day 119) for all recovery animals (Groups 1 and 4). Animals were fasted overnight prior to blood collection under anesthesia. The following parameters were analyzed in accordance with the applicable testing guidelines of OECD and FDA Redbook. Hematology: hemoglobin content (HGB); erythrocytes (RBC); leukocytes (WBC); neutrophilic granulocytes (Neut); reticulocytes (Reti); platelets (PLT); hematocrit value (HCT); differential blood count [relative and absolute neutrophilic, eosinophilic, and basophilic granulocytes, lymphocytes and monocytes]; large unstained cells (LUC); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); blood smears were prepared for potential future hematological and histopathological analysis; Coagulation: thromboplastin time (TPT); activated partial thromboplastin time (aPTT); Serum Chemistry: albumin; globulin; albumin/ globulin ratio; (Alb/Glo ratio, calculated); bile acids; bilirubin (total); cholesterol (total); creatinine; glucose; phosphate; protein (total) (TP); triglycerides; urea (in blood); calcium; chloride; potassium; sodium; alanine amino-transferase (ALAT); alkaline phosphatase (AP); aspartate aminotransferase (ASAT); lactate dehydrogenase (LDH); cholinesterase (CHE); gamma-glutamyl transpeptidase (gamma-GT); glutamate dehydrogenase (GLDH).
Urine samples were collected over a 16-h interval during study weeks 10 (Day 65) and 13 (Day 85) from all main study and recovery animals, and during study week 17 (Day 115) from all recovery animals (Groups 1 and 4). Urine pH, specific gravity, and volume were measured; color and turbidity were examined visually.
The following tests were performed using qualitative indicators of analyte concentration: protein, glucose, bilirubin, urobilinogen, ketones, hemoglobin (HGB) (approx. values), and nitrite. Microscopic examinations of sediment were also conducted.

Sacrifice and pathology:

Necropsy and pathology: On Day 91 (main study, one day after the last administration of the test article) or Day 119 (recovery phase, 28 days following cessation of test article administration), a complete gross necropsy was performed on all animals euthanized under CO2 anesthesia and exsanguinated. All superficial and subcutaneous tissues were examined and the cranial roofwas removed to allow observation of the brain, pituitary gland, and cranial nerves. The condition of the thoracic and abdominal viscera was noted. The adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, stomach (evacuated), thymus, testes, thyroid (including parathyroids), and uterus were weighed before fixation. Organ weights and the final body weight at necropsy of animals that died were recorded but not included in the calculation of mean values for comparison of control and test article-treated groups.
The following organs and tissues were preserved in the appropriate fixative (i.e. eyes were preserved in Davidson's solution; epididymides and testes were preserved in Bouin's fixative; remaining tissues were fixed in 10% buffered formalin): adrenal glands; aorta abdominalis; bone (os femoris); bone marrow (os femoris); bone marrow (sternum); brain (3 levels: cerebrum, cerebellum,
medulla/pons); caecum; coagulating gland; epididymides; eyes with optic nerves; gross lesions observed; head (without brain, eyes, and lower jaw), [for the preparation of the histological slides of the nose]; heart (3 levels: right and left ventricle, septum); harderian glands; intestine, large (colon,
rectum); intestine, small (duodenum, jejunum, ileum, including Peyer's patches), Swiss roll method; kidneys and ureters; liver (1 lobe); lungs [with mainstembronchi and bronchioles (preserved by inflation with fixative and then immersion)]; lymph node (1, cervical); lymph node (1, mesenteric); mammary gland (male and female); muscle (skeletal, leg); nerve (sciatic); esophagus; ovaries; oviduct; pancreas; pituitary; prostate; salivary glands (mandibular, sublingual, and parotid gland); seminal vesicle; skin (left flank); spinal cord (3 levels: cervical, mid-thoracic, lumbar); spleen;
stomach; testes; thymus; thyroids (including parathyroids; examined microscopically if in the plane of section); tissue masses or tumors (including regional lymph nodes); trachea (including larynx);
urinary bladder; uterus (including cervix and oviducts); and vagina. Blood smears prepared from all animals for hematological examination were retained for possible examination of pathological
changes.
The above-listed organs of all main study animals of Groups 1 and 4 and any prematurely deceased animals were examined microscopically after preparation of paraffin sections and hematoxylin-eosin (H-E) staining. The stomach of all main study animals in Groups 2 and 3 as well as of all recovery animals in Groups 1 and 4 was examined histologically due to observations in the stomach/forestomach of the main study animals in the highdose group (Group 4). In addition, frozen sections of the heart, liver, and one kidney were made, stained with Oil Red O, and examined microscopically for main study animals of Groups 1 and 4. A detailed microscopic examination was performed on one testis and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all main study males of Groups 1 and 4 following H-E and periodic acide Schiff (PAS) staining.
The histological slides of the nose of all main Group 1 and 4 study animals were prepared for microscopic evaluation of the nasal cavity, nasopharynx, and paranasal sinus.

Statistics:

The test article-treated groups (Groups 2 to 4) were compared to the control group using the following statistical tests for significance levels of 5% and 1%: body weight, food consumption, hematology, clinical chemistry, urinalysis, and relative and absolute organ weight data were subjected to the multiple t-test based on Dunnett (1964); all numerical functional tests were analyzed using the Student's t-test. Histopathology data were subjected to the exact test of Fisher (1948) (p < 0.05).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Transient observations of piloerection in a small number of high-dose (1.5% AM-1) treatment group males and females were noted on a limited number of test days during the first 7
weeks of the study. During the 4-week recovery period, piloerection was not observed in males or females previously treated with 1.5% AM-1 in drinking water

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the high-dose (1.5% AM-1) treatment group died during Day 14 after blood withdrawal for laboratory examinations. The death of this animal was regarded to be incidental and related to the stress caused by the isoflurane anesthesia during the blood withdrawal procedure.

Body weight and weight changes:

effects observed, treatment-related

Ophthalmological findings:

effects observed, non-treatment-related

Haematological findings:

no effects observed

Description (incidence and severity):

No test article-related effects were noted on the hematological or serum chemistry parameters evaluated on Days 14, 42, and 91 in the male and female animals treated with 0.15%, 0.5%, or 1.5% AM-1 via the drinking water for 90 days. Statistically significant differences
between test article treated animals and concurrent controls were generally within the laboratory's historical control ranges, were not associated with histopathological findings, and/or did not occur in a consistent pattern with respect to dose-response between measurement
intervals or between sexes that would suggest biological significance. Additionally, values for certain parameters in the control group animals were noted as being on the high-end or lowend of the historical control range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test article-related effects were noted on the hematological or serum chemistry parameters evaluated on Days 14, 42, and 91 in the male and female animals treated with 0.15%, 0.5%, or 1.5% AM-1 via the drinking water for 90 days. Statistically significant differences
between test article treated animals and concurrent controls were generally within the laboratory's historical control ranges, were not associated with histopathological findings, and/or did not occur in a consistent pattern with respect to dose-response between measurement
intervals or between sexes that would suggest biological significance. Additionally, values for certain parameters in the control group animals were noted as being on the high-end or lowend of the historical control range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test article-related effects were noted on the urinalysis parameters evaluated on Days 65 or 85 in the male and female animals treated with 0.15%, 0.5%, or 1.5% AM-1 via the drinking water for 90 days. Statistically significant differences between test article treated animals and concurrent controls were within the laboratory's historical control ranges, were not associated with histopathological findings, and/or did not occur in a consistent pattern with respect to dose-response between measurement intervals or between sexes that would suggest biological significance.
Urinary parameters for the male and female animals treated previously with 1.5% AM-1 via the drinking water for 90 days were similar to concurrent controls at the end of the 4-week recovery
period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The neurological screening did not reveal any test article-related effects on any of the parameters examined during the functional observation tests nor on the fore- and hind-limb grip strength or spontaneous motor activity assessments. Statistically significant differences in measured values for the fore- and hind-limb grip strength, spontaneous motor activity, and body temperature in study week 12 were not dose-related, not consistent between sexes, not correlated with histopathological findings, and/or were within the range of the laboratory's historical control data (mean ± 2 SD). With the exception of hind-limb grip strength in
high-dose (1.5% AM-1) treatment group males (statistically significantly higher than concurrent controls but within historical control range, therefore not considered biologically relevant), mean
values for these parameters were consistent with concurrent control values at the study week 16 (Recovery phase) evaluation

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test-article realted effects were noted on the relative ( to body weight at necropsy) and absolute organ weights of the male and females animals treated with 0.15%, 0.5%, or 1.5% AM-1 via the drinking water compared to the control group, neither at the terminal sacrifice at the end of the treatment period nor at the end of the recovery period. Statistically significant (at <=p 0.05 or p <= 0.01) increases in relative and absolute stomach weights were observed in the intermediate-and high-dse treatment group females compared to the control groups at the end of the treatment period but were not considered toxicologically relevant as they were not clearly dose-response related, not consistent between sexes, and consistent hsitopathological correlated were not noted. Futher, the values were within the range of the laboratory´s historical control data. Relative and absolute stomach weights of male
and female rats previously treated with 1.5% AM-1 via the drinking
water for 90 days were similar to concurrent controls at the end of
the 4-week recovery period. Statistically significant organ weight differences between Group 4 recovery males and females and control animals at the recovery necropsy (data not presented) were not considered test article related as the slight differences were without biological relevance and were not observed for Group 4 treated animals at the primary necropsy

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic investigation at necropsy did not reveal any test-article systemic changes in the organs or tissue of the animals teated with 0.15%, 0.5% or 1.5% AM_1 via the drinking water for 90 days, neither at terminal sacrifice at the end of the treatment period nor at the end of the 4-week treatment-free recovery periiod. A very small number of macorscopic changes were noted in the kidney (enlargeement), thymus (red disclored), uterus (dilated, filled with clear liquid), ovaries (cystic), and eyes (reduced in size), of individual animals of the test article-treated groups and control at terminal or recovery sacrifice. The changes were without histopathological correlates and are considered to be incidental findings due to the low number of animals affected and lack of adose-response relationship. The enlargement of the right kidney noted in 1 to 20 male animals treat woth 1.5% AM_1 via the drinking water at termnal sacrifice was histopathologically correlated with a benign nephroblastoma. However, this finding is considered to be incidental and without any toxicological releevance as only one animal of the high dose group was affected.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination of all scheduled organs and tissues (including all gross lesions) of the high-dose treatment group males and females, and of the stomach of the low- and intermediate-dose treatment groups males and females did not reveal any morphological changes that are considere to be related to the administraion of the test article. Lympho-histiocytic infiltrations in various organs (e.g. respiratory epithelium of the nasal cavity, heart, kidney, larynx, liver, and trachea) are considered to be spontaneous organ changes as they were seen in similar proportions of animals of the control and high-dose groups and are thus not considered test article-related. A minimal to mild focal thickening (hyperplesia) of the squamous cell epithelium near the mucosa with minimal hyperkeratosis was noted in the forestomach of male and female rats of all 3 AM-1 treatment groups as well as the control group. All other parts of the forestomach revealed a thin squamous epithelium. The male and female recovery animals showed no morphological differences in the forestomach between the controls and the high-dose animals. This
hyperplasia of the forestomach was regarded as a commonly occurring coincidental finding that is not considered relevant to human safety since the forestomach is not an organ with a human equivalent. Remaining coincidental findings in a small number of test animals (e.g. age associated involution of the thymus, fatty infiltration in the tubular epithelial cells of the kidneys and in the liver, and hemorrhage in the thymus, lungs and cervical lymph nodes related to terminal sacrifice procedures) are regarded as spontaneous organ changes which occurred with
similar incidence and severity in the control groups and thus are not considered test article-related.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Target concentrations of the test article in drinking water mixtures were confirmed via analysis of the relative composition (i.e. proportions of the four main glycolipid components) using a validated LC-MS analytical method (unpublished method developed on behalf of the sponsor) and were within the generally accepted range (±15% of nominal) or slightly lower (e.g., up to -17% for low-dose formulation, 1.5 mg/mL on Day 1). The measured concentrations of AM-1 in the drinking water samples obtained from the stock solution stored at animal laboratory conditions for 7 or 14 days did not reveal any signs of degradation of the test article in the drinking water solutions. The dose formulation samples were found to be stable and homogeneous for the duration of each weekly administration period. Analytically verified average test concentrations of 0.14%, 0.46%, or 1.35% AM-1 (overall mean for weeks 1 and 13) were achieved during the treatment phase. Given the limitation of accuracy (±15%) of the LC-MS-based quantification of the AM-1 glycolipid mixture in water, test article intake was calculated based on actual dose preparation records and procedures, which followed GLP standards and conditions using
validated laboratory instrumentation. This method of calculation is considered to provide higher accuracy and best reflects the actual test article concentrations in drinking water mixtures that were administered during the study. The calculation of the test article intake via the drinking water (i.e. based on measured drinking water consumption in mL/kg bw/day) confirmed a dose-dependent exposure of the male and female animals to the test article with overall mean values of 132, 413, and 1200 mg/kg-bw/day for males and 165, 501, and 1423 mg/kg-bw/day for females in the 0.15%, 0.5%, and 1.5% AM-1 dose groups, respectively

Key result

Dose descriptor:

NOAEL

Effect level:

1 201 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 423 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

The no-observed-adverseeffect level (NOAEL) for systemic toxicity was considered to be 1.5% AM-1 in the drinking water, equivalent to 1201 and 1423 mg/kg bw/day for male and female rats, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available study was conducted according to OECD guideline 408 and is of high quality.