D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Endpoint summary

Description of key information

Potential systemic toxicity of Glucamide OL was investigated in a subacute 28-day toxicity study in compliance with OECD test guideline 407. There were no adverse effects of Glucamide OL found up to the highest dose level tested of 650 mg/kg body weight/ day. No subchronic or chronic data are available with Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, available 90-day oral toxicity data from Glucamide 24 as source molecule can be used for evaluation purposes using read-across princliples (see also comprehensive read-across justification). Taking a conservative approach, the NOAEL of 200 mg/kg body weight per day revealed in the OECD guideline conform 90-day study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

Key value for chemical safety assessment

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:

2013-11-21 to 2014-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 8-9 weeks old, females: 8-9 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 217 - 283 g (mean: 250.60 g, ± 20% = 200.48 – 300.72 g)
females: 154 - 183 g (mean: 170.83 g, ± 20% = 136.67 – 205.00 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0801)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were housed in groups (5 animals/sex/cages) in type IV cages, polysulphone cages on Altromin saw fibre bedding
(lot no. 030713 and 260913)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed
any adverse clinical signs. Before the first administration all animals used for the study were weighed and assigned to the experimental
groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The doses for this study were chosen by the study monitor based on repeated dose toxicity study with the analogue test substance.
The doses used for the study with analogue test substance were 100, 250 and 625 mg/kg body weight/ day.
Similar dose levels were chosen for this study after discussion. The following doses were selected for the 3 dose groups
(LD = low dose, MD = medium dose, HD = high dose). The animals were treated with the test item formulation or vehicle on 7 days per week
for a period of 28 days. 5 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period).
5 animals per gender of the C and of the HD group were subjected to necropsy 14 days after the last administration (end of recovery period).

Control: 0 mg/kg body weight
Low Dose: 125 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 650 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence
of dose levels was selected with a view to demonstrate any dose related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume
as used for the high dose group.

Administration of Doses
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of
5 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples were retained from all groups once weekly during the
treatment period and stored between -15 and -35 °C.
Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose group samples of dosing
formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C.
In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared
high and low dose formulations and stored between -15 and -35 °C.
Each sample was retained twice (sample A, sample B, each of at least 5 mL).
At the end of the treatment period all A samples of dosing formulations were analysed at BSL BIOSERVICE Scientific Laboratories GmbH in
accordance with GLP under the reference number 136165.
The B samples were retained at BSL until the analysis had been performed, and will be discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

Frequency of treatment:

The animals were treated once daily.

Remarks:

Doses / Concentrations:
125 mg/kg bw, 300 mg/kg bw and 650 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

60 animals (30 males and 30 females) were included in the study (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random based on body weight
- Section schedule rationale : alle male animals together and all female animals together

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and
weekly during the treatment and recovery period.
Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an
additional period of 14 days following the last administration. General clinical observations were made once a day, preferably at the same
time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and
mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of
the treatment period as well as at the end of the recovery period in the recovery animals.

Functional Observations
Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period multiple detailed
behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted
in all animals.

Haematology
Haematological parameters (haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT),
white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells)
were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

Blood Coagulation
Coagulation parameters (prothrombin time (PT), activated partial thromboplastin time (aPTT)) were examined at the end of the treatment
and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry (alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP),
creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc),
sodium (Na), potassium (K)) were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters (specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood,
leukocytes) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL). Additionally, urine colour/ appearance were recorded.

Sacrifice and pathology:

Pathology
One day after the last administration (study day 29) all animals of the treatment period and 2 weeks after the last administration all animals
of the recovery period (study day 43) were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24664, expiry date: 30/06/2015 and xylazin,
Serumwerk, lot no. 00512, expiry date: 07/2014) and subjected to a detailed gross necropsy which includes careful examination of the external
surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries,heart) of all sacrificed animals was recorded as soon as possible.
Paired organs were weighed together.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with
coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes
(mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow,
lung and trachea, pituitary gland, mammary glands, oesophagus, skin) from all animals were preserved in 10% neutral buffered formalin
except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were
transferred to 70% ethanol.

Histopathology
The afore-listed organs (see above) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining
for the animals of the groups 1 and 4 sacrificed at the end of the treatment period.
Since microscopic changes that could be attributed to treatment with the test item were not observed in any animals of HD group in the
initial histopathologic examination, further histoprocessing and histopathology evaluation were not performed.
Any gross lesion macroscopically identified was examined microscopically in all animals.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the
GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The Study
phase from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005
[SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed
according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of
compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a
pathology phase report to the study director upon the completion of the study.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the
main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were
performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or IDBS E-WorkBook 9.4.0 software
(p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Mortality:

mortality observed, treatment-related

Description (incidence):

no adverse effects

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no relevance to test item treatment

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
No mortality occurred in the control or any of the dose groups during the treatment and the recovery periods of this study.

Clinical Observations
All male and female animals of MD and HD groups showed signs of salivation and moving the bedding, which were considered to be due to test
item treatment. These clinical signs were only observed immediately post dose administration, which suggests no systemic effect but more likely
a local effect. There were also isolated signs of diarrhea (male=C 1/5, HD 2/5, RHD 1/5; female= HD 1/5, RC 1/5), nasal discharge
(male= LD 1/5, RC 1/5; female= C 1/5), piloerection (male= HD 1/5), eschar (male= C 2/5, RC 2/5; female= RHD 2/5) and alopecia (female= MD 1/5).
These clinical signs were transient in appearance and were considered to have no toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, there were no changes of toxicological relevance noted in test item treated groups when compared to the corresponding
control groups during the treatment and the recovery periods. There were no statistically significant changes between the test item treated and
the control groups. The mean body weight increased with the progress of the study in the C, LD, MD and HD groups.
There were several transient changes (increase or decrease) in body weight gain without showing statistical significance in all dose groups during
the treatment and recovery period when compared to the corresponding control groups. But these changes were not considered to have any
biological relevance.

Food Consumption
In males and females, there were no changes of toxicological relevance for food consumption in the test item treated groups during the treatment
and the recovery period.
In females, the food consumption was higher during the treatment days 1-8, 15-22, which affected the cumulative food consumption of treatment
days 1-28 in LD, MD and HD groups when compared to corresponding control. This difference in food consumption was transient and had no
relevance to treatment.

Haematology and Blood Coagulation
In both males and females, there were no statistically or biologically significant changes of toxicological relevance noted for the measured
haematological and blood coagulation parameters. In males at the end of treatment there were moderately higher mean eosinophil values in LD, MD
and HD groups when compared to control. These differences were not statistically significant and had no dose response relation. At the end of
recovery there were slightly lower, not statistically significant, mean neutrophil and eosinophil values in male HD group. These differences were
due to higher value in single isolated males (animal 24 for neutrophil and animal 22 for eosinophil) of recovery control group (aberrant control)
and thus were not considered to be toxicologically relevant.
In females, there was marginally higher mean WBC count noted in LD, MD and HD groups. These differences were statistically insignificant and in
addition, the mean WBC of HD group was comparable to the mean WBC of recovery control group. Therefore, the differences were considered to
have no biological relevance.

Clinical Biochemistry
In both males and females, there were no adverse changes of toxicological relevance noted for the measured clinical biochemistry parameters.
However, there was statistically significantly higher mean AP value in male LD group, but there was no dose- response relation noted. There was
also statistically significantly lower mean ASAT in female MD and HD groups and mean TP in female HD group when compared to the corresponding
control. The decrease in TP value of HD group correlated to the slightly but not statistically significantly lower mean ALB of HD group.
Furthermore, in males at the end of treatment there were marginally higher mean AP in MD and HD groups and mean TBA in LD, MD and HD groups,
compared to controls. These changes were statistically insignificant and had no dose- response relation. At the end of recovery there was a higher,
not statistically significant, mean TBA value in HD group when compared to corresponding control.
In females, at the end of treatment there was marginally higher and lower, not statistically significant, mean TBA and Na, respectively in HD group
when compared to controls. There was also higher mean AP value in Recovery HD group.
The ALAT, ASAT, AP, TBA, Crea and Na values in main group animals (control and dose groups) were lower when compared to recovery groups
(control and dose groups). Although there were differences in clinical biochemistry parameters (mentioned above), these were not considered to
have adverse toxicological relevance due to the absence of any associated macroscopic or microscopic findings in liver and kidney.

Urinalysis
In males at the end of recovery higher erythrocyte level were found in 2 males (no.s 28 and 30) of recovery HD group. There were marginally
higher leukocyte level in 2 males (no.s 26 and 30) and marginally higher bilirubin, ketamine and protein levels in 1 male (no. 26) of recovery HD
group. In females at the end of treatment there was higher erythrocyte level in 1 female (no. 41) of MD group. There were also marginally higher
ketamine, erythrocyte and leucocyte value in 1 female (no. 46) of HD group. These differences were in comparison to concurrent control group.
In the absence of any associated pathological signs, the differences in males and females were not considered to be relevant to treatment.
Besides, all urinary parameters were in the normal range of variation and no noticeable differences between test item treated and control group
were observed.

Pathology
There were no gross lesions that could be attributed to treatment with the test item.
At the end of treatment, yellow focus on the epididymis was found in 2/ 5 control and 2/5 LD group males. Fluid distended uterus/ cervix was
found in 1/5 LD and 2/5 MD group females. At the end of recovery dilated pelvis was found in 1/5 HD group males.
All gross lesions recorded were considered to be within the range of normal background alterations which may be recorded in animals of this
strain and age.

Organ Weight
There were no treatment related effects on organ weights (absolute and relative to brain and terminal body weight) at the end of treatment
and recovery periods. Although, there were statistically significantly higher mean absolute brain weight in male recovery HD group and relative
brain (to terminal body weight) weights in female LD groups when compared to controls. But these changes were not associated with any
macroscopic or microscopic findings and was considered incidental.
In males there were slightly lower (in dose response relation) mean absolute and relative pituitary gland weights in LD, MD and HD groups
(-4% to -20% Vs Control). In females the mean absolute and relative pituitary weights were higher in all dose groups when compared to c
orresponding control. The absolute pituitary weights of males of all dose and control groups were within the histrocal control range and in
females the mean and most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of
macroscopical and histopathological findings no relevenace to test item treatment was considered.
Several other differences in mean absolute and/or relative organ weights (relative to terminal body weight and brain weight) between control and
treated groups were either statistically insignificant and/or there were no dose response relation. There were no macroscopic or microscopic
findings in any of the organs of treated and control groups. Therefore, the differences in organ weight data were not considered to have relevance
to test item treatment.

Histopathology
There were no microscopic lesions that could be attributed to treatment with the test item.
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study weeks 1, 2, 3 and 4 for all dose groups. The mean recoveries
observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 102.3%, 106.5% and 115.2% of the nominal concentration,
respectively. Nominal concentrations were confirmed for all dose groups, as mean of measured concentration did not differ from nominal
concentration by more than 20%.
Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD dose groups. After 6 hours storage
at room temperature recovery compared to starting value was 91.7% and 98.9%. All samples were stable, as concentration after storage did not
differ from start value by more than 20%.
Homogeneity of formulation samples was determined in study weeks 1 and 4 for the LD and HD dose groups. The mean recovery observed for
the LD dose group was 95.8% and 101.5% of the nominal value and 115.7% and 114.4% of the nominal value for HD dose group. The coefficients
of variation of the different sampling locations (top, middle, bottom) were 3.1% and 15.1% in LD dose group and 2.3% and 0.9% in HD dose group.
All samples were homogenous, as COV was below or equal 20%.

Dose descriptor:

NOAEL

Effect level:

>= 650 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day. The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.

Critical effects observed:

not specified

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with C18/C18 unsatd.-Glucamide in male and female Wistar rats with dose levels
of 125, 300, and 650 mg/kg body weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:
There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.
In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and most
individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological
findings no relevenace to test item treatment was considered.
There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day.
The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of C18/C18 unsatd.-Glucamide via

oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study.

The 4 groups comprised 5 male and 5 female Wistar rats. The animals were housed in groups (5 animals/sex/ cages).

The following doses were evaluated:

Control:                           0         mg/kg body weight/ day

Low Dose:                    125     mg/kg body weight/ day

Medium Dose:              300    mg/kg body weight/ day

High Dose:                   650    mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered

daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly

body weight measurements. During the period of administration, the animals were observed precisely each day for signs of toxicity.

All animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic

effects, animals in the recovery group were observed for a period of 14 days following the last administration.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was

taken and a set of organs/tissues was preserved. Haematological, blood coagulation and clinical biochemistry parameters were examined

from blood samples collected from an abdominal aorta of all terminally sacrificed animals.

Urinalysis was made on urine samples collected directly from the urinary bladder of all terminally sacrificed animals.

A full histopathological evaluation of the tissues was performed on high dose and control animals.Since microscopic changes that could

be attributed to treatment with the test item were not observed in any animals of HD group in the initial histopathologic examination,

further histoprocessing and histopathology evaluation were not performed.

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment or recovery period of this study.

All male and female animals of MD and HD groups showed signs of salivation and moving the bedding, which were considered to be due to

test item treatment. These clinical signs were only observed immediately post dose administration, which suggests no systemic effect but

more likely a local effect. Diarrhea, nasal discharge, piloerection, eschar and alopecia were transient in appearance or also occurred in the

control group. There was no considerable difference between the genders.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

There were no ophthalmoscopic findings in any of the animals of this study.

No significant differences were found in the body weight gain or food consumption of the dose groups of males and females when compared

to the corresponding control groups. No differences were found that were considered toxicologically relevantin the haematological and

blood coagulation parameters analysed in any of the treated groups when compared to the corresponding control group.

No toxicologically relevant effect was found in the clinical biochemistryparameters analysed in any of the treated groups when compared to

the corresponding control group. Statistically significant differences were not assumed to be biologically relevant.

There were no considerable test item-related differences in any of the urine parameters tested.

There were no gross lesions that could be attributed to treatment with the test item.

There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.

In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and

most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological findings no relevenace to test item treatment was considered. There were no microscopic lesions that could be attributed

to treatment with the test item. All microscopic findings recorded were within the range of normal background lesions which may be recorded

in animals of this strain and age. Formulation analysis was performed on samples of all dose groups collected at various intervals during

the study. The concentration verification of samples of all dose groups was investigated in study weeks 1, 2, 5 and 4.

The mean recoveries in LD, MD and HD groups were 102.3%, 106.5% and 115.2% of the nominal concentration, respectively.

The stability of formulation samples of LD and HD dose groups was investigated in study week 1. After 6 hours storage at room temperature recovery compared to starting value was 91.7 (for LD dose) and 98.9% (for HD dose).All samples were stable, as concentration after storage

did not differ from start value by more than 20%.

The Homogeneity of formulation samples of LD and HD dose groups was determined in study week 1 and 4. The mean recoveries observed

for LD dose group was between 95.8 % and 101.5% of the nominal value and between 115.7% and 114.4% of the nominal value for HD dose group. The coefficients of variation of the different sampling levels (top, middle and bottom) were between 3.1% and 15.1% in LD dose group, between 2.3% and 0.9% in HD dose group.

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with C18/C18 unsatd.-Glucamide in male and female Wistar rats with dose levels

of 125, 300, and 650 mg/kgbody weight/ day, followed by a recovery period of 14 days, the following conclusions can be made:

There were slightly lower and higher mean pituitary gland weights (absolute and relative) in males and females, respectively in all dose groups.

In males, the absolute pituitary weights of all dose and control groups were within the histrocal control range and in females the mean and most individual absolute pituitary weights were slightly higher than the historical control range. In the absence of macroscopical and histopathological findings no relevenace to test item treatment was considered.

There were no adverse effects of C18/C18 unsatd.-Glucamide found up to a dose level of 650 mg/kg body weight/ day.

The NOAEL of C18/C18 unsatd.-Glucamide in this study is considered to be 650 mg/kg body weight/ day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Feb. 18, 1991 to Sep. 30, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fuellinsdorf
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: The body weight range was: males: 187-225 grams and females: 151-184 grams- Housing: Individually in Makrolon type-3 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 343 (batch# 81/91, 83/91 and 85/91 rat maintenance diet) was available ad libitum. The results of analyses for contaminants were recorded.
- Water: Community tap-water from Fullinsdorf was available ad libitum. Results of bacteriological, chemical and contaminant analyses of representative samples were recorded.
- Acclimation period: 7 days under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22± 3°C
- Humidity: 40-70%
- Air changes: Air- conditioned room with 10-15 air changes/ hour
- Photoperiod: 12 hours fluorescent light/ 12 hours dark, music during the light period.

IN-LIFE DATES: From: Feb. 18, 1991 To: May 22, 1991

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test solution was prepared daily prior to each application. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using homogenizer. Homogeneity of the test substance in the vehicle was maintained during treatment using a magnetic stirrer.
Dose volume: 10 mL/kg bw
VEHICLE
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE PREPARATIONS: Concentrations, homogeneity and stability of the dose formulations were determined prior to pretest start. Further samples for analysis were taken during weeks 2, 7 and 11 of the test and subsequently analyzed. The analyses were performed in the Analytical Laboratories of RCC umweltchemie AG, according to a method which was supplied by the sponsor.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 10, 50, 200 and 500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 rats/sex/dose (Groups 1 to 5)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels end route of application used in this study were based on the data from a 14-day (range-finding) feeding toxicity study in rats (RCC 287458) and from a 14-day (range-finding) oral toxicity study (RCC 292195).
- Rationale for animal assignment: Animals were randomized by computer generated random algorithm.
- Rationale for selection of animal: Recognized by the international guidelines as the recommended test system.

TREATMENT GROUPS:
Group 1: Control (Bidistilled water)
Group 2: 10 mg/kg bw/day active test substance (equivalent to 22.2 mg/kg bw of test solution)
Group 3: 50 mg/kg bw/day active test substance (equivalent to 111.1 mg/kg bw of test solution)
Group 4: 200 mg/kg bw/day active test substance (equivalent to 444.4 mg/kg bw of test solution)
Group 5: 500 mg/kg bw/day active test substance (equivalent to 1111 mg/kg bw of test solution)
Recovery groups of 10 rats/sex/dose group were also utilized in this study. The recovery group rats were sacrificed after a 4-week recovery period following 13 week treatment with the test substance.

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Animals were observed twice daily for viability and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION :
- The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at treatment week 4 and treatment week 13 (shortly before the termination or treatment) and a third time on the recovery animals of group 1 and 5 at recovery week 4 (shortly before the termination of the recovery period). 10 to 90 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Ophthalmoscope BETA 200 (Eisenhut Vet. AG, CH-4123 Allschwil).
- Dose groups that were examined: On all surviving animals at Week 4 and 13. At week 17, on control and high dose (500 mg/kg bw) group animals.

HAEMATOLOGY: Yes
- Collection of blood samples: Blood samples were drawn from retro-orbital plexus.
- Time schedule for collection of blood: After 13 weeks of treatment (between hours of 06:30 and 09.00). Blood was collected from recovery group at week 17.
- Anaesthetic used for blood collection: Yes; light ether anesthesia.
- Animals fasted: Yes; animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes, Heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, coagulation: thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Collection of blood samples: Blood samples were drawn from retro-orbital plexus.
- Time schedule for collection of blood: After 13 weeks of treatment (between hours of 06:30 and 09.00). Blood was collected from recovery group at week 17.
- Animals fasted: Yes; animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters checked: Glucose, urea, creatinine, uric acid, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, total protein, albumin, globulin, albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-h fasting period into a specimen vial.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume (18 hour), specific gravity, color, pH, protein, glucose, ketones, bilirubin, urobilinogen, erythrocytes, urine sediment

Sacrifice and pathology:

SACRIFICE: Prior to necropsy, the animals were fasted for approximately 18 hours, but water was provided. Necropsies were performed by experienced prosectors. Each fasted individual as well as each moribund individual was anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination immediately before necropsy. The terminal body weight at necropsy was obtained from each animal immediately before its sacrifice. The recovery group was sacrificed after 17 weeks from the start of treatment.

GROSS PATHOLOGY: Yes;
- Necropsy: After treatment for 13 weeks, all animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
Adrenals; Aorta; Bone/Bone marrow (sternum, femur including distal joint); Brain (coronal sections of medulla/pons, cerebellar cortex and cerebral cortex); Cecum: Colon; Duodenum; Epididymides; Esophagus; Eyes with optic nerve and Harderian gland; Heart; Ileum; Jejunum; Kidneys; Larynx; Lacrimal gland, exorbital; Liver; Lung infused with formalin; Lymph nodes, mandibular, mesenteric; Mammary gland area; Nasal cavity; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland, mandibular; Sciatic nerve; Seminal vesicles; Skeletal muscle; Skin; Spinal cord, cervical, midthoracic, lumbar; Spleen; stomach; Testes; Thymus; Thyroid gland; Tongue; Trachea; Urinary bladder infused with formailn; uterus; Vagina; Gross lesions.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS: The following organ weights were taken from all animals necropsied at termination of treatment or recovery and their organ to terminal body weight ratios as well as organ to brain weight ratios determined:
Adrenals; Brain; Heart; Kidneys; Liver; Lung; Ovaries; Pituitary gland; Spleen; Testes with epididymides; Thyroid gland; Uterus; Prostate; Thymus; Seminal Vesicles.

HISTOPATHOLOGY: Yes;
- How many animals: Slides of all tissues collected at terminal sacrifice (end of treatment and treatment-free recovery) from the animals or the control and high-dose groups were examined by a board certified veterinary pathologist. The same applied to all occurring Gross Lesions and to all animals which died spontaneously or had to be terminated in extremis. All abnormalities were described and included in the report.
- Fixative used: The tissues were fixed in neutral phosphate buffered 4% formaldehyde solution
- Procedure: Tissues were processed embedded and cut at a thickness of 2 to 4 µm, and stained with hematoxylin and eosin.
- Tissues collected: Adrenal glands, aorta, bone marrow (femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, epididymides, esophagus, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands- mandibular and sublingual, sciatic nerve, seminal vesicles, skin, eyes with optic nerve, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland area, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina. As test substance-related morphologic changes were detected in the organs of high dose animals, the same organs (stomach) from animals of the mid-and low dose groups were examined.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and all ratios as well as clinical laboratory data:
- Univariate one-way analysis of variance was used to assess the significance or intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher test was not applied to the overall spontaneous mortality data nor to the overall ophthalmoscopy data, as there was a considerable mortality rate in group 5 in the absence of mortality in the control group and as only one rat showed an ophthalmoscopic abnormality.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
- Four male and two female rats of group 5 (500 mg a.i./kg bw) died or were killed in extremis between treatment days 27 and 91. The mortality of three of these males and of one of these females was considered to be treatment related.
- However, the causes of death of the other male and female of group 5 and of one male of each of groups 2 (10 mg a.i./kg bw) and 4 (200 mg a.i./kg bw) were found to be intubation errors rather than the test substance itself. No other death occurred.

CLINICAL SIGNS
- There was a dose related increase in incidence and severity of respiratory findings from group 3 (males only) to group 5. The main finding was breathing rales, which was accompanied by dyspnea and labored respiration in some rats of group 5.
- Slight to moderate sedation and emaciation was found in several rats and ruffled fur in all rats of group 5 and considered to reflect some general discomfort.
- All other findings were considered to be toxicologically irrelevant and/or treatment unrelated.
Further details on clinical signs are provided in the study report.

BODY WEIGHT AND WEIGHT GAIN
- No treatment-related effect on the body weight gain of any female rats treated at 10, 50, 200 or 500 mg/kg bw, nor on that of male rats treated at the 10 or 50 mg/kg bw dose levels.
- There was an apparently dose related retardation of body weight gain in male rats of groups 4 (200 mg/kg bw) and of group 5 (500 mg/kg bw). Body weights were statistically significantly lower than controls in males of group 4 at treatment weeks 9, 12 and 13 and in males of group 5 from treatment week 2 until termination of treatment in week 14. This effect lasted on at a statistically significant level in recovery males of group 5 until week 1 of the subsequent recovery period. Afterwards, the male rats of groups 4 and 5 gained weight at a rate similar to that of controls.

FOOD CONSUMPTION
- No effect on food consumption was observed at doses of 10, 50 and 200 mg a.i./kg bw.
- Food consumption of male rats of the 500 mg a.i./kg bw group was statistically significantly lower than that of controls during weeks 1-2 and 5 to 10 and that of females during weeks 1-2, 7-8 and 12-13. Male and occasionally female rats of the 500 mg a.i./kg bw group showed a similar, statistically non-significant trend during the remaining treatment period.
- During the treatment-free recovery period food consumption of the animals of the treatment groups was comparable to that of controls.

RELATIVE FOOD CONSUMPTION
- There was no toxicologically significant effect on the relative food consumption (i.e. food consumption related to body weight) of both sexes of groups 2 (10 mg a.i./kg bw) and 3 (50 mg a.i./kg bw) and of female rats or groups 4 (200 mg a.i./kg bw) and 5 (500 mg a.i./kg bw).
- The relative food consumption of group-4 males was statistically significantly higher than control during treatment week 13, and that of group-5 males during treatment weeks 10-11, 12-13 and 14-15 after having been significantly lower than control during treatment week 1-2. The relative food consumption of group-4 recovery males was statistically significantly higher than control during recovery week 2-3 that of group-5 recovery males throughout the recovery period.
- The findings in group-4 and group-5 males may partly reflect the treatment related retardation of body weight gain described below, and during the recovery period probably also some compensational response.

OPHTHALMOSCOPIC EXAMINATION
- There were no treatment-related effects on any ophthalmoscopy parameters. In the right eye of one female rat in 10 mg a.i./kg bw group, blood vessels were slightly visible at the 4- and 13-week observation points. This finding was considered to be spontaneous and therefore treatment unrelated.
- On recovery day 4, the left eye of one female rat in 50 mg a.i./kg bw group was found to be injured probably from blood collection.

HAEMATOLOGY
- The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment; however, the following effects were noted for the treated rats when compared to the controls:
i) slightly increased erythrocyte count (RBC) by 5% on average for both sexes of group 5 (500 mg a.i./kg bw)
ii) slightly increased hemoglobin concentration (HB) by 4 % on average for males of group 5
iii) slightly increased hematocrit (HCT) value by 6 to 7 % on average for both sexes of group 5 and by 3 % for males of group 4 (200 mg a.i./kg bw)
iv) slightly decreased mean corpuscular hemoglobin concentration (MCHC) by 2 to 4 % on average for both sexes of group 5 and by 2 % for females of group 4
v) slightly increased methemoglobin (MET-HB) concentration by 32 % on average for females of group 5.
- The changes noted reflect slight hemoconcentration and suggest changes in basal fluidity. From a functional point of view, these findings have no hematological implications. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.
- All other statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

CLINICAL CHEMISTRY:
- For clinical biochemical data the following effects were noted for rats of the treated groups at termination of the treatment when compared to the controls:
i) slightly increased uric acid and triglyceride concentration for females of group 5;
ii) slightly increased alanine aminotransferase and alkaline phosphatase activity for males or group 5;
iii) slightly decreased calcium concentration for males of all treated groups;
iv) slightly decreased chloride concentration for both sexes of group 5;
v) slightly increased total protein and globulin concentration, and slightly decreased A/G ratio for females or groups 3, 4 and 5.
- The findings noted were not considered to be of toxicological significance but more likely a reflection of metabolic adaptation due to an increased functional load on the liver. At termination of the treatment-free recovery period these findings were, with the exception of the chloride concentration and the A/G ratio for females of group 5, found to be reversed and comparable to those of the controls.
Further details on clinical chemistry are provided in the study report.
URINALYSIS
- Urinalysis data indicated no changes of toxicological significance at termination of the treatment. The only change noted was a slight increase in the overnight urinary output (Volume/18 hour) by 43 to 63 % on average for both sexes of group 5 (500 mg a.i./kg bw). The finding reflects an increased fluid intake. At termination of the treatment-free recovery period this finding was found to be reversed and comparable to that of the controls.
- All other differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

ORGAN WEIGHTS AND ORGAN WEIGHT RATIO
- After 13 weeks of treatment, the liver weights and their ratios to body weight and brain weight of female rats of group 5 (500 mg a.i./kg bw) as well as the liver to body weight ratio of males of this group were statistically significantly higher than the corresponding controls. These findings were considered to be treatment related, but no longer significant in recovery rats at termination of the 4-week recovery period following treatment.
- Additionally, the kidney to body weight ratios of group-5 females was slightly higher than the respective controls at termination of treatment. This may have been a reflection or their terminal body weights being slightly (statistically non-significantly) lower than that of controls or related to the concomitant slight increase in overnight urinary output.
- All other statistically significant differences noted at termination of the treatment period were considered to be either a reflection of the concomitant low terminal body weights in male rats of group-5 (statistically significantly below controls) or incidental and therefore treatment unrelated. The latter applied also to any statistically significant differences in absolute or relative kidney weights found at termination of the recovery period.
Further details on organ weights and organ weight ratio are provided in the study report.

GROSS PATHOLOGY:
- No treatment related macroscopic findings were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- The premature decedents from groups 2 (10 mg a.i./kg bw) and 4 (200 mg a.i./kg bw) showed lung changes clearly indicative of an accident at dosing, as did one male and one female from group 5 (500 mg a.i./kg bw).
- The remaining group-5 premature decedents gave no microscopic indication of the mechanism of toxicity, but the deaths are considered to be related to treatment. The group-5 survivors were affected by treatment but gave no microscopic evidence of toxicity.
- Several group-5 animals, particularly the premature decedents, showed exudation in the nasal cavity but this was thought to be due to the general poor condition of the animal and was certainly not of sufficient severity to cause death or morbidity. The term exudate was used to represent a range from simple mucous to frankly purulent which may be apparent in the same section. Vegetable matter was sometimes present in the exudate.
- Thymic atrophy which was largely cortical, and lung lesions were seen in some group-5 premature decedents but the changes are thought to be related to the poor general condition of the animals and not specifically treatment-related. Similarly, the small thymic hemorrhages identified are thought to be agonal and of no toxicological significance.
- Adrenocortical rest was seen in a number of animals. This lesion was congenital, consisted of normal cortex and may also be named 'Accessory cortical nodule'. The purpose of reporting this clearly incidental lesion was to ensure that there was no danger of confusion with the potentially significant 'Extruded hyperplastic nodule'. The distinction was easily made as the hyperplastic nodule was not completely surrounded by a fibrous capsule whereas the accessory nodule or rest has a complete capsule.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on mortality, food consumption, body weight and clinical signs observed at 500 mg a.i./kg bw dose group.

Critical effects observed:

not specified

Conclusions:

Glucamide 24 when administered by oral gavage daily for 13 weeks to male and female Wistar rats at dose levels of 0, 10, 50, 200 and 500 mg a.i./kg bw/day, revealed a no observed adverse effect level (NOAEL) at 200 mg a.i./kg bw/day for male and female rats.

Executive summary:

The repeated dose oral toxicity study of Glucamide 24 was performed by following method similar to the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

Male and female Wistar (HanIba, SPF bred) rats obtained from SRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fuellinsdorf were used in this study. The body weight range of animals was 187- 225 g (males) and 151-184 g (females). Animals were individually housed in Makrolon type-3 cages and maintained under controlled environmental conditions (Temperature: 22±3°C, Relative humidity: 40-70%, 10-15 air changes/ hour and 12 hours fluorescent light /12 hours dark). The study consisted of 4 test treatment groups and 1 control group containing 10 rats/sex/group. Test substance formulations were prepared in bi-distilled water. The animals of different groups were treated as follows: 

Group 1: Control (Bi-distilled water)

Group 2: 10 mg/kg bw/day active test substance (equivalent to 22.2 mg/kg bw of test solution)

Group 3: 50 mg/kg bw/day active test substance (equivalent to 111.1 mg/kg bw of test solution)

Group 4: 200 mg/kg bw/day active test substance (equivalent to 444.4 mg/kg bw of test solution)

Group 5: 500 mg/kg bw/day active test substance (equivalent to 1111 mg/kg bw of test solution)

Recovery groups of 10 rats/sex/dose group were also utilized in this study. The recovery group rats were sacrificed after a 4-week recovery period following 13 week treatment with the test substance.

The rats were observed for mortality and clinical signs throughout the study period. Animals were observed for ophthalmoscopic observation at pretest and after 4 and 13 weeks of treatment. Detailed observations, body weights and food consumption were recorded weekly. Urinalysis, hematology and clinical chemistry were performed at study termination prior to sacrifice.

After completion of treatment, all rats were sacrificed by exsanguination under sodium pentobarbitone anesthesia and necropsied. All animals were observed for gross pathological changes during necropsy and tissues were collected to perform histological examination. Absolute and relative organ weights were also determined.

There was no effect on food consumption at 10, 50 and 200 mg a.i./kg bw dose levels. In male and female rats of 500 mg a.i./kg bw, a statistically significant decrease in food consumption was observed when compared with the respective controls. There was no such effect in recovery rats during the treatment-free recovery period following treatment.

There was no toxicologically significant effect on the relative food consumption of either sex of groups 2 and 3, and of female rats of groups 4 and 5. The relative food consumption of group-4 males was statistically significantly higher than control during treatment week 13. Similarly, a statistically significant increase in relative food consumption (when compared to the control group) was observed in group-5 males during treatment weeks 10-11, 12-13 and 14-15 after having been significantly lower than control during treatment week 1-2. The relative food consumption of group-4 recovery males was statistically significantly higher than control during recovery week 2-3 than that of group-5 recovery males throughout the recovery period.

There were no effects on the body weight of any treated female rats or on that of males treated at the 10 or 50 mg a.i./kg bw dose levels. However, the body weight gain appeared to be retarded in a dose- related manner in male rats of groups 4 and 5. The body weights of group-4 males were significantly lower than the respective controls at treatment weeks 9, 12 and 13 and those of group-5 males from treatment week 2 until treatment week 14. This effect lasted in recovery males of group 5until week 1 of the subsequent treatment-free recovery period.

There was a dose related increase in incidence and severity of respiratory findings from group 3 (males only) to group 5 (males and females). The main finding was breathing rales, which was accompanied by dyspnea and labored respiration in some rats of group 5. Slight to moderate sedation and emaciation was found in several rats and ruffled fur in all rats of group 5and considered to reflect some general discomfort. All other findings were considered to be toxicologically irrelevant and/or not related to treatment.

Four male and two female rats of group 5 died or were euthanized in extremis between treatment days 27 and 91. The mortality of three of these males and of one of these females was considered to be treatment related. However, the causes of death of the other male and female of group 5 and of one male of each of groups 2 and 4 were found to be intubation errors rather than the test substance itself. No other death occurred.

There were no treatment related findings on ophthalmoscopy.

The hematological data reflected slight hemoconcentration and changes in basal fluidity. The findings were not considered to be of toxicological significance. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.

The clinical biochemical data noted were not considered to be of toxicological significance but more likely a reflection of metabolic adaptation due to an increased functional load on the liver. At termination of the treatment-free recovery period these findings were, with the exception of the chloride concentration and the A/G ratio for females of group 5, found to be reversed and comparable to those of the controls.

For urinalysis, no changes of toxicological significance were noted at termination of the treatment or at the end of the treatment-free recovery period. The only change noted was a slight increase in the overnight urinary output (Volume/18 hour) for both sexes of group 5 after 13 weeks. This finding was found to be reversed at termination of the treatment-free recovery period. The finding was considered to be related to an increased fluid intake.

After 13 weeks of treatment, the liver weights and their ratios to body weight and brain weight of female rats of group 5as well as the liver to body weight ratio of males of this group were significantly higher than the corresponding controls. These findings were considered to be treatment related, but no longer significant in recovery rats at termination of the 4-week recovery period following treatment.

There were no treatment related necropsy findings identified.

The group-2 and group-4 males which died showed clear microscopic evidence of death being due to an accident at dosing, as did some of the group-5 animals. The premature deaths of the remaining group-5 animals were considered to be treatment related, but there were no histological changes identified to indicate the mechanism of toxicity.

There was no treatment related death or morbidity in group 4 and so the oral administration of the test substance to rats for 13 weeks at the dose level of up to 200 mg a.i./kg bw/day was considered to produce no pathological evidence of toxicity.

Based on above,Glucamide 24 when administered by oral gavage daily for 13 weeks to male and female Wistar rats at dose levels of 0, 10, 50, 200 and 500 mg a.i./kg bw/day, revealed a no observed adverse effect level (NOAEL) at 200 mg a.i./kg bw/day for male and female rats.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH2-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

other: Mortality

Organ:

not specified

Treatment related:

yes

Conclusions:

Potential systemic toxicity of Glucamide OL was investigated in a subacute 28-day toxicity study in compliance with OECD test guideline 407. There were no adverse effects of Glucamide OL found up to the highest dose level tested of 650 mg/kg body weight/ day. No subchronic or chronic data are available with Glucamide OL. However, based on the very consistent toxicity profile of various Glucamides and taking the chemical and biological comparability into account, available 90-day oral toxicity data from Glucamide 24 as source molecule can be used for evaluation purposes using read-across princliples (see also comprehensive read-across justification). Taking a conservative approach, the NOAEL of 200 mg/kg body weight per day revealed in the OECD guideline conform 90-day study with Glucamide 24 is used also for Glucamide OL for assessment considerations.

Executive summary:

Glucamide OL is chemically and biologically equivalent to Glucamide 24, as they deviate only by the number of CH₂-units in the chain length of the respective alkyl moiety and thus read-across of available toxicological data from the source molecule to address data gaps of the target molecule is scientifically possible and toxicologically justified. (see also comprehensive read-across justification).

Repeated dose toxicity studies with the target molecule Glucamide OL (28-day study period) and the source molecule Glucamide 24 (90-day study period) revealed no distinct differences in the systemic toxicity profile despite the variation in carbon chain-lengths. Dominating are unspecific, local effects, predominantly in the forestomach of rats. This points to a concentration dependent irritative mode of action, common for surfactants, leading to downstream effects on feed intake and body weight at higher doses.

The performed read-across is scientifically and toxicologically valid not only based on shared structural and comparable functional similarities, but also due to common metabolic principles valid not just for the discussed source and target molecules, but for the entire class of glucamides.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data reliable and meet criteria for classification & labelling requirements.

System:

gastrointestinal tract

Organ:

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Repeated dose toxicity studies with Glucamide OL (28-day study period) and the read-across compound Glucamide 24 (90-day study period) revealed no distinct differences in the systemic toxicity profile. Dominating are nonspecific clinical effects and local effects, predominantly in the forestomach of rats. These points to a concentration dependent irritative mode of action, common for surfactants, leading to downstream effects on feed intake and body weight at the higher doses.

Additional information

The only primary test item related changes considered to be adverse to the rat were local forestomach effects in the high dose animals having received 500 mg/kg bw/day of the source chemical Glucamide 24. No adverse effects have been observed up to the highest dose of 650 mg/kg body weight per day tested in a 28 -day repeated dose toxicity study with Glucamide OL. 

Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. In assessing the relevance of such findings in rodents, aspects like the method of administration (e.g., gavage versus feeding) and the applicability of the forestomach to human organs (e.g., tissue concordance) have to be considered. Although findings in the forestomach of rats are not considered of biological relevance for humans (EFSA 2011; Proctor et al. 2007), the NOAEL of Glucamide OL following repeated administration to rats was conservatively established at 200 mg/kg bw/day using the subchronic 90 -day study from the source molecule Glucamide 24.

Justification for classification or non-classification

Based on the results from the available repeated dose toxicity data for Glucamide OL which revealed no adverse effects and given that no human relevance is deducible from the effects on the forestomach seen in rats with comparable Glucamides, it is concluded that Glucamide OL is not subject to classification and labelling according to the criteria of GHS and /or EU CLP.