Registration Dossier

Administrative data

Description of key information

Oral administration of the registered substance to Wistar rats at doses of 62.5, 250 and 1000 mg/kg/day, for 28 days resulted in two deaths due to aspiration of dosing formulations reflux, no clinical signs during daily or weekly observations, no findings during the functional observational battery or related tests (grip strength and locomotor activity), minor test item-unrelated differences in the mean daily food consumption of females (males were unaffected), no test item-related differences in mean body weights or their development, no test item-related changes in the hematology, clinical biochemistry or urinalysis parameters of males or females at any dose level, no differences of toxicological relevance in the mean absolute and relative organ weights. There were no late test item-related macroscopical changes in males or females after 4 weeks of treatment and 2 weeks of recovery.

Test item-related findings were generally restricted to microscopic changes in the forestomach of animals at 1000 mg/kg/day. These lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. These changes are considered to be `site of first contact` and thus local effects due to irritation rather than systemic toxic effects and are not considered relevant for human health hazard / risk assessment. After the 14 days recovery period, these changes were completely reversible. Comparable forestomach effects were not seen in the rats from the low- and mid-dose groups.

Since the forestomach effects are considered to be `site of first contact` and thus local effects which are not relevant for human hazard / risk assessment, the NOAEL of the registered substance with regard to systemic toxicity was established at 1000 mg/kg body weight per day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Hostapon SG and acylglycinate GCK-12H-T are structurally closely related UVCB substances, which differ predominantly in the counterion (sodium resp. potassium). According to the available experimental studies, both substances exhibit results in a comparable range. Therefore, it can be assumed that the results obtained with Hostapon SG for this specific endpoint also apply to acylglycinate GCK-12H-T.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
swissmedic; date of decision: 19-November-2010
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories B.V.,Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals:
Group 1: 10 males and 10 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 10 males and 10 females
Group 10: 1 male and 1 female
Age (at Delivery): Ca. 7 weeks
Body Weight Range
(at Acclimatization):
Males: 199 to 233 g (mean 214 g)
Females: 133 to 154 g (mean 144 g)
Identification:
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Randomly allocated to groups by body weight.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS

Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).

Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

Water: Community tap-water from Itingen was available ad libitum in water bottles.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method of administration: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily.
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 62.5 mg/kg/day
Group 3: 250 mg/kg/day
Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D43306.
Dose Volume: 10 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6.25 mg/mL/day
Group 3: 25 mg/mL/day
Group 4: 100 mg/mL/day
Duration of Pre-Randomization Phase: 1 day
Duration of Acclimatization Phase: At least 5 days
Duration of Treatment Phase: 28 days
Duration of Recovery Phase: 14 days

PREPARATION OF DOSING SOLUTIONS:
-For the purpose of this study the test material was prepared at the appropriate concentrations
as a solution in highly purified water.

VEHICLE
-Highly purified water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.
After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days).
The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The test item was used as analytical standard.
Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director after acceptance of the draft report.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
once daily on 7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 10 males and 10 females at 0 mg/kg bw/day
Group 2: 5 males and 5 females at 62.5 mg/kg bw/day
Group 3: 5 males and 5 females at 250 mg/kg bw/day
Group 4: 10 males and 10 females at 1000 mg/kg bw/day
Group 10: 1 male and 1 female (reserve animals)

Reserve animals were exchanged as needed during the acclimatization period and then removed from the study. Any raw data collected during the acclimatization period on reserve animals were not reported but retained in the raw data.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D43306.
Dose Volume: 10 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6.25 mg/mL/day
Group 3: 25 mg/mL/day
Group 4: 100 mg/mL/day
Duration of Pre-Randomization Phase: 1 day
Duration of Acclimatization Phase: At least 5 days
Duration of Treatment Phase: 28 days
Duration of Recovery Phase: 14 days
Positive control:
None
Observations and examinations performed and frequency:
Viability / Mortality: Observations for viability / mortality were recorded twice daily.
Daily Observations: The animals were observed daily for clinical signs before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period, and once daily during days 1 to 14 of the recovery period.
Weekly Behavioral Observations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.
Functional Observational Battery (Screen): During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results are present in the summary and individual tables of the weekly detailed clinical observations under week 4.
Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.
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 balance connected to the Harlan Laboratories computer.
Body Weights: Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

Clinical Laboratory Investigations
Blood and Urine Sampling:
After 4 Weeks: 20-Feb-2012 (allocation A and B)
After 6 Weeks: 05-Mar-2012 (allocation B)
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

Hematology
The following hematology parameters were determined:
-Complete Blood Cell Count
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium, high fluorescence)
Leukocyte count, total
Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count

-Hemoglobin Derivatives

Methemoglobin Heinz bodies (slides were prepared but not evaluated)
Coagulation

Prothrombin time (= Thromboplastin time) Activated partial Thromboplastin time

-Clinical Biochemistry
The following clinical biochemistry parameters were determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Alkaline phosphatase
Bile acids Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

-Urinalysis
The following urine parameters were determined:

-Physical Examination

Urine volume (18 hour)
Specific gravity (relative density)
Color
Appearance

-Chemical Examination

pH value
Nitrite
Protein
Glucose
Ketones Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

-Necropsy
Sacrifice:

After 4 Weeks: 20-Feb-2012 (allocation A)
After 6 Weeks: 05-Mar-2012 (allocation B)

All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

A blood sample was taken from each animal by heart puncture (ca. 2 mL) into appropriate tubes (Vacutainer glass tubes containing SST-Gel and Clot Activator or the equivalent) for serum preparation and then placed on ice or cool plates. Following centrifugation, the serum was divided in 2 aliquots of at least 350 µL and transferred to plastic (polypropylene) tubes. These samples were stored at approximately -80 °C and protected from light in case they were needed for analysis of hormone activity.

-Organ Weights
The organs from allocation A and B animals were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

-Histotechnique
All organ and tissue samples, as defined under Histopathology , were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

-Histopathology
Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Liver. Lungs and stomach of animals from the low and middle dose groups were also evaluated. Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.

A description of all abnormalities is attached (see Appendix VI ). Attempts were made to correlate gross observations with microscopic findings.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
-Viability / Mortality
- Organ Weights
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Only microscopic changes in the forestomach of animals at the limit dose of 1000 mg/kg body weight. These findings are not considered relevant for human health hazard / risk assessment.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, 250 mg/kg body weight/day of the registered substance was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of Hostapon SG dried as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

Oral administration of the registered substance to Wistar rats at doses of 62.5, 250 and 1000 mg/kg/day, for 28 days resulted in two deaths due to aspiration of dosing formulations reflux, no clinical signs during daily or weekly observations, no findings during the functional observational battery or related tests (grip strength and locomotor activity), minor test item-unrelated differences in the mean daily food consumption of females (males were unaffected), no test item-related differences in mean body weights or their development, no test item-related changes in the hematology, clinical biochemistry or urinalysis parameters of males or females at any dose level, no differences of toxicological relevance in the mean absolute and relative organ weights. There were no late test item-related macroscopical changes in males or females after 4 weeks of treatment and 2 weeks of recovery.

Test item-related findings were generally restricted to microscopic changes in the forestomach of animals at 1000 mg/kg/day. These lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. These changes are considered to be `site of first contact` and thus local effects due to irritation rather than systemic toxic effects and are not considered relevant for human health hazard / risk assessment. After the 14 days recovery period, these changes were completely reversible. Comparable forestomach effects were not seen in the rats from the low- and mid-dose groups.

Since the forestomach effects are considered to be `site of first contact` and thus local effects which are not relevant for human hazard / risk assessment, the NOAEL of the registered substance with regard to systemic toxicity was established at 1000 mg/kg body weight per day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The test item “Hostapon SG” was tested in a 28 day study in rats with oral dosing of 0, 62.5, 250 and 1000 mg/kg via gavage. Even at the highest dose level no adverse effects were reported and the NOAEL with regard to systemic toxicity was established at 1000 mg/kg body weight per day. Therefore, there is no need for classification and labelling of the test item according to CLP Regulation 1272/2008/EG.