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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
The toxicity of the test item Macrolex Rot E2G (CAS-No. 6829-22-7) following daily oral (gavage) administration for 4 weeks in the rat was determined.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity/Content: 99.9 %.
Appearance: Dark red powder.

Test animals

Species:
rat
Strain:
other: Wistar rats: Crl: WI (Han)
Details on species / strain selection:
One of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
Test system
Species/strain: Wistar rats: Crl: WI (Han)
Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
Number of animals in the study: 40 (20 males and 20 females). Four spare animals (2 males and 2 females) were purchased. As not used in this study, they were returned to stock
Age at initiation of treatment: Approximately 6 weeks.
Body weight range at initiation of - males: 148 to 176 g
treatment: - females: 137 to 153 g

Animal husbandry
Housing: One air-conditioned room in a barrier protected unit
Temperature: 22 + 3 °C (target range)
Relative humidity: 35 to 70 % (target range)
Air changes: At least 10 air changes per hour
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts)

Caging: Animals were housed in groups of 5 of the same sex and dose group in plastic cages with sawdust bedding, in compliance with European Regulations (Directive 2010/63/EU).
Bedding: Dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants. A small amount (handful) of shredded paper was provided as enrichment.
Diet: Rat pelleted complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
The animals were fasted for approximately 16 hours before clinical laboratory blood sampling, during urine collection and before necropsy.
Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system). Water is analysed twice a year for chemical and bacterial contaminants.
Animals were deprived of water during urine collection
Contaminants: No known contaminants were present in the bedding, drinking water or diet at levels which might have interfered with achieving the objectives of the study

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % (w/v) Carboxymethylcellulose, 400-800 centipoises.
Details on oral exposure:
Vehicle
Denomination: 1 % (w/v) Carboxymethylcellulose 400-800 centipoises.
Supplier: Sigma.
Storage: At room temperature (between +15 and +25 °C)
Water for injection: Supplier: Aguettant.
Storage: At room temperature (between +15 and +25 °C)
Frequency of preparation: Weekly.
Storage: At room temperature (between +15 and +25 °C)

Test item preparation
Preparation: The test item was prepared as a suspension in the vehicle at concentrations of 10, 30 and 100 mg/mL
Correction factor for active
ingredient: None.
Homogenecity of test item in
vehicle: Suspensions at concentrations of 1.00 to 100 mg/mL have been shown to be homogeneous
Stability of the test item in the
vehicle: Suspensions at concentrations of 1.00 to 100 mg/mL have been demonstrated to be stable for 24 hours and 8 days at room temperature (between +15 and +25 °C) protected from light or refrigerated
(between +2 and +8 °C)
Frequency of preparation: Weekly.
Storage of formulations: At room temperature (between +15 and +25 °C).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of preparations:
Four samples of 1 g were taken from each formulation including the control, used on the first day of treatment and on a suitable day during the last week of treatment. The samples were stored at room temperature (between +15 and +25 °C) until analysis.
One set of samples was analysed at the Test Facility within the stability period using a validated method.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males + 5 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for the dose selection: in preliminary study AB20659 conducted in the rat, the test item administered at the dose level of 500 and 1000 mg/kg bw/day for 5 days was well tolerated.

Examinations

Observations and examinations performed and frequency:
Morbidity/mortality, clinical signs, body weight, food consumpton, modified Irwin test (neurological function assessment), haematology, serum clinical chemistry, urine analysis
Sacrifice and pathology:
Pathology
On the day after the last dose (i.e. day 28), the animals were necropsied randomized. The animals were fasted overnight before necropsy.
The animals were killed by carbon dioxide inhalation and weighed and then exsanguinated.

All animals were submitted to full necropsy procedures including examination of the following:
external surface
all orifices
cranial cavity
the carcass
external surface of the brain and cervical spinal cord
thoracic and abdominal cavities and organs
cervical tissues and organs

Organ weights
The organs listed in the organ processing table were weighed at necropsy for all animals. Paired organs were weighed together.
Organs were weighed after dissection of fat and other contiguous tissues.
Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight).

Histopathology
The following organs/tissues for all animals in groups 1 (control) and 4 (high dose) killed at the end of the treatment period, were examined histologically:
Adrenal glands, Aorta, Bone (femur) and articulation , Bone (sternum) with bone marrow, Bone marrow smears, Brain, Bronchi (mainstem) , Caecum , Cervix, Colon, Duodenum , Epididymides, Eyes, Harderian glands, Heart, Ileum with Peyer‘s patches, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (mandibular), Lymph node (mesenteric), Mammary gland , Nasal cavity and Zymbal’s glands, Oesophagus, Optic nerves, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular, parotid, sublingual), Sciatic nerve (left), Seminal vesicles including coagulation gland, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Testes, Thymus, Thyroid glands, Tongue, Trachea, Urinary bladder, Uterus, Vagina.
Statistics:
The following parameters were analysed statistically on each occasion for males and females separately:
− body weights and body weight gains
− haematology, coagulation and serum clinical chemistry parameters, urine volume, specific gravity and urine pH
− terminal body weights, absolute and relative organ weights.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs were noted.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight was not affected by the administration of the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by the administration of the test item.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted in haematological parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted in blood biochemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related effects were noted in urinary parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
After 4 weeks of treatment, when compared with the vehicle control group, no relevant effect was noted on the behaviour and physiological parameters of the animals observed 1 hour after dosing with Macrolex Rot E2G at 100, 300 or 1000 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross observations that were considered to be test item-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histological observations that were considered to be test item-related.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no histological observations that were considered to be test item-related.
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related pathological changes.

Effect levels

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:
other: No test item-related in vivo effects and no test item-related histopathological changes were observed at the highest applied dose (1000 mg/kg bw/day).

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

No test item-related in vivo effects and no test item-related histopathological changes were observed.

The daily oral administration of Macrolex Rot E2G for 28 days to Wistar rats at the dose levels of 100, 300 or 1000 mg/kg bw/day did not induce any effects during the in vivo phase. No test item related histopathological changes were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered as a No Observed Adverse Effect Level (NOAEL).

Applicant's summary and conclusion

Conclusions:
The daily oral administration of Macrolex Rot E2G for 28 days to Wistar rats at the dose levels of 100, 300 or 1000 mg/kg bw/day did not induce any effects during the in vivo phase. No test item related histopathological changes were noted.
Executive summary:

The test item Macrolex Rot E2G (CAS-Nr. 6829-22-7) was applied daily per gavage for 4 consecutive weeks in doses of 0 (control), 100, 300 or 1000 mg/kg bw to male and female rats.

Morbidity/mortality checks were performed at least twice daily. Clinical observations were performed daily. A full clinical examination was performed weekly. Individual body weights were recorded weekly. Food consumption was measured weekly for each cage of animals. Modified Irwin test (Neurological Function Assessment) was performed pretest and in week 4. Clinical laboratory determinations were performed in week 4.

All animals were killed at the end of the treatment period and necropsied. Selected organs were weighed. Organ/tissue samples were fixed and preserved at necropsy for all animals. Selected organs/tissues from group 1 (0 mg/kg bw/day) and 4 (1000 mg/kg bw/day) animals killed at the end of the treatment period were examined histopathologically.

No test item-related in vivo effects and no test item-related histopathological changes were observed.

The daily oral administration of Macrolex Rot E2G for 28 days to Wistar rats at the dose levels of 100, 300 or 1000 mg/kg bw/day did not induce any effects during the in vivo phase. No test item related histopathological changes were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered as a No Observed Adverse Effect Level (NOAEL).