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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other:

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
No guideline or details of method given in the publication
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
none given

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Species: Rat
Strain: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Age at dosing: 6-8 wks
Weight at dosing: Males: 266.1-339.5g; Females: 186.2-246.0g
Source: Charles River Laboratories (Kent, UK)
Acclimation period: 7 days
Diet: Certified rodent diet 5003, ad libitum
Water: Municipal water, ad libitum
Housing: 2-3/sex

Environmental conditions:
Temperature: 19-23°C
Functional observation battery animals: 22.0-22.7°C
Humidity: 45-65%
Air changes: 15-20 air changes/h
Photoperiod: 12 h light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
After an acclimatisation period of ca. 7 days, rats were allocated to groups by computer-based stratified random sampling. The test article, MeTHF was administered orally via gavage to groups of rats for a period of 90 d. Animals (10/sex/gp), recieved a single dose daily at concentrations of 0, 80, 250, 500, 1000 mgkg bw/d. A further group of rats (5/sex/group) which received the control and high dose group were retained for a further month free of administration of test article, for a 1 month recovery period. Following 90 d of treatment (or 1 month post dosing) animals were subjected to complete necropsy. Body weight, food consumption were measured at regular intervals. Clinical pathology evaluations (haematology, clinical chemistry and urinalysis) were performed with all surviving animals subjected to complete gross necropsy and full histopathology.
Vehicle:
water
Details on oral exposure:
Animals recieved a single oral gavage dose of test article formulated in water at 0, 80, 250, 500 or 1000 mg/kg bw/d, employing a dose volume of 10 mL/kg bw. The formulation was stble for up to 18 d when refrigerated (2-8°C), therefore dosing solutions were prepared on 9 occasions.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Samples of the test article and vehicle control formulations were taken from formulations prepared for dosing day 1 and during wks 5 and 12. All formulation prepared were within the limits of acceptability (90-110% of nomnal).
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
water
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main study: 10/sex/gp
Recovery period: 5/sex/gp (control and high dose only)
Control animals:
yes, concurrent vehicle
not specified
Details on study design:
refer above
Positive control:
n/a

Examinations

Observations and examinations performed and frequency:
Animals were inspected once daily during the pre-treatment period, pre-dose and again 4 h post dosing. During the recovery period animals were assessed daily.
Sacrifice and pathology:

Organ weights: Adrenal glands, brain, epididymides, heart, kidney, liver, ovary, prostate, spleen, thymus, testis
Sacrifice and pathology: Gross pathological examination was performed on all animals and included examination of the external surface, all orifices and associated tissues.
The following tissues were preserved in 10% neutral buffered formalin for subsequent histopathological examination (with the exception of eyes and testes which were fixed with a formaldehyde-glutaraldehyde solution and Modified Davidson's solution, respectively) and performed on control and high dose group animals. The liver, pituitary, thyroids and adrenal glands which showed changes at the high dose were also examined at the mid and low dose groups:
Accessory sex glands (¿:epididymides, prostate, seminal vesicle (coagulating gland), testes; ¿: ovary, oviduct, uterus, vagina), aorta, bone (sternum, femur + marrow), brain, eyes (+optic nerve & Harderian gland), gastrointestinal tract (oesophagus, stomach, intestine (caecum, colon duodenum, ileum, jejunum, rectum)), heart, kidney, knee joint, lacrimal gland (extraorbital), liver, lymph nodes (inguinofemoral), respiratory system (larynx, lung), salivary glands, skeletal muscle, skin, spinal cord, spleen, thymus, urinary bladder
Other endocrine producing/sensitive glands (adrenals, mammary gland (¿ only), pancreas, pituitary, thyroid (+parathyroid))
Other examinations:
Body weights: Animals were weighed at initiation of dosing and weekly during administration and at study termination.
Food consumption: weekly
Water consumption: daily
Ophthalmological examination: Eyes were examined before administration (all animals) and in wk 5 and 12 (control and high dose animals)
Neurological functional examinations: Not conducted

Haematology and clinical chemistry:
Conducted during wks 5/6 and the end of the treatment period (wk 13). Animals were fasted overnight prior to blood sampling.
Haematology: red blood cell parameters (haematocrit (commonly termed PCV), haemoglobin concentration (Hb), mean haemoglobin concentration (MHC), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), erythrocyte count, platelet count, reticulocyte count), white blood cell parameters (total and differential (neutrophils, lymphocytes, eosinophils, basophils, monocytes) leukocyte count), coagulation parameters (activated partial thromboplastin time (APTT), fibrinogen, prothrombin time (PT)).

Clinical chemistry: electrolytes (sodium, potassium, calcium, chloride, inorganic phosphorus), kidney function test (creatinine, urea), glucose, liver function tests (albumin, globulin, albumin:globulin, alkaline phosphatase (ALP), alanine aminotransferase (ALT [commonly referred to as glutamic pyruvic transaminase (GPT)]), aspartate aminotransferase (AST [commonly referred to as glutamic oxaloacetic transaminase (GOT), ¿-glutamyl transferase (¿-GT)]), total bilirubin (T.Bili), total protein (TP), lipid profile (total cholesterol)

Urinalysis:
Conducted during wks 5/ from main study and recovery animals and the end of the treatment period (wk 13). Animals housed in metabolism cages for 24 h. During collection, animals were given water, but food was withheld. The following urinary parameters were measured: specific gravity, pH, total volume, protein, glucose, ketones, bilirubin, blood, urobilinogen, leukocytes, colour, sediment
Statistics:
Conducted separately for each sex. Each parameter was analysed either parametrically or non-parametrically. Testing for each parameter was either one-sided for increases or decreases, or two-sided. To compare each dose group to the control group, sequential trend tests were performed.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
increased incidence of salivation were observed pre-dose or immediately post dose at 500 and 1000 mg/kg bw/d. The frequency and incidence increased with dose, and is commonly assoicated with test article that have taste aversion.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
3 premature deaths were observed during the course of the study. 1 female (500 mg/kg bw/d) on day 1 and 1 male on day 40 (1000 mg/kg bw/d) were euthanised following a dosing error. On day 37, 1 female (80 mg/kg bw/d) did not recover from anaethesia following the clinical pathology blood sample collections. These deaths were incidence and not test article related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: reduction in bwt was observed in all dose gps, including the controls in wks 5/6 and wk 13. This transient drop in bwt was considered due to the wk 13 bwt being collected shortly after the animals had been fasted overnight, and therefore not considered test article related.
A slight decrease in overall bwt gain was noted for both male and female animals when compared to the control gp, and was most evident for males dosed at 1000 mgkg bw/d (15.7 and 13.4% lower than controls for males and females, respectively). Evidence of reversibility was oted during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
effects observed, non-treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in serum cholesterol were observed in both sexes at 1000 mg/kg bw/d (14.4 - 70.3% above the concurrent control) in wks 6 and 13. Complete reversibility was noted at the end of the recovery period.
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article related absolute and relative kidney weight were observed at dose levels of 500 mg/kg bw/d and greater in both sexes (4-20% above concurrent controls). In the absence of histopathological changes, these changes were considered to be of no toxicological concern.

Increased absolute and relative liver weights were observed in both sexes at dose levels of 500 mg/kg bw/d and greater in both sexes (9-32% above concurrent controls). There were no test article related differences in organ weights for recovery animals. All other organ weights were considered to be within normal ranges.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological effects were limited to the liver, which were deemed test article related. Hepatocellular centrilobular hypertrophy was present in main study animals at 1000 mg/kg bw/d animals at a minimal grade in 8/10 males and 6/10 females and a mild grade in 1/10 males. Therefore were no microscopic effects observed at 250 or 500 mg/kg bw/d treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the NOAEL is deemed to be 250 mg/kg bw/day for both males and females based on increased absolute and relative liver weights (at equal to and greater than 500 mg/kg bw/day), with associated histopathology (hepatocellular centrilobular hypertrophy at 1000 mg/kg bw/day).
Executive summary:

In this study, MeTHF was administered orally via gavage for 90 days to Sprague Dawley rats. Animals (10/sex/group) were administered concentrations, once daily at 0 (water), 80, 250, 500 and 1000 mg/kg bw/day. A further group of rats (5/sex/group) which received the control and high dose group were retained for a further month free of administration of test article, for a 1 month recovery period. Following 90 d of treatment (or 1 month post dosing) animals were subjected to complete necropsy. Body weight, food consumption were measured at regular intervals. Clinical pathology evaluations (haematology, clinical chemistry and urinalysis) were performed with all surviving animals subjected to complete gross necropsy and full histopathology.

Increases in serum cholesterol were observed in both sexes at 1000 mg/kg bw/d (14.4 - 70.3% above the concurrent control) in weeks 6 and 13. Complete reversibility was noted at the end of the recovery period.

Test article related absolute and relative kidney weight were observed at dose levels of 500 mg/kg bw/d and greater in both sexes (4-20% above concurrent controls). In the absence of histopathological changes, these changes were considered to be of no toxicological concern.

Histopathological effects were limited to the liver, which were deemed test article related. Hepatocellular centrilobular hypertrophy was present in main study animals at 1000 mg/kg bw/d animals at a minimal grade in 8/10 males and 6/10 females and a mild grade in 1/10 males. Therefore were no microscopic effects observed at 250 or 500 mg/kg bw/d treated animals.

Increased absolute and relative liver weights were observed in both sexes at dose levels of 500 mg/kg bw/d and greater in both sexes (9-32% above concurrent controls). There were no test article related differences in organ weights for recovery animals. All other organ weights were considered to be within normal ranges.

Under the conditions of this study the NOAEL is deemedto be 250 mg/kg bw/day for both males and females based on increased absolute and relative liver weights (at qual to and greater than 500 mg/kg bw/day), with associated histopathology (hepatocellular centrilobular hypertrophy at 1000 mg/kg bw/day).