Registration Dossier

Administrative data

Description of key information

Repeat dose oral:

A very limited published report of a three-month oral study in rats, conducted to GLP but with no stated guideline, found no toxicity at the highest tested dose of 26 mg/kg bw/day.

 

In a robust, 90-day oral gavage study in rats, MeTHF was administered once daily at dose levels of 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 dose levels equal to and greater than 500 mg.kg bw/day), with associated histopathology (hepatocellular centrilobular hypertrophy at 1000 mg/kg bw/day).

 

Repeat dose inhalation:

The potential repeat dose toxicity of MeTHF following sub-chronic (90 day) repeat dose inhalation exposure to young male and female Han Wistar rats (10 animals/sex/group) was investigated. Following a range-finder assessment, MeTHF was administered via nose only inhalation at doses of 0, 2, 4.5, 10 mg/L, 6 hours/day, 5 (weeks 1 to 12) or 7 (week 13) days a week.

 

Under the conditions of this study the NOAEC is deemed to be >9.96 mg/L (deemed to be the maximum tolerated dose) in males and females based on no adverse effects observed. Clinical signs (abnormal gait, salivation and higher low beam activity scores) and a slight effect on body weight, food consumption and water consumption was seen for animals exposed to 9.96 mg/L. In addition, irregular oestrous cycles were seen in all treated groups and a shift in the regular cycle length, from 4 to 5 days, was observed for females exposed to 9.96 mg/L. All changes were considered non-adverse. There were no microscopic changes attributable to the test item.

 

Repeat dose dermal:

No study has been conducted.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
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
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
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
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
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.
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
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
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
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).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
KL.1
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2018 to 20 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test Material: MeTHF (2-Methyltetrahydrofuran)
Description: Colorless liquid
Lot/Batch No.: 2-7E25S
Purity: 99.98%
CAS No.: 96-47-9
Stability of test compound: Confirmed stable for the duration of the study (25 May 2019 (two years from date of manufacture, manufacture date: 25 May 2017))
Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Age: 9 -11 wks at dosing
Weight at dosing: M: 232-294g; F: 172-194g
Source: Envigo RMS Limited, UK
Acclimation period: 11 days
Diet: Teklad 2014C Diet, ad libitum (except overnight before blood sampling for hematology or blood chemistry and during exposure)
Water: Municipal water, ad libitum (except during exposure)
Housing: Groups of 5 of the same sex

Environmental conditions:
Temperature: 20-24°C
Humidity: 40-70%
Air changes: not stated
Photoperiod: 12 hours light/dark
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only chamber. Aluminium alloy construction comprising a base unit, three animal exposure
sections, a top section and a pre-chamber
- Method of holding animals in test chamber: animals were restrained in a plastic snout-only tube. Animals on study were acclimatized to the method of restraint for three consecutive days preceding the first test item exposure
- Source and rate of air: From in-house compressed air system at 19 L/min
- Method of conditioning air: filtered
- System of generating particulates/aerosols: Glass sintered vaporizer. The test item was supplied to the generator, via a feed line, from a syringe (all glass) driven at a constant rate by a syringe pump (KD Scientific)
- Temperature, humidity, pressure in air chamber: refer to Table 7.5.2/01-1
- Air flow rate: 19 L/min
- Method of particle size determination:
- Treatment of exhaust air: Drawn by in-house vacuum system at 20 L/min

TEST ATMOSPHERE
- Brief description of analytical method used:
Vapor samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler) set up in series
Sample solvent: Acetonitrile
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: Minimum of 1 sample from Group 1/day (taken at approximately 60 minutes during exposure) 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test sample (solvent trap collected in acetonitrile): Transfer to volumetric flask using acetonitrile to provide a solution containing MeTHF at a nominal concentration in the range 200 – 2000 µg/mL. Inject onto the GC. The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis.
Duration of treatment / exposure:
6 h/d, 5 days/week (weeks 1-12) or 7 days/week (week 13)
Frequency of treatment:
either 5 days/week or 7 days/week
Dose / conc.:
0 mg/L air (analytical)
Dose / conc.:
2.07 mg/L air (analytical)
Dose / conc.:
4.62 mg/L air (analytical)
Dose / conc.:
9.96 mg/L air (analytical)
Dose / conc.:
0 mg/L air
Dose / conc.:
2 mg/L air
Dose / conc.:
4.5 mg/L air
Dose / conc.:
10 mg/L air
No. of animals per sex per dose:
10/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
The conduct of the study generally followed OECD 413 (2017). Four groups of 10 male and 10 female Han Wistar rats (randomised by bodyweight) received the test item by nose only inhalation, exposed daily for 6 h/d, 5 days/week (weeks 1-12) or 7 days/week (week 13) at target concentrations of 0, 2.0, 4.5, 10 mg/L, with different dose levels achieved by varying the concentration of test item in the exposure system, whilst keeping the duraation of exposure constant. A vehicle control (air ) group was also included. Body weight gain, water and food consumption were measured at regular intervals.

All animals were subjected to ophthalmoscopy, haematology, clinical chemistry (including thyroid hormones), sperm analysis, oestrus cycle, FOB assessment, organ weights, and macropathology. Histopathological examinations were carried out on the negative and vehicle control and high dose animals.
Positive control:
n/a
Observations and examinations performed and frequency:
1.Observations: Mortality and moribundity: observed twice daily, once in the morning and once in the afternoon
Clinical observations: recorded for all animals twice daily. A detailed physical examination was performed weekly. From week 11, arena observations were also performed, the examinations were performed at approximately the same time of day (before dosing), by an observer unaware of the experimental group identities (refer below to point 5 below)

2. Body weights: Recorded twice weekly, starting 1 week prior to dose administration. Body weight was also recorded on the first day of dosing.

3. Food consumption: Recorded weekly.

4. Water consumption: Daily during Weeks 3 to 13 of treatment; measured per cage.

5. Functional observation battery (FOB): Recorded for all animals during each week from week 11. Observations included:
- Detailed physical examinations. after removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
- Sensory reactivity and grip strength: sensory reactivity and grip strength assessments were performed on all animals during Week 12 of treatment. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:
- Approach response: a blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction
- Pinna reflex: the inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response
- Auditory startle reflex: the animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor
- Tail pinch response: The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression
- Grip strength: forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

6. Locomotor activity:
- Motor activity: During Week 12 of treatment, the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

7. Ophthalmic examination: All animals examined pre-treatment, with the control and high dose groups examined prior to necropsy. The adrexae, conjunctivae, cornea, sclera, anterior chamber, iris (pupils dilated), lens, vitreous and fundus were examined.

8. Haematology and clinical chemistry: Conducted in week 13 of treatment. Sampling was from the sublingual vein.
- Haematology: red blood cell parameters (haematocrit, haemoglobin concentration, mean cell haemoglobin concentration, mean cell haemoglobin, mean cell volume, erythrocyte count, red cell distribution), white blood cell parameters (total and differential (neutrophils, lymphocytes, eosinophils, basophils, monocytes,large unstained cells) leukocyte count), clotting parameters (platelet count, prothrombin, activated partial thromboplastin time).
- Clinical chemistry: electrolytes (sodium, potassium, calcium, chloride, inorganic phosphorus), urea, glucose, creatinine, liver function tests (alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T.Bili), total protein, albumin/globulin),lipid profile (total triglyceride, total cholesterol), thyroid function (triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH))

9. Urinalysis: Not conducted

10. Oestrous cycle: Vaginal smears performed for 14 days during weeks 12 and 13 using cotton swabs.
Sacrifice and pathology:
11. Gross pathology: Gross pathological examination was performed on all animals and included examination of the external surface and all orifices.

12. Histopathology : The following tissues were preserved in 4% formaldehyde for subsequent histopathological examination (with the exception of eyes and optic nerve which were preserved in Davidson's fluid) nd the testes which were preserved in modified Davidson's fluid: Adrenals, aorta (thoracic), brain (including sections of cerebrum, cerebellum, and medulla/pons), bone marrow, digestive system (caecum, colon, duodenum, epiglottis, oesophagus, ileum (including Peyer's patches), jejunum, rectum, stomach, tongue), eyes (+optic nerves, lachrymal and Harderian glands), femoral bone (+marrow), heart (including auricular and ventricular regions), kidneys, liver (2 lobes), lymph nodes (mesenteric, tracheo-bronchial, left axillary, hilar), respiratory system (larynx ( 5 sections), lungs (including bronchi), nasal turbinates (4 levels including teeth), trachea), (accessory) sex organs (epididymides, ovaries, prostate, seminal vesicles, testes, uterus (with cervix), vagina), pancreas, pituitary, salivary glands (submandibular/sublingual), sciatic nerves, skeletal muscle, skin + mammary gland (inguinal area), spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, sternal bone and marrow, thymus, thyroid (+parathyroids), urinary bladder. Histopathological analysis was conducted for all listed tissues, with the exception of eye, Harderian gland, lachrymal gland, mesenteric lymph node, optic nerve, left sciatic nerve and the tongue.
The right lung was used for Bronchoalveolar Lavage (BAL) sampling and the left lung was processed for histology and light microscopy.

13. Organ weights: The following organs were trimmed of adherent tissue and weighed: adrenals, brain, epididymides (left and right), heart, kidneys, liver, lungs (including bronchi), ovaries, spleen, testes (left and right), thymus, thyroids (+parathyroids), uterus (+cervix).

14. Bone marrow smears: Bone marrow smears were prepared, for all animals, immediately following death. Smears were air dried and subsequently fixed in methanol. No examinations were performed, however, the smears were retained for possible future examination.

15. Sperm analysis: Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
-Sperm mortality: A sample of sperm was expressed from the vas deferens and, where possible, at least 200 sperm per animal analysed.
-Sperm morphology: An aliquot of the sperm/medium mixture was diluted with 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male of groups 1 and 4, where possible.
-Sperm count: The left cauda epididymis of each male for groups 1 and 4 was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portion was allowed to thaw then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
-Homogenisation resistant spermatid count: After removal of the tunica, the left testis of each male for groups 1 and 4 was frozen. Prior to analysis, the testis was allowed to thaw then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT
stain tube before being assessed for homogenisation-resistant spermatid count using CASA.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
Grip strength and motor activity, body weight (using gains over appropriate study periods), haematology, blood chemistry, sperm analysis, oestrous cycles, organ weights (absolute and adjusted for terminal body weight).

For grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests were performed.

For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups, using scores appropriate to the severity of the observation assuming 4 day cycles to be normal.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs during the detailed weekly physical examination.

Unsteady gait was observed for all Group 4 animals on return to the home cage, in addition excessive salivation was noted, on occasion. These signs, where observed, had typically resolved by the end of day observation.

Clinical signs associated with the dosing procedure included wet fur and red staining (of the head, nose or eyes), on occasion, on return to cage for all groups including control and were considered to be associated with the method of restraint.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weight gains were apparent for males exposed to 9.96 mg/L when compared with the control group (0.85X control). There were no effects for males exposed to 2.07 or 4.62 mg/L. The lower body weight gain for males exposed to 9.96 mg/L was not associated with any effect on food consumption and was inconsistent with the effect for females and was therefore considered incidental.

Body weight gains for all treated female groups were variable. No body weight gain was apparent for females exposed to 9.96 mg/L following the first week of exposures, thereafter higher gains were observed resulting in overall higher mean gain (1.4X control after 13 weeks). Gains for females exposed to 2.07 mg/L were also higher than control (1.3X control), whilst gains for females exposed to 4.62 mg/L were similar to control (1.1Xcontrol). The higher body weight gain for females exposed to 9.96 mg/L correlated with the slight increase in food consumption for this group. The magnitude of the changes was low and therefore these changes were considered non-adverse.
(refer to Table 7.5.2/01-2)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Weekly food consumption was slightly higher for females exposed to 9.96 mg/L when compared with control. There were no apparent effects for females exposed to 2.07 or 4.62 mg/L or treated males. This however was considered non-adverse and was inconsistent with the effect for males and was therefore considered incidental.
Food efficiency:
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption measurements commenced from Week 3. Higher weekly water consumption was observed for both sexes exposed to 9.96 mg/L when compared with control, with less of an effect from Week 9. This however was considered non-adverse due to the low magnitude of the change and taking into consideration that the effect lessened over the course of the study.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean white cell counts were higher than control for females exposed to 9.96 mg/L (1.3X control), principally due to higher monocyte (1.7X) and lymphocyte (1.3X) counts. Higher monocyte counts were also observed for males exposed to 4.62 or 9.96 mg/L (up to 1.4X control). However the majority of the values were within the control range and therefore these differences were attributed to normal variation.

All other differences from control, including those which attained a degree of statistical significance, were generally small, inconsistent between the sexes or considered to be due to high intra-group variation and therefore were considered not to be test item related.

(refer to Table 7.5.2/01-5)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean ALT concentrations were higher than control for all treated groups (up to 1.2X, 1.5X and 1.6X control, Group 2, 3 and 4 respectively).

Mean alkaline phosphatase was higher than control for females exposed to 9.96 mg/L (1.8X), the majority of individual values were only slightly above the control range, however two females had notably higher concentrations (#137, #140).

All other differences from control, including those which attained a degree of statistical significance, were generally small, inconsistent between the sexes or considered to be due to high intra-group variation and therefore were considered not to be test item related.

There were no microscopic correlates for the higher liver enzymes (ALT, ALP concentrations) observed for treated groups and the higher liver weights for animals exposed to 9.96 mg/L. and Ttherefore these changes were considered non adverse.

(refer to Table 7.5.2/01-5)
Urinalysis findings:
not examined
Description (incidence and severity):
n/a
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Observations
- Detailed physical examination and arena observations:
Detailed physical examination and arena observations commenced from Week 11. There were no test item related clinical signs during the detailed physical examination.
During the arena observations elevated gait was seen for 2/10 females exposed to 9.96 mg/L (#137, #138) from Week 11. In addition flattened gait was seen for one female in Weeks 12 and 13 (#136) and flattened posture was seen for one female in Week 11 (#140).

- Sensory reactivity and grip strength:
There were no test item related changes.
Group mean fore and hindlimb grip strength was lower than control for all treated male groups (not exposure related) and mean hindlimb grip strength was lower than control for females exposed to 9.96 mg/L, however, all were within the range of the historical control data therefore this was considered incidental.

- Motor activity:
Total low beam activity scores for males exposed to 4.62 or 9.96 mg/L were higher than the control (1.3X and 1.4X control, respectively).
There were no apparent effects on the high beam activity for males or activity scores for females. A small number of differences at individual timepoints attained statistical significance, however these were isolated and as there was no effect on the total scores these differences were attributed to normal variation.

These effects during the arena observations or motor activity assessments were seen in a small number of animals or were observed in one sex and there were no effects on the remaining behavioural assessments (high beam activity, sensory reactivity and grip strength), and therefore these changes in isolation and without associated histopathology were also considered non-adverse.
Immunological findings:
not examined
Description (incidence and severity):
n/a
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Mean liver weights (absolute and adjusted for terminal body weight) were higher than control for both sexes exposed to 9.96 mg/L (1.2X control), without associated histopathology and therefore were considered adaptive.

All other differences from control were generally small, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.

(refer to Table 7.5.2/01-2)
Gross pathological findings:
no effects observed
Description (incidence and severity):
n/a
Neuropathological findings:
not examined
Description (incidence and severity):
n/a
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes for animals exposed to 9.96 mg/L.

The incidence and distribution of all findings were incidental and unrelated to treatment.

The microscopic findings in the testis (atrophy) and epididymis (reduced luminal sperm/luminal cell debris) were observed in males exposed air only and 4.62 or 9.96 mg/L MeTHF. However, this finding was not observed in all males from these groups and the severity was mostly minimal. Testicular atrophy is often observed in control rats used for inhalation studies (Lee et al., 1993) and is attributed to increased body temperatures (thermal stress) and correlated with the small size of the testes and epididymides seen in some animals macroscopically.

(refer to Table 7.5.2/01-2)
Reference:
Lee KP, Frame SR, Sykes GP, Valentine R. Testicular degeneration and spermatid retention in young male rats. Toxicol Pathol 1993;21:293-302.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
n/a
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Bronchoalveolar Lavage (BAL):
- Cell counts:
There were no test item related changes.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

- Total protein and lactate dehydrogenase:
There were no test item related changes.
Group mean concentrations were variable when compared with control, however individual values for test animals were typically within the control range and therefore all differences were attributed to normal biological variation.

Sperm Analysis:
All groups showed a lower percentage motile sperm than expected and for the majority of samples it was not possible to assess 200 sperm. Percentage motile sperm of males exposed to 9.96 mg/L appeared lower than that of the control group. The relationship to treatment was uncertain as this change was not statistically significant and no clear trends were observed in the individual data.
The percentage normal sperm was lower than expected in control and treated groups; the majority of abnormal sperm were decapitate. Slight changes compared with controls were observed for males exposed to 9.96 mg/L. Changes in percentage normal and total abnormal sperm were not statistically significant. Tail abnormalities were statistically significantly higher in treated animals than in the control group. The relationship to treatment was uncertain.
These findings correlated with the small size of the testes and epididymides seen in some animals macroscopically and may account for the effect on sperm motility and higher percentage of abnormal sperm (decapitated).
Sperm numbers in cauda epididymis and testis were lower than expected in control and treated groups. Testicular sperm numbers for males exposed to 9.96 mg/L were similar to those of the control group, whilst cauda epididymal sperm numbers appeared lower than those of the control group. This change was not statistically significant and was considered unrelated to treatment with MeTHF, being due to one high value in the control group. (refer to Table 7.5.2/01-3).

Oestrous cycles:
Oestrous cycles were evaluated during Weeks 12 and 13, a proportion of animals in the treated groups had irregular cycles, 2 or 3 females per test group compared with no irregular cycles in control.
Remaining animals were having regular 4 to 5 day cycles, however, for females exposed to 9.96 mg/L there was a higher proportion of 5 day cycles than 4 day cycles. Four females exposed to 9.96 mg/L had 5 day cycles compared with 1 in the control group; no 4 day cycles were recorded for females exposed to 9.96 mg/L compared with 5 in the control group. As all females continued to cycle these changes were considered non adverse. (refer to Table 7.5.2/01-4).
Dose descriptor:
NOAEC
Effect level:
> 9.96 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no adverse effects when tested up to the maximum tolerated dose
Critical effects observed:
no

Table 7.5.2/01-2
Rat 90-day inhalation study: body weight, selected organ weight and histopathology parameters

Parameters

¿ (mg/L)

¿ (mg/L)

0

2

4.5

10

0

2

4.5

10

Ter. body wts (g)

362

362

359

348

213

223

220

228*

Body wt change(g) (1-92)

+97

+94

+96

+82

+33

+42

+36

+46*

Organ weights (abs: g adjusted values for bwt: g)

- Liver:     abs
                   adj.

11.053
10.915

11.299
11.148

11.763
11.705*

12.895
13.242**

7.388
7.702

8.264
8.175

8.343
8.404*

9.606
9.321**

- Testes:   abs
                   adj.

2.602
2.553

2.806
2.751

2.697
2.676

2.630
2.755

-

-

-

-

- U+C:      abs
                   adj.

-

-

-

-

0.699
0.725

0.617
0.586

0.635
0.589

0.510
0.467**

Histopathology: [total number examined [minimal, slight, moderate, marked]]

Liver,infiltrate, inflammatory cell

10
[0,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

10
[1,0,0,0]

10
[1,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

Testes, atrophy

10
[4,2,0,0]

-

2
[1,0,1,0]

10
[4,0,0,0]

-

-

-

-

U+C:

-

-

-

-

10
No A.H.

-

-

10
No A.H.

*p=0.05; **p<0.01

abs: absolute

adj: terminal weight adjusted

U+C: uterus + cervix

No A.H.: no adverse histopathology reported

Table 7.5.2/01-3
Rat 90-day inhalation study: sperm analysis

Parameters

¿ (mg/L)

0

2

4.5

10

Spermanalysis - group mean values

- Motile sperm (%)

30

26

22

13

- Progressively motile sperm (%)

10

11

4

8

-Cauda epididymis

- Weight (g)

0.153

-

-

0.133

- Sperm count (millions/g)

384

-

-

262

- Total (million)

64

-

-

38

Testis

- Weight (g)

1.32

-

-

1.31

- Sperm count (millions/g)

56

-

-

56

- Total (million)

83

-

-

80

Spermanalysis – morphology, group mean values

Total no. of sperm analysed

1563

-

-

1108

Normal (n [%])

76 [44.5]

-

-

59 [33.5]

Total abnormal
(n [%])

81 [55.5]

-

-

59 [66.5]

Decapitate ([%])

70 [49.3]

-

-

43 [54.7]

Head abnormal
(n [%])

7 [4.2]

-

-

13 [8.4]

Tail abnormal
(n [%])

7 [3.3]

-

-

14 [15.5]*

Table 7.5.2/01-4
Rat 90-day inhalation study: oestrous cyclicity parameters

Parameters

¿ (mg/L)

0

2

5

10

Oestrous cycles - group values (no. of animals/total animals)

Regular cycles

- 4 day

5/10

3/10

4/10

0/10

- 4/5 day

4/10

4/10

4/10

3/10

- 5 day

1/10

0/10

0/10

4/10

Irregular cycle

0/10

3/10

2/10

3/10

Acyclic

0/10

0/10

0/10

0/10

Total no. with regular cycles

10/10

7/10

8/10

7/10

Total no. with irregular cycles

0/10

3/10

2/10

3/10

Irregular: at least one cycle of 2, 3 or 6 to 10 days

 

Table 7.5.2/01-5
Rat 90-day inhalation study: selected haematology and clinical chemistry parameters

Parameters

¿ (mg/L)

¿ (mg/L)

0

2

4.5

10

0

2

4.5

10

Haematology

Hct (L/L)

0.455

0.453

0.455

0.464

0.429

0.440

0.453**

0.455**

Hb (g/dL)

15.5

15.7

15.9

16.0*

14.1

14.6*

14.9**

15.0**

MCHC

33.9

34.6*

34.9*

34.5*

32.8

33.2

32.9

33.1

WBC (x109/L)

4.82

4.87

5.03

5.17

3.37

3.85

3.93

4.28*

N (x109/L)

1.09

1.15

1.12

1.29

0.65

0.72

0.71

0.73

L (x109/L)

3.55

3.56

3.72

3.68

2.60

3.00

3.11

3.39*

E (x109/L)

0.06

0.05

0.05

0.05

0.04

0.04

0.03

0.04

B (x109/L)

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01*

M (x109/L)

0.07

0.07

0.09

0.10*

0.06

0.08

0.06

0.10**

LUC (x109/L)

0.03

0.04

0.04

0.04

0.01

0.02

0.02

0.02

PT (sec)

23.1

22.1

22.1

20.6**

20.8

21.4

21.0

20.7

APTT (sec)

19.9

20.8

21.7*

19.7

19.7

20.7

20.5

19.5

Clinical chemistry

ALP (IU/L)

100

101

105

97

49

48

55

89**

ALT (IU/L)

58

72

70

86**

44

54

65**

72**

Chol (mmol/L)

1.98

2.41**

2.30**

2.19**

2.12

2.39

1.98

2.04

Trig (mmol/L)

1.71

1.37

1.49

1.39

0.88

0.85

1.13

0.92

*p=0.05; **p<0.01

Hct: haematocrit

Hb: haemoglobin

MCHC: mean cell Hb concentration

WBC: total white blood cell

N: neutrophils

L:lymphocytes

E: eosinophils

B: basophils

M: monocytes

LUC: large unstained cells

PT: prothrombin time

APTT: activated partial thromboplastin time

ALP: Alkaline phosphatase

ALT: Alanine aminotransferase

Chol: Total cholesterol

Trig: Triglycerides

Conclusions:
Under the conditions of this study the NOAEC is deemed to be >9.96 mg/L (deemed to be the maximum tolerated dose) in males and females based on no adverse effects observed. Clinical signs (abnormal gait, salivation and higher low beam activity scores) and a slight effect on body weight, food consumption and water consumption was seen for animals exposed to 9.96 mg/L. In addition, irregular oestrous cycles were seen in all treated groups and a shift in the regular cycle length, from 4 to 5 days, was observed for females exposed to 9.96 mg/L. All changes were considered non-adverse. There were no microscopic changes attributable to the test item.
Executive summary:

The potential repeat dose toxicity of MeTHF following sub-chronic (90 day) repeat dose inhalation exposure to young male and female Han Wistar rats (10 animals/sex/group) was investigated. Following a range-finder assessment, MeTHF was administered via nose only inhalation at doses of 0, 2, 4.5, 10 mg/L, 6 hours/day, 5 (weeks 1 to 12) or 7 (week 13) days a week.

 

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, oestrous cycle, body weight, food consumption, water consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry (including thyroid hormone assessment), organ weight, sperm analysis, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

 

The mean achieved concentrations, following 13 weeks of exposure, were 2.07, 4.62 and 9.96 mg/L (104, 103 and 100% of the target concentrations) for Groups 2, 3 and 4, respectively.

 

Test item related clinical signs, in relation to dosing, included unsteady gait and salivation for animals exposed to 9.96 mg/L on return to the home cage. In addition abnormal gait (flattened/elevated) or flattened posture was seen at the weekly arena observations in a small number of females exposed to 9.96 mg/L. Low beam activity scores were higher than control for males exposed to 4.62 or 9.96 mg/L.These effects during the arena observations or motor activity assessments were seen in a small number of animals or were observed in one sex and there were no effects on the remaining behavioural assessments (high beam activity, sensory reactivity and grip strength), and therefore these changes in isolation and without associated histopathology were also considered non-adverse. 

 

Irregular oestrous cycles were observed for a proportion of females from all treated groups and a shift in the regular cycle length, from 4 to 5 days, was seen for females exposed to 9.96 mg/L.As all females continued to cycle these changes were considered non-adverse. 

 

Higher body weight gain and food consumption was observed for females exposed to 9.96 mg/L and higher water consumption was observed for both sexes exposed to 9.96 mg/L.The magnitude of the body weight gain and food consumption changes were low and therefore these changes were considered non-adverse. The higher water consumption observed was considered non-adverse due to the low magnitude of the change and taking into consideration that the effect lessened over the course of the study. 

 

Alanine aminotransferase concentrations were higher than control for all treated groups and alkaline phosphatase was higher than control for females exposed to 9.96 mg/L; liver weights

were higher than control for both sexes exposed to 9.96 mg/L.There were no microscopic correlates for the higher liver enzymes (alanine aminotransferase and alkaline phosphatase concentrations) observed for treated groups and the higher liver weights for animals exposed to 9.96 mg/L. Therefore these changes were considered non-adverse.

 

Under the conditions of this study the NOAEC is deemed to be >9.96 mg/L (deemed to be the maximum tolerated dose) in males and females based on no adverse effects observed. Clinical signs (abnormal gait, salivation and higher low beam activity scores) and a slight effect on body weight, food consumption and water consumption was seen for animals exposed to 9.96 mg/L. In addition, irregular oestrous cycles were seen in all treated groups and a shift in the regular cycle length, from 4 to 5 days, was observed for females exposed to 9.96 mg/L. All changes were considered non-adverse. There were no microscopic changes attributable to the test item.

Endpoint conclusion
Endpoint conclusion:
no study available
Dose descriptor:
NOAEC
9 960 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
A GLP, test guideline compliant (OECD 413) sub-chronic (90 day) repeat dose inhalation study
Organ:
other: Effects observed were considered non-adverse

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2018 to 20 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test Material: MeTHF (2-Methyltetrahydrofuran)
Description: Colorless liquid
Lot/Batch No.: 2-7E25S
Purity: 99.98%
CAS No.: 96-47-9
Stability of test compound: Confirmed stable for the duration of the study (25 May 2019 (two years from date of manufacture, manufacture date: 25 May 2017))
Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Age: 9 -11 wks at dosing
Weight at dosing: M: 232-294g; F: 172-194g
Source: Envigo RMS Limited, UK
Acclimation period: 11 days
Diet: Teklad 2014C Diet, ad libitum (except overnight before blood sampling for hematology or blood chemistry and during exposure)
Water: Municipal water, ad libitum (except during exposure)
Housing: Groups of 5 of the same sex

Environmental conditions:
Temperature: 20-24°C
Humidity: 40-70%
Air changes: not stated
Photoperiod: 12 hours light/dark
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only chamber. Aluminium alloy construction comprising a base unit, three animal exposure
sections, a top section and a pre-chamber
- Method of holding animals in test chamber: animals were restrained in a plastic snout-only tube. Animals on study were acclimatized to the method of restraint for three consecutive days preceding the first test item exposure
- Source and rate of air: From in-house compressed air system at 19 L/min
- Method of conditioning air: filtered
- System of generating particulates/aerosols: Glass sintered vaporizer. The test item was supplied to the generator, via a feed line, from a syringe (all glass) driven at a constant rate by a syringe pump (KD Scientific)
- Temperature, humidity, pressure in air chamber: refer to Table 7.5.2/01-1
- Air flow rate: 19 L/min
- Method of particle size determination:
- Treatment of exhaust air: Drawn by in-house vacuum system at 20 L/min

TEST ATMOSPHERE
- Brief description of analytical method used:
Vapor samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler) set up in series
Sample solvent: Acetonitrile
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: Minimum of 1 sample from Group 1/day (taken at approximately 60 minutes during exposure) 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test sample (solvent trap collected in acetonitrile): Transfer to volumetric flask using acetonitrile to provide a solution containing MeTHF at a nominal concentration in the range 200 – 2000 µg/mL. Inject onto the GC. The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis.
Duration of treatment / exposure:
6 h/d, 5 days/week (weeks 1-12) or 7 days/week (week 13)
Frequency of treatment:
either 5 days/week or 7 days/week
Dose / conc.:
0 mg/L air (analytical)
Dose / conc.:
2.07 mg/L air (analytical)
Dose / conc.:
4.62 mg/L air (analytical)
Dose / conc.:
9.96 mg/L air (analytical)
Dose / conc.:
0 mg/L air
Dose / conc.:
2 mg/L air
Dose / conc.:
4.5 mg/L air
Dose / conc.:
10 mg/L air
No. of animals per sex per dose:
10/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
The conduct of the study generally followed OECD 413 (2017). Four groups of 10 male and 10 female Han Wistar rats (randomised by bodyweight) received the test item by nose only inhalation, exposed daily for 6 h/d, 5 days/week (weeks 1-12) or 7 days/week (week 13) at target concentrations of 0, 2.0, 4.5, 10 mg/L, with different dose levels achieved by varying the concentration of test item in the exposure system, whilst keeping the duraation of exposure constant. A vehicle control (air ) group was also included. Body weight gain, water and food consumption were measured at regular intervals.

All animals were subjected to ophthalmoscopy, haematology, clinical chemistry (including thyroid hormones), sperm analysis, oestrus cycle, FOB assessment, organ weights, and macropathology. Histopathological examinations were carried out on the negative and vehicle control and high dose animals.
Positive control:
n/a
Observations and examinations performed and frequency:
1.Observations: Mortality and moribundity: observed twice daily, once in the morning and once in the afternoon
Clinical observations: recorded for all animals twice daily. A detailed physical examination was performed weekly. From week 11, arena observations were also performed, the examinations were performed at approximately the same time of day (before dosing), by an observer unaware of the experimental group identities (refer below to point 5 below)

2. Body weights: Recorded twice weekly, starting 1 week prior to dose administration. Body weight was also recorded on the first day of dosing.

3. Food consumption: Recorded weekly.

4. Water consumption: Daily during Weeks 3 to 13 of treatment; measured per cage.

5. Functional observation battery (FOB): Recorded for all animals during each week from week 11. Observations included:
- Detailed physical examinations. after removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
- Sensory reactivity and grip strength: sensory reactivity and grip strength assessments were performed on all animals during Week 12 of treatment. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:
- Approach response: a blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction
- Pinna reflex: the inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response
- Auditory startle reflex: the animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor
- Tail pinch response: The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression
- Grip strength: forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

6. Locomotor activity:
- Motor activity: During Week 12 of treatment, the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

7. Ophthalmic examination: All animals examined pre-treatment, with the control and high dose groups examined prior to necropsy. The adrexae, conjunctivae, cornea, sclera, anterior chamber, iris (pupils dilated), lens, vitreous and fundus were examined.

8. Haematology and clinical chemistry: Conducted in week 13 of treatment. Sampling was from the sublingual vein.
- Haematology: red blood cell parameters (haematocrit, haemoglobin concentration, mean cell haemoglobin concentration, mean cell haemoglobin, mean cell volume, erythrocyte count, red cell distribution), white blood cell parameters (total and differential (neutrophils, lymphocytes, eosinophils, basophils, monocytes,large unstained cells) leukocyte count), clotting parameters (platelet count, prothrombin, activated partial thromboplastin time).
- Clinical chemistry: electrolytes (sodium, potassium, calcium, chloride, inorganic phosphorus), urea, glucose, creatinine, liver function tests (alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T.Bili), total protein, albumin/globulin),lipid profile (total triglyceride, total cholesterol), thyroid function (triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH))

9. Urinalysis: Not conducted

10. Oestrous cycle: Vaginal smears performed for 14 days during weeks 12 and 13 using cotton swabs.
Sacrifice and pathology:
11. Gross pathology: Gross pathological examination was performed on all animals and included examination of the external surface and all orifices.

12. Histopathology : The following tissues were preserved in 4% formaldehyde for subsequent histopathological examination (with the exception of eyes and optic nerve which were preserved in Davidson's fluid) nd the testes which were preserved in modified Davidson's fluid: Adrenals, aorta (thoracic), brain (including sections of cerebrum, cerebellum, and medulla/pons), bone marrow, digestive system (caecum, colon, duodenum, epiglottis, oesophagus, ileum (including Peyer's patches), jejunum, rectum, stomach, tongue), eyes (+optic nerves, lachrymal and Harderian glands), femoral bone (+marrow), heart (including auricular and ventricular regions), kidneys, liver (2 lobes), lymph nodes (mesenteric, tracheo-bronchial, left axillary, hilar), respiratory system (larynx ( 5 sections), lungs (including bronchi), nasal turbinates (4 levels including teeth), trachea), (accessory) sex organs (epididymides, ovaries, prostate, seminal vesicles, testes, uterus (with cervix), vagina), pancreas, pituitary, salivary glands (submandibular/sublingual), sciatic nerves, skeletal muscle, skin + mammary gland (inguinal area), spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, sternal bone and marrow, thymus, thyroid (+parathyroids), urinary bladder. Histopathological analysis was conducted for all listed tissues, with the exception of eye, Harderian gland, lachrymal gland, mesenteric lymph node, optic nerve, left sciatic nerve and the tongue.
The right lung was used for Bronchoalveolar Lavage (BAL) sampling and the left lung was processed for histology and light microscopy.

13. Organ weights: The following organs were trimmed of adherent tissue and weighed: adrenals, brain, epididymides (left and right), heart, kidneys, liver, lungs (including bronchi), ovaries, spleen, testes (left and right), thymus, thyroids (+parathyroids), uterus (+cervix).

14. Bone marrow smears: Bone marrow smears were prepared, for all animals, immediately following death. Smears were air dried and subsequently fixed in methanol. No examinations were performed, however, the smears were retained for possible future examination.

15. Sperm analysis: Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
-Sperm mortality: A sample of sperm was expressed from the vas deferens and, where possible, at least 200 sperm per animal analysed.
-Sperm morphology: An aliquot of the sperm/medium mixture was diluted with 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male of groups 1 and 4, where possible.
-Sperm count: The left cauda epididymis of each male for groups 1 and 4 was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portion was allowed to thaw then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
-Homogenisation resistant spermatid count: After removal of the tunica, the left testis of each male for groups 1 and 4 was frozen. Prior to analysis, the testis was allowed to thaw then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT
stain tube before being assessed for homogenisation-resistant spermatid count using CASA.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
Grip strength and motor activity, body weight (using gains over appropriate study periods), haematology, blood chemistry, sperm analysis, oestrous cycles, organ weights (absolute and adjusted for terminal body weight).

For grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests were performed.

For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups, using scores appropriate to the severity of the observation assuming 4 day cycles to be normal.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs during the detailed weekly physical examination.

Unsteady gait was observed for all Group 4 animals on return to the home cage, in addition excessive salivation was noted, on occasion. These signs, where observed, had typically resolved by the end of day observation.

Clinical signs associated with the dosing procedure included wet fur and red staining (of the head, nose or eyes), on occasion, on return to cage for all groups including control and were considered to be associated with the method of restraint.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weight gains were apparent for males exposed to 9.96 mg/L when compared with the control group (0.85X control). There were no effects for males exposed to 2.07 or 4.62 mg/L. The lower body weight gain for males exposed to 9.96 mg/L was not associated with any effect on food consumption and was inconsistent with the effect for females and was therefore considered incidental.

Body weight gains for all treated female groups were variable. No body weight gain was apparent for females exposed to 9.96 mg/L following the first week of exposures, thereafter higher gains were observed resulting in overall higher mean gain (1.4X control after 13 weeks). Gains for females exposed to 2.07 mg/L were also higher than control (1.3X control), whilst gains for females exposed to 4.62 mg/L were similar to control (1.1Xcontrol). The higher body weight gain for females exposed to 9.96 mg/L correlated with the slight increase in food consumption for this group. The magnitude of the changes was low and therefore these changes were considered non-adverse.
(refer to Table 7.5.2/01-2)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Weekly food consumption was slightly higher for females exposed to 9.96 mg/L when compared with control. There were no apparent effects for females exposed to 2.07 or 4.62 mg/L or treated males. This however was considered non-adverse and was inconsistent with the effect for males and was therefore considered incidental.
Food efficiency:
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption measurements commenced from Week 3. Higher weekly water consumption was observed for both sexes exposed to 9.96 mg/L when compared with control, with less of an effect from Week 9. This however was considered non-adverse due to the low magnitude of the change and taking into consideration that the effect lessened over the course of the study.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean white cell counts were higher than control for females exposed to 9.96 mg/L (1.3X control), principally due to higher monocyte (1.7X) and lymphocyte (1.3X) counts. Higher monocyte counts were also observed for males exposed to 4.62 or 9.96 mg/L (up to 1.4X control). However the majority of the values were within the control range and therefore these differences were attributed to normal variation.

All other differences from control, including those which attained a degree of statistical significance, were generally small, inconsistent between the sexes or considered to be due to high intra-group variation and therefore were considered not to be test item related.

(refer to Table 7.5.2/01-5)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean ALT concentrations were higher than control for all treated groups (up to 1.2X, 1.5X and 1.6X control, Group 2, 3 and 4 respectively).

Mean alkaline phosphatase was higher than control for females exposed to 9.96 mg/L (1.8X), the majority of individual values were only slightly above the control range, however two females had notably higher concentrations (#137, #140).

All other differences from control, including those which attained a degree of statistical significance, were generally small, inconsistent between the sexes or considered to be due to high intra-group variation and therefore were considered not to be test item related.

There were no microscopic correlates for the higher liver enzymes (ALT, ALP concentrations) observed for treated groups and the higher liver weights for animals exposed to 9.96 mg/L. and Ttherefore these changes were considered non adverse.

(refer to Table 7.5.2/01-5)
Urinalysis findings:
not examined
Description (incidence and severity):
n/a
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Observations
- Detailed physical examination and arena observations:
Detailed physical examination and arena observations commenced from Week 11. There were no test item related clinical signs during the detailed physical examination.
During the arena observations elevated gait was seen for 2/10 females exposed to 9.96 mg/L (#137, #138) from Week 11. In addition flattened gait was seen for one female in Weeks 12 and 13 (#136) and flattened posture was seen for one female in Week 11 (#140).

- Sensory reactivity and grip strength:
There were no test item related changes.
Group mean fore and hindlimb grip strength was lower than control for all treated male groups (not exposure related) and mean hindlimb grip strength was lower than control for females exposed to 9.96 mg/L, however, all were within the range of the historical control data therefore this was considered incidental.

- Motor activity:
Total low beam activity scores for males exposed to 4.62 or 9.96 mg/L were higher than the control (1.3X and 1.4X control, respectively).
There were no apparent effects on the high beam activity for males or activity scores for females. A small number of differences at individual timepoints attained statistical significance, however these were isolated and as there was no effect on the total scores these differences were attributed to normal variation.

These effects during the arena observations or motor activity assessments were seen in a small number of animals or were observed in one sex and there were no effects on the remaining behavioural assessments (high beam activity, sensory reactivity and grip strength), and therefore these changes in isolation and without associated histopathology were also considered non-adverse.
Immunological findings:
not examined
Description (incidence and severity):
n/a
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Mean liver weights (absolute and adjusted for terminal body weight) were higher than control for both sexes exposed to 9.96 mg/L (1.2X control), without associated histopathology and therefore were considered adaptive.

All other differences from control were generally small, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.

(refer to Table 7.5.2/01-2)
Gross pathological findings:
no effects observed
Description (incidence and severity):
n/a
Neuropathological findings:
not examined
Description (incidence and severity):
n/a
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes for animals exposed to 9.96 mg/L.

The incidence and distribution of all findings were incidental and unrelated to treatment.

The microscopic findings in the testis (atrophy) and epididymis (reduced luminal sperm/luminal cell debris) were observed in males exposed air only and 4.62 or 9.96 mg/L MeTHF. However, this finding was not observed in all males from these groups and the severity was mostly minimal. Testicular atrophy is often observed in control rats used for inhalation studies (Lee et al., 1993) and is attributed to increased body temperatures (thermal stress) and correlated with the small size of the testes and epididymides seen in some animals macroscopically.

(refer to Table 7.5.2/01-2)
Reference:
Lee KP, Frame SR, Sykes GP, Valentine R. Testicular degeneration and spermatid retention in young male rats. Toxicol Pathol 1993;21:293-302.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
n/a
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Bronchoalveolar Lavage (BAL):
- Cell counts:
There were no test item related changes.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

- Total protein and lactate dehydrogenase:
There were no test item related changes.
Group mean concentrations were variable when compared with control, however individual values for test animals were typically within the control range and therefore all differences were attributed to normal biological variation.

Sperm Analysis:
All groups showed a lower percentage motile sperm than expected and for the majority of samples it was not possible to assess 200 sperm. Percentage motile sperm of males exposed to 9.96 mg/L appeared lower than that of the control group. The relationship to treatment was uncertain as this change was not statistically significant and no clear trends were observed in the individual data.
The percentage normal sperm was lower than expected in control and treated groups; the majority of abnormal sperm were decapitate. Slight changes compared with controls were observed for males exposed to 9.96 mg/L. Changes in percentage normal and total abnormal sperm were not statistically significant. Tail abnormalities were statistically significantly higher in treated animals than in the control group. The relationship to treatment was uncertain.
These findings correlated with the small size of the testes and epididymides seen in some animals macroscopically and may account for the effect on sperm motility and higher percentage of abnormal sperm (decapitated).
Sperm numbers in cauda epididymis and testis were lower than expected in control and treated groups. Testicular sperm numbers for males exposed to 9.96 mg/L were similar to those of the control group, whilst cauda epididymal sperm numbers appeared lower than those of the control group. This change was not statistically significant and was considered unrelated to treatment with MeTHF, being due to one high value in the control group. (refer to Table 7.5.2/01-3).

Oestrous cycles:
Oestrous cycles were evaluated during Weeks 12 and 13, a proportion of animals in the treated groups had irregular cycles, 2 or 3 females per test group compared with no irregular cycles in control.
Remaining animals were having regular 4 to 5 day cycles, however, for females exposed to 9.96 mg/L there was a higher proportion of 5 day cycles than 4 day cycles. Four females exposed to 9.96 mg/L had 5 day cycles compared with 1 in the control group; no 4 day cycles were recorded for females exposed to 9.96 mg/L compared with 5 in the control group. As all females continued to cycle these changes were considered non adverse. (refer to Table 7.5.2/01-4).
Dose descriptor:
NOAEC
Effect level:
> 9.96 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no adverse effects when tested up to the maximum tolerated dose
Critical effects observed:
no

Table 7.5.2/01-2
Rat 90-day inhalation study: body weight, selected organ weight and histopathology parameters

Parameters

¿ (mg/L)

¿ (mg/L)

0

2

4.5

10

0

2

4.5

10

Ter. body wts (g)

362

362

359

348

213

223

220

228*

Body wt change(g) (1-92)

+97

+94

+96

+82

+33

+42

+36

+46*

Organ weights (abs: g adjusted values for bwt: g)

- Liver:     abs
                   adj.

11.053
10.915

11.299
11.148

11.763
11.705*

12.895
13.242**

7.388
7.702

8.264
8.175

8.343
8.404*

9.606
9.321**

- Testes:   abs
                   adj.

2.602
2.553

2.806
2.751

2.697
2.676

2.630
2.755

-

-

-

-

- U+C:      abs
                   adj.

-

-

-

-

0.699
0.725

0.617
0.586

0.635
0.589

0.510
0.467**

Histopathology: [total number examined [minimal, slight, moderate, marked]]

Liver,infiltrate, inflammatory cell

10
[0,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

10
[1,0,0,0]

10
[1,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

10
[0,0,0,0]

Testes, atrophy

10
[4,2,0,0]

-

2
[1,0,1,0]

10
[4,0,0,0]

-

-

-

-

U+C:

-

-

-

-

10
No A.H.

-

-

10
No A.H.

*p=0.05; **p<0.01

abs: absolute

adj: terminal weight adjusted

U+C: uterus + cervix

No A.H.: no adverse histopathology reported

Table 7.5.2/01-3
Rat 90-day inhalation study: sperm analysis

Parameters

¿ (mg/L)

0

2

4.5

10

Spermanalysis - group mean values

- Motile sperm (%)

30

26

22

13

- Progressively motile sperm (%)

10

11

4

8

-Cauda epididymis

- Weight (g)

0.153

-

-

0.133

- Sperm count (millions/g)

384

-

-

262

- Total (million)

64

-

-

38

Testis

- Weight (g)

1.32

-

-

1.31

- Sperm count (millions/g)

56

-

-

56

- Total (million)

83

-

-

80

Spermanalysis – morphology, group mean values

Total no. of sperm analysed

1563

-

-

1108

Normal (n [%])

76 [44.5]

-

-

59 [33.5]

Total abnormal
(n [%])

81 [55.5]

-

-

59 [66.5]

Decapitate ([%])

70 [49.3]

-

-

43 [54.7]

Head abnormal
(n [%])

7 [4.2]

-

-

13 [8.4]

Tail abnormal
(n [%])

7 [3.3]

-

-

14 [15.5]*

Table 7.5.2/01-4
Rat 90-day inhalation study: oestrous cyclicity parameters

Parameters

¿ (mg/L)

0

2

5

10

Oestrous cycles - group values (no. of animals/total animals)

Regular cycles

- 4 day

5/10

3/10

4/10

0/10

- 4/5 day

4/10

4/10

4/10

3/10

- 5 day

1/10

0/10

0/10

4/10

Irregular cycle

0/10

3/10

2/10

3/10

Acyclic

0/10

0/10

0/10

0/10

Total no. with regular cycles

10/10

7/10

8/10

7/10

Total no. with irregular cycles

0/10

3/10

2/10

3/10

Irregular: at least one cycle of 2, 3 or 6 to 10 days

 

Table 7.5.2/01-5
Rat 90-day inhalation study: selected haematology and clinical chemistry parameters

Parameters

¿ (mg/L)

¿ (mg/L)

0

2

4.5

10

0

2

4.5

10

Haematology

Hct (L/L)

0.455

0.453

0.455

0.464

0.429

0.440

0.453**

0.455**

Hb (g/dL)

15.5

15.7

15.9

16.0*

14.1

14.6*

14.9**

15.0**

MCHC

33.9

34.6*

34.9*

34.5*

32.8

33.2

32.9

33.1

WBC (x109/L)

4.82

4.87

5.03

5.17

3.37

3.85

3.93

4.28*

N (x109/L)

1.09

1.15

1.12

1.29

0.65

0.72

0.71

0.73

L (x109/L)

3.55

3.56

3.72

3.68

2.60

3.00

3.11

3.39*

E (x109/L)

0.06

0.05

0.05

0.05

0.04

0.04

0.03

0.04

B (x109/L)

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01*

M (x109/L)

0.07

0.07

0.09

0.10*

0.06

0.08

0.06

0.10**

LUC (x109/L)

0.03

0.04

0.04

0.04

0.01

0.02

0.02

0.02

PT (sec)

23.1

22.1

22.1

20.6**

20.8

21.4

21.0

20.7

APTT (sec)

19.9

20.8

21.7*

19.7

19.7

20.7

20.5

19.5

Clinical chemistry

ALP (IU/L)

100

101

105

97

49

48

55

89**

ALT (IU/L)

58

72

70

86**

44

54

65**

72**

Chol (mmol/L)

1.98

2.41**

2.30**

2.19**

2.12

2.39

1.98

2.04

Trig (mmol/L)

1.71

1.37

1.49

1.39

0.88

0.85

1.13

0.92

*p=0.05; **p<0.01

Hct: haematocrit

Hb: haemoglobin

MCHC: mean cell Hb concentration

WBC: total white blood cell

N: neutrophils

L:lymphocytes

E: eosinophils

B: basophils

M: monocytes

LUC: large unstained cells

PT: prothrombin time

APTT: activated partial thromboplastin time

ALP: Alkaline phosphatase

ALT: Alanine aminotransferase

Chol: Total cholesterol

Trig: Triglycerides

Conclusions:
Under the conditions of this study the NOAEC is deemed to be >9.96 mg/L (deemed to be the maximum tolerated dose) in males and females based on no adverse effects observed. Clinical signs (abnormal gait, salivation and higher low beam activity scores) and a slight effect on body weight, food consumption and water consumption was seen for animals exposed to 9.96 mg/L. In addition, irregular oestrous cycles were seen in all treated groups and a shift in the regular cycle length, from 4 to 5 days, was observed for females exposed to 9.96 mg/L. All changes were considered non-adverse. There were no microscopic changes attributable to the test item.
Executive summary:

The potential repeat dose toxicity of MeTHF following sub-chronic (90 day) repeat dose inhalation exposure to young male and female Han Wistar rats (10 animals/sex/group) was investigated. Following a range-finder assessment, MeTHF was administered via nose only inhalation at doses of 0, 2, 4.5, 10 mg/L, 6 hours/day, 5 (weeks 1 to 12) or 7 (week 13) days a week.

 

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, oestrous cycle, body weight, food consumption, water consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry (including thyroid hormone assessment), organ weight, sperm analysis, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

 

The mean achieved concentrations, following 13 weeks of exposure, were 2.07, 4.62 and 9.96 mg/L (104, 103 and 100% of the target concentrations) for Groups 2, 3 and 4, respectively.

 

Test item related clinical signs, in relation to dosing, included unsteady gait and salivation for animals exposed to 9.96 mg/L on return to the home cage. In addition abnormal gait (flattened/elevated) or flattened posture was seen at the weekly arena observations in a small number of females exposed to 9.96 mg/L. Low beam activity scores were higher than control for males exposed to 4.62 or 9.96 mg/L.These effects during the arena observations or motor activity assessments were seen in a small number of animals or were observed in one sex and there were no effects on the remaining behavioural assessments (high beam activity, sensory reactivity and grip strength), and therefore these changes in isolation and without associated histopathology were also considered non-adverse. 

 

Irregular oestrous cycles were observed for a proportion of females from all treated groups and a shift in the regular cycle length, from 4 to 5 days, was seen for females exposed to 9.96 mg/L.As all females continued to cycle these changes were considered non-adverse. 

 

Higher body weight gain and food consumption was observed for females exposed to 9.96 mg/L and higher water consumption was observed for both sexes exposed to 9.96 mg/L.The magnitude of the body weight gain and food consumption changes were low and therefore these changes were considered non-adverse. The higher water consumption observed was considered non-adverse due to the low magnitude of the change and taking into consideration that the effect lessened over the course of the study. 

 

Alanine aminotransferase concentrations were higher than control for all treated groups and alkaline phosphatase was higher than control for females exposed to 9.96 mg/L; liver weights

were higher than control for both sexes exposed to 9.96 mg/L.There were no microscopic correlates for the higher liver enzymes (alanine aminotransferase and alkaline phosphatase concentrations) observed for treated groups and the higher liver weights for animals exposed to 9.96 mg/L. Therefore these changes were considered non-adverse.

 

Under the conditions of this study the NOAEC is deemed to be >9.96 mg/L (deemed to be the maximum tolerated dose) in males and females based on no adverse effects observed. Clinical signs (abnormal gait, salivation and higher low beam activity scores) and a slight effect on body weight, food consumption and water consumption was seen for animals exposed to 9.96 mg/L. In addition, irregular oestrous cycles were seen in all treated groups and a shift in the regular cycle length, from 4 to 5 days, was observed for females exposed to 9.96 mg/L. All changes were considered non-adverse. There were no microscopic changes attributable to the test item.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
9 960 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
A GLP. test guideline complinat (OECD 413) sub-chronic (90 day) repeat dose inhalation study

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

Comparison with the CLP criteria

As described above in the rat repeat dose inhalation toxicity study, no significant toxic effects occurred at any dose. No significant toxic effects of relevance to humans were seen in the rats; where non-adverse effects were observed these occurred at dose levels well in excess of the specified guidance values for classification with STOT-RE Category 2.

 

On this basis, classification of MeTHF with STOT-RE is not warranted.

Justification for classification or non-classification

Based on the available data Methyltetrahydrofuran is not classified for adverse effects following repeated exposures according to Regulation (EC) No. 1272/2008.