Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-methyltetrahydrofuran; MeTHF CAS 96-47-9
- Substance type: Monoconstituent substance
- Physical state: no information
- Analytical purity: no information
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))

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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).

Effect levels

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

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.