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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-04-10 to 2019-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2019-02-11 to 2019-04-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
only 14 days of exposure (performed as a preliminary study to OECD 422)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
only 14 days of exposure (performed as a preliminary study to OECD 422)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST rat of Wistar origin
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 49-54 days (males), 36-41 days (females)
- Weight at study initiation: 216-231 g for males, 132-147 g for females
- Fasting period before study: no data
- Housing: 5 animals of the same sex per cage (Type IV polypropylene/ polycarbonate) with Certified laboratory wood bedding
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: Above 10 air-exchanges/ hour
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: from February 07, 2019 to February 25, 2019
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared not longer than for three days and stored at 5 ± 3 °C until use.

VEHICLE
- Concentration in vehicle: 20, 60 and 180/150 mg/mL
- Amount of vehicle: 5 mL kg/bw
- Lot/batch no. (if required): 181001
- Purity: distilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical dose verification of the formulations was performed once during this DRF study.
Measured concentrations of formulations applied in the study varied in the acceptable range (between 98 % and 101 % of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.
Duration of treatment / exposure:
14 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
900, then reduced to 750 on Day 4
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in agreement with the Sponsor and based on the oral LD50 value of 2040 mg/kg bw/day
- Fasting period before blood sampling for clinical biochemistry: yes (food deprivation for approximately 16 hours)
- Post-exposure recovery period in satellite groups: none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 (prior to study start) and twice weekly on Days 0, 3, 7, 10 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined with the measurement of non-consumed diet once weekly


WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 14 of treatment
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: WBC, RBC, HGB, MCV, MCH, MCHC, PLT, RET, differential white blood cell count, blood clotting time (APTT, PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 14 of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters checked: ALT, AST, GGT, ALP, TBIL, CREA, UREA, GLUC, CHOL, Pi, Ca++, Na+, K+, Cl-, ALB, TPPROT, A/G ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal.
The following organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes and epididymides, which were preserved in modified Davidson solution and then stored in 4 % formaldehyde solution:
Adrenal glands, Aorta, Bone with bone marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Mammary gland, Heart, Kidneys, Large intestines (caecum, colon, rectum), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Sexual organs (testes with epididymides, seminal vesicles with coagulating gland, ovaries, uterus with cervix and oviducts, vagina), Skin, Small intestines (duodenum, ileum, jejunum, rectum, including Peyer’s patches), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder
HISTOPATHOLOGY: No
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data: Body weight, Hematology and blood coagulation, Clinical chemistry, Organ weight.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Frequency of clinical signs, ophthalmoscopy, pathological and histopathological findings by sex and dose was calculated.
Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2,5-Dimethoxytetrahydrofuran caused clinical signs in slight or moderated degree at 900 mg/kg bw/day (decreased activity, squatting position, incoordination and prone position) and at 750mg/kg bw/day (decreased activity incoordination) in male and female animals.
The behavior and physical condition of all animals were normal in male and female animals in the control group and at 100 and 300 mg/kg bw/day during the course of 14-day administration.
At 900 mg/kg bw/day, slight or moderate decreased activity, squatting position, incoordination and prone position were detected in male (5/5) and female (5/5) animals 5-60 minutes after the administration on Days 0, 1 and 2.
After the dose reduction to 750 mg/kg bw/day, decreased activity and incoordination were noted for each animal during the remaining days of treatment period (5/5 male and 5/5 female).
All animals recovered – were normal – on Day 3 (washout day).
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 100, 300 or 900/750 mg/kg bw/day groups during the 14-day treatment period (male and female).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant body weight loss was detected in male and female animals administered with 900 mg/kg bw/day between Days 0 and 3.
The body weight development was undisturbed in male and female animals after the dose reduction to 750 mg/kg bw/day (between Days 4 and 13).
The body weight and body weight gain of the male and female animals were unaffected at 100 mg/kg bw/day and in female animals at 300 mg/kg bw/day throughout the entire observation period.
In the male animals at 300 mg/kg bw/day, the mean body weight gain was significantly lower than in the control group between Days 0 and 3 resulting in significantly lower mean body weight from Day 3 up to the termination of the study (-5 %, -7 %).
At 900 mg/kg bw/day, the body weight decreased in each male and female animal between Days 0 and 3.
After the dose reduction, the mean body weight gain exceeded the control value reaching statistical significance between Days 3 and 7 (male and female) and between Day 7-10 (female). However, the summarized body weight gain remained significantly lower than in the control group in male animals and was lower – no statistical significance – than in the control group in female animals.
As a consequence, the mean body weight was lower than in the control in male animals from Day 3 up to the end of the study (minus 22 % – minus 13 %) and in female animals on Days 3 and 7 (-16, -7 %). The mean body weight of female animals at 900/750 mg/kg bw/day was similar to their control by the end of the treatment period (Day 13).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was reduced in male and female animals at 900/750 mg/kg bw/day during the first week of the study in full compliance with the body weight changes.
The mean daily food consumption was comparable in the control and 100, 300 mg/kg bw/day (male and female) during the 14-day observation period.
At 900/750 mg/kg bw/day, the daily mean food intake was significantly lower than in the control in male and female animals during week 1 and it was similar to the control during week 2 (male and female).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related elevation in the mean percentage of neutrophil granulocytes (NEU), monocytes (MONO) and reticulocytes (RET) were observed in male and female animals at 900/750 mg/kg bw/day along with lowered mean percentage of lymphocytes (LYM).
In the male animals at 100 and 300 mg/kg bw/day, the examined hematological parameters were comparable to their control.
In the female animals, statistical significances were detected at the higher mean white blood cell count (WBC) and percentage of reticulocytes at 100 mg/kg bw/day and at the higher mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) at 300 mg/kg bw/day with respect to their control.
At 900/750 mg/kg bw/day, statistically significant differences were observed with respect to the appropriate control at the higher mean percentage of neutrophil granulocytes, monocytes and reticulocytes, at the lower mean percentage of lymphocytes in male and female animals as well as at the higher mean white blood cell count in female animals.
Additionally, statistical significance was noted for the lower mean hematocrit value (HCT) and shorter mean prothrombin time (PT) in male animals at 900/750 mg/kg bw/day.
The slight, sporadic statistical differences compared to the control (HCT, PT in male animals at 900/750 mg/kg bw/day; WBC, RET and MCV, MCH in female animals at 100 and 300 mg/kg bw/day, respectively) were with minor degree and values remained within the historical control ranges. Therefore, these findings were judged to be toxicologically not relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slight elevation in activity of alanine aminotransferase (ALT) in male animals and elevated cholesterol concentration (CHOL) in male and female animals at 900/750mg/kg bw/day and in male animals at 300 mg/kg bw/day suggest an effect on the hepatic function.
At 100 mg/kg bw/day, statistical significance was detected at the slightly higher mean concentration of chloride (Cl-) in male animals and all examined parameters were comparable to their control in female animals.
At 300 mg/kg bw/day, higher mean concentrations of cholesterol, sodium (Na+) and chloride were observed in male animals and lower mean concentration of creatinine (CREA) and higher mean concentration of glucose (GLUC) were detected in female animals when compared to their control.
Compared to their control, statistical significances were noted for the higher mean activity of alanine aminotransferase in male animals, higher mean concentration of cholesterol in male and female animals and total protein (TPROT) in female animals. Lower mean level of chloride and creatinine and lower mean albumin: globulin ratio (A/G) were also detected in female animals at 900/750 mg/kg bw/day when compared to their control.
Statistically significant differences in some parameters were considered to be of little or no biological significance as the mean values correlated well with the historical control values (creatinine, glucose, total protein, A/G, Na+, Cl), the profile of change has no biological significance (creatinine) or findings were only detected at the lower doses (glucose, Cl, Na+).
Urinalysis findings:
not examined
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):
Evaluation of absolute and relative organ weights revealed slight elevation of kidneys weight in female animals and of liver weight in male and female animals at 900/750 mg/kg bw/day, which were judged to be treatment related. Histopathological examinations are needed to reveal the nature of these findings nevertheless, these might be an indication of the adaptation process.
There were no statistically or biologically significant differences between the control and 100 mg/kg bw/day treated male or female animals in the weights of the examined organs.
In the male animals at 300 mg/kg bw/day, statistically significant difference with respect to the control was detected at the slightly higher mean liver weight relative to body weight
In the female animals at 300 mg/kg bw/day, the liver weight relative to body and brain weights and thymus weight relative to body weight exceeded the control value.
At 900/750 mg/kg bw/day, statistically significant difference with respect to the control was detected at the lower mean body weight (absolute and relative to brain weight), at the higher mean liver weight (absolute and relative to body and brain weights), at the lower mean weights of heart (absolute) and seminal vesicles with coagulating gland and prostate (absolute and relative to brain weigh) and at the higher weights of brain and adrenal gland (both relative to body weight) in male animals.
In the female animals at 900/750 mg/kg bw/day, the mean weights of liver (absolute and relative to body and brain weights), kidneys (relative to body and brain weights) and thymus (relative to body and brain weights) were higher than in the control group.
Changes in the liver weights in male and female animals and in kidney weights in female animals at 300 or 900/750 mg/kg bw/day might be related to a test item influence on hepatic and renal function taking into account the changes in some clinical chemistry parameters (ALT, cholesterol). The degree of all of these changes was small therefore these might be considered to be indication of the adaptation process.
Statistical significances at the thymus weight was due to the relative low control value in female animals and all individual values remained well within the historical control ranges.
Statistical significances noted for some organs (brain, heart, seminal vesicles, adrenal glands) in male animals were with small degree and were originated from the low fasted body weight.
Therefore, these findings in organ weights (adrenal glands, brain, heart, seminal vesicles, thymus) were considered to be of little or no biological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any macroscopic changes, which were related to treatment with test item in male or female animals (100, 300 or 900/750 mg/kg bw/day).
Renal pyelectasia – right side or both sides – was observed in the control group (1/5 male, 1/5 female), at 100 mg/kg bw/day (1/5 male), at 300 mg/kg bw/day (1/5 male and 1/5 female) and at 900/750 mg/kg bw/day (1/5 female).
Pyelectasia is a common finding in this strain of experimental rats. There were no inflammatory or other pathological signs related to this finding and therefore it was not attributed to test item treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
mortality
organ weights and organ / body weight ratios

see attached document

Conclusions:
Under the condition of the present study 2,5-Dimethoxytetrahydrofuran caused clinical signs, reduction in body weight development and food consumption after 13 days oral administration to male or female Han:WIST rats at 900/750 mg/kg bw/day.
At 900/750 mg/kg bw/day, a test item influence was observed in white blood cell parameters (NEU, MONO, LYM) and percentage of reticulocytes (male and female), in concentrations of cholesterol (male and female) and ALT activity (male), changes in in organ weights (liver, male and female; kidneys, female).
At 300 mg/kg bw/day, lesser but significant changes in some of these parameters were observed: body weight, cholesterol concentration in male animas, liver weight in male and female animals.
At 100 mg/kg bw/day, there were no test item related findings.
Executive summary:

In a subacute toxicity study 2,5-Dimethoxytetrahydrofuran was administered to 5 rats/sex/dose in water by gavage at dose levels of 0, 100, 300 and 900/750  mg/kg bw/day.

Detailed clinical observations were performed daily after the treatment. Body weights were recorded twice weekly in groups of control, 100 and 300 mg/kg bw/day and daily in high dose groups from Day 2. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed.

There was no mortality in control, 100, 300 or 900/750 mg/kg bw/day groups.

Test item related clinical signs were observed in slight or moderated degree at 900 mg/kg bw/day (decreased activity, squatting position, incoordination and prone position on Days 0, 1, 2) and at 750mg/kg bw/day (decreased activity incoordination, from Day 4 to Day 13) in male and female animals.

Significant body weight loss was detected in male and female animals administered with 900 mg/kg bw/day between Days 0 and 3. The body weight development was undisturbed in male and female animals after the dose reduction to 750 mg/kg bw/day – between Days 4 and 13.

The mean daily food consumption was reduced in male and female animals at 900/750 mg/kg bw/day during the first week of the study in full compliance with the body weight changes.

Test item related elevation in the mean percentage of neutrophil granulocytes (NEU), monocytes (MONO) and reticulocytes (RET) were observed in male and female animals at 900/750 mg/kg bw/day along with lowered mean percentage of lymphocytes (LYM).

Slight elevation in activity of alanine aminotransferase (ALT) in male animals and elevated cholesterol concentration (CHOL) in male and female animals at 900/750mg/kg bw/day and in male animals at 300 mg/kg bw/day suggest an effect on the hepatic function.

Necropsy examinations did not reveal any macroscopic changes, which were related to treatment with test item in male or female animals (100, 300 or 900/750 mg/kg bw/day).

Evaluation of absolute and relative organ weights revealed slight elevation of kidneys weight in female animals and of liver weight in male and female animals at 900/750 mg/kg bw/day, which were judged to be treatment related. Histopathological examinations are needed to reveal the nature of these findings nevertheless, these might be an indication of the adaptation process.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2,5-dimethoxyfuran
EC Number:
211-797-1
EC Name:
Tetrahydro-2,5-dimethoxyfuran
Cas Number:
696-59-3
Molecular formula:
C6H12O3
IUPAC Name:
tetrahydro-2,5-dimethoxyfuran
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST rat of Wistar origin
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 75-80 days (males and females)
- Weight at study initiation: 315-366 g for males, 204-238 g for females
- Fasting period before study: no data
- Housing: Type III polypropylene/ polycarbonate, with Certified laboratory wood bedding
> Before mating: 2 animals of the same sex/cage
> Mating: 1 male and 1 female / cage
> Pregnant females: individually
> Males after mating: 2 animals / cage
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: Above 10 air-exchanges/ hour
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: from April 18, 2019 to June 28, 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared not longer than for three days and stored at 5 ± 3 °C until use.

VEHICLE
- Concentration in vehicle: 20, 50 and 100 mg/mL
- Amount of vehicle: 5 mL kg/bw
- Lot/batch no. (if required): 191001
- Purity: distilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study.
Five aliquots of 5 mL of each formulation (20, 50 and 100 mg/mL) and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C until analysis.
Concentration of the test item in the dosing formulations varied between the range of 96 % and 105 % in comparison to the nominal values.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified. The recovery of the test item from the vehicle was within the acceptance criteria.
2,5-Dimethoxytetrahydrofuran proved to be stable in distilled water at the intended concentrations at room temperature and at 5 ± 3 °C for three days.
Duration of treatment / exposure:
Males: 48 days (14 days pre-mating and 1-14 days mating in animals plus 20-33 days of post-mating period).
Females: 14 days pre-mating, through 1-14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice except for one dam, which was administered up to and including lactation day 12 (altogether for 51-58 days).
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on the basis of the results of a preliminary dose range finding study with 2,5-Dimethoxytetrahydrofuran in rats (Study no. 855-400-4394)
- Fasting period before blood sampling for clinical biochemistry: yes (food deprivation for approximately 16 hours)
- Post-exposure recovery period in satellite groups: none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, after the administration at approximately the same time

DETAILED CLINICAL OBSERVATIONS: Yes
at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated. Body weight was measured on the day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated according to measurement days and for the study overall.

FOOD CONSUMPTION: yes
The food consumption was determined weekly by weighing the given and non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating period: Days 7, 13, and post-mating period: Days 20, 27, 34, 41 and 47 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment (i.e. on the day of necropsy)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: in five male and five female animals randomly selected from each group.
- Parameters checked: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, RET, Differential white blood cell count (Neutrophil, Lymphocyte, Eosinophil, Monocyte, Basophil), blood clotting times (APTT and PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
one day after the last treatment (i.e. on the day of necropsy)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals:
in five male and five female animals randomly selected from each group.
- Parameters checked: ALT, AST, TBIL, CREA, UREA, GLUC, CHOL, Na+, K+, ALB, TPROT

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once prior to the first exposure and once weekly thereafter.
- Dose groups that were examined: all animals
- Battery of functions tested: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling (on Day 48 for male and female animals). General physical condition and behavior of animals were tested. A modified Irwin test was performed.

IMMUNOLOGY: No

OTHER:
- thyroid hormones (FT4 and TSH): in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Blood samples were collected from animals as follows:
> from 2-6 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter)
> from all dams and from 1-7 pups per litter on post-partum/ post-natal day 13;
> from all parent male animals at termination on Day 48.

- Examination of placental sign (see section 7.8.1 for details)
- Observation of the delivery process (see section 7.8.1 for details)
- Observation of the offspring (see section 7.8.1 for details)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal.
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows:
− Parental male animals: after the optionally extended post-mating period on Day 48.
− Dams on post-partum day 13 or shortly thereafter (between Days 51 and 58).
− One dam (no. 324) of the 250 mg/kg bw/day-dose group was euthanized on gestation day 13 (Day 30) because of severe wounding on the skin of head, neck and upper part of the back.
− Not mated control female animal on Day 30;
− Offspring: on post-natal day 13 or shortly thereafter.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating glands of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

HISTOPATHOLOGY: Yes
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group.
List of organs preserved:
Adrenal glands, Aorta, Bone with bone marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Mammary gland, Heart, Kidneys, Large intestines (caecum, colon, rectum), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pituitary, Pancreas, Salivary glands (submandibular), Sciatic nerve, Sex organs (testes, epididymides, prostate seminal vesicle with coagulating gland, ovaries, uterus with cervix and fallopian tube, vagina), Skin, Small intestines (duodenum, ileum, jejunum, including Peyer’s patches), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (500 mg/kg bw/day) groups and in euthanized female animal at 250 mg/kg bw/day.
Based on the necropsy observations, the histological processing and evaluation were performed for the following organs:
- liver: 1/12 female at 100 mg/kg bw/day, 1/12 male at 250 mg/kg bw/day and 1/12 male at 500 mg/kg bw/day;
- kidneys: 2/12 male at 100 mg/kg bw/day, 5/12 male at 250 mg/kg bw/day and 2/12 male at 500 mg/kg bw/day; 1/12 female at 100 mg/kg bw/day; 1/12 female at 500 mg/kg bw/day; three pups in control group; 3 pups at 100 mg/kg bw/day; 5 pups at 250 mg/kg bw/day;
- spleen: one pup at 250 mg/kg bw/day.

ORGAN WEIGHT!
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined.
Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight was not determined as no related changes were detected.
Paired organs were weighed together.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs (decreased activity, piloerection or incoordination) were detected in male and female animals at 500 mg/kg bw/day after the daily administration during the first week of the study.
There were no test item related clinical signs in parental animals (male and female) in the control, 100, 250 or 500 mg/kg bw/day groups at the detailed weekly clinical observations. All animals exhibited normal behavior and physical condition with no abnormalities except for one dam (wounds on the skin of the head, neck, on upper part of the back) from Day 13 up to gestation day 7.
There were no clinical signs in male or female animals in control, 100 or 250 mg/kg bw/day group except for one female animal (1/12) at 250 mg/kg bw/day during the entire observation period. In this female animal, wounds were detected on the head, neck or the upper part of the back from Day 8 up to gestation day 12.
Wounds on the skin is a dermal change in experimental rats of this strain similar age and was considered as individual lesion in this study. Similar finding was not detected in other animals and at the high dose treated animal.
At 500 mg/kg bw/day, decreased activity (12/12 male and 12/12 female), piloerection (7/12 male and 12/12 female) and incoordination (2/12 male and 12/12 female) were observed with variable incidence after the daily treatment during week 1 (between Days 1 and 7). All animals showed normal behavior from Day 8 up to the termination of the treatment.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 100, 250 or 500 mg/kg bw/day groups during the course of study (male and female).
One female animal administered with 250 mg/kg bw/day was euthanized on gestation day 13 because of individual lesion. Severe wounding was detected on the skin of head, neck and upper part of the back from Day 8 up to the day of the euthanasia. Histological investigation of skin revealed exudative dermatitis in this animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in male animals at 500 mg/kg bw/day during the observation period and in female animals at 500 mg/kg bw/day during the lactation period.
The mean body weight was comparable in male animals in the control and 100 mg/kg bw/day groups during the pre-mating, mating and post-mating periods. Although, statistical significance with respect to the control group was detected at the slightly lower mean body weight gain during the pre-mating period (between Days 0 and 13) and during the last 2 weeks of the treatment period (between Days 34 and 47) and at the summarized body weight gain (between Days 0 and 47) in in male animals at 100 mg/kg bw/day.
At 250 mg/kg bw/day, the mean body weight remained below the control value in male animals on Days 41 and 47 due to the lower mean body weight gain of these animals during the entire observation period reaching statistical significances between Days 0-7, 34-41 and 0-47.
The mean body weight was statistically significantly lower with respect to the control in the male animals at 500 mg/kg bw/day from Day 7 up to the termination of the treatment. The mean body weight gain was also lower than in the control group in high dose treated male animals between Days 0-7 and 27-41 as well as if summarized (between days 0 and 47).
In the female animals, the mean body weight was comparable with their control during the pre-mating and gestation period at 100, 250 and 500 mg/kg bw/day groups.
Statistically significant difference with respect to the control was observed at the lower mean body weight of dams at 500 mg/kg bw/day on lactation days 0, 4 and 13, as well as at the lower mean body weight gain between Days 0 and 7 during premating period and between gestation days 14 and 21.
These changes in body mean weight gain at 100 and 250 mg/kg bw/day were sporadic and of minor degree and resulted in only slight changes in the mean body weight. Therefore, these findings were considered to be toxicologically not relevant.
The reduced body weight gain seen in the high dose male animals resulted in significant changes of the mean body weight mainly during the first weeks and last two weeks of the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was slightly reduced at 500 mg/kg during the pre-mating and post-mating period in male animals as well as in female animals during the first week of the study and lactation periods.
The mean daily food consumption was slightly lower than in the control group in the male animals at 100 mg/kg bw/day between Days 34 and 41 as well as at 250 mg/kg bw/day during the pre-mating period (Day 0-13) and between Days 34 and 41.
In the male animals 500 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean daily food consumption during the entire observation period except for week between day 20 and 27.
In the female animals, lower mean daily food consumption was detected at 100 mg/kg bw/day between Days 7-13 and at 250 mg/kg bw/day during the pre-mating period and between lactation days 0-4.
In the female animals 500 mg/kg bw/day, statistical significance with respect to the control was noted for the lower mean daily food consumption during the first week of administration (Day 0-7) and during the lactation period (between lactation days 0 and 13).
Based on these observations, test item administration influenced the mean food consumption in male and female animals administered with 500 mg/kg bw/day.
Slight changes in food intake at the lower doses were considered to be toxicologically not relevant because of the minor degree or sporadic occurrence.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 250 or 500 mg/kg bw/day.
Statistically significant difference with respect their control was detected at the slightly higher mean platelet count (PLT) in male animals at 100 and 500 mg/kg bw/day. However, all individual values were well within the historical control range. Statistical significance was presumably originated from the relative low values of control male animals.
In female animals of the 100 mg/kg bw/day group, statistically significantly lower mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) was observed.
At 250 mg/kg bw/day, the mean red blood cell count (RBC) was higher, the mean corpuscular (erythrocyte) hemoglobin concentration and mean hemoglobin content (MCH) were slightly lower and the mean activated prothrombin time (APTT) was slightly longer than in the control group in female animals.
In the female animals at 500 mg/kg bw/day, statistically significant difference with respect to the control was detected at the higher mean red blood cell count, higher mean hematocrit (HCT), lower mean corpuscular volume (MCV), mean hemoglobin content and lower mean corpuscular (erythrocyte) hemoglobin concentration.
These differences were considered to be toxicologically not relevant due to the minor degree and because of all values met the historical control data (RBC, HCT, MCV, MCHC, PLT, APTT).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 250 or 500 mg/kg bw/day (male or female).
Slight changes in cholesterol (male and female), urea (male) or total bilirubin (female) at 500 mg/kg bw/day might be related to the test item effect however these findings were not accompanied by alterations in organ pathology, therefore were considered to be toxicologically not relevant.
The examined clinical chemistry parameters were comparable with the control at 100 mg/kg bw/day.
Statistically significant differences with respect to the control were detected in the male animals at the higher mean concentrations of cholesterol (CHOL) at 250 and 500 mg/kg bw/day, at higher mean urea concentration at 250 mg/kg bw/day and at the higher mean concentration of albumin (ALB) at 500 mg/kg bw/day. The mean urea concentration exceeded the control value in male animals at 500 mg/kg bw/day (no statistical significance was detected).
In the female animals at 500 mg/kg bw/day, elevated concentrations of total bilirubin (TBIL), cholesterol, albumin and total protein were observed when compared to their control. At 250 mg/kg bw/day, slightly elevated concentration of total bilirubin and cholesterol was also detected in female animals, but values of female animals remained well within the historical control range and did not reach statistical significances.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 100, 250 or 500 mg/kg bw/day groups at the end of the treatment period (on Day 48).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no adverse effects on the weights of examined organs in male or female animals at any dose level (100, 250 and 500 mg/kg bw/day).
Slight changes in the weights of several organs with respect to the control were mainly originated from the lower mean fasted body weight in male animals at 100, 250 or 500 mg/kg bw/day – reaching statistical significances at the mid and high doses. In the lack of related clinical chemistry or histopathological alterations the following findings were judged to be toxicologically not relevant in male animals:
- lower mean heart weight (absolute) at 100, 250 and 500 mg/kg bw/day;
- higher mean testes weight relative to body weight at 100, 250 and 500 mg/kg bw/day;
- lower mean absolute weights of brain and seminal vesicles, higher mean liver weight relative to body weight at 500 mg/kg bw/day;
The fasted mean body weight was lower than in the control group in male animals at 250 and 500 mg/kg bw/day.
In the female animals, some sporadic statistically significant difference compared to their control were considered to be of no biological relevance: lower mean thymus weights (absolute and relative to brain weight) at 100 mg/kg bw/day, lower mean spleen weight (absolute) and higher mean weights of brain and kidneys both relative to body weight at 500 mg/kg bw/day.
Histopathological examinations revealed no lesions and there were no test item-related adverse effects in clinical pathology parameters indicative for an impairment of function of these organs.
Slight changes in weights of some organs – heart, testes, brain, liver and seminal vesicle with coagulating gland in male animals and thymus, spleen and kidneys in female animals – were considered to be toxicologically not relevant because there were no supporting histological findings and dose relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Specific macroscopic alterations were not detected in the organs or tissues at any dose levels (100, 250 or 500 mg/kg bw/day) at the necropsy.
In the male animals in control group, smaller than normal large liver lobe (1/12), smaller than normal testes (2/12), larger than normal testes (1/12), smaller than normal seminal vesicle (1/12) and prostate (1/12) and soft tissue formation in one side kidney (1/12) were observed.
In the male animals administered with the test item, one or both sided pyelectasia (2/12 at 100 mg/kg bw/day; 5/12 at 250 mg/kg bw/day; 2/12 at 500 mg/kg bw/day) and Hernia diaphragmatica (1/12 at 250 mg/kg bw/day and 1/12 at 500 mg/kg bw/day) were observed at the terminal necropsy.
In the female animal subjected to early necropsy (1/12 at 250 mg/kg bw/day) on gestational day 13, larger than normal spleen and submandibular lymph nodes, dead fetus in the uterine horn and wounds on the skin of the head, neck and upper part of the back (1/1, each) were detected as individual lesions.
In dams, one side pyelectasia was observed in control (1/11), 100 mg/kg bw/day (1/12) and 500 mg/kg bw/day (1/12) groups. Hernia diaphragmatica was noted for single dam at 100 mg/kg bw/day (1/12).
Smaller than normal liver lobe, testes, seminal vesicle, prostate, larger than normal testes, soft tissue formation in one side kidney were individual changes in control male animals.
Wounds on the skin are common finding in experimental rats of this strain with similar age and is considered as individual lesion.
Hernia diaphragmatica and pyelectasia are frequent observations in untreated experimental rats of this strain. In the lack of related inflammatory or other pathological histopathological alterations, pyelectasia and hydrometra were judged to be toxicologically not relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations did not reveal any toxic or other test item related lesions in the investigated reproductive and other organs of experimental male and female animals at 500 mg/kg bw/day.
In the male animals of control (12/12) and 500 mg/kg bw/day (12/12) groups, the investigated organs of reproductive system (testes, epididymides, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of control – except for one animal – and test item treated animals.
However, in one animal in control group (1/12), the one side testis was smaller than normal and lack of mature spermatozoa in the one side testis and epididymis was detected. The other cells of these organs – the spermatogonia, the spermatocytes and the spermatids – were normal in this control animal. These findings were considered as individual disorder, without toxicological significance.
In another control animal (No.: 103) for which smaller than normal testes, prostate and seminal vesicles were noted at the necropsy, the histological picture of testes and seminal vesicles was normal, while decreased amount of the secretum was observed in the prostate.
In the female animals belonging to the high dose (500 mg/kg bw/day) treated and control groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (12/12, in control and high dose groups). The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5, in both groups) animals.
In one control male animal (1/12), nephroblastoma (embryonal nephroma, Wilms tumor) was detected – in correlation with macroscopic finding in one side kidney. Nephroblastoma is a rare neoplasia in rats arising from the primordial metanephric blastemal and occurs mainly in young animals, as an individual disorder.
Diaphragmatic hernia (including part of liver lobe) is a developmental disorder, without toxicological significance. Focal fibrosis in the Glisson capsule was observed in these animals: 1/1 male at 250 mg/kg bw/day; 1/6 male at 500 mg/kg bw/day; 1/1 female at 100 mg/kg bw/day). The focal fibrosis in the Glisson’s capsule of the liver is a consequence of the mechanical irritation due to the diaphragmatic hernia.
One or both sided pyelectasia was detected in several animals in the kidneys without degenerative, inflammatory or other histological (fibrotic etc.) lesion. Therefore, this renal change was considered as a common finding in laboratory rats without toxicological significance (2/2 male at 100 mg/kg bw/day; 5/5 male at 250 mg/kg bw/day; 2/7 at 500 mg/kg bw/day; 1/5 female control; 1/1 female at 100 mg/kg bw/day).
In one female animal at 250 mg/kg bw/day (1/1), severe exudative dermatitis was observed – in accordance with the wounds on the skin as macroscopic finding. The exudative dermatitis is a subchronic inflammation probably by bacterial origin. No fungal infection or parasites were observed in this dermal lesion. Moderate hyperplasia of the spleen and submandibular lymph nodes (without tumor cells) were also observed in this animal in connection with long lasting antigen stimulus due to bacterial dermatitis.
In animals selected for full histological examinations, no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum thyroid hormones:
The thyroid hormone (free FT4 and TSH) levels were not affected by the test item in parental male animals (100, 250 and 500 mg/kg bw/day) or in offspring sampled on post-natal day 13.
Statistical significance was detected at the lower mean concentration of free-thyroid hormone level (FT4) of parental male animals at 250 and 500 mg/kg bw/day compared to their control. The concentrations of FT4 were within the ranges of the historical control data, not related to doses and not accompanied by changes in TSH levels. Therefore, findings in FT4 levels in ale animals were considered to be toxicologically not relevant.

Estrous cycle, Delivery data of dams, Reproductive performance: see section 7.8.1
F1 (Offspring) Generation: see section 7.8.1

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

See tables in the attached document

Applicant's summary and conclusion

Conclusions:
the No Observed Adverse Effect Level (NOAEL) of the test item is 250 mg/kg/day for both male and female rats
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening according to OECD TG 422, 2,5-Dimethoxytetrahydrofuran was administered orally (by gavage) once daily at 0 (vehicle only), 100, 250 and 500 mg/kg body weight doses to four groups of Han:WIST rats consisting of 12 animals per sex per group. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

2,5-Dimethoxytetrahydrofuran was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature and in a refrigerator (at 5 ± 3 °C) for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. 2,5-Dimethoxytetrahydrofuran concentrations in the dosing formulations varied within the range of 96 % and 105 % in comparison to the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 48 days). Females were additionally exposed through the gestation period and up to lactation days 12-15, i.e. up to the day before necropsy (altogether for 51-58 days) except for two female animals subjected to early necropsy on Day 30.

Observations included mortality, clinical signs, body weight, food consumption (as well as mating, pregnancy, delivery process, and development of F1 offspring, estrous cycle not detailed here, see section 7.8.1).

The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Data on litters are not reported here (see section 7.8.1).

Blood samples were collected for determination of serum levels of thyroid hormones (FT4 and TSH) from all dams and from all parent male animals at termination. Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five male and female animals randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid glands were preserved from all adult males and females for the intended subsequent histopathological examination. Histopathology examination was performed on ovaries, uterus with oviduct and cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in dam subjected to early necropsy at 250 mg/kg bw/day. In addition, organs showing macroscopic findings were processed and examined histologically based on the macroscopic findings at the necropsy.

Results:

There was no test item related mortality at any dose level (100, 250 and 500 mg/kg bw/day).

Clinical signs of systemic toxicity related to the test item were detected in male and female animals administered with 500 mg/kg bw/day at the daily clinical observations during the first week of the study. Decreased activity, piloerection or incoordination were observed after the daily administration between Days 1 and 7.

There were no clinical signs at the detailed weekly clinical observations or changes in behavior, physical condition or in reaction to different type of stimuli at the functional observations.

The body weight development was slightly depressed at 500 mg/kg bw/day during the entire treatment period (pre-mating, mating and post-mating period in male animals and during the lactation period in female animals).

The mean daily food consumption was reduced in male animals at 500 mg/kg bw/day during the entire observation period and in female animals at 500 mg/kg bw/day during the first week of the study and during the lactation period.

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 250 or 500 mg/kg bw/day (male or female).

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose.

Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 250 or 500 mg/kg bw/day.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides or seminal vesicles with coagulating gland as a whole in male animals or in the examined weights of animals (male and female) selected for toxicity examinations at any dose level.

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 500 mg/kg bw/day.

Conclusion:

Under the conditions of the present study, 2,5-Dimethoxytetrahydrofuran administered at 100, 250 and 500 mg/kg bw/day by oral gavage caused signs of systemic toxicity – clinical signs reduced body weight development and food consumption – at 500 mg/kg bw/day in male and female Han:WIST rats.

There were no adverse effects at 100 or 250 mg/kg bw/day (male and female).

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male and female rats: 250 mg/kg bw/day