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Description of key information

Data for repeated dose toxicity are available from a 28-day oral toxicity study in rats performed according to OECD and EU guidelines as well as GLP principles. Based on this study, a NOAEL of 50 mg/kg bw/d was established based on effects on liver in rats at 150 mg/kg bw/day and higher. As supporting information, the results of a screening study performed according to OECD guideline 421 are included as well.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June - 3 September, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: Males 169-212 g; Females 147-176 g
- Fasting period before study: Not applicable
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type; during overnight activity monitoring individual housing in MIII type) with sterilised sawdust as bedding material and paper as cage-enrichment. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 23.7°C
- Humidity (%): 42 - 83%; Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12; Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dosed undiluted at 150 and 500 mg/kg/day. To allow accurate dosing at 50 mg/kg/day in undiluted form, water was used as vehicle for this dose group. Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenised to visually acceptable levels. No correction was made for the purity of the test substance.

VEHICLE
- Concentration in vehicle: 5 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of group 1 (0 mg/kg bw/d) and 2 (50 mg/kg bw/d) prepared for use on Day 12 were analysed to check homogeneity (group 2 only) and accuracy of preparation. The analytical method used (GC-FID) can be found in section 8 of this IUCLID file.

Test substance formulations in water (i.e. Group 2) formed a homogeneous suspension. For the formulation of Group 2, the concentrations analysed for some samples were below the target concentrations (79% to 87% of target). For all other samples from Group 2, the concentrations analysed were in agreement with target concentrations (91% to 94% of target).
Since the GC method developed proved to be prone to variation due to the great number of water injections made, the obtained results were the best achievable and, thus, were accepted.
Duration of treatment / exposure:
At least 28 days.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 5 hours difference between the earliest and latest dose.

Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels are based on results of an 8-day dose range finding study with Ditetrahydrofurylpropane

Animals at 150 mg/kg/day (Group 3) were inadvertently dosed on day 7 with a dose volume being significantly higher than intended (i.e. approximately 5 ml/kg instead of 0.15 ml/kg). It was evaluated that this adversely affected the study integrity, due to mortality/moribundity that subsequently occurred in this group. Therefore, a new group of animals (Group 5) was added to the study to restore the study integrity. These animals were treated at the same dose level as the initial Group 3 animals for 28 days, and the results of Group 5 replaced the results of Group 3.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations included: Mortality / Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane )
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all surviving animals
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all surviving animals
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin Identification marks: not processed, Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, Eyes with optic nerve [if detectable], (Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical/midthoracic/lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes, recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY: Yes
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- all tissues from all animals of all dose groups which died spontaneously or were terminated in extremis,
- all gross lesions.
Based on (possible) treatment-related changes in the liver and kidneys the histological examination was extended to those particular organs of all animals of groups 2 and 5 (males and/or females). All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lethargy, flat posture, uncoordinated movements, abnormal gait and/or ptosis were noted among animals treated at 500 mg/kg/day between days 1 and 3.
There were no clinical signs of toxicity noted in animals at 0, 50 and 150 mg/kg/day during the observation period.
Salivation observed in animals of the 150 and 500 mg/kg/day dose groups was considered to be a physiological response to the taste of the test substance rather than a sign of systemic toxicity, considering the nature and minor severity of the effect and its time of occurence (i.e. after dosing). Alopecia and scabs are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
The death of one male (no.10), at 50 mg/kg/day was considered to be due to a gavage accident (i.e. based on fluid noted in the thoracic cavity and microscopic abnormalities). No further mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals were considered to have been unaffected by treatment with the test substance.

The higher body weight gain of animals at 150 mg/kg/day (achieving a level of statistical significance for females) was considered to be due to the fact that these animals were not of the same delivery as group 1, 2 and 4 animals, as they were added to the study to replace the initial group 3 animals. Means were also within the normal range for rats of this age and strain. Therefore, these changes were considered not related to treatment with the test substance. No toxicological significance was ascribed to the slightly higher body weight gain (statistically significant) of females at 50 and 500 mg/kg/day in the first week of the study, as these changes were of a temporary and mild nature. Body weight gain of these females during the study remained well within normal limits for female rats of this age and strain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.

Relative food intake of females at 500 mg/kg/day was slightly reduced during the first two weeks of treatment. Since this change was of a temporary nature, it was considered to be of no toxicological relevance.

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):
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Any statistically significant changes at 150 mg/kg/day were considered to be of no toxicological significance as the changes occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These changes included increased haemoglobin levels in males, increased mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in males and females and reduced relative monocyte counts in males and females (not statistically significant for females).

The minor statistically significant reduction in prothrombin time (PT) in females at 500 mg/kg/day was considered not to represent a change of toxicological significance, because the mean remained well within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Reduced aspartate aminotransferase (ASAT) levels in females at 500 mg/kg/day,
- Increased alanine aminotransferase activity (ALAT) in one male (no. 19) at 500 mg/kg/day,
- Increased total protein levels in females at 150 and 500 mg/kg/day,
- Increased albumin levels in females at 150 and 500 mg/kg/day,
- Reduced total bilirubin levels in males at 150 and 500 mg/kg/day,
- Reduced glucose levels in males at 500 mg/kg/day and increased glucose level in one female (no. 38) at 500 mg/kg/day,
- Increased cholesterol level in one female at 500 mg/kg/day (no. 38),
- Increased potassium level in one female at 500 mg/kg/day (no. 38),
- Increased calcium levels in females at 150 and 500 mg/kg/day (not statistically significant at 150 mg/kg/day),
- Increased inorganic phosphate levels in males at 500 mg/kg/day, and in one female (no. 38) at 500 mg/kg/day.

No toxicological significance was ascribed to the slight but statistically significant higher calcium level in males at 150 mg/kg/day. This change occurred in the absence of a dose-related distribution and the mean value remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weights (not statistically significant in males) and liver to body weight ratios were observed in males at 500 mg/kg/day and in females at 150 and 500 mg/kg/day. The increase in liver weight was more pronounced in females than in males, i.e. approximately 20 and 43% for males and females respectively (corrected for body weight). In addition, increased kidney weights and kidney to body weight ratios were observed in males at 500 mg/kg/day.
No toxicological significance was ascribed to the increased kidney weight and kidney to body weight ratio in females at 150 mg/kg/day. This change occurred in the absence of a dose-related distribution and mean values remained within the range considered normal for rats of this age and strain.
Gross pathological findings:
no effects observed
Description (incidence and severity):
An accentuated lobular pattern of the liver was observed in one male (no. 19) at 500 mg/kg/day.

In the male at 50 mg/kg/day that died spontaneously on day 2, watery-clear fluid and many yellowish nodules grown together with the lungs were observed in the thoracic cavity. These findings indicate a gavage accident and were considered not related to the test substance. Other findings in this animal consisted of a reduced size of epididymides and seminal vesicles, and enlargement of the thymus. Incidental findings among other control and treated animals included enlargement of the mandibular lymph nodes, a sore in the throat region of the skin, reddish foci on the lungs, gray-white foci on the adrenals, fluid in the uterus and a watery-clear cyst in the right horn of the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a dose-related distribution they were considered changes of no toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment related microscopic findings were noted:
- Midzonal/centrilobular hypertrophy (minimal or slight degree) in the liver of 4/5 males and 4/5 females at 500 mg/kg bw/day.
- Increased incidence and severity of cortical hyaline droplets in males at 150 mg/kg bw/day (slight degree) and males at 500 mg/kg bw/day (moderate degree).
- Increased incidence of slight or moderate degrees of corticomedullary tubular basophilia in 2/5 males at 150 mg/kg bw/day and 4/5 males at 500 mg/kg bw/day.

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on effects on liver and kidneys in animals at 150 mg/kg/day and higher.
Critical effects observed:
not specified
Conclusions:
Based on a repeated dose toxicity study performed according to OECD/EC guidelines and GLP principes, a No Observed Adverse Effect Level (NOAEL) for Ditetrahydrofurylpropane of 50 mg/kg/day was established, based on effects on liver and kidneys in animals at 150 mg/kg bw/day and higher.
Executive summary:

Wistar rats were treated with Ditetrahydrofurylpropane for 28 consecutive days by daily oral gavage at dose levels up to 500 mg/kg/day.

No signs of toxicity were noted during the in-life phase, except for the first few days during which lethargy, flat posture, uncoordinated movements, abnormal gait and/or ptosis were noted among animals treated at 500 mg/kg/day.

Increased liver weights were noted at necropsy in males at 500 mg/kg/day and females at 150 and 500 mg/kg/day, along with a single occasion of an accentuated lobular pattern of the liver in one of these males. At 500 mg/kg/day, this was supported by midzonal/centrilobular hypertrophy of the liver in both sexes. The location of the hypertrophy in centrilobular and midzonal hepatocytes and absence of any morphological lesions indicative of hepatocytotoxicity was consistent with that of an adaptive change. The lower total bilirubin levels and aspartate aminotransferase activity at 500 mg/kg/day could reflect adaptive changes in the liver. However, considering the magnitude of the increase in liver weight in these animals (i.e. approximately 20 and 43% for males and females respectively), the increased liver weight was considered to be of an adverse nature. In addition, a number of clinical biochemistry parameters were observed that suggest an effect of the test substance on liver function. These parameters included increased total protein, albumin, glucose and cholesterol levels in (individual) females at 150 and 500 mg/kg/day and reduced glucose levels in males at 500 mg/kg/day.

In the kidneys of male rats at 150 and 500 mg/kg/day, an increase in the incidence and severity of cortical hyaline droplets was observed. These droplets were considered to represent alpha-2u-globulin, a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular epithelial cell damage (hyaline droplet nephropathy) which was evident in this study as an increase in the severity of corticomedullary tubular basophilia in males at 150 and 500 mg/kg/day. Kidney weights were also increased at these dose levels. An increase in the incidence/severity of cortical hyaline droplets is a specific male rat response which is not observed in normal female rats and higher species of either sex, including humans.

Other changes in clinical biochemistry parameters included increased calcium levels in females at 150 and 500 mg/kg/day, increased inorganic phosphate levels in males at 500 mg/kg/day, and an increased inorganic phosphate and potassium level in one female at 500 mg/kg/day.

The increase in calcium levels in females at 150 and 500 mg/kg/day coincides with an increase in albumin levels in these animals. As albumin is a major calcium binding protein and no correlating morphological changes of these animals were observed, this change probably reflects the increase in albumin levels and is considered of no toxicological significance.

From the results presented in this report, a definitive No Observed Adverse Effect Level (NOAEL) for Ditetrahydrofurylpropane of 50 mg/kg/day was established, based on effects on liver and kidneys in animals at 150 mg/kg/day and higher.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 September - 28 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 316 - 323 gr (males) or 201 - 204 gr (females).
- Fasting period before study: All males were deprived of food before necropsy. Fasting does not adversely affect interpretation of the examined parameters. Females were not fasted before necropsy.
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 – 22.2°C
- Humidity (%): 43 - 81%
Temporary deviations from the maximum level of relative humidity occurred in the animal room. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 28 September - 28 November 2010
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

Storage conditions of formulations: At ambient temperature.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX Project 484749). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Some females (1, 2, 1,1 in Group 1,2,3,4 respectively) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-day oral toxicity study with Ditetrahydrofurylpropane (NOTOX Project 484766)
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity. On Day 3 of the mating period all animals were observed under Project number 495179 instead of Project number 495227. Clinical signs recorded under the incorrect number were transferred to the correct number afterwards.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.
Inadvertently, the body weight from one female of Group 1 was determined on Lactation Day 2 instead of lactation Day 1. Given that recording of body weight occurred only one day later than intended, this is considered to provide sufficient information on body weight development of this animal during the lactation phase.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
(Average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
No

CLINICAL CHEMISTRY
No

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
No
Sacrifice and pathology:
GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
Females which delivered on lactation Days 5 and 6. Females which failed to deliver (Group 1; 1 and Group 4;1) on post-coitum Day 26 (females showed evidence of mating). Males: following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks (not processed) , Cervix, Clitoral gland, Coagulation gland, Epididymides <1>, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes <1>, Uterus, Vagina, All gross lesions.

<1> Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
<2 > In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from all F0 –males on the scheduled day of necropsy: Epididymides, Testes.

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

The preputial gland, seminal vesicles and prostate from all group 1 and 4 males were processed histologically. No histopathology was required on these tissues (except for those males that failed to sire).

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 for staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups:
Group 1: one male and female that had no offspring.
Group 4: one male and female that had no offspring.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

One ovary from one female of Group 4 was not available for histopathology. Reason for missing this tissue is that this tissue was not discernable at trimming. Sufficient data was available for evaluation.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 50 and 150 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.

Alopecia and scales observed among females at 50 and 150 mg/kg w/day occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption after allowance for body weight was similar between treated and control animals.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations.

Incidental findings among control and/or treated animals included red-brown discolouration of the epididymides, a yellowish nodule on the preputial glands, greenish nodules on the epididymides, reduced size of the prostate and seminal vesicles, a tan/red/brown focus on the clitoral glands and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
not specified
Conclusions:
In a reproduction/developmental toxicity screening test with Ditetrahydrofurylpropane performed according to OECD guideline 421 and GLP principles, no parental, reproduction or developmental toxicity was observed up to the highest dose level tested (150 mg/kg bw/day).
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.
Executive summary:

A reproduction/developmental toxicity screening test with Ditetrahydrofurylpropane in rats by oral gavage was performed according to OECD guideline 421 and GLP principles was performed. In a 28-day oral toxicity study with Ditetrahydrofurylpropane (NOTOX project 484766), a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established, based on effects in liver and kidneys at 150 and 500 mg/kg bw/day. Based on these results, the dose levels for this reproduction/developmental toxicity screening test were selected to be 15, 50 and 150 mg/kg bw/day. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 15, 50 and 150 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 -55 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation. The following parameters were evaluated: mortality / viability, clinical signs, body weights, food consumption, reproduction/developmental parameters, observations pups, macroscopy, organ weights, and histopathology. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction or developmental toxicity was observed up to the highest dose level tested (150 mg/kg bw/day).

Based on the parameters assessed in this study, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Bot the repeated dose study and the screening study were performed according to the relevant OECD/EC guidelines and GLP rinciples (Klimisch 1 studies).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

In a repeated dose toxicity study, performed according to OECD 407 test guidelines, Wistar rats were treated with Ditetrahydrofurylpropane for 28 consecutive days by daily oral gavage at dose levels 0, 50, 150 and 500 mg/kg bw/day. Formulation analyses confirmed that formulations were prepared accurately and homogenously. No signs of toxicity were noted during the in-life phase, except for the first few days during which lethargy, flat posture, uncoordinated movements, abnormal gait and/or ptosis were noted among animals treated at 500 mg/kg bw/day. Body weights and food intake were unaffected by treatment and functional observation tests revealed no abnormalities. No toxicologically relevant changes occurred in haematological parameters.

Increased liver weights were noted at necropsy in males at 500 mg/kg/day and females at 150 and 500 mg/kg/day, along with a single occasion of an accentuated lobular pattern of the liver in one of these males. At 500 mg/kg/day, this was supported by midzonal/centrilobular hypertrophy of the liver in both sexes. The location of the hypertrophy in centrilobular and midzonal hepatocytes and absence of any morphological lesions indicative of hepatocytotoxicity was consistent with that of an adaptive change. The lower total bilirubin levels and aspartate aminotransferase activity at 500 mg/kg/day could reflect adaptive changes in the liver. However, considering the magnitude of the increase in liver weight in these animals (i.e. approximately 20% and 23%/43% for males and females respectively), the increased liver weight was considered to be of an adverse nature. In addition, a number of clinical biochemistry parameters were observed that suggest an effect of the test substance on liver function. These parameters included increased total protein, albumin, glucose and cholesterol levels in (individual) females at 150 and 500 mg/kg/day and reduced glucose levels in males at 500 mg/kg/day.

In the kidneys of male rats at 150 and 500 mg/kg/day, an increase in the incidence and severity of cortical hyaline droplets was observed. These droplets were considered to represent alpha-2u-globulin, a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular epithelial cell damage (hyaline droplet nephropathy) which was evident in this study as an increase in the severity of corticomedullary tubular basophilia in males at 150 and 500 mg/kg/day. Kidney weight was only increased at the highest dose level. An increase in the incidence/severity of cortical hyaline droplets is a specific male rat response which is not observed in normal female rats and higher species of either sex, including humans.

Other changes in clinical biochemistry parameters included increased calcium levels in females at 150 and 500 mg/kg/day, increased inorganic phosphate levels in males at 500 mg/kg/day, and an increased inorganic phosphate and potassium level in one female at 500 mg/kg/day. The increase in calcium levels in females at 150 and 500 mg/kg/day coincides with an increase in albumin levels in these animals. As albumin is a major calcium binding protein and no correlating morphological changes of these animals were observed, this change probably reflects the increase in albumin levels and is considered of no toxicological significance.

From the results presented in this report, a definitive No Observed Adverse Effect Level (NOAEL) for Ditetrahydrofurylpropane of 50 mg/kg/day was established, based on effects on liver in rats starting at 150 mg/kg/day and higher. The effects on kidneys at 150 mg/kg bw/day are considered to be not relevant for humans, see discussion above, and therefore not considered a critical effect.

With regards to classification according to CLP Regulation (EC) 1272/2008, the effects observed were judged to be not sufficient to justify classification for single target organ toxicity after repeated exposure (STOT) due to lack of significant toxicity.

Justification for classification or non-classification

Based on the available data, it is not required to classify the substance for repeated dose toxicity according to the CLP Regulation (EC) 1272/2008.