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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Information from published industry source (USEPA), minor limitations in design and/or reporting but otherwise adequate for assessment

Data source

Reference Type:
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
according to
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): 2,5-Dihydrofuran
- Lot No.: X23849-84
- Physical state: Clear colorless liquid
- Purity: 97.5 - 99.9%

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Kingston (Stone Ridge, NY)
- Age at study initiation: 52 days
- Weight at study initiation: 264.3 ± 7 or 197.8 ± 12 g (mean ± SD), for male and female rats respectively
- Housing: single housing in stainless-steel, wire-mesh cages
- Diet: ad libitum, Certified Rodent Diet (Agway Prolab RMH 3200, ground chow)
- Water: ad libitum

- Temperature (°C): 19.4 - 22.8 (67 - 73 °F)
- Humidity (%): 55 - 66
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
clean air
Details on inhalation exposure:
The inhalation exposures were conducted in 420 L stainless-steel and glass inhalation chambers. Animals were singly housed during the exposure. The exposure chambers were maintained under negative pressure relative to the room air. The air flow, temperature, and humidity were recorded every 30 minutes. The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Nitrogen gas was passed through the glass bead-packed column at 5 Lpm. The resultant vapour was directed via glass tubing to a thee just upstream of the 420 L inhalation chamber where it was mixed with filtered, condition dilution air to produce a total airflow of 94 to 102 Lpm (13 to 15 air changer per hour).

- Brief description of analytical method used: Chamber vapour concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a single MIRAN IA infrared gas anaylsyzer (Wilks Foxbor Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve (Valco instruments, Houston, TX). Chamber vapour samples were continuously collected from each chamber. The valve position was periodically changed to dample from each chamber at least once each hour. Voltage data were converted to concentration by linear intrapolation between the calibration data point immediately on each side of the sampled data.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Animals were exposed to target concentrations of 0, 125, 400, or 1250 ppm for 22 (males) or 23 (females) exposures. The mean time-weighted average exposure concentrations (± standard deviation) were 129 ± 10, 403 ± 24, and 1249 ± 51 ppm for male rats and were 128 ± 11, 403 ± 24, and 1247 ± 51 ppm for female rats. No test substance was detected in the control chamber. Daily TWA exposure concentrations were within 11% of the target concentration for the mid- and high-concentration and within 19% of for the low-concentration. Nominal concentrations were 151 ± 12, 396 ± 26, and 2098 ± for male rats and were 151 ± 12, 397 ± 26, and 2113 ± 220 ppm for female rats the same groups.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week, for 4 weeks, and an additional 2 (males) or 3 days (females) of the 5th week.
Doses / concentrations
Doses / Concentrations:
129, 403, 1249 ppm (male); 128, 403, 1247 ppm (female)
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure concentrations were selected based on the results of a one-week probe study during which two male and two female rats per group were exposed to 1250, 2500, 5000, or 7500 ppm of the test substance 6 hours/day for 5 days. Based on mortality at 5000 and 7500 ppm and convulsions at 2500 ppm, test substance concentrations of 0, 125, 400, and 1250 ppm were selected for the four-week study.
- Necropsy: All surviving animals were euthanatized and necropsied on the day following the last exposure.


Observations and examinations performed and frequency:
Rats visible through chamber windows were observed during exposure. Tapping sounds were made to assess the animals activity level. Before and after exposure, each rat was removed from its cage and examined. Cageside observations were conducted one a day on weekends. Observations included, but were not limited to, examination of the hair, skin, eyes and mucous membranes, motor activity, faeces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behaviour patterns.

Body weights were measured prior to exposure on Days 0, 4, 7, 14, 21, and 28. Fasted body weight were collected at termination after perfusion.

Feed consumption was measured on Days 4, 7, 14, 21, and 28. Animals were fasted the day prior to necropsy.

- Time schedule for collection of blood: after the last exposure
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: haemoglobin concentration, haematocrit, red blood cell count, white blood cell count, red blood cell indices, and platelet count. Slides containing blood smears were examined for cellular morphology and differential white blood cell count.

- Time schedule for collection of blood: after the last exposure
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, creatinine, urea nitrogen, glucose, total bilirubin, total protein, albumin, albumin/globulin ratio, gamma-glutamyltransferase, calcium, phosphorus, sodium, potassium, and chloride

Animals were observed the Friday prior to the first exposure (Day -3) and prior to exposure on Days 8, 15, 22, and 29 of the study. Animals were evaluated at random. The animals were observed for the following endpoints: severity of convulsions and tremors, ranking of reactivity, alertness, coordination of movement, sensory function (vision and pain perception, pinna reflex, righting reflex, approach response, touch response. Descriptive categories of differing behaviour were noted for the following endpoints: pupillary size, description and induction of convulsions and tremors, alertness, auditory orientation, vocalization, description of body position and gait abnormalities, stereotype and bizarre behaviour, unusual respiration, muscle tone

A quantitative assessment of forelimb and hindlimb strength grip strength was performed using an apparatus equipped with a digital push-pull gauge (Model DFIS, John Chatillon & Sons, Inc., Kew Gardens, NY_ the rat was placed on a rectangular screen and then lifted back and upwardly until it released its grip. The grip strength was repeated for a total of 3 readings.

A quantitative assessment of foot splay was performed by releasing a rat 32 cm above a bench, in a horizontal position and allowing it to drop on a sheet of paper (Edwards and Parker, 1977). The foot splay was determined by measuring the distance between marks caused by ink placed on the outside digit of each hindfoot with a water soluble marker.
Sacrifice and pathology:

The following tissues were fixed in 10% neutral buffered formalin: nasal passages, trachea, lungs, larynx, heart, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal gland, thyroid glands, parathyroid glands, thymus, spleen, mesenteric lymph nodes, bone marrow (femoral), brain, spin cord, testes, right sciatic nerve, right tibial nerve, epididymides, males accessory sex glands, ovaries, vagina, uterus, Fallopian tubus, and gross lesions.

All tissues, except for the peripheral nervous system were embed in paraffin, sectioned at 5 µm and stained. All tissues from the control and high-concentration groups, the nasal passages, thymus, spleen, bone marrow, testes, epididymides, and accessory sex glands from the mid-concentration groups; and the nasal passages and thymus form the low-concentration group were examined microscopically.
Other examinations:
The lungs, liver, kidneys, adrenal glands, testes, spleen, thymus, and brain were weighed. Paired organs were weighed together.
Mean values were calculated for analytical concentration, chamber temperature, chamber relative humidity, chamber air flow, body weight and body weight change. Feed consumption, body weight , and body weight change were evaluated using the following statistical tests: Barlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Duncan's multiple range test (feed consumption and body weight) (p≤0.05) or Dunnett’s t-test (body weight change) (p ≤ 0.05) to indicate statistical significance.

Continuous FOB data an behaviour score were analysed using a repeated measures analysis of variance/multivariate analysis. Baseline (pretest) values were subtracted from test-day values to normalize the variance. Data from individual test days were also analysed in using one-way analysis of variance an pairwise comparison made to control group values using a Dunnett’s t-test. Categorical data were analysed using a two-way and multiway frequency table/log-lineair model. Time points indication significant changer were further analysed using Fisher’s Exact test. A probability of p ≤ 0.05 two-tailed was used to determine significance.

When variances of the means were not consider equal by the Bartlett’t test (p ≤ 0.01), the data were evaluated using a Kruskal-Wallis H-test and Mann-Whitney U-test.

Results and discussion

Results of examinations

Details on results:
- One 1250 ppm male rat was found dead prior to exposure on Day 6 and two 1250 ppm female rates were found dead prior to exposure on Day 19. No other spontaneous mortality was observed during the study. During exposure, the 1250 ppm group had reduced activity and partially-closed eyes. All female 1250 ppm rats also exhibited head shaking movements and three of five animals had chewing motions during exposure. Other clinical sings consisted of unkempt haircoats, porphyrin nasal discharges, and piloerection. Additional sign of toxicity for the 1250 ppm group were observed 18 hours after most exposure with animals exhibiting tremors and gait disturbances. Two 1250 ppm females had brief convulsion induced by handling on Days 7 or 20. One of these animals exhibited abnormal behaviour shortly before and after the convulsion; this animals also exhibited tremors with pawing motions and head shaking. Other clinical signs for this group were considered spurious.
- Animals in the 400 ppm group also had reduced activity and partially-closed eyes during exposure, but the frequency and severity were less than for the 1250 group. No other clinical signs of toxicity during exposure were noted for the 400 ppm groups, and no clinical signs of toxicity were seen after exposure.
- The 125 ppm group exhibited minimal reduced activity during exposure only on Days 0, 1, and 2, and partially-closed eyes were observed during the first exposure for the 125 ppm group. No clinical signs of toxicity were noted for this group, except for single incidence of porphyrin nasal discharge which were observed in two female rats.

Mean body weight reflect the decrease feed consumption with significantly lower body weight for all treated groups on Day 4, and lower mean body weight for all but the 125 ppm group thereafter. Mean terminal body weights were reduced in a concentration-dependent manner for all test substance-exposed groups compared to the control group. This reduction was significant (p ≤ 0.05) for the 400 and 1250 ppm groups.

Mean feed consumption values for all test substance exposed groups were significantly lower (p ≤ 0.05) than for the control group on Day 4, and was reduced for all but the 125 ppm group thereafter.

No toxicologically significant changes were observed in haematology.

No toxicologically significant changes were observed in serum chemistry endpoints.

- The FOB indicated that animals in the 1250 ppm group had increased incidence and severity of tremors. Additional finding included a higher incidence (p ≤ 0.05) of reduced muscle tone (soft, flabby muscles), and abnormal gait (splayed walking), and an elevated (p ≤ 0.05) hindlimb hypotonic gait score.
- One 400 ppm male rate had tremors on Day 29. A reduced (p ≤ 0.05) response to touch was noted on Day 15 for the 400 and 1250 ppm male rats when the data was normalized to the based response, but this was not considered to be toxicologically significant.

The lower body weights for the 400 and 1250 ppm groups influenced the weights (absolute and relative to body weight) of the brain, kidneys (males only), thymus, spleen, and testes.

Exposure-related changes observed for the 1250 ppm male rat that died on Day 6 consisted of moderate atrophy of the spleen , moderate atrophy of the subcutaneous and abdominal adipose tissue, minimal yellowish-red discharge on the hair of the face, and minor dry urine stains on the inguinal hair. For the female rats that died on Day 19 consisted of yellow mucous through the intestinal tract, abdominal hair wet with saliva, and a minor porphyrin nasal discharge. Agonal changes included gas in the stomach of a rat, and minimal to minor thymus haemorrhage. No exposure-related changes were observed for the rats that survived to necropsy.

There were no exposure-related gross or microscopic observation the nervous system of animals on this study. Exposure-related changes observed in the nasal passages consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and serocellular exudate in the middle or dorsal meatus for the 400 and 1250 ppm groups.

Effect levels

Dose descriptor:
Effect level:
125 ppm
Based on:
test mat.
Basis for effect level:
other: Absence of the histopathological changes in the nasal passages and absence of any clinical signs of neurotoxicity observed in the 400 ppm dose group.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion