Tetrahydro-3-methylfuran

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

16 June 1987 to 24 August 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although no specific test guideline was identified, the study was conducted according to valid and internationally recognized test procedures that followed recognized GLP standards.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1992

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Although no specific testing guideline was identified, the study was conducted generally according to the method specified in OECD Guideline 414. Pregnant Swiss (CD-1) female mice were exposed to vapor concentrations of tetrahydrofuran of 0, 600, 1800 or 5000 ppm for 6 hours/day, 7 days/week on days of gestation (DG) 6-17. Developmental evaluations were performed on pregnant mice euthanized on DG 18.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrofuran

EC Number:

203-726-8

EC Name:

Tetrahydrofuran

Cas Number:

109-99-9

IUPAC Name:

tetrahydrofuran

Details on test material:

- Name of test material: tetrahydrofuran
- Supplier: ChemCentral, Kansas City, MO, USA
- Lot No.: BNW 51437-15
- Analytical purity: 99.3 to 100.2% (bulk chemical, gas chromatography); analyzed on 2/6/87, 3/19/87, 5/1/87 and 9/9/87
- Storage conditions: under nitrogen at 65 deg F in grounded containers
- Stability under test conditions: tetrahydrofuran was monitored during exposures with mean measured concentrations in the exposure chambers within 2% of target concentrations. Tetrahydrofuran was found to be stable in the generator reservoir and the exposure chambers
- Peroxide content: tetrahydrofuran was analyzed once/month after opening for peroxide formation and was found to contain less than 100 ppm at each analysis. Chamber atmospheres were analyzed for organic peroxides (p-hydroxyphenylacetic acid-coupling reaction) and levels were below the limit of detection.
- Degradation products: chamber atmospheres were also tested for the presence of potential breakdown products including furan, isopropanol, and butyraldehyde. None of these were detected at concentrations in excess of 0.1%.

Test animals

Species:

mouse

Strain:

other:

Details on test animals or test system and environmental conditions:

- Source: Charles River, Raleigh, NC
- Age at study initiation: 11 weeks
- Fasting period before study: none
- Housing: stainless steel wire racks equipped with automatic watering system (10-11 mice/cage)
- Diet: pelleted NIH-07 (Ziegler Bros. Inc., Gardners, PA) ad libitum except during 6-hour exposure periods
- Water (ad libitum): ad libitum throughout the study including exposure periods
- Acclimation period: quarantined 20 days prior to the start of exposures


ENVIRONMENTAL CONDITIONS
- Temperature (°F): during quarantine, 72 +/- 3; during exposures, 75 +/- 3
- Humidity (%): during quarantine, 50 +/- 15; during exposures, 55 +/- 15
- Chamber airflows: 12 to 18 CFM (average in all chambers, 14.0 to 15.4 CFM)
- Photoperiod (hrs dark / hrs light): 12/12

In-Life Dates: 8 July 1987 (mating) to 24 August 1987 (fetal exams)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: conditioned air

Details on exposure:

Exposure Chambers:
Batelle-designed inhalation exposure chambers (Harford Systems; Lab Products Inc., Aberdeen, MD) were employed. The 2-3 cubic meter stainless-steel chambers (1.7 cubic meters active mixing volume) contained three levels of caging. The cage units accomodated individual animal cages, feed troughs, and automatic watering systems.

Vapor Generation/Exposure System:
Tetrahydrofuran was pumped from a bulk storage reservoir at a steady rate by a liquid micrometering pump into a rotating flask (100 rpm) partially immersed in a hot water bath at 175 deg F. Vapor from the flask was carried by a metered stream of nitrogen. The vapor entered a short distribution manifold from which individual delivery lines carried metered amounts of the vapor to each exposure chamber. Target vapor concentrations were achieved by diluting with filtered air immediately before entering the chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Vapor Concentration Monitoring:
Vapor concentrations were monitored during animal exposures at approximately 30-minute intervals by gas chromatography (Hewlett-Packard 5840 GC) using a 1/8 in. o.d. X 1.0 foot nickel column packed with 1% SP-1000 on 60/80 mesh Carbopack B operated at 145 deg C. An 8-port valve allowed measurements of concentrations of the test material in the three exposure chambers, the control chamber, the holding chamber, the exposure room, an on-line standard, and nitrogen blank. A 400 ppm tetrahydrofuran certified standard in nitrogen (MG Industries Scientific Gases, Los Angeles, CA) was used to check instrument drift throughout the exposure day.

The precision of the on-line concentration monitoring was estimated from 17 consecutive measurements of the 400-ppm on-line THF standard. A 0.26% coefficient of variation was observed. The limit of detection of tetrahydrofuran was 0.04 ppm.

Vapor Concentration Uniformity:
The uniformity of chamber atmosphere vapor concentrations were measured prior to the study and once during the study. All chambers were acceptable with percentage relative standard deviations of

Details on mating procedure:

- Impregnation procedure: co-housed with male mice
- Ratio F/M per cage: 2 or 3 females/male
- Length of cohabitation: overnight
- Mating was conducted for 5 consecutive nights to obtain approximately 33 pregnant animals/group.
- Proof of pregnancy: confirmed by the presence of a vaginal plug, referred to as day of gestation 0 of pregnancy (0 DG)

Duration of treatment / exposure:

12 consecutive days (6-17 DG for mated mice)

Frequency of treatment:

6 hours/day

Duration of test:

18 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (filtered air), 600, 1800 or 5000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

10 virgin (unmated) females and approximately 33 positively mated females

Control animals:

yes

Details on study design:

Virgin female mice were included to provide a reference for any effects that the state of pregnancy may have had on the toxic response. These animals were exposed concurrently with mated animals.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Animals were observed daily for mortality, morbidity and signs of toxicity. The date and time of death or euthanasia of moribund animals was recorded and the animals necropsied.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Mated female mice were weighed on DG 0, 6, 9, 12, 15 and 18; virgin mice were weighed prior to the start of exposures and on exposure days 1, 5 and 10 and at euthanization.

POST-MORTEM EXAMINATIONS: Yes
Mice were killed by CO2 asphyxiation, weighed and examined grossly for signs of maternal toxicity.

Ovaries and uterine content:

Both ovaries from each female were saved for sectioning and quantitative follicle counts. Apparently nongravid uteri from positively mated females were stained with 10% ammonium sulfide to detect possible implantation sites. The number, position and status of implants were recorded for each gravid uterus. Placentas were examined and discarded unless abnormal.

Fetal examinations:

- External examinations: Live fetuses were weighed and examined for gross defects.
- Soft tissue examinations: The sex of live fetuses was determined by internal examinations of the gonads after injection of Nembutal(TM) (sodium pentobarbital). Fifty percent of live fetuses and any fetuses with gross external abnormalities were examined for visceral defects by dissection of fresh tissue.
- Skeletal examinations: All fetal carcasses, with and without heads, were prepared for skeletal staining. Cartilage as well as ossified bone were visualized by double-staining using Alcian bue and alizarin red S.
- Head examinations: The heads of 50% of the live fetuses were removed and placed in Bouin’s fixative. After fixation, the heads were serially sectioned with a razor blade and examined for soft-tissue craniofacial abnormalities.

Statistics:

All means and standard deviations for animal data were calculated. Mean body weights (as a mean of litter means for fetal data) analyzed by an analysis of variance (ANOVA) model for unbalanced data. Response variables, either body weights or the arcsin transformations of proportional incidence data, were analyzed against the class variable, treatment, in a one-way ANOVA model. A Tukey’s t-test (two-tailed) was used to assess statistically significant differences between the control and exposed groups. If appropriate, a dose-response relationship was determined by means of an orthogonal trend test (Winer, 1971). In the case of proportional data this test was performed on transformed variables. The litter was used as the basis for analysis of fetal variables.

Indices:

The following parameters, expressed as mean +/- SE, when appropriate, were computed from data for all pregnant animals and their litters:
- Number of dead maternal animals, animals removed from the study and reason for removal
- Summary of maternal toxicity, including incidences of changes detected during clinical observations
- Number and percentage of pregnant animals
- Maternal body weights
- Weights of gravid uterus
- Extragestational weight and weight gain
- Number of implantation sites/litter
- Number of litters with live fetuses
- Number and percent of live fetuses/litter
- Body weight of live fetuses/litter
- Body weight of male and female fetuses/litter
- Sex ratio of fetuses/litter
- Number and percent of early and late resorptions/litter
- Number and percent of non-live/litter (early and late resorptions and dead fetuses)
- Listings of malformations and variations observed in fetuses/litters
- Number and percent of malformed fetuses
- Number and percent of litters with malformed fetuses

Historical control data:

Contemporary control data (based on N=83 litters) for Swiss CD-1 mice was available in addition to the unexposed (0 ppm) control animals:
Maternal Wt (18 DG): 54.4 +/- 5.6
Gravid Uterine Wt: 20.2 +/- 3.6
Extragestational Weight Gain: 6.6 +/- 3.0
Implants: 12.6 +/- 2.1
Live Fetuses: 11.7 +/- 2.2 (93.5 +/- 7.3%)
Early Resorptions: 0.6 +/- 0.8 (4.6 +/- 6.3%)
Late Resorptions: 0.2 +/- 0.5 (1.9 +/- 3.7%)
Dead Fetuses: 0.0 +/- 0.0 (0%)
Total Intrauterine Death: 0.8 +/- 1.0 (6.5 +/- 7.3%)
All Fetuses ( Weight, g): 1.36 +/- 0.11
male (g): 1.39 +/- 0.11
female (g): 1.34 +/- 0.10

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Unexpectedly high maternal mortality was observed at the 5000 ppm exposure concentration with 27% of mated females dying between DG 6 and 11 and 30% of virgin females dying during exposure days 1 through 5. Severe depression of the central nervous system may have been a contributing cause since all animals at the 5000 ppm dose level and some at the 1800 ppm dose level were sedated for up to 1 hour following exposures. Sedation at the 1800 ppm exposure level was a clear sign of maternal toxicity. Total numbers of exposures at the 5000 ppm dose level were reduced after 6 days of exposure by placing surviving females in exposure chambers supplied with fresh air until scheduled euthanization.

Mean body weights of virgin females at the highest dose level were significantly less than control animals by exposure day 5, however, this group recovered most of its weight by euthanization. Mean body weights (g) for virgin female mice at the 5000 ppm exposure concentration on exposure days 1, 5, 10 and at sacrifice were:
- 5000 ppm (N = 6 to 8): 26.7 +/- 2.6; 20.5 +/- 2.2*; 25.8 +/- 4.7*; 25.0 +/- 0.9*
Mean body weights for pregnant mice at the 5000 ppm exposure level were also significantly less than controls, but remained low throughout the study. Also, a significant reduction in mean body weights was also observed in the 1800 ppm exposed animals by DG 15. The body weight depression at the 5000 ppm exposure level can be attributed in part to the elevated intrauterine mortality in this group. At the 1800 ppm exposure level, the primary contribution to the reduced mean body weights was a deficit in uterine weight. Mean body weights (g) for control, 1800 ppm-exposed and 5000 ppm-exposed mice on DG 6, 9, 12, 15 and 18 were as follows:
- Control (N=30): 29.3 +/- 1.7; 31.5 +/- 2.1; 36.4 +/- 2.5; 45.5 +/- 2.7; 54.3 +/- 3.8
- 1800 ppm (N=27): 29.3 +/- 1.8; 31.1 +/- 2.5; 35.3 +/- 4.1; 41.7 +/- 6.7*; 49.0 +/- 10.1*
- 5000 ppm (N=20); 26.8 +/- 1.3; 26.8 +/- 2.6*; 27.8 +/- 2.8*; 29.8 +/- 3.3*; 29.4 +/- 5.0*
* significantly different from control groups, p<0.05

Uterine weights (g) were reduced in the 1800 ppm and 5000 ppm exposure groups versus controls. Extragestational weight gains (EGWG: body weight at sacrifice - uterine weight - DG0 weight) were reduced in the 5000 ppm exposed mice:
- Uterine Wts: 20.5 +/- 3.3 (control); 15.6 +/- 7.1* (1800 ppm); 1.0 +/- 3.8* (5000 ppm)
- EGWG: 7.2 +/- 1.9 (control); 1.4 +/- 1.7* (5000 ppm)
* significantly different from control groups, p<0.05

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

600 ppm (nominal)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

600 ppm (nominal)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Each exposure group consisted of 33 plug-positive mice and 10 virgin female mice. Exposure to tetrahydrofuran had no effect on the number of implantations per dam. Mean number and percentage of live fetuses/litter at the 1800 and 5000 ppm exposure concentrations were significantly decreased compared to control. Corresponding significant increases in the number and percentage of resorptions/litter in these same groups were reported. Female mice surviving to euthanization had litters with 95% incidence of early resorption. Refer to Table 1.

Neither fetal weights nor fetal sex ratios were affected at the 600 or 1800 ppm exposure concentrations (Table 1). Fetal body weights (as means of litter means) were reduced for the 5000 ppm exposure group when compared to controls. The percentage of male fetuses was reduced at the 5000 ppm exposure concentration (Table 1):
Fetal body weights:
- All fetuses (g): 1.3 +/- 0.1 (control); 1.0 (5000 ppm, only a single litter)
- Males (g): 1.4 +/- 0.1 (control); 1.0 (5000 ppm)
- Females (g): 1.3 +/- 0.1 (control); 1.0 (5000 ppm)

Fetal Malformations:
The incidence of fetal malformations was not significantly affected following gestational exposures to tetrahydrofuran. However, several malformations were observed at low incidences in the 1800 ppm group that were not present either in the 0 ppm controls or contemporary controls: cleft palate (5 fetuses, 2.0%), edema (1 fetus, 0.4%), ectopic ovaries (1 fetus, 0.8%), and undescended testes (2 fetuses, 1.6%). These latter malformations were all observed in fetuses from a single low-weight litter that also had a 54% incidence of resorptions. There was an increased incidence of reduced ossifications of the sternebrae in the 1800 ppm exposure group and these correlated with increasing exposure concentrations. Statistical analysis of the 5000 ppm exposure group was not possible since only a single litter with live fetuses was present at this exposure concentration.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Reproductive Measures for Mice Exposed to Tetrahydrofuran

	Tetrahydrofuran exposure conc. (ppm)
	0	600	1800	5000
Number of
Plug-positive females	33	33	33	33
Pregnant	31	28	27	27
Implantations/dam	12.8±1.8	12.0±2.2	12.0±1.9	12.6±2.8
Live fetuses/litter^1	11.9±1.9	11.1±3.2	9.3±4.4*	0.6±2.7*
Resorptions/litter
Total	0.9±1.1	0.9±1.8	2.7±4.1	12.0±3.7*
Early	0.6±1.0	0.7±1.8	1.8±3.9	11.9±4.0*
Late	0.3±0.5	0.2±0.5	0.9±2.2	0.1±0.5
Litters with >3 resorptions	1	1	5	19*
Percent of
Pregnant	94	85	82	83
Live fetuses/litter	93.1±8.2	91.2±19.6	77.4±34.8**	4.3±19.2**
Resorptions/litter
Total	6.9±8.2	8.4±19.5	22.6±34.8**	95.7±19.2**
Early	4.7±7.6	6.7±19.4	14.6±31.4**	95.0±3.2
Late	2.1±3.7	1.72±4.5	8.0±21.5	0.7±1.5
Litters with resorptions	60	50	63	100***
Male fetuses	46.6±15.8	47.0±15.0	50.7±16.2	16.7^2

1 Significantly correlated with exposure level (p < 0.05).

2 Based on a single litter.

* Significantly different from controls (p < 0.05).

** Significantly different from controls (p < 0.05, arcsine transformation).

*** Significantly different from controls (p < 0.05, chi-square transformation).

Applicant's summary and conclusion

Conclusions:

Exposure of Swiss CD-1 mice to 0, 600, 1800 or 5000 ppm of tetrahydrofuran resulted in significant mortality of pregnant and virgin females (5000 ppm) and was embryotoxic at the 1800 and 5000 ppm exposure concentrations. The embroytoxic effects occurred in the presence of significant maternal toxicity that was expressed as maternal mortality (5000 ppm), significantly prolonged sedation (1800 and 5000 ppm) and reduced mean uterine and body weights (1800 and 5000 ppm). No significant increases in frank malformations or reductions in fetal body weights of live fetuses at the 1800 ppm exposure concentration were observed.

Executive summary:

Pregnant and virgin female Swiss (CD-1) mice were exposed to 0, 600, 1800 or 5000 ppm of tetrahydrofuran vapors whole-body for 6 hours/day, 7 days/week on days of gestation (DG) 6 to 17. Due to an unexpectedly high rate of toxicity (27% between DG 6 and 11) at the highest exposure concentration, exposures were discontinued on DG 11 (total of 6 exposures). A total of 30% of exposed virgin female mice died during exposure days 1 through 5. Severe central nervous system depression was proposed as causing or contributing to death. Sedation lasting for up to 1 hour following exposures was observed in all animals in the 5000 ppm exposure group as well as some animals in the 1800 ppm exposure group. Exposures to THF had no effect on the number of implantations/dam. The number and percentage of live fetuses/litter in both the 1800 and 5000 ppm exposure groups were reduced compared to controls. Neither fetal weights nor fetal sex ratios were affected by exposures at 1800 or 5000 ppm. Because only a single litter with live fetuses was present at the 5000 ppm exposure concentration, statistical analysis at this level was not possible. Although not statistically significant, several malformations were observed at low incidences in the 1800 ppm group that were not present either in the 0 ppm controls or contemporary controls: cleft palate, edema, ectopic ovaries, and undescended testes. These latter malformations were all observed in fetuses from a single low-weight litter that also had a 54% incidence of resorptions. There was an increased incidence of reduced ossifications of the sternebrae in the 1800 ppm exposure group and these correlated with increasing exposure concentrations. Tetrahydrofuran was not judged to be a selective developmental toxicant in Swiss CD-1 mice based on the clear presence of maternal toxicity at embryotoxic exposure concentrations. The NOAEL for both maternal and developmental toxicity in the current study was 600 ppm.