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Repeated dose toxicity: inhalation

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Administrative data

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:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-methoxyethyl) ether
EC Number:
EC Name:
Bis(2-methoxyethyl) ether
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Diethylene glycol dimethyl ether
Diethylene glycol dimethyl ether
Constituent 3
Reference substance name:
Constituent 4
Reference substance name:
Details on test material:
Source: Aldrich Chemical Company
Lot: 1617HK
Purity: <99%

Test animals

other: Crl:CD BR
Details on test animals or test system and environmental conditions:
Obtained from Charles River Breeding Laboratories, Kingston, New York.
Upon arrival, rats were individually housed in 5" x 11" x 7" suspended, stainless steel, wire mesh cages.
Animals were quarantined for one week prior to study initiation, and were weighed and observed 4 times during the quarantine period. Rats were assigned to 5 treatment groups using a computer-based randomization program such that the groups mean body weights 2 days prior to the first exposure were similar. After grouping, each rat was assigned a unique 6-digit identification number and a 1-3 digit identification number which was tattooed on each rat's tail; both sets of identification numbers were recorded on a card affixed to the cage.
After grouping and during the exposure period, rats were housed in pairs in 8" x 14" x 8" suspended, stainless steel,, wore mesh cages; during the recovery period rats were housed individually in similar cages. At study initiation, rats were approximately 8 weeks old and weighed between 215-269 g. Except during exposure, Purina Certified Rodent Chow #5002 and water were available ad libitum.
Animal rooms were maintained on a timer-controlled, 12 h/12 h light/dark cycle. Environmental conditions of the room were targeted for a temperature of 23 +/- 2 °C and relative humidity of 50 +/- 10 %. Excursions outside these ranges were judged to have been of insufficient magnitude and/or duration to have adversely affected the validity of the study.

Administration / exposure

Route of administration:
Type of inhalation exposure:
nose only
Details on inhalation exposure:
Rats were individually restrained in perforated, stainless steel cylinders with conical nose pieces. Each restrainer was inserted into Plexiglas and stainless steel face plates that were attached to a glass 39.L exposure chamber such that only the nose of each rat protruded into the chamber.
Test atmospheres of digylme were generated by vaporization. For the 30 and 100 ppm design concentrations, diglyme vapors were generated by pumping the liquid test material with Harvard Model 22 compact infusion pumps into Instatherm 3.neck, glass, round bottom mixing flasks. The flasks were electrically heated to a temperature of approx. 95-110 °C to facilitate evaporation of the test material. Nitrogen was added to the mixing flasks at approx. 0.25 - 0.86 L/min to sweep the vapor into unheated glass connecting tubes. For the 3.0 and 10 ppm design concentrations , the test material was contained in glass reservoirs that were immersed in a water bath maintained at a temperature of approx. 29-39 °C. Conditioned, filtered houseline air was passed over the surface of the test material at approx. 0.08 - 0.9 L/min to sweep the vapors into unheated glass connecting tubes. For all test chambers, conditioned, filtered houseline air was added in the connecting tubes at approx. 34-43 L/min. All resulting vapor/air mixtures were swept into cylindrical, 38 liter glass exposure chambers and dispersed with glass dispersion funnels upon entering the exposure chambers. Exposure chamber concentrations were adjusted by varying either the test material feed rates into the mixing flasks, the nitrogen or air flow rates and/or the water bath temperature. Chamber atmospheres were exhausted through water scrubbers, dry ice cold traps, and MSA charcoal/HEPA cartridge filters prior to discharge into fume hoods. Rats from the procedural control group were exposed to conditioned, filtered houseline air (approx. 38L/min) using the same type of exposure chamber.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The test chamber concentrations of diglyme were determined at approx. 45 min intervals during each exposure by gas chromatography. Known volumes of chamber atmospheres were drawn from the rats breathing zone through midget glass impingers using acetone as the trapping solvent. A single impinger was used for the 3.0 and 10 ppm design levels while tandem impingers were used for the 30 and 98 ppm design levels. Based in replicate analysses, the vapor collection efficiency for each impinger was determined to be 95 %; values cited in this report are uncorrected for collection efficiency. Impinger samples were analyzed with replicate injections using a Hewlett Packard 5880A gas chromatograph equipped with a flame ionization detector. The chromatograph was operated isothermally at 110 °C using a 30 m x 0.53 mm i.d. fused silica open bore column coated to a nominal film thickness of 5 µm with polydimethyl siloxane. The atmospheric concentrations of diglyme were calculated by comparing peal areas with standard curves prepared daily. Standards were prepared weekly by diluting known amounts of diglyme in acetone. During each exposure, chamber temperatures were measured using thermocouple probes, relative humidities were measured with a Bendix Model 566 wet/dry bulb psychrometer, and chamber oxygen concentrations were measured with a Biosystems Model 3100R oxygen monitor.
Duration of treatment / exposure:
6 hours/day
5 days/week
Frequency of treatment:
Two weeks followed by 14 days of recovery
Doses / concentrationsopen allclose all
Dose / conc.:
3 ppm
measured concentration: 3.1 ppm corresponding to 17.3 mg/m³
Dose / conc.:
10 ppm
measured concentration: 9.9 ppm corresponding to 55.2 mg/m³
Dose / conc.:
30 ppm
measured concentration: 30 ppm corresponding to 167 mg/m³
Dose / conc.:
100 ppm
measured concentration: 98 ppm corresponding to 547 mg/m³
No. of animals per sex per dose:
Control animals:


Observations and examinations performed and frequency:
Body weights and clinical observations:
During the exposure periond, all rats were weighed and observed for clinical signs of toxicity before each exposure; observations for clinical signs were also taken immediately following each exposure. During the 14-day recovery period, all rats were weighed and observed daily, weekends excluded.
Sacrifice and pathology:
Each group of 20 rats was divided into 2 subgroups of 10 rats using computer-generated random number tables. Ten rats per group were killed by sodium pentobarbital anesthesia and exsanguination after the 10th exposure; the remaining rats were killed on the 14th day of recovery. All rats received gross and histopathologic examinations. The testes, epididymides, prostate and seminal vesicles were weighed at necropsy. Microscopic examinations were limited to representative tissues of the reproductive tract, including the testes, epididymides, prostate and seminal vesicles.
Group mean body weights and body weights gains fir the test rats were compared to controls during the exposure and recovery periods. Data were statistically analyzed by one-way analysis of variance. Data from the test groups were compared to controls by the least significant among-to-with group variation. Significance was judged at the 0.05 probability level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Effects common for restrained rats were observed immediately after exposure (diarrhea, red nasal/ocular discharges) in all groups including controls. Several additional clinical signs of toxicity were observed in rats from all groups during the study. However, due to the low incidence and the types if clinical signs observed, the signs were considered to be incidental and not compound-related.
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of rats exposed to 30 or 98 ppm diglyme were significantly lower than controls for study days 9-15. Significantly lower body weights were also noted in the 3.1 and 9.9 ppm groups for study das 9-10 and 10-11, respectively. However, the weight depressions in the 3.1 and 9.9 ppm groups were considered to be incidental and not compound related since: a) the magnitude of the weight depression was low (typically 2-3 % lower than control values); b) the period when body weights were depressed was not consistent and appeared at different times during the second week of exposures; and c) the duration of weight depression was not longer than 2 days.
Mean body weight gains for the 98 ppm group were significantly lower than controls when evaluated for study days 1-5 and 1-12. Although some statistically significant differences in daily mean body weight gains were noted in the 3.1, 9.9 and 30 ppm groups during the second week of exposure and the first few days of the recovery period, the differences were small and did not appear to be concentration-related.
In contrast to the 98 ppm group, the weight depression in rats exposed to 30 ppm dud not appear to be compound-related. For example, the daily mean body weights of rats from the 98 ppm group were not consistently lower than those from the 30 ppm group during the study. Moreover, the magnitude of the weight depression was similar for both groups despite a 3-fold difference in concentration. Although the duration and magnitude of the body weight depressions for the 30 and 98 ppm groups were similar, the actual differences for the 30 ppm group were small and not considered to be biologically relevant.
The body weight differences in diglyme-exposed rats were smaller than expected compared to the results of the previous study with diglyme (Valentine, 1999). For example, group mean body weights among rats exposed to 11ß ppm diglyme were approx. 8 % lower than controls after the 10th exposure and remained significantly lower than controls until study day 25. In this study, the mean body weight of rats from the 98 ppm group was indistinguishable from controls by study day 16. Such differences in response are probably due to the group-to-group variability associated with small sample populations.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Weights of testes, seminal vesicles, prostate, and epididymes were similar to those of controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Upon gross examination, the Only changes noted in the reproductive organs included large testes (98 ppm), and epididymal mass (30 ppm), small prostate (control) and small seminal vesicles (3 ppm); these changes were found in only 1 rat from each of the groups indicated. The epididymal mass observations were considered to be incidental and not compound-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopic examination, minimal, unilateral testicular atrophy was observed after the 10th exposure in 1 rat from each of the 0, 9.9 and 98 ppm groups. After 14 days of recovery, 2 of 10 rats from the 98 ppm group had minimal or mild, unilateral or bilateral testicular atrophy. Exfoliated degenerative germ cells were found in some epididymal tubules of atrophic testes in rats from the 9.9 and 98 ppm groups. The prostatitis and spermatic granuloma found in some test groups was considered unrelated to diglyme exposure. Rats exposed to 3.1, 9.9 or 30 ppm diglyme had no exposure-related pathological lesions. All other observed changes were considered to be incidental and unrelated to the test material.
The amount of testicular injury produced by 98 ppm diglyme was considerably lower than that found in the 110 ppm group from the previous study with diglyme (Valentine, 1999). The incidence of testicular injury was not only lower in the present study (3/20 at 98 ppm vs 4/10 at 110 ppm by the 14th day of recovery) but the lesions were also less severe and tended to be unilateral rather than bilateral in distribution. The incidence of this type of testicular lesion is somewhat variable in control animals bit is approx. 15 % in 10-12- week old male rats. The data from the present study suggest that the incidence of testicular atrophy among all groups is within the range of historic control values for rats of similar age.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Dose descriptor:
Effect level:
30 ppm
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
Lowest effective dose / conc.:
100 ppm
male reproductive system
other: prostate, epididymis
Treatment related:
Dose response relationship:

Applicant's summary and conclusion

A NOAEC of 30 ppm was set based on slight body weight depression and possible testicular effects noted at 9 ppm.
Executive summary:

Repeated exposure to 98 ppm digylme caused slight depressions in body weights and body weight gains during the exposure period. Although some significant body weight effects were also noted in the 3.1, 9.9 and 30 ppm groups, the differences were small, did not exhibit the same onset and duration, and did not appear concentration-related; for these reasons, weight changes in the other groups were not considered to be compound-related.

The incidence, severity and persistence of testicular lesions observed in animals exposed to dilyme at concentration up to 98 ppm were comparbale to those normalyly associated with control populations of unexposed rats. However, since the tsesticular effects observed at 98 ppm were slightly more severe than the other test groups, this concentration may reflect a minimal effect level.