Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable reported study performed analogue to current OECD guideline.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
received from Specialty Chemicals and Aldrich Chemicals, WI, USA
colorless liquid
Purity: >99%

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, NY, USA
- Age at study initiation: 7 weeks
- Weight at study initiation: males - 244 g; females - 189 g
- Diet (e.g. ad libitum): Purina Certified Rodent Chows #5002 ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2 °C
- Humidity (%): 50 +/- 10%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: n.a.
Details on inhalation exposure:
- Exposure protocol
Five froups of 20 male and 10 female rats were used. Three groups of male and female rats were exposed nose-only to concentrations of 100, 300 or 1000 ppm Diglyme in air. A positive control group, consisting of 20 male and 10 female rats, was exposed to 2-Methoxyethanol at 300 ppm in air. A control group was exposed to air only. Rats were assigned to treatment groups using a computer-based randomization program such that the mean body weights of the groups 5 days prior to the start of exposure were similar.
Rats were individually restrained in perforated, stainless-steel cylinders with conical nose pieces. Each restrainer was inserted into face-plates on stainless-steel and glass 150-litre exposre chambers such that only the nose of each rat protruded into the chamber. Exposures were conducted for 6 hours/day, 5 days/week for 2 weeks, and were followed by a recovery period lasting up to 84 days after the last exposure. Five randomly-selected male and female rats were killed after the 10th exposure and after 2 weeks recovery. In addition, five male rats per group were killed after 6 and 12 weeks of recovery.

- Atmosphere generation
Diglyme or Methoxyethanol vapours were generated by pumping the liquid test material through Teflon tubing with Harvard Model 975 compact infusion pumps into Instatherm three-neck, glass, round-bottomed mixing flasks. For Diglyme and 2-Methoxyethanol generation, the flasks were electrically heated to 111-123°C, respectively, to facilitate evaporation of the test materials. Conditioned, filtered house line air was added to the mixing flak at approx. 35-46 litre/min to dilute and sweep the vapour/air mixtures through unheated glass connecting tubes into the inlets of the 150-litre stainless-steel and glass exposure chambers. The chambers were cubical with pyramidal tops and bottoms. exposure chamber concentrations of Diglyme or 2-methoxyethanol were adjusted by varying the test material feed rate into the mixing flasks. To promote uniform distribution, the vapour/air mixtures were dispersed with baffles on entering the exposure chambers. The control group was exposed in a similar 150-litre stainless-steel and glass exposure chamber, operated at an airflow of approx. 37 litres/min. Chamber atmospheres were exhausted through water scrubbers, dry ice cold traps, and MSA charcoal and HERA chartridge filters prior to discharge into fume hoods.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The exposure chamber concentrations were determined at approx. 30-min intervals during each exposure. For diglyme analysis, known volumes of the chamber atmospheres were drawn from rats' breathing zone through tandem midget glass impingers containing Acetone as the trapping solvent. For 2-Methoxyethanol analysis, replicate gas samples (approx. 0.5 mL) were collected from the breathing zone of the rats with a gas-tight syringe. Impinger or gas samples were analysed by gas chromatography using a flame ionisation detector. For Diglyme, samples were analysed on a Hewlett Packard 5790A gas chromatograph operated isothermally at 110°C using a 3 ft x 2 mm i.d. glass column packed with 10% SE-30 on 80/100 mesh Supelcoport. For 2-Methoxyethanol, gas samples were injected directly into Hewlett Packard 5710A gas chromatograph operated isothermally at 175°C using a 6 ft x 2 mm i.d. glass column packed with 3% OV-225 on 80/100 mesh Supelcoport. The atmospheric concentrations were calculated with standard curves prepared daily. Standard Diglyme soluntions were prepared weekly by diluting known amounts of Diglyme in Acetone. Vapour 2-Methoxyethanol standards were prepared daily by evaporating known amounts of 2-Methoxyethanol in calibrated gas bottles.
Chamber temperatures were measured regularly during each exposure with mercury thermometers, relative humidities were measured with a Vaisala HMI 31F Temperature and Humidity Indicator, and chamber oxygen concentrations were measured with a Biosystems Model 3100R oxygen monitor.
Duration of treatment / exposure:
6 h/d
Frequency of treatment:
5 d/week for 2 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
110 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
370 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1100 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
20 males/dose, 10 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Please refer to "Details on inhalation exposure"
Positive control:
None

Examinations

Observations and examinations performed and frequency:
- Body weights and clinical observations
All rats were weighed and observed for clinical signs daily and for the 14-day post-exposure recovery period. Male rats assigned to the extended recovery groups were also weighed and observed at least once a week for upp to 12 weeks after exposure.

- Clinical pathology
Urine samples were collected only from those male and female rats to be killed by design within 24 hours of urine collection. Urine specimens were collected overnight from five male and five female rats per exposure group after the 9th exposure and on the 13th day of recovery, from five male rats per group on the 41st day of recovery, and from the remaining five male rats per group on the 83rd day of recovery. Samples were analysed for volume, osmolality, urobilinogen, pH, haemoglobin or occult blood, glucose, protein, bilirubin and ketone.
Blood samples were taken from the orbital sinus of five male and five female rats per group after the 10th exposure and on the 14th day of recovery, from five male rats per group on the 42nd day of recovery, and from the remaining five male rats per group on the 84th days of recovery. Blood samples were analysed for erythrocyte count, haemoglobin concentration, haematocrit, platelet count, leucocyte count, and relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, eosinophils, monocytes and basophils. Mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were calculated from the erythrocyte data. Serum activities of alkaline phosphatase (AP), alanine aminotransferase (ALT), and aspartate aminotranferase (AST), amd serum concentrations of urea nitrogen, creatinine, total protein and cholesterol were also maesured.
Sacrifice and pathology:
- Pathology
Each test group of rats was divided into subgroups of five rats based on computer-generated random number tables. Five male and five female rats per group were killed after the 10th exposure and on the 14th day of recovery, and five male rats per group were killed on the 42nd and 84th days of recovery by Sodium pentobarbital anaesthesia and exsanguination for gross and histopathological examinations. The lungs, kidneys, spleen and male reproductive organs (testes, epididymides, seminal vesicles and prostate) were weighed at necropsy. Bone, eyes, testes and epididymides were fixed in Bouin's solution. All other organs and tissues were fixed in 10% Formalin solution. Paraffin sections were prepared according to routine histological techinques. All sections were steined with Haematoxylin and Eosin. In addition, all testes were stained with the Periodic acid-Schiff (PAS) method. Representative samples of the following tissues were prepared for microscopic examination: heart, lungs, mesentric and mediastinal lymphh nodes, nasal cavities, trachea, liver, pancreas, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, kedneys, urinary bladder, bone/bone marrow (sternal), spleen, thymus, thyroid gland, adrenal glands, brain, eyes, testes, epididymides, prostate, seminal vesicles, vagina, ovaries, uterus, and any other organs or tissues with gross lesions.
Other examinations:
none
Statistics:
Mean body weights, body weight gains, absolute organ weights, and relative organ weights (organ to body weight ration) for exposed rats were compared with control rats during the exposure and recovery periods. Data were statistically analysed by one-way analysis of variance. exposure group values were compared with controls by the least significant difference or Dunnett's tests when the ration of variance (F) indicated a significant among-to-within group variation. Significant differences were considered at the 0.05 probability level. Clinical pathology data were analysed by one-way analysis of variance and Bartlett's test. When the F-test was significant, the Dunnett test was used to compare means from the control group with each exposure group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
- Clinical observations:
Among the 1100 ppm group Diglyme rats, seven males had coloured ocular discharge and 17 males and one female has diarrhoea during the exposure phase. Diarrhoea and ocular discharge were generally transient clinical signs first noted during week 1 or 2 of exposure. No other chemical-related clinical observations were observed. Rats from all groups including controls showed red nasal and ocular discharge immediately following exposures. These effects are commonly seen in rats under restraint and were not considered to be compound-related.
Two rats (one of each sex from the 1100 ppm group) died during the study. One was injured during the last exposure and was sacrificed; the other died after 35 days of recovery with severe inflammation of the kidney and prostate resulting from a large mineralised mass in the urinary bladder. Neither of these deaths was attributed to Diglyme.

- Body weights:
Mean body weights of male rats from all test groups were significantly lower than control values throughout the exposure period. The magnitude of the body weight reduction was inversely related to the concentration of diglyme. Weight recovery began after the 10th exposure such that mean body weights were comparable to control values by recovery day 14 for the 110 ppm groups rats, by recovery day 28 for the 370 ppm group, and by recovery day 42 for the 1100 ppm Diglyme and 300 ppm 2-Methoxyethanol groups. No significant body weight differences were observed in female rats throughout the study.

-Clinical pathology:
Male and female rats exposed to 1100 ppm Diglyme were moderately anaemic after the 10th exposure; however, by recovery day 14, clinical signs of anaemia were nit detected in any treament group.
Mean platelet counts of male and female rats from all test groups were similar to control values after the 10th exposure. However, after 14 days of recovery, the mean platelet counts for several of the Diglyme groups were reduced. For example, thrmbocytopaenia was considered marked in males and females from the 1100 ppm Diglyme group, and mild to moderate in males exposed to 370 ppm Diglyme. Platelet counts returned to control values by 42 days of recovery in male rats. Compared with control values, maen leucocyte counts were reduced in male rats from all test groups and in female rats from the 1100 ppm group. The leukopaenia in Diglyme-exposed males was primarily due to a restriction in the number of circulating lymphocytes compared with controls. The lymphopaenic effect was concentration-related, with the most severe effects seen in the 1100 ppm Diglyme group. Lymphopaenia was also observed in the 1100 ppm females. A marked monocytopaenia, observed in rats exposed to 1100 ppm, also contributed to the leikopaenia. Females from the 1100 ppm group had marked monocytopaenia and neutropaenia. Several additional differences in neutrophil counts for all male test groups and female rats from 110 and 370 ppm Diglyme groups, anf monocyte counts of male and female rats from the 110 and 370 ppm Diglyme groups were also observed; however, in comparison with reference ranges for these parameters in Crl:CD BR rats, the biological significance of these effects was considered equivocal. After a 14-day recovery period, only mild lymphopaenia was observed in male and female rats from the 1100 ppm Diglyme group. The serum enzyme activities of ALT, AST and AP and total protein concentrations were significantly reduced in male and female rats exposed to 1100 ppm Diglyme compared with controls after the 10th exposure. After 14 days of recovery, serum hepatic enzyme activities and serum protein concentrations were similar to control values in all test groups.

- Organ weights:
Mean absolute and relative weights of male reproductive organs were significantly lower in Diglyme exposed rats compared with controls. The magnitude of the weight effects was concentration-related, with the most severe effects observed in the 1100 ppm group. A concentration-related reduction in mean absolute prostate and seminal vesicle weights was seen in the 370 ppm and 1100 ppm groups. Absolute and relative testes weights were reduced in the 1100 ppm group as were the relative weights after the 10th exposure. After 14 days of recovery, mean absolute testes and epididymides weights in the 370 and 1100 ppm groups and prostate weights in the 1100 ppm group were significantly lower than controls.Mean relative testes and epididymides weights for the 1100 ppm group were also lower than controls. After 42 days and 84 days of recovery, the mean absolute and relative testes weights in the 1100 ppm group were significantly lower than compared with control values; mean absolute testes weights were also lower than controls in the 110 and 370 ppm groups after 42 days of recovery and mean relative epididymides weigths were lower than controls in the 370 ppm group after 84 days of recovery.
Among female rats, the only significant weight changes associated with Diglyme exposure included increased mean absolute and relative liver weights in the 1100 ppm group and increased mean absolute liver weights in the 370 ppm group after the 10th exposure. Additional organ weight differences in male or female rats were not biologically significant and were considered unrelated to the test item.

- Pathology:
Upon gross examination after the 10th exposure, the thymus and tissues of the reproductive organs in male rats exposed to 1100 ppm Diglyme were compared with controls. The prostate, seminal vesicles and thymus appeared normal at necropsy by 42 days of recovery. The small size of the testes and epididymides in rats from this group was a persistent observation throughout the study. Overall, the gross lesions in male rats were consistent with the microscopic observations. These observations were noted in rats exposed to either 1100 or 300 ppm Diglyme. No compound-related gross lesions were noted at necropsy in female rats.
After exposure to 110 ppm Diglyme for 2 weeks slight testicular atrophy was observed microscopically in two of five rats, while two of five controls had minimal testicular atrophy. The epididymal tubules of both groups contained exfoliated degenerative germ cells. None of the control rats had testicular atrophy following 14, 42 or 84 days of recovery. After 14 days of recovery, two of five rats exposed to 110 ppm Diglyme had slight testicular atrophy. After 42 days of recovery, two of five rats from this group showed minimal testicular injury involving a small number of atrophic tubules; most seminiferous tubules had normal germinal epithelium. By 84 days of recovery, the morphology of the testicular germinal epithelium was normal.
After exposure to 370 ppm Diglyme for 2 weeks, slight testicular atrophy afftecting primarily spermatocytes and immature spermatids were seen in all males. After 14 days of recovery, minimal to moderate testicular atrophy was present in all five rats. Immature spermatids and spermatocytey were found, but mature spermatozoa had not yet developed. Exfoliated degenerative germ cells and a slightly reduced number of spermatozoa were found in the epididymal tubules. After 42 days of recovery, the testes of only two of five rats exhibited very slight testicular atrophy. After 84 da of recovery, the morphology of the testicular germinal epithelium was considered normal. Although a very small fraction of tubules were lined with Sertoli cells only, the remaining tubules had normal germinal epithelial morphology.
All male rats exposed to 1100 ppm Diglyme had severe testicular atrophy after the 10th exposure. The germinal epithelium was extensively damaged and all spermatogenic stages of germ cells were affected; numerous spermatid giant cells were also present. the epididymal tubules showed munerous exfoliated degenerative germinal cells and slight to moderate oligospermia. After 14 days of recovery, the seminiferous tubules showed slight regenration of spermatocytes and spermatids. Some seminiferous tubules were lined with only Sertoli cells and a few spermatogonia. Slight Leydig cell hyperplasia was present in all rats. The epididymidal tubules contained numerous spermatid giant cells with only a few spermatozoa (moderate to severe oligospermia). After 42 days of recovery, many tubules showed a regenerating germinal epithelium consisting of spermatocytes and immature spermatids although minimal to moderate Leydig cells hyperplasia persisted. The epididymal tubules contained numerous exfoliated germinal cells and few spermatozoa (moderate to moderate oligospermia). After 84 days of recovery, three of four rats had almost normal germinal epithelium, but the remaining rat showed moderate testicular atrophy with only partially regenerated germinal epithelium. In addition to the testicular effects, the seminal vesicles and prostate were atrophic after 2 weeks of exposure but these changes were absent by 14 and 42 days of recovery, respectively.
Minimal to severe bone marrow hyperplasia and lymphoid tissue atrophy of the spleen and thymus were apparent in both male and female rats exposed to 1100 ppm Diglyme. While the atrophic changes in the haematopoetic tissues of rats from this group had resolved after 14 days of recovery, extramedullary haematopoetic foci were evident in the liver of rats from both sexes and in the spleen of males. Evidence of haematopoetic effects in males was essentially absent after 42 days of recovery.

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
370 ppm
Sex:
female
Basis for effect level:
other: changes in haematopoetic system
Dose descriptor:
NOAEC
Effect level:
110 ppm
Sex:
male
Basis for effect level:
other: slight testicular atrophy

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the observed changes in the haematopoetic system of female rats the NOEC (Diethylene glycol dimethyl ether, female) is considered to be 370 ppm. No NOEC (Diethylene glycol dimethyl ether) could be derived for male rats since effects on the reproductive organs were observed at all dose levels tested.
Executive summary:

Triethylene glycol dimethyl ether and Diethylene glycol dimethyl ether, which is tested for its subacute inhalation toxicity, belong to the glycol ether family. These substances have been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile. Due to the fact that Triethylene glycol dimethyl ether and Diethylene glycol dimethyl ether have nearly the same chemical structure (especialy with reference to the functional groups):

1. Triethylene glycol dimethyl ether:H3C-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3

2. Diethylene glycol dimethyl ether:H3C-O-CH2-CH2-O-CH2-CH2-O-CH3

the same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. Furthermore, it can be shown that Ethylene glycol dimethyl ether, Diethylene glycol dimethyl ether and Triethylene glycol dimethyl are are not acute orally toxic and neither Ethylene glycol dimethyl ether nor Diethylene glycol dimethyl ether was found to be acute toxic by inhalation. Therefore, a read-across from Triethylene glycol dimethyl ether to data obtained with Diethylene glycol dimethyl ether is scientifically justified.

To assess the inhalation toxicity of Diglyme, groups of 20 male and 10 female rats were exposed by nose-only inhalation 6 hours/day, 5 days/week for 2 weeks to either 0 (control), 110, 370 or 1100 ppm Diglyme. 2-Methoxyethanol was applied as positive control. Rats were sacrificed immediately following exposure, after a 14-day recovery period, or after 42 and 84 days of recovery (males only). Parameters investigated included in-life observations and body weights, clinical pathology, and histopathology with organ weights.

Exposure to Diglyme produced a variety of concentration-related changes. The most striking effect produced in all test groups was cellular injury involving the testes, seminal vesicles, epididymides and prostate. Although these effects were more severe at the higher concentrations tested, partial or complete recovery was seen by 84 days post-exposure. Changes in the haematopoetic system occurred in both sexes and involved the bone marrow, spleen, thymus, leucocytes and erythrocytes. The testicular effects of Diglyme were somewhat less pronounced than those seen with 2 -Methoxyethanol. The NOEC for repeated inhalation exposure to Diglyme in female rats is 370 ppm. For males, the NOAEC was derived at 110 ppm.