Registration Dossier

Administrative data

Description of key information

Oral: 
NOAEL (28 days, gavage; rat): 250 mg/kg bw
Main target organ: testes, thymus, hematopoetic system
Oral (read across to triglyme):
NOAEL (28 days, gavage; rat): 250 mg/kg bw
Main target organ: testes, thymus, hematopoetic system
By inhalation (read across to diglyme):
NOAEL (14 days; rat): 110 ppm (650 mg/kg bw)
Main target organ: testes, thymus, hematopoetic system

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-10-10 to 1991-11-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study; GLP study; scientifically well performed study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF-Zucht
- Age at study initiation: ca. 6 months
- Housing: In groups of five in Makrolon type-4 cages, in air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
water
Duration of treatment / exposure:
28 days
Frequency of treatment:
once per day
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during working days. Once daily during weekends or holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice twice daily during working days. Once daily during weekends or holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100g body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Once weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once weekly.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of the test.
- Anaesthetic used for blood collection: Yes (Ketanest)
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Leukocyte count, Differential leukocyte count, Reticulocyte count, Heinz bodies, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after the collection of blood.
- Animals fasted: Yes
- How many animals: All.
- Parameters checked: Sodium, Potassium, Phosphorus, Uric acid, Total bilirubin, Direct bilirubin, Creatinine, Serum glucose, Uric nitrogen, Calcium, Chloride, ASAT, ALAT, AP, CGT, Total protein, Albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: On the night of test day 26/27.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, color, pH, Haemoglobin, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Density, Sediment.

NEUROBEHAVIOURAL EXAMINATION: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced throbocytes in females at 1000 mg/kg bw
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
reduced alkaline phosphatase in males and females at 1000 mg/kg bw
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight increas for females at 1000 mg/kg bw; reduced tested and reduced thymus in males at 1000 mg/kg bw
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
findings in testes and thymus at 1000 mg/kg bw
Details on results:
BODY WEIGHT AND WEIGHT GAIN
At the beginning of the test, significant difference of body weight was observed in female animals at 62.5 mg/kg bw. No difference was observed in other groups. This is not considered to be treatment related because there is no dose-effect relationship.

HAEMATOLOGY
The clotting time was decreased in male animals of all treated groups without dose-effect relationship. The thrombocyte counts were slightly decreased in female animals at 1000 mg/kg bw.

CLINICAL CHEMISTRY
At 1000 mg/kg bw, the AP activity was significantly decreased, and the Creatine values were increased. In male animals at 62.5 mg/kg bw, the phosphorus values were signficantly increased. In male animals at 250 mg/kg bw, the sodium values were decreased. In male animals of 62.5 and 250 mg/kg bw , the calcium levels were increased and the urea levels were decreased. All the effect showed no dose-response relationship.

ORGAN WEIGHTS
In male and female animals at 1000 mg/kg bw, the thymus weights were decreased. In males the tested weight was decreased as well.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hign dose group:
In the thymus of 4 males and 2 females, narrowed and loosend cortex with high reduction of lymphocytes were observed. In testes of 2 males, degradation of germinal epithelium and increased single cell necrosis were found. The number of matured sperm cells was significantly reduced.
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical chemistry; organ weights; histopathology
Critical effects observed:
not specified

Justification of use of the reported study as key study for repeated dose toxicity:

The reported study is qualified to be used as key study for the repeated dose toxicity based on the metabolism, kinectis and mode of action considerations.

It has been shown that tetraglyme induces toxicity via systemic exposure to toxic metabolites 2-methoxy ethanol and 2-methoxy acetic acid, indicating that the repeated dose toxicity of tetraglyme is the result of systemic exposure to steady state toxic metabolites. Considering that tetragylme undergoes extensive metabolism and its toxic metabolites are highly hydrophilic, it can be derived that the 28-day exposure is likely to reflect the steady state toxic metabolites and thus the subchronic/chronic toxicity.

The steady-state toxic metabolites premises no metabolic overload. According to the reported 28 -day study, tetraglyme did not induce any effects at dose levels of 62,5 and 250 mg/kg bw and it is not likely that a metabolic overload could occur upon prolonged exposure. The NOEL obtained in this study is considered to reflect the NOEL of subchronic/chronic toxicity.

In conclusion, the available data (physico-chemical data, metabolism/kinetic data, mode of toxicity, 28-day toxicity study) supports that the results obtained the 28-day study on tetraglyme can be used as key study for the endpoint repeated dose toxicity.

Conclusions:
The no observed effect level (NOEL) after 28-day exposure of rats is determined to be 250 mg/kg bw/day. The NOEL obtained in this study is considered to be valid for subchronic/chronic toxicity in combination of data on metabolism, kinetics, and mode of action of glymes.
Executive summary:

Tetraglyme was administereddaily by oral gavage to Wistar rats of male and female at dose levels of 62.5, 250 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle - water only.

In all the groups, the behavior, clinical sign, body weight, food consumption and water consumption were recorded. The Haematology and clinical chemistry analyses, and urinalysis tests were performed at the end of the treatment. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues.

All animals survived during the treatment period. No clinical signs, body weight, food and water consumption changes were noted. Haematological analysis showed that the thrombocyte counts were slightly decreased in females of high dose group. The clinical chemistry test showed that the decreased AP activity and elevated creatinine were found in both males and females at 1000 mg/kg bw. The urinalysis showed no compound related toxicity. In male and female animals of high dose group, the thymus weights were decreased.

Histopothology investigation showed several treatment related effect in animals of high dose group. In the thymus of 4 males and 2 females, narrowed and loosened cortex with high reduction of lymphocytes were observed. In testes of 2 males, degradation of germinal epithelium and increased single cell necrosis were found. The number of matured sperm cells was significantly reduced.

Based on the effects of clinical chemistry, organ weight and histopathology, the no observed effect level (NOEL) of the test item is 250 mg/kg bw/day for male and female rats.

The reported 28 -day study is considered to be qualified to be used as key study for the repeated dose toxicity together with the data on metabolism, kinectics and mode of action of glymes. It can reasonably assumed that the NOEL obtained in this 28-day reflect the steady state of toxic metabolites and thus the NOEL of subchronic/chronic toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable and robust; 2 studies/reports are available on target or source chemicals. The study results are in line with other study types such as reproduction toxicity studies.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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: see 'Remark'
Remarks:
Justification of read-across: The target chemical belongs to the homologues series of glymes, where there is an incremental increase in the number of CH2CH2O units. Therefore, it can be assumed that target and other glyme members (mono-, di-, and triglyme) share the same toxic mode action. Justification for reliability of 2: scientifically well-performed study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
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
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
Remarks:
Doses / Concentrations:
110, 370, 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
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.
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.
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
Critical effects observed:
not specified
Conclusions:
Diglyme and 2-methoxyethanol induced testicular damage and changes in the haematopoetic system upon inhalation exposure. The NOEC for repeated inhalation exposure to diglyme in female rats is 370 ppm. For males, the NOAEC was derived at 110 ppm.
Executive summary:

The subacute inhalation toxicity of tetraglyme was assessed based on the analogue approach using diglyme as a read-across supporting substance. The toxicity profile observed upon inhalation did not differ much when compared to oral toxicity.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
650 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable study on source chemicals.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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: see 'Remark'
Remarks:
Justification of read-across: The target chemical belongs to the homologues series of glymes, where there is an incremental increase in the number of CH2CH2O units. Therefore, it can be assumed that target and other glyme members (mono-, di-, and triglyme) share the same toxic mode action. Justification for reliability of 2: scientifically well-performed study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
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
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
Remarks:
Doses / Concentrations:
110, 370, 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
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.
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.
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
Critical effects observed:
not specified
Conclusions:
Diglyme and 2-methoxyethanol induced testicular damage and changes in the haematopoetic system upon inhalation exposure. The NOEC for repeated inhalation exposure to diglyme in female rats is 370 ppm. For males, the NOAEC was derived at 110 ppm.
Executive summary:

The subacute inhalation toxicity of tetraglyme was assessed based on the analogue approach using diglyme as a read-across supporting substance. The toxicity profile observed upon inhalation did not differ much when compared to oral toxicity.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

In order to investigate the repeated dose toxicity of tetraglyme a subacute oral toxicity study was performed in rats according to OECD 407. The animals were exposed to at dose levels of 0, 62.5, 250 and 1000 mg/kg bw.

All animals survived during the treatment period. No clinical signs, body weight, food and water consumption changes were noted. Haematological analysis showed that the thrombocyte counts were slightly decreased in females of high dose group. The clinical chemistry test showed that the decreased alkaline phosphatase activity and elevated creatinine in both males and females at 1000 mg/kg bw. The urinalysis showed no compound related toxicity. At 1000 mg/kg bw the thymus weights were decreased.

Histopothology investigation showed several treatment related effect in animals at 1000 mg/kg bw. In the thymus of 4 males and 2 females, narrowed and loosened cortex with high reduction of lymphocytes were observed. In testes of 2 males, degradation of germinal epithelium and increased single cell necrosis were found. The number of matured sperm cells was significantly reduced.

Based on the effects of clinical chemistry, organ weight and histopathology, the NOEL of tetraglyme is 250 mg/kg bw/day for male and female rats.

The reported study on tetraglyme is qualified to be used as key study for the repeated dose toxicity based on the scientific rationales and from the regulatory point of view: 

a)     Scientic rationales:

It has been shown that tetraglyme induces toxicity via systemic exposure to toxic metabolites 2-methoxy ethanol and 2-methoxy acetic acid, indicating that the repeated dose toxicity of tetraglyme is the result of systemic exposure to steady state toxic metabolites. Considering that tetragylme undergoes extensive metabolism and its toxic metabolites are highly hydrophilic, it can be derived that the 28-day exposure is likely to reflect the steady state toxic metabolites and thus the subchronic/chronic toxicity.

The steady-state toxic metabolites premises no metabolic overload. According to the reported 28 -day study, tetraglyme did not induce any effects at dose levels of 62,5 and 250 mg/kg bw and it is not likely that a metabolic overload could occur upon prolonged exposure. The NOEL obtained in this study is considered to reflect the NOEL of subchronic/chronic toxicity.

With regard to target organ identification, clear toxic effects on testes, thymus and thrombocytes count were identified in the 28-day study. Upon subchronic/chronic exposure at dose level of 1000 mg/kg bw it can be predicted that effects found in thymus and thrombocytes could lead to generally poor health status of treated animals.

In conclusion, the available data (physico-chemical data, metabolism/kinetic data, mode of toxicity, 28-day toxicity study) supports that the results obtained the 28-day study on tetraglyme can be used as key study for the endpoint repeated dose toxicity.

b)     Regulatory point of view:

In accordance with REACH Annex IX, there is sufficient information leading to the conclusion that tetraglyme has to be classified as reprotoxic category 2, R61 and reprotoxic category 3, R62, because

- Tetraglyme caused changes in testes and fertility impairment in rats.

- Tetraglyme and its homologues substances (tri-, di- and monoglyme) caused developmental toxic effect in rabbits, mice and rats in absence of significant maternal toxicity.

It is proposed to perform the risk assessment based on the developmental toxicity data (rabbit study; read-across from triglyme). Such approach is considered to be sufficiently robust, also to cover the risk that may associated with subchronic/chronic exposure. The NOAEL obtained for the developmental toxicity is 75 mg/kg bw and the NOEL for the subacute toxicity is 250 mg/kg bw. Further animal testing, such as 90-day toxicity study, is not likely to add more confidence with respect to the risk assessment.

For the purpose of read-across justification further studies (28 -day oral toxicity study on triglyme and 14 -day inhalation toxicity study on diglyme) are provided.

A subacute oral toxicity study of triglyme was performed in rats. The animals were exposed to 0, 62.5, 250 and 1000 mg/kg bw.

All animals survived and no clinical signs were noted at any dose level. No neurological or ophthalmological effects or changes in mucosa were noted. Except of the males of the 1000 mg/kg bw/d group which showed a decreased body weight gain, the body weight gain of all animals was not affected. There were no effects upon the mean daily food and water consumption observed in all test groups. There were no changes in haematology and clinical chemistry noted at 62.5 and 250 mg/kg bw dose groups. At 1000 mg/kg bw the leucocyte count was decreased in male rats; the thrombocyte count was decreased in all animals of the high dose group. At 1000 mg/kg bw/d the bilirubin and creatinin concentration was significantly increased. Relative organ weights were within the control range with the exception of a decreased testis and epididymides weight (absolute and relative) of the males in the high dose group. The absolute and relative thymus weight was also significantly decreased in all animals of the 1000 mg/kg bw/d group. Females of the 250 mg/kg bw group had decreased thymus weight. The size of testes was reduced in males of the 1000 mg/kg bw dose group. No microscopic changes occurred at any dose level with the exception of changes of the testes, epididymides and spermatogenesis in male rats of the 1000 mg/kg bw/d dose group which caused oligo- and aspermia. In both males and females involution of thymus was observed in the high dose group.

Based on the effects of clinical chemistry, organ weight and histopathology, the NOEL is 62.5 mg/kg bw and NOAEL is 250 mg/kg bw.

The reported study on triglyme is to be used to demonstrate validity of proposed read-across approach using homologues substances (tri-, di-, and monoglyme) and the decreasing toxicity tendency from tri- to tetraglyme.

Both tri- and tetraglyme did not induce any treatment effect at 62.5 mg/kg bw. Triglyme induced reduced thymus weight in females at 250 mg/kg bw. Tetraglyme did not induce any effect at 250 mg/kg bw. At 1000 mg/kg bw, comparable findings were obtained for tri- and tetraglymes, indentifying testes, thymus and blood system as targets. But the degree of effects was more pronounced for triglyme.

Rats were exposed to diglyme by inhalation for 14-days (6h/d, 5d/w). The major findings were testicular toxicity and changes in hematopoetic system. The reported study demonstrates that the toxicity profile of diglyme does not differ to that of tri- or tetraglyme and that the exposure routes (oral or by inhalation) do not influence the toxicity of glymes.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The toxicity profile could be clearly identified and the NOEL obtained is considered to be valid for the repeated dose toxicity.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Scientifically well performed study

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Repeated dose toxicity: dermal - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

It is concluded that no classification is warranted regarding repeated dose toxicity according to 67/548/EEC and Regulation 1272/2008/EC. In the 28-day study toxicity on male sexual organs was observed, supporting the proposed classification of tetraglyme as reprotoxicant.