Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: EPA Testing Consent Order 40 CFR 799.5000
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals were uniquely identified by a toe clip procedure.

TEST ANIMALS
- Source: Charles River Breeding Laboratory Inc., Portage, MI.
- Age at study initiation: 8 weeks at fist dose.
- Weight at study initiation: 291.3 - 339.7 g (males); 177.4 - 218.0 g (females)
- Housing: individually in stainless-steel cages mounted in a stainless-steel Maxi-Rack. During the period of motor activity testing they were housed in chamber C, a modified Relocatable Containment System® in room 164 of the CHF Building of BRRC. A layer of Deotized Animal Cage Board@ was kept under each cage and changed at least three times per week.
- Diet: Ground Purina Certified Rodent Chow #5002 was available ad libitum except during periods of motor activity testing.
- Water: ad libitum except during motor activity tests on days 1, 30, 60 and 90 after initiation of TGME treatment. Water was provided by Nalgene bottles.

ENVIRONMENTAL CONDITIONS
Room temperature and relative humidity were monitored continuously by a Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model #8368-00.
- Temperature: 66 – 77°F
- Humidity: 40 – 70%
- Photoperiod: 12 hour light/12 hour dark cycle. (0500 – 1700 light)
- White noise levels in 164C were routinely maintained between 59 dBA and 63 dBA during motor activity testing sessions.
- White noise generated during the motor activity test sessions was monitored with a Realistic® Sound Level Meter, Model #33-1050 and a Varian strip chart recorder, Model #A25 or a Graphtec® Microservo SR6402 chart recorder. The sound level meter was calibrated daily with a GenRad® Sound-Level Calibrator, Model #GR1562.

IN-LIFE DATES: From: July 24, 1989 To: October 25, 1989
Route of administration:
oral: drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The drinking water solutions were prepared by direct addition of TGME to tap water. A concentrated solution was prepared from which the dosing solutions were made to help facilitate distribution of the test material with a minimum of mixing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the actual dosing solutions were taken and analyzed for TGME concentration (prior to being administered to the animals) during study Weeks 1 through 4. In subsequent weeks, the samples were stored refrigerated, and one sample from each preparation (concentrations being selected sequentially) were analyzed in Weeks 8 and 13 with one control.
Duration of treatment / exposure:
90 days
Remarks:
Doses / Concentrations:
0, 0.42, 1.24, 4.3 TGME/kg/day (males); 0, 0.42, 1.29, 4.1 TGME/kg/day (females)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 0.4, 1.2, 4.0 mg/kg/day
Basis:
other: (target doses)
No. of animals per sex per dose:
Each dosage group was randomly divided into two blocks. Block 1 and Block 2.
Block 1: 7 males/7 females (control and mid-dose); 8 males/8 females (low dose and high dose)
Block 2: 8 males/8 females (control and mid-dose); 7 males/7 females (low dose and high dose).
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a 14-Day dose range-finding study conducted at Bushy Run Research Center (BRRC Project Report '52-606). Dose levels were chosen to produce toxicity or clear behavioral effects at the high dose and graded dose dependent effects in at least two of the doses tested including the high dose.
- Rationale for animal assignment: Animals were assigned to test groups based on body weight, by a computer generated non-stratified randomization procedure. Once rats were randomized into dosage groups, each dosage group was randomly divided into two blocks. Block 1 contained 7 male and 7 females from each of the control and mid-dose treatment groups and 8 males and 8 females from each of the low-dose and high-dose treatment groups. Block 2 contained 8 males and 8 females from each of the control and mid-dose treatment groups and 7 males and 7 females from each of the low-dose and high-dose treatment groups. The first day of exposure to test drinking water solutions was Monday, July 24, 1989 for Block 1 animals and Tuesday, July 25, 1989 for Block 2 animals. Rats not selected for the study were removed from the room.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked included mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Day 1 and weekly thereafter. Due to the size of the water bottles, it was necessary to collect full and empty water bottle weights twice a week, but these weights were combined and reported on a weekly interval basis.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Battery of functions tested:
- (motor activity) Ten animals/sex/group were evaluated for neurobehavioural function using a functional observational battery prior to their first exposure, on the day after their first overnight exposure to TGME, and on the 8th, 15th, 30th, 60th, and 90th day after their first day of exposure to TGME.
Specific biochemical examinations:
No further data available.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked were: Convulsions, piloerection, urinary pattern, pupil response to light, mouthbreathing, visual placing, catatonic pose. toe pinch, body weight, tremors, respiratory pattern, startle response, excessive vocalization, lacrimation, muscle tone, treadmill, tailflick, stereotypy, gait, pupil size, salivation, diarrhea, grip strength, positive geotropism, rectal temperature.
- Description of procedures: The conduct of an FOB involved handling each animal, evaluating, and recording the absence or presence of a predetermined set of behavioral signs. This record also included the degree of severity of a behavioral sign when this was appropriate. During the examination, the animal was placed in a clean Plexiglasm cage and evaluated for approximately 2 minutes for convulsions, tremors, stereotypy, piloerection, respiration, urination, gait, and acoustic startle response. The animal was then held and evaluated for pupil size, pupil response to light, vocalization, salivation, mouthbreathing, lacrimation, diarrhea, visual placing, and muscle tone. Catatonia, fore-limb and hind-limb grip strength, performance on a rotating treadmill, positive geotropism ("Inclined screen turn"), toe and tail withdrawal reflexes, rectal temperature and body weight were subsequently evaluated.
- Minimization of bias:
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: No data

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: automated recording apparatus designed to measure activity in a novel environment (San Diego Instruments Inc. San Diego, CA}.
- Length of session, number and length of subsessions: All test sessions were 90 minutes in duration. For purposes of statistical evaluation, intrasession intervals were 10 minutes in duration.
- Parameters measured: Data for· ambulatory activity, fine motor activity, rearing activity, and the sum of these individual types of activities (sum of all counters or total activity) were collected. Rearing activity was collected in two sets based on the animals position in the testing cage (front cage and back cage) at the time of rearing.
Sacrifice and (histo)pathology:
NEUROANATOMIC PATHOLOGY
- Time point of sacrifice: After 91 days of treatment were anesthetized with sodium pentobarbital.
- Number of animals sacrificed: 10 animals/sex/group (the first 5 animals/group/block for each sex)
- Procedures for perfusion: Tissues were fixed by intracardiac perfusion, first with a phosphate buffered solution of methanol-free electron microscopic grade 5% formaldehyde and then with a phosphate buffered solution. of 5% glutaraldehyde. After perfusion, the animals were placed in the refrigerator for at least three hours prior to further dissection.
- Number of animals perfused: 10 animals/sex/group (the first 5 animals/group/block for each sex)
- Tissues evaluated: All animals scheduled for neuroanatomic pathology were given a complete gross necropsy examination, and the following additional tissues were saved: Lungs, liver, kidneys, testes/ovaries, spleen and adrenals.

ANATOMIC PATHOLOGY
- Time point of sacrifice: 93 days of treatment (for Block 1 animals) or 92 days of treatment (for Block 2 animals).
- Number of animals sacrificed: the remaining 5 animals/sex/group were anesthetized with methoxyflurane and killed by severing the brachial vessels to permit exsanguination, and given a complete gross necropsy examination.
- Parameters measured:
- Organs weighed: liver, kidneys, brain, lungs, adrenals, and testes (males).
-Tissues evaluated: Brain (same sections as those taken for Neuroanatomic Pathology), spinal Cord, cervical, thoracic, lumbar, liver (2 sections), lungs (perfused with formalin Via the trachea), testes/ovaries, kidneys, adrenals, spleen were fixed in 10% neutral buffered formalin.

HISTOLOGIC PATHOLOGY
Brains and spinal cords that were examined by light microscopy were processed for paraffin embedding, sectioned at 5-6 microns, and stained with hematoxylin and eosin, as well as with the luxol fast blue and Bielschowsky's techniques. Peripheral nerves from one hind leg (i.e. the proximal sciatic nerve from above the knee level, as well as the tibial, common peroneal and sural nerves from below the knee) were embedded in glycol methacrylate, sectioned at 2 microns, and stained with hematoxylin and eosin. Histologic examinations were performed on all of the tissues listed below for 6 animals/sex/group by light microscopy. Lesions were graded as to severity, where possible, into 5 categories (minimal, mild, moderate, marked, and severe).
Forebrain (cross sections)
Center of the Cerebrum (cross sections)
Center of the Midbrain (cross sections)
Cerebellum and Pons (cross sections)
Medulla Oblongata (cross sections)
Spinal cord (cervical and lumbar; cross and longitudinal sections)
Dorsal Root Ganglia (longitudinal sections)
Dorsal and Ventral Root Fibers (longitudinal sections)
Proximal Sciatic Nerve (cross and longitudinal sections below the knee)
Tibial Nerve (cross and longitudinal sections below the knee)

-Light microscopic examinations were performed on livers from all animals and testes from all male animals. Livers were processed for paraffin embedding, sectioned at 5-6 microns, and stained with hematoxylin and eosin.
-Testes were processed for embedding in glycol-methacrylate, sectioned at 2.5microns, and stained with Periodic Acid Schiff's-hematoxylin.
Other examinations:
No further examinations.
Positive control:
No
Statistics:
The accepted level of Type 1 error rate was set to an approximate overall value of 0.05 within separate test domains, but individual p values were reported without correction for multiple testing. To reduce the increased false positives associated with repeated significance testing, the correction procedure suggested by Mantel (1980) was used when testing for overall significance. Evaluations were divided into 8 domains; body weights, body weight gains, food consumption, water consumption, functional observational battery, motor activity, organ weights, and microscopic diagnosis. The critical level of significance was adjusted for each test domain by dividing the overall critical level of significance for the test domain (α = 0.05) by the square root of the number of significance tests within the test domain. These levels were subsequently rounded up to either 0.005 or 0.01. All statistical tests, except the frequency comparisons for pathology findings were performed using BMDP Statistical Software (Dixon, 1985 and 1988). The frequency data tests for pathology findings are described by Sokal et ale (1969).

The data for water consumption, food consumption, body weight, body weight gain, and organ weights were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differ from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests.

Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Details on results:
Treatment with TGME did not result in clinical signs of toxicity, alterations in the functional observational battery, or gross or microscopic lesions in the nervous system. One female animal from the high-dose treatment group died on study Day 37 without apparent cause. Treatment with TGME resulted in decreases in mean food consumption, body weight, and body weight gain for males and females in the high-dose treatment group and trends for decreased food consumption and body weight for males in the mid-dose treatment group. Water consumption tended to be decreased at each measurement period for females in the high-dose treatment group. Small decreases in mean food consumption, body weight, and body weight gain were also apparent for females in the mid-dose treatment group.



FOB
There were no treatment-related differences observed in the FOB for males or females during the study. Statistically significant decreases in body
weight observed for males at the time of the FOB were consistent with the weekly body weight findings discussed previously. A statistically significant
decrease in mean fore-limb grip strength observed for males in the high dose treatment group at the Day 90 evaluation was considered to be a result of elevated mean fore-limb grip strength for the control group during this evaluation. This conclusion was based on the range of fore-limb grip strength values collected for the control group at other evaluations and the lack of a dose-response relationship for TGME-treated males at the Day 90 evaluation.

MOTOR ACTIVITY: For males, a statistically significant decrease in mean cumulative test session activity was observed on Day 60 for animals in the high-dose treatment group (29% decrease, Figure 4C). Significant dose effects were observed by repeated measures analysis on Day 60 and 90 and a significant interaction between time and dose was observed on Day 90. Subsequent group comparisons for total activity at within session intervals detected statistically significant decreases in mean activity for the high dose treatment group on Day 60 (60 to 70 minutes) and on Day 90 (30 to 40 minutes). These findings were associated with ·trends for decreased mean activity for males in the high-dose group at additional time points during these test sessions (Figure 7C and 7D). Additional findings for males in the high-dose treatment group included statistically significant dose effects (repeated-measures analysis) for ambulation on Day 30 that were associated with statistically significant decreases in mean ambulation during the 10 to 20 and 20 to 30 minute test session intervals (Appendix 9, Table· 3). For females, statistically significant decreases in mean cumulative test session activity were observed on Day 90 (30% decrease, Figure 4D) in the high dose treatment group. Intergroup statistical comparisons at discrete test session intervals were not performed (according to the protocol) since statistically significant dose or time by dose effects were not observed by repeated measures analysis. However, there was a trend for decreased mean activity for females in the high-dose group during the first 60 minutes of the Day 90 test session (Figure 8D). No other potential treatment-related alterations in motor activity were observed.

Decreases in motor activity observed for males and females in the high-dose treatment group at the Day 60 (males only) and Day 90 evaluation periods were of small magnitude and are considered to be of minor significance. Because motor activity is an apical test, by nature it can be altered by a number of factors affecting the physiology and/or morphology of living organisms. In the absence of signs or context of nervous system involvement, the motor activity data are difficult to interpret as an expresslon of neurotoxicity.
Dose descriptor:
NOAEL
Effect level:
4 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: absence of neurotoxicity
Remarks on result:
other:
Conclusions:
The decreases in motor activity observed for this study were not considered to be neurotoxicologically significant based on the small magnitude of the changes, the parallel changes observed in body weights at these evaluation periods, which suggest an association between body weight and motor activity, the lack of behavioral effects observed with the FOB evaluations, which suggest no effect on select components of the nervous system, and finally the lack of effects observed histologically in tissues examined from the central or peripheral nervous systems.

Based on the results of this study, treatment of Sprague-Dawley rats with TGME via the drinking water for 13 weeks did not produce neurotoxicity at doses as high as 4.0 g/kg/day. Based on the results of this study, the no observable effect level for neurotoxicity for TGME is at least 4.0 g/kg/day.
Executive summary:

In a guideline (OECD 408) and GLP study, exposure to TGME in the drinking water of rats at doses up to 4 g/kg/day for 90 days did not result in clinical signs of toxicity, alterations in the functional observational battery, or gross or microscopic lesions in the nervous system. Minor decreases in motor activity were observed in the 4 g/kg/day treatment group at the Day 60 (males only) and Day 90 (males and females) evaluation periods. These decreases in motor activity were not considered to be neurotoxicologically significant based on the small magnitude of the changes, the parallel changes observed in body weights at these evaluation periods, and the lack of corroborative behavioral effects from the FOB evaluations or histological changes in central or peripheral nervous system tissues. Based on these findings 4 g/kg/day is a subchronic NOAEL for TGME neurotoxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
Study primarily a neurotoxicity study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triethylene glycol monomethyl ether (TGME)
- Physical state: Clear, colorless, nonviscous liquid
- Analytical purity: 99.3%
- Storage condition of test material: Room temperature
- Supplier: Union Carbide Chemicals and Plastics company.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals were uniquely identified by a toe clip procedure.

TEST ANIMALS
- Source: Charles River Breeding Laboratory Inc., Portage, MI.
- Age at study initiation: 8 weeks at first dose.
- Weight at study initiation: 291.3 - 339.7 g (males); 177.4 - 218.0 g (females)
- Housing: individually in stainless-steel cages mounted in a stainless-steel Maxi-Rack. During the period of motor activity testing they were housed in chamber C, a modified Relocatable Containment System® in room 164 of the CHF Building of BRRC. A layer of Deotized Animal Cage Board@ was kept under each cage and changed at least three times per week.
- Diet: Ground Purina Certified Rodent Chow #5002 was available ad libitum except during periods of motor activity testing.
- Water: ad libitum except during motor activity tests on days 1, 30, 60 and 90 after initiation of TGME treatment. Water was provided by Nalgene bottles.

ENVIRONMENTAL CONDITIONS
Room temperature and relative humidity were monitored continuously by a Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model #8368-00.
- Temperature: 66 – 77°F
- Humidity: 40 – 70%
- Photoperiod: 12 hour light/12 hour dark cycle. (0500 – 1700 light)
- Other: White noise levels in 164C were routinely maintained between 59 dBA and 63 dBA during motor activity testing sessions. White noise generated during the motor activity test sessions was monitored with a Realistic® Sound Level Meter, Model #33-1050 and a Varian strip chart recorder, Model #A25 or a Graphtec® Microservo SR6402 chart recorder. The sound level meter was calibrated daily with a GenRad® Sound-Level Calibrator, Model #GR1562.

IN-LIFE DATES: From: July 24, 1989 To: October 25, 1989

Administration / exposure

Route of administration:
oral: drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The drinking water solutions were prepared by direct addition of TGME to tap water. A concentrated solution was prepared from which the dosing solutions were made to help facilitate distribution of the test material with a minimum of mixing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the actual dosing solutions were taken and analyzed for TGME concentration (prior to being administered to the animals) during study Weeks 1 through 4. In subsequent weeks, the samples were stored refrigerated, and one sample from each preparation (concentrations being selected sequentially) were analyzed in Weeks 8 and 13 with one control.
Duration of treatment / exposure:
90 days.
Doses / concentrationsopen allclose all
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
actual 420mg/kgbw/day (males and females)
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Remarks:
actual 1240mg/kgbw/day (males);1290 mg/kgbw/day (females)
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
actual 4300mg/kgbw/day (males); 4100mg/kgbw/day (females)
No. of animals per sex per dose:
Each dosage group was randomly divided into two blocks. Block 1 and Block 2.
Block 1: 7 males/7 females (control and mid-dose); 8 males/8 females (low dose and high dose)
Block 2: 8 males/8 females (control and mid-dose); 7 males/7 females (low dose and high dose).
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a 14-Day dose range-finding study conducted at Bushy Run Research Center (BRRC Project Report '52-606). Dose levels were chosen to produce toxicity or clear behavioral effects at the high dose and graded dose dependent effects in at least two of the doses tested including the high dose.
- Rationale for animal assignment: Animals were assigned to test groups based on body weight, by a computer generated non-stratified randomization procedure. Once rats were randomized into dosage groups, each dosage group was randomly divided into two blocks. Block 1 contained 7 male and 7 females from each of the control and mid-dose treatment groups and 8 males and 8 females from each of the low-dose and high-dose treatment groups. Block 2 contained 8 males and 8 females from each of the control and mid-dose treatment groups and 7 males and 7 females from each of the low-dose and high-dose treatment groups. The first day of exposure to test drinking water solutions was Monday, July 24, 1989 for Block 1 animals and Tuesday, July 25, 1989 for Block 2 animals. Rats not selected for the study were removed from the room.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked included mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Day 1 and weekly thereafter. Due to the size of the water bottles, it was necessary to collect full and empty water bottle weights twice a week, but these weights were combined and reported on a weekly interval basis.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Battery of functions tested:
- (motor activity) Ten animals/sex/group were evaluated for neurobehavioural function using a functional observational battery prior to their first exposure, on the day after their first overnight exposure to TGME, and on the 8th, 15th, 30th, 60th, and 90th day after their first day of exposure to TGME.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals scheduled for neuroanatomic pathology were given a complete gross necropsy examination, and the following additional tissues were saved: Lungs, liver, kidneys, testes/ovaries, spleen and adrenals.
HISTOPATHOLOGY: Yes, Forebrain (cross sections), center of the cerebrum (cross sections), center of the midbrain (cross sections), cerebellum and pons (cross sections), medulla oblongata (cross sections), spinal cord (cervical and lumbar; cross and longitudinal sections), dorsal root ganglia (longitudinal sections), dorsal and ventral root fibers (longitudinal sections), proximal sciatic nerve (cross and longitudinal sections below the knee), tibial nerve (cross and longitudinal sections below the knee). Testes were also examined.
Other examinations:
See also IUCLID 7.9.1 Neurotoxicity - BRRC (1990)
Statistics:
The accepted level of Type 1 error rate was set to an approximate overall value of 0.05 within separate test domains, but individual p values were reported without correction for multiple testing. To reduce the increased false positives associated with repeated significance testing, the correction procedure suggested by Mantel (1980) was used when testing for overall significance. Evaluations were divided into 8 domains; body weights, body weight gains, food consumption, water consumption, functional observational battery, motor activity, organ weights, and microscopic diagnosis. The critical level of significance was adjusted for each test domain by dividing the overall critical level of significance for the test domain (α = 0.05) by the square root of the number of significance tests within the test domain. These levels were subsequently rounded up to either 0.005 or 0.01. All statistical tests, except the frequency comparisons for pathology findings were performed using BMDP Statistical Software (Dixon, 1985 and 1988). The frequency data tests for pathology findings are described by Sokal et al. (1969).

The data for water consumption, food consumption, body weight, body weight gain, and organ weights were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differ from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
One female from the high-dose treatment group died on study day 37.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in mean body weight and body weight gain were observed for males in the high-dose treatment group throughout the study. Body weight decrement for males in the high-dose treatment group became more pronounced as the study progressed and mean body weight was ultimately 123 grams less than the mean for the control group at the end of the study. Mean body weight and body weight gain for males in the mid dose treatment group also tended to be decreased (2 to 8%) compared to the mean for the control group. Statistically significant decreases in mean body weight gain were observed for females in the high-dose treatment group at several measurement periods. Decreases in body weight gain were observed as early as the Week 0 to 1 measurement period (9% decrease) and lasted throughout the study (22% decrease for the Week 0 to 13 measurement period). Apparent dose-related decreases in body weight gain were observed for females in the low-dose treatment group beginning at Week 5 (3 to 8%) and in the mid-dose treatment group for most of the study (6 to 13%). Small dose-related decreases in mean body weight were observed for females in all three treatment groups. These changes were not statistically different from the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in mean food consumption were observed for males and females in the high-dose treatment group through most of the study. The decreases observed were generally in the range of 10 to 23% for males and 8 to 15% for females. In addition, a tendency for decreased mean food consumption for males in the mid-dose treatment group throughout the study (generally 3 to 4%) and an apparent dose-related decrease in mean food consumption for females in the low-dose (generally 4 to 7%) and mid dose (generally 7 to 10%) treatment groups were observed. Statistically significant changes for animals in the low-dose and mid-dose treatment groups were limited to Week 5 for females from the mid-dose treatment group.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Unexpectedly high values for water consumption were observed for males (approximately 60 grams/animal/day) and females (approximately 52 grams/animal/day) in all treatment groups (including controls) during the first week of the study. These are in contrast to pre-treatment water consumption measurements evaluated under similar conditions over a 4-day period of approximately 40 grams/animal/day for males and approximately 29
grams/animal/day for females. It is unclear what contribution spills made to the high values observed during study Week 1 since excessive spills were not observed. Mean water consumption values were lower for all groups during study Week 2 than in study Week 1 and water consumption remained relatively constant during the remainder of the study. There were no treatment-related alterations' in water consumption for males during this study. A treatment-related decrease in mean water consumption was observed for females in the high-dose treatment group throughout the study (generally 15% to 29%). These values were statistically significant only during study Week 4. A statistically significant increase in mean water consumption for males in the high-dose treatment group during study Week 13 was not considered to be biologically significant based on the lack of similar findings during the study.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increases in mean liver weight were observed for male animals in all three TGME treatment groups. Mean brain weight for perfused male animals and mean kidney weight for non-perfused male and female animals were statistically increased when expressed as percent of final body weight. Decreases in absolute brain or kidney weights were not observed and the findings for relative organ weight were considered to be a result of decreased body weight. Decreases in mean absolute testes weights were observed for the non-perfused males in the mid-dose (9%) and high-dose (19%) treatment groups (n=5males/group). However, these changes were not statistically significant. Size decrease was observed for a single male in the mid-dose treatment group (approximately 50% size decrease) at necropsy.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy of the deceased female revealed size increase for kidneys and adrenals and diffuse color change of various tissues. A specific cause of death was not apparent. There were no gross or microscopic lesions found in the nervous system of any rats that were attributed to TEGME ingestion.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal or mild hepatocellular hypertrophy was observed for 10 females in the high-dose treatment group. Fibrous connective tissue was observed around a small number of the bile ducts (cholangiofibrosis) for 7 males in the high-dose treatment group. All lesions were of minimal severity and were not considered to be physiologically significant. Cholangiofibrosis was not observed for males in any other treatment group or for females. Vacuolization or hypertrophy (minimal or mild severity) were also observed in the livers of some males in the low-dose and mid-dose treatment groups and females in the high-dose treatment group. Hypertrophy can be considered a physiological adaptation due to large subchronic doses of the test material, which is likely to be metabolized by alcohol dehydrogenase and aldehyde oxidase in the liver.

Treatment-related degeneration and/or atrophy of seminiferous tubules were observed for most males in the high dose treatment group. The cell types affected in the tubules were spermatocytes and developing spermatids. Similar lesions were not observed for males in the low-dose or mid-dose treatment groups. At the Sponsor's request, testes from all males on the study were examined histologically. Significant seminiferous tubule degeneration and/or atrophy were observed in 12 of 15 males in the high dose treatment group. The target cells in the tubules appeared to be spermatocytes and developing spermatids. Treatment-related lesions were not observed in the low-dose and mid-dose groups. No other potential treatment-related alterations in organ weights, gross pathology, or histopathology were observed in this study.

Effect levels

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no
Lowest effective dose / conc.:
1 200 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, treatment of Sprague-Dawley rats with TGME via the drinking water for 13 weeks produces moderate toxicity at 4.0
g/kg/day and minimal to mild toxicity at 1.2 g/kg/day.
Executive summary:

In a guideline (OECD 408) and GLP study, exposure to triethylene glycol methyl ether (TGME) in the drinking water of rats for 90 days resulted in decreases in mean food consumption, body weight, and body weight gain for males and females in the 4 g/kg/day treatment group and trends for decreased food consumption and body weight for males in the 1.2 g/kg/day treatment group. Water consumption tended to be decreased at each measurement period for females in the 4 g/kg/day treatment group. Small decreases in mean food consumption, body weight, and body weight gain were also apparent for females in the 1.2 g/kg/day treatment group. One female animal from the 4 g/kg/day treatment group died on study Day 37 without apparent cause. Dose-related increases in mean liver weight were observed for male animals in all three TGME treatment groups. Associated microscopic changes included hepatocellular cytoplasmic vacuolization and/or hepatocellular hypertrophy, for males in the 4 g/kg/day treatment group. Vacuolization or hypertrophy (minimal or mild severity) were also observed in the livers of some males in the 0.4 g/kg/day and 1.2 g/kg/day treatment groups and females in the 4 g/kg/day treatment group. Hypertrophy can be considered a physiological adaptation due to large subchronic doses of the test material, which is likely to be metabolized by alcohol dehydrogenase and aldehyde oxidase in the liver. Treatment-related degeneration and/or atrophy of seminiferous tubules were observed for most males in the 4 g/kg/day treatment group. The cell types affected in the tubules were spermatocytes and developing spermatids. Similar lesions were not observed for males in the 0.4 g/kg/day or 1.2 g/kg/day treatment groups. On the basis of these findings 1200 mg/kg bw/day l is considered to be a subchronic LOAEL for TGME and 400mg/kg bw/day a NOAEL under the conditions of this study.