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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study methodology followed was equivalent or similar to OECD TG 413 and in accordance with the Principles of GLP. Also, the report contains sufficient information to permit a meaningful evaluation of study results.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
A density of 0.913 g/mI, was used for calculation of concentration instead of a value of 0.908 g/ml, which was the density given in the Material Safety Data Sheet. This is a difference of only 0.6% therefore, the difference in concentration was negligible
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
yes
Remarks:
as described above
Qualifier:
according to
Guideline:
other: EPA 40 CFR Parts 798.6050 and EPA 40 CFR Parts 798.6400.
Deviations:
yes
Remarks:
as described above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethylene Glycol Monopropyl Ether
- Physical state: liquid
- Analytical purity: 99.8-99.9 % (gas chromatography with flame ionization detection)
- Impurities (identity and concentrations): not specified
- Lot/batch No.: not specified
- Expiration date of the lot/batch: not specified
- Stability under test conditions: not specified
- Storage condition of test material: not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approximately 7 weeks of age
- Weight at study initiation: 236 ± 6 grams (males); 177 ± 8 grams (females)
- Fasting period before study: none
- Housing: singly housed
- Diet (e.g. ad libitum): ad libitum during non exposure periods
- Water (e.g. ad libitum): ad libitum through an automatic watering system
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70 - 75 °F
- Humidity (%): 41-58 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4.2 m3 stainless steel and glass chamber inhalation chambers
- Method of holding animals in test chamber: in individual cages
- Source and rate of air: dried, oil-free air
- Method of conditioning air: not specified
- Temperature, humidity, pressure in air chamber: 22.2 - 22.5 °C, 54 - 63.9 %
- Air flow rate: 9, 24, 28, 33 liters/minute
- Air change rate: not specified
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber vapor concentrations were monitored with an automated multipositional air sampling and analysis system. The system consisted of a MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT), a Perkin-Elmer SIGMA 15 ,data station, and a four-port multipositional environmental sampling valve (Valco Instruments, Houston, TX).

Chamber vapor samples were continuously collected, from a fixed reference position within each chamber, through the four port valve using TEFLON tubing (3/16" ID). The voltage output of a sampled atmosphere was integrated over a one minute interval following a five minute flushing period. The Perkin-Elmer SIGMA 15 data station calculated the vapor concentration by analog-to-digital conversion and linear interpolation between selected calibration data points. An AATS program called "MIRAN", is used by the SIGMA 15 microprocessor for automated storage and retrieval of ca1ibration data. Vapor concentration is calculated by simple linear interpolation between specific data points on the calibration curve. Then the multipositional environmental sampling valve was automatically advanced to the next valve position, and the procedure repeated until termination of the six hour exposure.

Total chamber flow was monitored via a MAGNEHELIC~ gauge (Dwyer Instruments, Inc., Michigan City, IN) which measured the differential pressure across an orifice 1ocated in the chamber exhaust duct. The chamber flow corresponding to the gauge value was determined weekly, using a TSI Air Velocity Meter (Model 1.650) to determine the linear velocity of the supply air.

- Samples taken from breathing zone: yes

- A Royco Model 225 or Model 4100 Aerosol Particle Counter (HIAC/Royco Instruments Division, Pacific Scientific, Menlo Park, CA) was used to measure nongaseous airborne material within the inhalation chamber. The particle counter sampled the chamber atmosphere once each week during exposure from a fixed reference position at 0.01 cfm for one minute, and counted airborne particles greater than 0.3 pm (Model 225) of 0.5 pm (Model 4100). The results were compared to that of all chamber for the first day of exposure for males, and the air control chamber for all other days of particle size determination. The results indicated that an aerosol of EP was not present


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentrations of the EP vapor for the 100, 200, and 400 ppm exposure groups were (mean ± SD) 100 ± 5, 203 ± 7, and 402 ± 7 ppm for male groups and 100 ± 5, 202 ± 7, and 402 ± 6 ppm for female groups, respectively. The nominal concentrations were 111 ± 4, 195 ± 10, and 392 ± 9 ppm for male groups and 111 ± 3, 197 ± 11, and 391 ± 9 ppm for female groups.
Overall mean chamber temperature and relative humidity varied from 22.0 to 22.5 °C and 54.0 to 63.9%, respectively. Particle count data indicated that an aerosol of the test material was not present.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day, 5 days/week for 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200 and 400 ppm (nominal concentrations)
Basis:
other: The analytical concentrations of the EP vapor for the 100, 200, and 400 ppm exposure groups were (mean ± SD) 100 ± 5, 203 ± 7, and 402 ± 7 ppm for male groups and 100 ± 5, 202 ± 7, and 402 ± 6 ppm for female groups, respectively.
No. of animals per sex per dose:
10 male + 10 female
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on previous studies
- Rationale for animal assignment: random
Positive control:
Historical positive control data were used to demonstrate the sensitivity of the Functional Observation Battery

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 4, 7 and weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pretreatment day (-4), days 4, 10, 32, 60 and 95 after initial exposure
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity and grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
HISTOPATHOLOGY: Yes
Other examinations:
not applicable
Statistics:
Mean values with standard deviations were calculated where appropriate. Data were evaluated using the following computer generated statistical tests where appropriate: Bartlett's test (p ≤ 0.01), one-way analysis of variance (ANOVA, p ≤ 0.05), and Duncan's multiple range test (p ≤ 0.05). Prior to conducting statistical analyses on counts of defecations, urinations, and vocalizations, the data were transformed by addition of 0.5 to each count, and then calculation of the square root of the sum. This transformation was performed to make the variances independent of the means.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY - No mortality occurred, during the study. No clinical abnormalities were observed during exposure for males or females in any group.

Red discolored urine was observed for three female rats shortly after the first exposure to 400 ppm EP, for all male and female rats on the next day prior to the second exposure to 400 ppm EP, and then generally once each week thereafter in 1-3 rats. Red discolored urine was observed less frequently and in fewer animals in the 200 ppm male and female groups. One female rat from the 100 ppm group exhibited red discolored urine on a single occasion as did a single control male (Rat-2); the control animal also exhibited bilateral hydronephrosis. Red discolored urine was generally seen following the first exposure after a weekend without EP exposure, except in the 100 ppm and control animals.

Other compound-related clinical signs in males consisted of red discoloration of the facial hair, porphyrin nasal discharges, porphyrin tears, and sialorrhea. Discoloration of facial hair was seen from one to six times in four 200 ppm animals and two 400 ppm animals. Single incidences of porphyrin nasal discharges were seen in three 400 ppm animals and a single incidence of porphyrin tears in one 400 ppm animal. Three or four incidences of sialorrhea were observed in two 400 ppm animals. These clinical signs were probably a response to the irritation caused by the inhalation of EP. No other clinical abnormalities were observed for males from any exposure level.

Other compound related clinical signs in females consisted of red discoloration of the facial hair, porphyrin nasal discharges, sialorrhea, and lacrimation. A single incidence of red discoloration of facial hair was observed in one control. Discoloration of facial hair was observed once to five times in 9 females exposed to 100 ppm and 8 females exposed to 200 ppm and 1 to 13 times in 4 females exposed to 400 ppm. Porphyrin nasal discharges were seen twice each in single females from the 200 and 400 ppm groups. A single incidence of sialorrhea was observed for a 200 ppm female, and one female from the 400 ppm group was seen to have sialorrhea on seven occasions. A single incidence of lacrimation was observed for the 400 ppm group. These clinical signs were probably a response to the irritation caused by the inhalation of EP.

Orange discoloration of abdominal hair observed in one female from the 400 ppm group was probably related to staining with red urine.
A wound in a single 200 ppm female rat was unrelated to exposure. Alopecia was observed in 1 male from the control group, 1 male from the 200 ppm group,4 males from the 400 ppm group, 2 females from the control group, 3 females from the 100 ppm group, and 2 females from the 200 ppm group. These clinical abnormalities were not considered compound-related

BODY WEIGHT AND WEIGHT GAIN - Mean body weights of male and female animals exposed to 400 ppm EP were slightly lower than those of the control groups. At no time during the study were these difference statistically significant. Mean body weights of male and females animals exposed to 100 or 200 ppm EP were comparable to those of the control groups.

NEUROBEHAVIOUR - The FOB examinations performed on all animals prior to the first exposure to EP and all FOB examinations on all control animals throughout the study were unremarkable. For those statistically significant differences in behavior were observed for the EP-treated animals when compared to the control animals, these differences appeared to be random in nature and were not related to exposure level and did not correlate with increasing exposure time.

Visual orientation by the 100 and 200 ppm male group occurred at a lower rate than for the other males. In general, the control and 400 ppm male groups did not appear to habituate to the visual stimulus to as large a degree as the 100 and 200 ppm male groups. A statistically significant increase in no response occurred during the Day 10 and Day 95 FOBs for the 100 ppm male group and during the Day 10, Day 32, and Day 95 FOBs for the 200 ppm male group.

Auditory orientation was significantly lower for the 100 (1 of 10) and 200 ppm (1 of 10) male group at Day 95 when compared to the controls (6 of 10). On Day 32, the pinna response for the 100 ppm male group was statistically significantly reduced (2 of 10) when compared to the control animals (7 of 10).

The usual response to approach by a probe consisted of the animal slowly approaching and sniffing the probe or no reaction to probe. For males at the pre-exposure FOB, a statistically significantly higher incidence of no reaction was observed for the 100 ( 5 of 10) and 400 ppm (7 of 10) groups when compared to the controls (0 of 10). On Day 32 for the 400 ppm male group, the incidence of slowly approaching and sniffing the probe was significantly lower (0 of 10) when compared to the controls (4 of 10). On Day 60, for the 400 ppm male group, the incidence of slowly approaching and sniffing the probe was significantly lower for the 400 ppm male group (1 of 10) when compared to the controls (6 of l0), and the incidence of no reaction was significantly higher (9 of 10) when compared to the controls (4 of 10). The incidence of responses to touch was significantly lower for the 200 ppm female group at Day 4, the 400 ppm female group at Day 10, and the 100 ppm female group at Day 95 and was significantly lower for the 400 ppm female group at Day 60.

During the Day 95 FOB, the 400 ppm female group had fewer (p ≤ 0.01) animals which were considered normally alert during handling (6 of l0), but also had a higher incidence (p ≤ 0.05) of animals which attempted to escape (3 of 10) when compared to the controls where 10 of 10 animals were normally alert and none attempted to escape.

Statistically significant changes observed in response to approach by a probe for females at the pre-exposure FOB, consisted of the 100 ppm female group having a higher incidence of no reaction to the probe (5 of 10) and a lower incidence of approaching and sniffing the probe (3 of 10) and the 200 ppm female group having a lower incidence of approaching and sniffing the probe (1 of 10) and a higher incidence of pulling away slightly (3 of 10) when compared to the controls, where 9 of 10 rats approached and sniffed the probe. On Day 10, the 200 ppm female group had a statistically significant higher incidence of slowly approaching and sniffing the probe (10 of 10) when compared to the controls (5 of 10).

A number of FOB differences approached but did not attain statistical significance. There were no statistically significant differences in mean forelimb or mean hindlimb grip strength between the treatment and control groups during the study.

ORGAN WEIGHTS - Mean terminal body weights of perfused male rats were comparable among all groups. Mean terminal body weights of unperfused male rats at all concentrations of EP were lower than that of the control group, although the differences were not statistically significant and not in an exposure-related pattern.

Mean terminal body weights of perfused female rats at all concentrations of EP were lower than that of the control group. No clear exposure-related pattern or statistically significant differences were seen. Mean terminal body weights of unperfused female rats were comparable among all groups.

Mean absolute and relative (to body weight) spleen weights for unperfused females exposed to 400 ppm were statistically significantly increased when compared to the control group. The mean absolute and relative spleen weights for the other 400 ppm animals (the perfused female rats and the perfused and unperfused male rats) were biologically significantly higher than that of the control groups. No treatment-related spleen weight changes were observed for the perfused and unperfused animals, male and female, at the 200 and 100 ppm exposure levels.

Mean absolute and relative liver weights of perfused and unperfused female rats exposed to 400 or 200 ppm EP were slightly heavier when compared to the control group, although, the differences were not statistically significant.

Mean absolute and relative liver weights of perfused and unperfused female rats exposed to 100 ppm EP and of perfused and unperfused male rats exposed to 400, 200, or 100 ppm EP were comparable to that of the control groups.

The mean relative, but not absolute, kidney weight of unperfused male rats and the mean absolute and relative kidney weights of perfused female rats exposed to 400 ppm EP were slightly, but not statistically significantly elevated when compared to the control groups. These weight changes may be a reflection of the slightly lower mean terminal body weight seen in these animals. Mean relative and absolute kidney weights of the 200 and 100 ppm unperfused male and of the 400, 200, and 100 ppm perfused male rats were comparable to that of the control groups. Mean relative and absolute kidney weights of the 200 and 100 ppm perfused female and of the 400, 200, and 100 ppm unperfused female rats were comparable to that of the control groups.

Mean absolute and relative brain weights of perfused and unperfused male and female rats at all EP exposure levels were comparable to the control groups.

GROSS PATHOLOGY - No treatment-related gross pathology or neuropathology changes were observed for any animals exposed to EP.

HISTOPATHOLOGY: NON-NEOPLASTIC - Treatment-related changes consisted of hemosiderosis in the Kupffer cells of the liver at 400 (4/5-rnales, 5/5-female) and 200 ppm (3/5-males, 5/5-female), hemosiderosis in the proximal convoluted renal tubules at 400 (3/5-males, 5/5-female) and 200 ppm (5/5-males, 5/5-female), and increased severity of hemosiderosis in the splenic red pulp at 400 (5/5-males, 5/5-female) and 200 ppm (5/5-males, 4/5-female). Hemosiderin was identified, in hematoxylin eosin stained sections, as a golden globular pigment in the kidneys and Kupffer cells of the liver, of both male and female rats exposed to 200 or 400 ppm of the test material. The most likely source of the pigment was from increased red blood cell destruction and metabolism of heme pigments in the kidneys and liver. The spleen, which normally contains hemosiderin, had increased amounts present in male and female rats exposed to 200 and 400 ppm of the test material. No cellular degeneration was associated with pigment deposition. No other treatment-related histopathological changes were observed for males and females from the 400 and 200 ppm groups.
No treatment-related histopathological changes were observed for males and females from the 100 ppm group

Effect levels

Dose descriptor:
NOEL
Effect level:
> 400 ppm
Sex:
male/female
Basis for effect level:
other: based on no neurological effects observed at the highest level tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

not applicable

Applicant's summary and conclusion

Conclusions:
From the results of the study, it was concluded that Ethylene Glycol Monopropyl Ether did not cause neurologic effects at vapor concentrations at the highest tested level of 400 ppm.
Executive summary:

Groups of 10 male and 10 female rats were exposed to concentrations of 0, 100, 200, and 400 ppm ethylene glycol monopropyl ether (EP) 6 hours/day, 5 days/week for fourteen weeks. Parameters examined during the study included clinical signs and body weight gain. A Functional Observational Battery (FOB) was used to detect functional impairment of the nervous system four days prior to exposure to EP and at 4, 10, 32, 60, and 95 days after the initial exposure. The FOB consisted of an observational procedure to detect unusual responses in activity, coordination, behavior, and changes in sensory function. Forelimb and hind limb grip strength were also quantitatively evaluated.

At the end of the exposure period, five male and five female rats from each group were fixed by intravascular perfusion and central nervous system (CNS) and peripheral nervous system (PNS) tissues were examined after staining with hematoxylin and eosin or Luxol Fast Blue and Bodian silver impregnation techniques. The PNS was also examined following plastic embedment and toluidine blue staining of the sciatic and tibial nerves. All 10 male and 10 female rats from each exposure group were necropsied and abdominal and thoracic organs were examined for macroscopic lesions and brain, liver, kidney, and spleen weights were measured.

 

Clinical signs indicative of irritation due to EP consisted of red discoloration of facial hair, porphyrin nasal discharges and tears, and sialorrhea in rats exposed to 200 or 400 ppm EP and red discolored facial hair only in female rats exposed to 100 ppm. At exposure concentrations of 200 and 400 ppm, males and females exhibited signs of red blood cell damage including red discolored urine, pigment deposition in the kidneys and Kupffer cells of the liver, and increased amounts of pigment in the spleen. Spleen weights were biologically significantly increased for the 400 ppm male and female groups, reaching statistical significance only for the unperfused female group. One female rat exposed to 100 ppm EP exhibited a single incidence of red discolored urine, as did one control male. This single incidence of red urine at the 100 ppm level was not considered compound-related.

 

The mean liver weights of perfused and unperfused female rats exposed to 200 or 400 ppm EP, were slightly heavier than those of the control groups. Kidney weight changes observed at 400 ppm in unperfused male rats and in perfused female rats were considered to be a reflection of slightly lower mean terminal body weights, rather than a direct toxic effect.

 

At exposure levels which were systemically toxic to male and female rats, no neurologic deficits in motor or sensory function were detected. Neuropathological lesions were not observed in either the CNS or PNS of rats exposed to 400 ppm EP for fourteen weeks.

The no-observed-effect level for neurological effects, was greater than 400 ppm, the highest level tested. A site of toxic action was the red blood cell, with secondary involvement of the spleen, liver, and kidneys. The NOEL for toxicity was 100 ppm based upon the lack of evidence of red blood cell effects or liver weight changes.

Relevance of Hemolytic Effects in Rodents to Humans

The hemolytic activity in certain animal species of ethylene glycol ethers such as ethylene glycol monopropyl ether and ethylene glycol monobutyl ether (2 -butoxyethanol; CAS No. 111 -76 -2) have been shown to be due to the alkoxyacetic acid metabolites, 2 -propoxyacetic acid and 2 -butoxyacetic acid (Ghanayem et al., 1987 and 1989; Foster et al., 1987; Ghanayem and Sullivan, 1993; Boatman et al., 1993). The red blood cell hemolysis seen in sensitive species such as rats and mice produces secondary effects in the liver, kidney and spleen. In the case of the most well studied of these glycol ethers, 2 -butoxyethanol, blood from humans, including the elderly and those with blood disorders, has been shown to be far less susceptible to this hemolytic effect, with human sensitivity estimated to be 150x less than that seen in the rat (Udden and Patton, 1994; Udden, 1996).

Ghanayem, B.I., Burka, L.T., and Mathews, H.B. (1987). Metabolic basis of ethylene glycol monobutyl ether (2 -butoxyethanol toxicity: Role of alcohol and aldehyde dehydrogenass. J. Pharmacol. Exp. Ther. 242:222 -231.

Ghanayem, B.I., Burka, L.T., and Mathews, H.B. (1989). Structure-activity relationships for the in vitro hematoxicity of N-alkoxyacetic acids, the toxic metabolites of glycol ethers. Chem.-Biol. Inter. 70:339 -352.

Foster, P.M.D., Lloyd, S.C. and Blackburn, D.M. (1987). Comparison of the in vivo and in vitro testicular effects produced by methoxy-, ethoxy- and n-butoxyacetic acids in the rat. Toxicology 43:17 -30.

Ghanayem, B.I. and Sullivan, C.A. (1993). Assessment of the haemolytic activity of 2 -butoxyethanol and its major metabolite, butoxyacetic acid, in various mammals including humans. Hum. Exp. Toxicol. 12:305 -311.

Boatman, R.J., Perry, L.G. and Bialecki, V.E. (1993). The in vitro hemolytic activity of propoxyacetic acid in both rat and human blood. The Toxicologist 13, 364 (Abstract 1424).