Registration Dossier

Administrative data

Description of key information

oral studies

OECD 422, diet: NOAEL = 300mg/kg (based on liver and body weight effects), Dow 2004

6 weeks, gavage: LOAEL 891mg/kg (based on reduced body weight), Eastman Kodak, 1982, RA substance DEGBE

OECD 408, drinking water, 90 -days: NOAEL = 250mg/kg (based on liver and body weight effects), Johnson et al. 2005, RA substance DEGBE

dermal studies

90 days, dermal: NOAEL = 2000mg/kg (highest dose tested), LOAEL for local effects = 200mg/kg, Auletta et al. 1993, RA substance DEGBE

inhalation studies

5 weeks, vapour: NOAEC = 18ppm, app. 0.12mg/L (highest dose tested), Dow 1981, RA substance DEGBE

OECD 414, vapour, 90 days: NOAEC = 14ppm, app. 0.09 mg/L (highest dose tested), BASF 1992, RA substance DEGBE

OECD 413, vapour, 90 days: NOAEC = 41ppm, app. 0.25mg/L (based on liver and body weight effects), Dow, 1985, RA substance EGHE

Corrected for differences in molecular weight (146 vs. 190 g/mol) between EGHE and DEGHE results in a NOAEC for DEGHE of 0.33 mg/L.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Remarks:
Toxicology & Environmental Research and Consulting, The Dow Chemical Company
Limit test:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Diethylene glycol hexyl ether, 2- [2-(hexyloxy)ethoxy] ethanol
- Synonyms: DEGHE, HEXYL CARBITOL
- Physical state: liquid
- Purity of the test material was 96.8%.
Species:
rat
Strain:
other: CD rats (Cr1 :CD(SD)IGS BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC, USA)
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: Males: 267 - 302 g; Females: 181 - 205 g
- Housing: stainless steel cages (prebreeding: 1 per cage, breeding: one male + one female per cage), plastic cages (dams and litter)
- Diet: LabDiet® Certified Rodent Diet #5002 (PMI NutritionInternational, St . Louis, Missouri) in meal form. ad libitum
- Water: municipal water. ad libitum
- Acclimation period: at least 1 week


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 1°C
- Humidity: 40-70%
- Air changes: 12-15 times per hr
- Photoperiod: 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: July 31st, 2003 To: Sept. 2nd, 2003 (males) and Sept. 9th, 2003 (females)
Route of administration:
oral: feed
Details on route of administration:
Route justification:
Oral was the preferred route of exposure according to OECD Guideline 422. Exposure by diet was selected in the event that a positive finding warranted a subseyuent two-generation reproductive toxicity study that would be conducted using the dietary route.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Pre-mixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for two weeks prior to breeding of the P1 adults. Following breeding, the male diet was prepared weekly until necropsy. During gestation and lactation, females from each dose group were provided with the appropriate concentration fo the the test substance given during breeding.
- Mixing appropriate amounts with (Type of food): Serial ditution of a concentrated test material-feed mixture (premix) with ground feed. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data. To avoid potential overdosing during the breeding period, co-housed animals were provided with the female diet, which was of lower concentration.
- Storage temperature of food: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Measurement of test material concentration revealed mean concentrations ranging from 92.5 to 104% of targeted concentrations. Analysis of low-dose female and high-dose male diet indicated that the test material was distributed homogeneously. Stability analysis confirmed that the premix was stable for 14 days and that the test diets were stable for eight days at the concentrations used in the current study.
Duration of treatment / exposure:
males: at least 14 days prior to mating, continuing throughout mating for a total of 33 days
females: 14 days prior to breeding, continuing through breeding (until pregnancy occured or up to two weeks), gestation (three weeks) and lactation (four days) (total 39-52 days)
Frequency of treatment:
not applicable - dosed via diet
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
- Dose selection rationale:
1000 mg/kg/day was selected as the high-dose level based upon data obtained from a preliminary range-finding study where minimal toxicity was seen at this dose level . The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a no-observed-effect-level (NOEL).
- Post-exposure period: none

- Mating Procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1 :1 mating) until pregnancy occurred or two weeks had elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0 . The sperm- or plug-positive (presumed pregnant) females were then separated from the male and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further oppotiunity for mating.
Observations and examinations performed and frequency:
DAILY IN-LIFE OBSERVATIONS:
Twice each day, a cage-side examination was conducted and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions) and animal behavior, moribundity, mortality, availability of feed and water, and litter observations for lactating females.
Females were observed for signs of parturition beginning on or about day GD 20. In addition, females that delivered litters received clinical examinations on lactation days (LD) 0, 1 and 4. Females that failed to mate or failed to deliver litters were examined weekly. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behavior.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and LD 3. The DCO was conducted on all animals at approximately the same time each examination day according to an established format. The examination included cage-side, hand-held and open-field observations that were recorded categorically or used explicitly defined scales (scored).

FUNCTIONAL TESTS
The functional tests included a sensory evaluation, rectal temperature, grip performance and motor activity. Functional tests were conducted pre-exposure and during the last week of the treatment period. For females, this took place on LD 4.
- Sensory Evaluation: Evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box.
- Rectal Temperature: Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp RET-2, T-type) approximately 4 cm into the rectum for about 15-20 seconds. Temperature was then recorded. The thermistor was validated at 37 °C before and after the study. The instrument was re-calibrated if the validation temperature recordings differ from the reference thermometer by more than ± 0.5 °C.
- Grip Performance: Hind-limp grip performance was tested. The observer placed the animal's forelegs on a plastic bench and the hind-feet were set on a horizontal screen attached to an electronic strain gauge. The observer then smoothly but firmly pulled backward on the tail until the animal's grip on the screen was broken. An electronic strain gauge reading was used to record the animal's resistance to the pull in grams. The average of three trials was used for statistical analysis. Fore-limb grip performance was similarly tested . In this application, a bench was not used, and the animal was placed so that the fore-feet were on the screen and the hind-feet were suspended approximately 10 cm above the smooth horizontal plastic surface.
- Motor activity: An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each test session consisted of ten 8-minute epochs, totalling 80 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 70-80 minutes in CD rats. Activity counts for each epoch were recorded.

BODY WEIGHT:
All rats were weighed pre-exposure, twice during the first week of study and once during the second week. Male body weights continued to be recorded weekly throughout the study. Presumed pregnant females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases.

FOOD CONSUMPTION:
For males and females, feed consumed was determined pre-exposure and twice during the first week by weighing feed crocks at the start and end of a measurement cycle. Thereafter, feed crocks were measured weekly during the pre-breeding phase. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly for males . For mated females, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation: Feed consumption (g/day) = (initial weight of crock - final weight of crock) / (# of days in measurement cycle).

HEMATOLOGY:
Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA) and smears were prepared, stained with Wright's stain, and archived for potential future evaluation, if warranted. Hematologic parameters were assayed using a Technicon H•1E Hematology Analyzer. The following assays were performed. Hematocrit (HCT), Hemoglobin (HgB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Platelet (PLAT) count, Differential WBC count, Red blood cell indices (MCH, MCV and MCHC). For Coagulation Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000. The Prothrombin time (PT) assay was performed.

CLINICAL CHEMISTRY:
Serum was separated from cells as soon as possible following blood collection. Enzyme Activities of Alkaline phosphatase (AP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and concentrations of Albumin (ALB), Cholesterol (CHOL), Creatinine (CREAT), Electrolytes (Na, K, PO4, Cl and Ca), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Urea nitrogen (UN) were determined.

URINALYSIS:
A timed urine volume was obtained from all male rats in each dose group (nonfasted) during the week prior to the scheduled necropsy (test day 28). Animals were housed in metabolism cages and urine collected overnight (approximately 16 hours). The following assays/analysis were performed. pH, Bilirubin, Glucose, Proteins, Ketones, Blood, Urobilinogen.
Urine was also collected by manual compression of the bladder . The urine was pooled for each group and the microsediment characterized microscopically.
Sacrifice and pathology:
CLINICAL PATHOLOGY:
On the day prior to the scheduled necropsy, all males and females in each dose group were fasted overnight. At necropsy, the animals were anesthetized with CO2, and then blood samples collected from the orbital sinus. Blood samples were not obtained from females that failed to deliver a litter.

ANATOMIC PATHOLOGY:
A complete necropsy of all the adults was performed. Male rats were necropsied on test day 34 while females that delivered litters were necropsied on post-partum day 5. Dosing continued until the day prior to sacrifice. Female rats that did not deliver a litter were necropsied at least 24 days after the last day of the mating period. Both males and females were fasted overnight prior to necropsy. Fasted adult rats submitted alive for a necropsy were anesthetized by CO2 vapors, weighed, bled (via orbital sinus), their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened, and the brain, pituitary, and adjacent cervical tissues examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle. The skin was reflected from the carcass, the thoracic and abdominal cavities opened, and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues incised. The uteri of all females were examined and the number of implantation sites recorded. The uteri of females that did not deliver litters were stained with a 10 % solution of sodium sulfide in order to verify pregnancy status.

The following tissues were trimmed and weighed: testes, epididymides, seminal vesicles with coagulating glands (and seminal fluid), prostate, ovaries, liver, kidneys, adrenals, thymus, spleen, brain, thyroid/parathyroid (after fixation), and heart. The organ to body weight ratios were calculated. Representative samples of tissues listed below were collected and preserved in neutral, phosphate-buffered 10% formalin, except that testes and epididymis were preserved by immersion in Bouin's fixative. Transponders were removed and placed in jars with the tissues.

HISTOPATHOLOGY:
Histologic examination of the tissues listed below and tissues with relevant gross lesions were conducted on all adult rats from the control and high-dose groups.
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at five μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis was qualitatively evaluated. Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes was examined for the presence of degenerative changes (e.g., a vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Examination of tissues from the remaining groups was limited to liver and salivary glands in males and, liver and mesenteric tissues in females. Paraffin-embedded tissues were sectioned approximately six μm thick, stained with hematoxylin and eosin, and examined by a using a light microscope.

Tissues Collected and Preserved at Necropsy:
Adrenals, kidneys, prostate, aorta, lacrimal/harderian glands, rectum, auditory sebaceous glands, larynx, salivary glands, bone (including joint ), liver, seminal vesicles , bone marrow, lungs, skeletal muscle, brain (cerebrum, brainstem, cerebellum), mammary gland - females only, skin and subcutis, cecum, mediastinal lymph node, spinal cord (cervical, thoracic, lumbar), cervix, mediastinal tissues, spleen, coagulating glands, mesenteric lymph node, stomach, colon , mesenteric tissues, testes, cranial nerve - optic, nasal issues/pharynx, thymus, duodenum, oral tissues, thyroid gland, epididymides, ovaries, tongue, esophagus, oviducts, trachea, eyes, pancreas, urinary bladder, gross lesions, parathyroid glands, uterus, heart, peripheral nerve -tibial, vagina, ileum (with peyer's patch), pituitary, jejunum.
Statistics:
Statistical analyses: Parental body weights, gestation and lactation body  weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data,  hematological data and organ weights were first evaluated by Bartlett's test for homogeneity. Parametric and nonparametric data were then  analyzed by the appropriate analysis of variance (ANOVA). If the ANOVA  was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05) or the  Wilcoxon Rank Sum test with Bonferroni's correction was performed. Feed consumption data were excluded if the feed was spilled or scratched.  Females failing to deliver a litter were excluded from analyses of gestation and lactation body weight, feed consumption and organ weights. Detailed clinical observation and sensory evaluation scores were analyzed using a z-test of proportions (at alpha = 0.05). Rectal temperature and grip performance were analyzed by an analysis of covariance. Motor activity was analyzed by a repeated measure design.
Clinical signs:
no effects observed
Description (incidence and severity):
Examinations performed on all animals weekly throughout the study revealed no treatment-related or statistically significant findings. A number of incidental observations bearing no relationship to treatment occurred during the study. The excessive forelimb hair loss recorded in both males and females was attributed to excessive grooming or licking.
Mortality:
no mortality observed
Description (incidence):
All animals survived until termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although not statistically identified, body weights of the high-dose males on test day 33 were slightly decreased (5.6%) when compared to their respective controls. Body weights of the mid- and low-dose males were not different from controls. Pre-mating body weights of dams given 1000 mg/kg/day were statistically identified as decreased (5.2%) on test day 14. Body weights of dams given 1000 mg/kg/day also were decreased throughout gestation with statistically significant differences identified on GD 7, 14 and 20. Lactation body weights in the high dose group were significantly decreased on LD 1 and 4. Consistent with body weight effects during gestation, high-dose dams had decreased body weight gains durin gestation which were statistically identified on GD 14-20, as well as decreased overall gain throughout the gestation period. Lactation body weight gains, although decreased in test substance treated animals, were not considered treatment related due to the lack of statistical difference and the normal variability inherent in this endpoint during lactation (control lactation body weight gains range was 11.8 g to 30.4 g). Furthermore, the 100, 300 and 1000 mg/kg/day groups were within the range of historical control values from recently completed OECD 422 studies.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically identified decreases in feed consumption were noted during the pre-breeding phase on days 1-4 in the 1000 mg/kg/day males and on days 1-4 and 4-7 in the 1000 mg/kg/day females. Feed consumption of females given 300 mg/kg/day was decreased and statistically identified on days 1-4. The decrease in feed consumption of dams given 300 mg/kg/day was not considered toxicologically significant as the decrease was minimal and there was not a corresponding effect on body weight. During the pre-mating period, there were no significant differences in the amount of food consumed by males given 300 mg/kg/day and males and females given 100 mg/kg/day when compared to their respective controls. During gestation, statistically identified decreases in feed consumption were observed on GD 0-7, 7-14 and 14-20 in the 1000 mg/kg/day dose group. There were no significant differences in the amount of feed consumed by the 300 mg/kg/day dose group during the gestation period when compared to their respective controls. The statistically identified decrease in GD 14-20 feed consumption in the 100 mg/kg/day females was not considered treatment related, as the difference was not dose related. There were no significant differences in the amount of feed consumed by any of the dose groups when compared to their respective controls during the lactation period.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related hematologic effects, or effects on coagulation in males or females at any dose level .
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females given 1000 mg/kg/day had statistically identified and treatment-related increases in blood urea nitrogen and serum ALT and ALP activities. The increases in serum ALP activity correlated with histological observation of panlobular hepatocyte hypertrophy. Although frank hepatocyte necrosis was not observed, increased ALT activity may be associated with sublethal hepatocyte injury. The slight increase in blood urea nitrogen in females given 1000 mg/kg/day was not due to renal disease as there were no histopathologic changes in the kidney or the urinary tract . Additionally, serum electrolytes and creatinine levels were largely unaffected indicating normal renal function. The minor increase in blood urea nitrogen was interpreted to reflect increased protein catabolism associated with lowered body weight and body weight gains in females given 1000 mg/kg/d.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related change was the lowering of urinary pH in males given 1000 mg/kg/day. While majority of the control urine samples were within the pH range of 7 .5 - 8.5 and none less than pH 7.0, five out of
twelve males given 1000 mg/kg/day had urinary pH of 6.0 - 6 .5. This lowering of urinary pH was considered to be due to probable urinary excretion of acidic metabolite(s) of the test substance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects on behavior or demeanor were observed at any dose level during the lactation period. There were no notable observations made during the cageside observations.
Regarding sensory evaluation, examinations performed on males and females at termination revealed no treatment-related findings.
Regarding rectal temperature, no treatment related effects.
Regarding Grip performance, there were no treatment effects on hindlimb grip performance either in males (p = 0 .6187) or females (p = 0.3386). Similarly, there were no treatment effects on forelimb grip performance either in males (p = 0.5423) or females (p = 0.7255).
Regarding motor activity, treatment did not affect motor activity total counts (treatment-by-time interaction) either in males (p = 0.2404) or in females (p = 0.6761). Similarly, the distribution of the motor activity counts within session (treatment x time x epoch interaction) was not affected by treatment either in males (p = 0.1436) or in females (p = 0.9577).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Male rats given 1000 mg/kg/day had a treatment-related, statistically identified, 15% increase in relative liver weight over that of the controls, which correlated with centrilobular hypertrophy observed histologically . Although relative kidney weight was statistically increased (12.9%), there was no histopathological correlate and therefore, this was not interpreted to be of toxicological significance. A very slight increase in the relative brain weights in males given 1000 mg/kg/day was reflective of the treatment-related lower body weight in this group. There was a statistically identified, 18% decrease in the mean absolute weight of seminal vesicles (with coagulating glands) in males given 1000 mg/kg/day over that of the controls. Mean relative seminal vesicle weight was not significantly altered. Relative seminal vesicle weights appear to be less sensitive to body weight changes, as decreases in absolute seminal vesicle weights have been reported in the presence of body weight changes. The performing laboratory has limited historical control data for seminal vesicle weights in OECD 422 studies, because they only recently began measuring this endpoint. Historical control values for one OECD 422 study that included seminal vesicle weights were 1.464 g (absolute) and 0.378 g/100 g body weight (relative) for control animals with a mean body weight of 390.5 g. The high-dose seminal vesicle weights in the current study do not differ appreciably from these values. Histologically, there was no correlate for the decreased seminal vesicle weights in majority (11 out 12) of the animals except for one that showed a slight reduction in overall size with decreased amounts of secretory material. Also, this isolated finding was neither accompanied by any histopathologic changes in other male reproductive organs nor any decrements in reproductive performance, and thus may have been spurious.

Female rats given 1000 mg/kg/day had a treatment-related, statistically identified, 23.5% decrease in thymic weight compared to controls. The reduction in thymic weight was interpreted to be stress related in the high dose group. However, there was no histopathologic correlate to this weight decrement. There was a statistically identified, treatment-related, 22.6% increase in relative liver weight in females given 1000 mg/kg/day over that of the controls and this alteration correlated wit h histological observation of panlobular hepatocyte hypertrophy. A very slight but statistically identified decrement in the absolute heart weight in females given 1000 mg/kg/day was interpreted to be reflective of the treatment-related lower body weights in this group. The relative heart weights of high-dose females were not statistically different from control values. Statistically identified decrements in the absolute adrenal weights in females given 100 and 1000 mg/kg/day, and statistically identified decrements in the absolute ovarian weights in females given 100 mg/kg/day were considered spurious and not treatment related due to lack of dose-response relationship .
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related gross pathologic observations. Two males given 1000 mg/kg/day had bilateral alopecia of the forelimbs, two females given 1000 mg/kg/day showed focal alopecia of the thorax, one female given 1000 mg/kg/day had alopecia of the left forelimb, and one female given 100 mg/kg/day had bilateral alopecia of the forelimbs. The alopecia was largely attributed to excessive grooming and/or licking and considered not treatment related because the majority of the skin samples from the affected sites were histologically within normal limits. Erosions in the glandular mucosa of the stomach were grossly observed in one control female, two females given 300 mg/kg/day and in three females given 1000 mg/kg/day. These effects were deemed to be spontaneous and not treatment related. All other gross pathologic observations, such as aspirated blood in the lung, thickened wall of the urinary bladder with calculi and, jejunal diverticulum, were considered spontaneous alterations, unassociated with exposure to the test substance
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no adverse effects of the test substance on neurological function.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The liver was identified as the primary target organ. Very slight periportal hepatocyte hypertrophy was seen in males given 1000 mg/kg/day and a very slight panlobular hepatocyte hypertrophy was seen in females given 1000 mg/kg/day. Treatment related, but secondary effects seen were very slight diffuse acinar hypertrophy of the submandibular salivary gland in majority of the males given 1000 mg/kg/day and a very slight atrophy of the mesenteric adipose tissue in majority of the females given 1000 mg/kg/day. Hypertrophy of the submandibular salivary gland is mediated through an adrenergic mechanism and is considered likely to be an exaggerated physiological response. The very slight atrophy seen in the mesenteric adipose tissue in females given 1000 mg/kg/day was consistent with lower body weight of this group. The seminal vesicle of 1 out 12 males given 1000 mg/kg/day had a slight overall reduction in size, slightly decreased content of secretory material and, a slight increase in the amount of pyknotic material in the epithelium. This was interpreted to be not treatment related due to the low incidence. Histologically, the skin samples from animals that showed alopecia on gross examination (forelimb or thorax) were largely within normal limits except for one female given 1000 mg/kg/day that showed slight atrophy of hair follicles (thoracic skin sample) which was considered not treatment related. Histologically, focal erosions on the glandular mucosa of the stomach were seen in one control female, two females given 300 mg/kg/day and in one female given 1000 mg/kg/day. Two other females given 1000 mg/kg/day showed erosions in the stomach glandular mucosa on gross examination, however, they could not be confirmed histologically. Erosions of the glandular mucosa of the stomach were interpreted to be spontaneous and not related to the test substance treatment. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to the test substance.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: general toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No neurological effects observed at the hightest dose tested.
Remarks on result:
other: Neurological effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Analyses of all test diets from the first mix of the main study revealed  mean concentrations ranging from 92.5 to 104% of targeted concentrations.  

Analyses of the low dose female and high dose male diets indicated that  the diets were homogeneous.  Analyses confirmed that the 10% premix was stable for 14 days and that the test diets were stable for 8 days at concentrations ranging from 0.120 to 0.359%, which encompass the range of dietary concentrations used in the study.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
: 6 week test period. Not all end points examined.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: CR, COBS, CD, BR albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Weight at study initiation: 235.7+/-15.1g
- Housing: individual in suspended wire bottomed cages.
- Diet (e.g. ad libitum): ad libitum, Purina rodent chow 5001
- Water (e.g. ad libitum): ad libitum via automatic watering system
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- no data
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Doses recalculated weekly to allow for animal growth.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
daily, 5 days per week
Remarks:
Doses / Concentrations:
891, 1781 or 3564 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
- Dose selection rationale: based on 50%, 25% and 12.5% of the fasted LD50.
Positive control:
A number of other glycol ethers were assessed in the same study effectively acting as reference controls.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, except weekends.
- Cage side observations: signs of systemic toxicity, appearance and behaviour. Urine and faeces appearance. Mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 6, 13, 20, 27, 34, 41.

FOOD CONSUMPTION AND COMPOUND INTAKE - measured at same times as body weight.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from vena cava immediately prior to autopsy.
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No data
- Parameters checked : Hgb, Hct, RBC count and indices, total and relative WBC count,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from vena cava immediately prior to autopsy.
- Animals fasted: No data
- Parameters checked: glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, ALP, LDH, BUN, creatinine and glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Animals that died spontaneously were autopsied as soon as possible. Moribund animals were sacrificed and similarly autopsied.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Survivors killed by CO2 inhalation. Tissues examined: lung, heart, thymus, kidney, liver, spleen, brain, salivary glands, stomach, cecum, colon, duodenum, jejenum , ileum, pancreas, esophagus, adrenals, pituitary, thyroid, parathyroid, trachea, mesenteric lymph nodes, testes, epididymides, prostate, seminal vesicles, coagulating gland, bone marrow, tongue, nasal cavities, eyes.
Statistics:
No information
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Significant toxic effects in top dose (only 4 animals at end of study - 4 intubation errors and 2 sacrifices moribund). The only other effects seen in more than the odd high dose animal were dyspnea, prostration and unkempt hair. No clinical effects were seen in the intermediate and low dose groups.

BODY WEIGHT AND WEIGHT GAIN: Significant reduction seen in top dose from day 3 onwards. No significant body weight gain changes in mid and low dose groups. Note however that a separate part of the study (organ/body weight comparisons) reports significant body weight changes at all dose levels and reports different terminal body weights. Despite the report presenting results from individual animals, it is not possible to reconcile this contradiction unless the second set of results are carcass weights minus organs. However, a conservative approach needs to be taken with the assumption that reduced terminal body weight was seen at all doses.

FOOD CONSUMPTION: significant reduction seen in top dose group. No significant changes in mid and low dose groups.

HAEMATOLOGY: Decreased Hgb, MCHC and total RBC and increased MCV and MCH at high and intermediate dose.

CLINICAL CHEMISTRY: Decreased serum glucose at high and intermediate dose. No other effects.

ORGAN WEIGHTS: Absolute and relative increases in spleen and liver weight were seen in the intermediate and high dose groups. Other organ weight changes were not dose related or were attributed as secondary to body weight changes.

GROSS PATHOLOGY: Enlarged dark spleens in some high dose animals.

HISTOPATHOLOGY: NON-NEOPLASTIC: Splenic congestion in a small number of high dose animals, accompanied by red pulp hypocellularity and hemosiderin like pigmentation in one case. Stomach hyperkeratosis was seen in virtually all animals at all doses. This effect is likely to be at least partly related to the dosing method, so the findings are uncertain in terms of relevance. Similarly, renal effects, included hyaline droplet degeneration, proteinaceous casts and hemosiderin i the proximal convoluted tubles were seen. The latter two appear to be compound related and were significant in the mid and high dose groups. A single incidence was seen in the low dose group. However, the hyaline droplet degeneration was also seen in all control animals, rendering its significance uncertain.
Dose descriptor:
NOAEL
Effect level:
< 891 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Reduced terminal body weight
Critical effects observed:
yes
Lowest effective dose / conc.:
891 mg/kg bw/day (actual dose received)
System:
other: General: reduced body weight
Treatment related:
not specified

4 intubation errors reported in high dose, 2 in mid dose and one in low dose group. Top dose may have exceeded MTD.

Conclusions:
The most significant toxic effects seen were splenic congestion, kidney microscopic effects (proteinaceous casts and hemosiderin that did not translate to gross pathological effects), changes to liver gross pathology and body weight. Effects seen at the highest dose suggested that this may have exceeded the MTPD. The limited effects seen at the lowest dose could not be attributed directly to treatment and therefore this is deemed to be the no effect level.
Executive summary:

In a 6 week sub-acute gavage study that broadly followed the standards for such tests pertaining at the time, standard toxicological end points were studied for rats in doses up to 3564mg/kg bw/day. Significant toxicity was seen in the high dose group, suggesting that the MTD may have been exceeded. The only effects seen in the low dose group were equivocal changes in the kidney (proteinaceous casts and hemosiderin) and changes attributed to the dosing method (stomach hyperkeratosis). Possible effects were reported on body weight reduction in this group (8%) but since there was some discrepancy in the reporting of this (other data indicated no significant change at the low dose) this could not be interpreted definitively. The lowest dose of 891mg/kg bw /day was therefore identified as a no effect level.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Housing: individually in stainless steel cages.
- Age at study initiation: 5 weeks
- Diet (ad libitum): Certified rodent diet #5002 (PMI Nutrition)
- Water (ad libitum): tap water.
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-22.5
- Humidity (%): 48-51
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared weekly, serial dilution for lower dose levels. Concentration changed weekly to accomodate animal growth. Solutions found to be stable for at least 9 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were between 98.3-102% of target. Test material intake calculated from drinking water consumption to be between 100.7 and 104.8% of target levels for all doses over study period. Analysis by GC/FID.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuous via drinking water.
Remarks:
Doses / Concentrations:
50, 250, 1000mg/kg
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on two week range finder study. Other routes (incorporation into diet or inhalation) not practical.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: skin, fur, mucous membranes, respiration, nervous system function, behaviour, moribundness, mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, including hand held and open field observations

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-study and during last week of treatment

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to necropsy from orbital sinus.
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes
- Parameters checked: Hct, Hgb, RBC count, WBC count, platelet count, differential WBC count, RBC indices and morphology, reticulocyte count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to necropsy
- Animals fasted: Yes
- Parameters checked: ALP, ALT, AST, serum albumin, chlolesterol, creatinine, glucose, total bilirubin, total protein, BUN, electrolytes (Ca, Cl, PO4, K, Na)


URINALYSIS: Yes
- Time schedule for collection of urine: overnight during week prior to necropsy
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: No
- Parameters checked: colour, appearance, sg, volume, pH, bilirubin, glucose, protein, ketones, blood, urobilinogen. Urine sediment (pooled per sex/dose) also examined microscopically.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-study and during last week of treatment
- Battery of functions tested: sensory evaluation (nociception test, startle response), grip strength, motor activity, rectaul temperature.+


OTHER: Sperm analysis: For control and high dose animals, total spermatid count per testis, total sperm counts per epididymidis, total counts per gram of tissue, sperm motility. 200 sperm per animal assessed for morphology. Liver metabolic enzymes from high dose and control animals to assess the levels of mixed function oxidases (CYP1A1, CYP1A2, CYP2B1/2, CYP2E1) and microsomal UGT. Positive controls were used in enzyme assays.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. All organs from high dose and control animals plus a selected number from low and mid dose animals. Rats fasted over night then anesthetised with CO2 and complete necropsy on all animals. Tissues examined: brain, liver, kidneys, heart, spleen, adrenals, testes, epididymides, uterus, ovaries, thymus. Bone marrow smears from femurs of all animals. Histological analysis of high and control animals of tissues examined; also lungs, liver, kidneys and spleen (females only) and relevant gross lesions from the middle and low dose groups processed and examined.
Other examinations:
no additional examinations beyond those described above.
Statistics:
Means and standard deviations for continuous data. All parameters examined tested for equality of variance (Bartlett's test, alpha=0.01) and if significant, transformed to obtain equality of variance. Alpha levels set at 0.05 for interaction between designated variables for dose related effects. Alpha levels also set at 0.05 for interaction terms (with Bonferroni's correction.)
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):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN: Slight reduction in high dose group of both parameters observed over whole study period (10% males, 6% females). All within historical control ranges.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) Reduced consumption (7-8%) in high dose group, particularly in males

HAEMATOLOGY. RBC and Hgb slightly but statistically significantly reduced in mid and high dose animals (both sexes- 3-8%). HCT similarly slightly reduced in high dose groups. Changes showed a dose response relationship, particularly for males. Results for mid dose group all within laboratory historical control values for drinking water studies all (except male RBC value, which was within historical control for all dietary and drinking water studies.) Note that control values were higher than historical control ranges. Also note that no changes to RBC indices or morphology were observed. A small but significant sex-by-dose interaction with reticulocyte was attributed to random variation and not treatment. There were no effects on WBC parameters.

CLINICAL CHEMISTRY: Slight but statistically significant decreases in serum levels of AST, total protein and cholesterol where observed in both sexes at the highest dose.

URINALYSIS: Consistent with the decreased water consumption, urine sg was increased in the high dose group. Urine pH also decreased with dose but this was attributed to excretion of the metabolite 2-(2-butoxyethoxy)acetic acid. There were no other effects that could be related to the slightly decreased RBC parameters.

ORGAN WEIGHTS: Liver (relative) and kidney (relative and absolute) weights increased in the high dose group. Spleen weight increases observed were not dose dependent and for the mid and low dose animals within the historical control ranges for dietary studies (but not drinking water studies). No treatment related and dose dependent organ weight changes were seen in the mid and high dose groups.

GROSS PATHOLOGY: No changes attributed to treatment observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: The only finding attributed to treatment was in female livers in the high dose groups where slight or very slight lesions were recorded (foci of aggregates of macrophages/histiocytes which are common in F344 rats and were observed in greater numbers in the high dose females.) Very slight hypertrophy of periportal hepatocytes was also seen in 6 high dose females. A slightly greater incidence of renal cortical tubule degeneration was also seen (very slight in nature) was seen in the high dose animals. Since this is routinely found at low incidence in F344 rats of this age, the observation could not be regarded as definitive. No treatment related histopathological effects were found in the bone marrow or spleen.

HISTORICAL CONTROL DATA:Discussion on historical control data is included above in the discussions on the individual findings.

OTHER FINDINGS: Liver enzyme analysis: Activities of all mixed function oxidases generally slightly increased. As only the top dose was examined, it is not possible to relate this to the NOAEL. Sperm analysis: No effects seen.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects water consumption; haematology; clinical chemistry; organ weights; histopathology; liver enzyme changes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The small changes in haematological parameters seen in the mid dose group were within historical control ranges and since they were close to the concurrent control values (within 3%) and these themselves were unusually high, changes were not deemed to be an adverse effect.
Executive summary:

In a well reported 90 day sub-chronic guideline and GLP drinking water study, standard toxicological end points, supplemented by additional examinations, were studied for Fischer 344 rats in doses up to 1000mg/kg. Multiple, albeit mild, effects were seen in the high dose group. The only treatment related effect seen in the mid dose group were equivocal changes (decreases of around 2 -3%) in erythron (RBC count, Hgb and Hct) that were statistically significant to unusually high concurrent controls but within historical control ranges.

This dose level of 250mg/kg/day was therefore considered to be the no adverse effect level.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. Kinston, NY
- Age at study initiation: Approximately 6 week
- Fasting period before study: None
- Housing: Rats were house 2/cage in stainless steel wire-mesh cages
- Diet (ad libitum): Except during exposure
- Water ( ad libitum): Except during exposure
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Standard conditions
- Humidity (%): Standard conditions
- Air changes (per hr): Standard conditions
- Photoperiod: (12 hrs dark / 12 hrs light):


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
See the attachment-1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See the attachment-1
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day/week
Dose / conc.:
20 ppm (nominal)
Dose / conc.:
40 ppm (nominal)
Dose / conc.:
85 mg/m³ air (nominal)
No. of animals per sex per dose:
20/sex/exposure group
Control animals:
yes
Details on study design:
- Rationale for animal assignment: Random
- Post-exposure recovery period in satellite groups: Four week recovery period
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to, during, and following each exposure for signs of toxic effects. Animals were observed once a day on weekends and holidays during the fourteen-week exposure regimen. Animals held for the four-week recovery period were observed at least once a day.
DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure and weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: During exposure week 14 and after 4 weeks of recovery
OPHTHALMOSCOPIC EXAMINATION: Yes

- Time schedule for examinations: Prior to exposure and at sacrifice
- Dose groups that were examined: All dose groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to sacrifice during exposure week fourteen and after 4 weeks of recovery
- Anesthetic used for blood collection: Yes (identity) Methoxyflurane
- Animals fasted: Yes
- How many animals: 10/sex/exposure group
- Parameters checked in table [No.7,8, 9, 10] were examined.


CLINICAL CHEMISTRY: - Time schedule for collection of blood: Prior to sacrifice during exposure week fourteen and after 4 weeks of recovery
- Anesthetic used for blood collection: Yes (identity) Methoxyflurane
- Animals fasted: Yes
- How many animals: 10/sex/exposure group
- Parameters checked in table [No.1, 2, 3, 4] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During exposure week 14 and after 4 weeks of recovery
- Metabolism cages used for collection of urine: Yes
- Parameters checked in table [No.13, 14] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Ten rats per exposure group were sacrificed on October 10 (males) and 10 (females), after receiving 66 and 67 exposures, respectively. The 4-week recovery rats, ten males per exposure group and ten females per exposure group were sacrificed after 27 and 28 post exposure days, respectively. The rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. Gross examinations were performed and selected tissues were fixed in 10% neutral buffered formalin for possible future histologic evaluation

HISTOPATHOLOGY: Yes
Histologic evaluation was performed on selected tissues from animals in the high Concentration and control groups. No histologic evaluations were performed on the low and intermediate hexyl CELLOSOLVER groups at the end of either the exposure regimen or the recovery period.
Other examinations:
Organ weights: The brain, liver, kidneys, spleen, lungs, thymus gland, and testes (males only) from all animals were weighed at sacrifice. Organ weights were recorded as absolute weights and as a percentage of both body weight and brain weight.

Statistics:
Results of quantitative continuous variables (such as body weight changes) were intercompared among the three concentration groups and one control group by use of Bartlett's homogeneity of variance (Sokal and Rohlf, 1969), analysis of variance (ANOVA) (Sokal and Rohlf, 1969), and Duncan's multiple range tests (Snedecor and Cochran, 1967). The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett's test indicated heterogeneous variances, all groups were compared by ANOVA for unequal variances (Welch or Brown and Forsythe, 1974) followed if necessary by t-tests. Corrected Bonferroni probabilities were used for t-test comparisons. Statistical procedures for hematologic continuous variables were similar and details of the procedures can be found
in the Clinical Pathology Report (Appendix 2). The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The principal sign observed in males that was considered to be exposure related was a urogenital wetness in the 71 ppm group. Urogenital wetness was also observed in female rats, but in a dose-related manner among all exposed groups. On a daily basis, the urogenital wetness was principally observed after the exposure rather than in the morning (before the exposure). The urogenital wetness was primarily observed beginning during week 6 of the study and continued until the termination of exposures. Also, beginning about week 9 and continuing until the termination of exposures, rats of the 71 ppm group were observed lying on their sides during the daily exposures. There were no significant signs observed during the recovery period.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Consistent statistically significant decreases in body weight gain were observed in the 71 ppm males (weeks 5-12) and females (weeks 5-14), and in the 41 ppm females (weeks 6-12). There were no exposure-related decreases in body weight gain observed in any group during the recovery period. There were no statistically significant differences in absolute body weight for any of the groups at the end of the exposure or recovery period
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant Increases in food and water consumption were observed in the 71 ppm males at the end of the exposure regimen (day 93). A significant increase in food consumption was observed in the 71 ppm females at the end of both the exposure regimen (day 94) and the recovery period (day 122). Other statistically significant differences were considered to be spurious as they were observed in the 20 ppm group with no effects observed in the 41 ppm group, i.e., no dose-response effect
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no exposure-related ocular effects observed during the study.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no biologically significant alterations were observed in hematology parameters for rats monitored at 14 weeks or following a four week recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no biologically significant alterations were observed in serum chemistry parameters for male rats monitored at 14 weeks or following a four week recovery period.
However, decreases in transaminases (AST and ALT) and sorbitol dehydrogenase (SDH), and Increases in alkaline phosphatase (ALP) were observed at the end of the exposure period for female rats exposed to 71 ppm vapor. No similar effects were observed on these enzyme levels at the end of the recovery period for female rats exposed to 71 ppm. However, there was a statistically significant increase in gamma-glutamyl -transferase for the 71 ppm females at the end of the recovery period.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no biologically significant alterations were observed in urinalysis parameters for rats monitored at 14 weeks or following a four week recovery period.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were two organs for which possible exposure-related effects were observed. For males, statistically significant increases in absolute and relative kidney weights were observed for the 41 and 71 ppm groups at the end of the exposure regimen. A statistically significant Increase in the kidney/body weight ratio was also observed for the 20 ppm males. Similar effects were not observed for males after the recovery period. Females of the 71 ppm group exhibited slight Increases in absolute and relative kidney weights, with the kidney/body weight value achieving statistical significance. Absolute and relative liver weights were statistically significantly increased for females of the 71 ppm group at the end of the exposure regimen.

The liver/body weight ratio was also significantly increased for the 41 ppm group females. However, there were apparent dose-related Increases in absolute and relative liver weights for all exposed groups of females at the end of the exposure regimen. Absolute and relative liver weights were also significantly increased at the end of the recovery period for the 71 ppm females. For males, statistically significant Increases in relative (both to body and brain weight) liver weights were observed for the 71 ppm group. However, dose-related increases were evident for the liver/brain weight data for exposed males. Liver/body weight ratios were statistically significantly increased for the 41 and 71 ppm males at the end of the recovery period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions found that were attributable to exposure to the test substance.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histologic lesions observed that were attributable to the test substance vapor exposure.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
41 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Critical effects observed:
no

None

Conclusions:
Based on the data from this study, 41 ppm is considered to be the concentration at which no biologically significant toxic effects were observed. Although the toxic effects observed at 71 ppm were generally mild, the spectrum of body weight, clinical chemistry, and organ weight effects, some of which persisted into the recovery period, are considered to be treatment related and thus to be biologically relevant in assessing potential health effects from this material.

Executive summary:

Four groups, each consisting of 20 Fischer 344 rats per sex, were exposed for 6 hours per day, 5 days per week, for 14 weeks to the vapor of ethylene glycol hexyl ether) at target concentrations of 0 (control), 20, 40, or 85 ppm and followed by 4 week recovery period.. Actual mean concentrations obtained for this study were 20, 41, and 71 ppm ethylene glycol hexyl ether.

 

Rats were received from breeding Laboratories. Rats were kept for acclimatization for 5 days. At study initiation rats were of approximately 9 weeks old. Rats were housed 2/cage in stainless steel cages with wire mash bottoms. Rats were provided ad libitum feed and municipal water except during exposure. The animals were kept on a 12-hour photoperiod throughout the study.

 

Monitors for toxic effects included clinical observations, body weight, food and water consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations.

  

The principal sign observed in males that was considered to be exposure related was a urogenital wetness in the 71 ppm group. Urogenital wetness was also observed in female rats, but in a dose-related manner among ethylene glycol hexyl ether-exposed groups. On a daily basis, the urogenital wetness was principally observed after the exposure rather than in the morning (before the exposure). The urogenital wetness was primarily observed beginning during week 6 of the study and continued until the termination of exposures. Also, beginning about week 9 and continuing until the termination of exposures, rats of the 71 ppm group were observed lying on their sides during the daily exposures. There were no significant signs observed during the recovery period. There were no mortalities during the study.

 

Consistent statistically significant decreases in body weight gain were observed in the 71 ppm males (weeks 5-12) and females (weeks 5-14), and in the 41 ppm females (weeks 6-12). There were no exposure-related decreases in body weight gain observed in any group during the recovery period. There were no statistically significant differences in absolute body weight for any of the groups at the end of the exposure or recovery period.

 

Statistically significant Increases in food and water consumption were observed in the 71 ppm males at the end of the exposure regimen (day 93). A significant increase in food consumption was observed in the 71 ppm females at the end of both the exposure regimen (day 94) and the recovery period (day 122). Other statistically significant differences were considered to be spurious as they were observed in the 20 ppm group with no effects observed in the 41 ppm group, i.e. no dose-response effect.

 

There were no exposure-related ocular effects observed during the study. There were no biologically significant alterations were observed in hematology parameters for rats monitored at 14 weeks or following a four week recovery period. There were no biologically significant alterations were observed in urinalysis parameters for rats monitored at 14 weeks or following a four week recovery period. There were no biologically significant alterations were observed in serum chemistry parameters for male rats monitored at 14 weeks or following a four week recovery period.

However, decreases in transaminases (AST and ALT) and sorbitol dehydrogenase (SDH), and Increases in alkaline phosphatase (ALP) were observed at the end of the exposure period for female rats exposed to 71 ppm ethylene glycol hexyl ether vapor. No similar effects were observed on these enzyme levels at the end of the recovery period for female rats exposed to 71 ppm of ethylene glycol hexyl ether vapor. However, there was a statistically significant increase in gamma-glutamyl-transferase for the 71 ppm females at the end of the recovery period.

 

Increased absolute and/or relative liver and kidney weights in both sexes of the 71 ppm group and, to a lesser extent, in the 41 ppm group. However, there were no exposure-related macroscopic or microscopic abnormalities found in this study. There were no exposure-related effects on the potential primary target tissues for glycol ethers of blood and testes. The changes observed at 41 ppm and below were not considered to be biologically significant, while the effects observed at 71 ppm, although not severe, were considered to be related to exposure to ethylene glycol hexyl ether vapor and thus to have biological relevance.

Based on the data from this study, 41 ppm is considered to be the concentration at which no biologically significant toxic effects were observed. Although the toxic effects observed at 71 ppm were generally mild, the spectrum of body weight, clinical chemistry, and organ weight effects, some of which persisted into the recovery period, are considered to be treatment related and thus to be biologically relevant in assessing potential health effects from this material.

 

 

 

 

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr K Thomae GmbH, Biberach
- Weight at study initiation: Male: approx 236g. female approx 171g
- Housing: in same sex pairs, wire cages. Cages also used for exposure period (2 per chamber)
- Diet : KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum (during exposure food was withdrawn). Checked for contaminants
- Water: ad libitum except during exposure. Checked for contaminants.
- Acclimation period: Animals sham exposed for 5 days before exposure period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 25/7/89 To: 6/12/89
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
- Exposure apparatus: Glass/steel exposure chamber volume ~1.1m3
- Method of conditioning air: Glass heat exchanger. Heated glass evaporator for solvent vapour plus two component atomiser with compressed air and counterstreamwise to supply air. The exhaust air system was set lower than the supply air system (positive pressure). This ensured that no laboratory air reached the control animals. The exhaust air system was set higher than the supply air system (negative pressure). This ensured that the laboratory was not contaminated as the result of any leakages from the inhalation chambers.
- Air flow rate: 21-23 m3/h
- Temperature, humidity, pressure in air chamber: 24-26C, 35-45% RH, -10Pa differential pressure to atmosphere (+10Pa for control)
- Method of particle size determination: HIgh dose group checked on days 10 and 66 for evidence of aerosols in test atmosphere using Polytec analyser. - Method of particle size determination: The following equipment was used: particle size analyzer HC 15 (Polytec), particle counter unit (BASF), millivolt writer (Kipp & Zonen), vacuum pump (Millipore), sampling probe (glass) internal diameter 4 mm.

TEST ATMOSPHERE
- Brief description of analytical method used: Daily by total hydrocarbon analysers and gas chromatography.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations in the inhalation chambers of test groups 2 (6 ppm) and 3 (14 ppm) and partially of test group 1 (2 ppm) were monitored by means of total hydrocarbon analyzers. The total hydrocarbon analyzers were calibrated with mixtures of the test substance in air that were generated in the inhalation chambers not containing animals and analyzed by gas chromatography. The concentration of test group 1 (2 ppm) was analyzed partially by gas chromatography after absorption of DGBE in propanol-2. Sampling period was 20 minutes. For measured concentrations, see remarks below.
Duration of treatment / exposure:
90 days plus satellite recovery group (4 weeks)
Frequency of treatment:
6hrs per day.
Remarks:
Doses / Concentrations:
2, 6 and 14 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0.013, 0.04, 0.094mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: 4 weeks
- Dose selection rationale: 2 week range finder (reported in separate record. Top dose is saturated vapour pressure at max temperature recommended in guideline for exposure conditions, mid and low dose as previously used in Gushow study (included as separate record) where liver effects reported at mid dose.
- Post-exposure recovery period in satellite groups: 4 weeks, all dose groups
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during exposure (3x time) and recovery period (1x day). A groupwise observation of clinical signs was performed during exposure; clinical signs and findings were also recorded after exposure, before exposure and during the preflow and post-exposure observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Before acclimation, before exposure then weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before acclimation and at end of study
- Dose groups that were examined: Top dose and control using handslit lamp

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At end of study, from retro-orbital venous plexus
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Blood was taken from all animals (main groups) at the end of the exposure period.
- The following parameters were determined using a particle counter (S Plus model, by Coulter, Krefeld, FRG):
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- differential cell count
- thromboplastin time
- reticulocytes
The data obtained were transferred to a computer (VAX 11/780; supplied by DEC, Munich, FRG).
The differential blood count was evaluated visually. The reticulocytes were counted using an automatic differential system (HEMATRAK 480 model by Geometric Data, Munich, FRG). The data were transferred to the computer.

CLINICAL CHEMISTRY: Yes
- Numerous clinicochemical parameters and various enzyme activities were measured and a clotting time analysis was performed.
- Time schedule for collection of blood: At end of study, from retro-orbital venous plexus
- Animals fasted: Yes
- The following parameters were determined:
1. Enzymes
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
2. Blood chemistry
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: At end of study, overnight collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
The following examinations were carried out:
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- sediment
The sediment was evaluated microscopically.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Sacrifice by exsanguination from the abdominal aorta/vena cava under anaesthesia. Moribund animals sacrificed and necropsied as soon as possible. A complete necropsy of all animals including weighing of certain organs and a gross-pathologic evaluation was performed. Selected organs/tissues were examined histopathologically.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Organs examined for control and top dose group: Heart, liver, kidneys, testes, ovaries, skin, jejenum, rectum, mediastinal & mandibular lymph node, musculature, sternum, extraorbital lachrimal glands, nasal cavity & paranasal sinus, brain, thymus, aorta, spleen, adrenals, prostate, seminal vesicle, esophagus, ileum, urinary bladder, nervus ischiadicus, femur/knee joint/bone marrow, pituitary gland, trachea, salivary glands, pancreas, stomach, cecum, eyes, mammary gland, cervical/thoracic/lumbar cord, thyroid, lungs, uterus, duodenum, colon plus all gross lesions
Organs examined for mid and low dose group: lungs, nasal cavity, salivary (mandibular/sublingual) glands, liver, mandibular lymph nodes plus all gross lesions.
Organs examined for recovery group animals: salivary (mandibular/sublingual) glands, liver, mandibular lymph nodes plus all gross lesions.
Other examinations:
none
Statistics:
The statistical evaluation of the data was carried out on the computer systems of the Department of Toxicology.

Clinical examinations:
Means and standard deviation were calculated for the variables (body weight and body weight change) of the animals of each test group for the statistical evaluation of the study and printed in tables together with the individual values (body weight). Statistical relevance was established using methods of ANOVA (2), DUNNETT (3, 4).
Significances were shown in the tables (* for p <= 0.05 and ** for p <= 0.01) .

Clinical chemistry, hematology and urinalysis:
- Clinical chemistry and hematology:
Means and standard deviations have been calculated for each test group and tabulated together with the individual values. In order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups, excepting the differential blood count, were compared with those for the control group using the analysis of variance (ANOVA and DUNNETT's test 2, 3, 4).
Significances resulting from the statistical comparison have been indicated in the tables on means.

- Urinalyses
The assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi2 test in appropriate two by two contingency tables. Significances which resulted from this chi2 test have been indicated in the tables (* for S >= 95%, ** for S >= 99%).
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Isolated spontaneous findings unrelated to exposure. No mortality. During the preflow period and the post-exposure observation period the animals showed no clinical signs and findings different from normal. Animal No. 4 (male) from the control group showed on day 84 swellings in the region of the neck, and the anogenital region was smeared with urine, and the animal showed intermittent breathing. Animal No. 97 (male) from test group (2 ppm) showed on day 84 swellings in the region of the neck. Animal No. 39 (male; 14 ppm) showed on day 3 and on day 7 reddish discharge from the right eye and both eyes, respectively, after exposure (blood test positive). Animal No. 116 (male, 14 ppm) showed on days 0 - 3 and on day 6 after exposure reddish discharge from the left eye (blood test positive) as well as on days 1 - 3 before exposure reddish crusts on the eye margins (blood test positive) and on days 1 - 3 crusts on the eye margins during exposure. Animal No. 153 (female; 14 ppm) showed on day 8 after exposure reddish discharge from the left eye (blood test positive) and on day 9 before, during, and after exposure reddish crusts on the margin of the left eye (blood test positive). No deaths were recorded during the post-exposure observation period.The animals No. 4 (male) from control group and animal No. 97 (male) from satellite group (2 ppm) were sacrificed in a moribund state on day 84. Due to the results of histopathologic examinations of these animals the moribund state of animals No. 97 and No. 4 was not caused by the treatment with the substance.

BODY WEIGHT: There were occasionally reported significant differences between dose groups and controls but these appeared to be random occasional occurences with no consistent pattern and therefore deemed not biologically significant. The body weight of the males of satellite group (6 ppm) during the exposure period had arithmetically significantly increased on day 14 and that of the males of satellite group (14 ppm) on day 49 when compared to the control group (p
BODY WEIGHT CHANGE: During exposure period: In the males of test group 3 (14 ppm) there was on day -7 an arithmetically significant influence (p
HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS: Some random effects across all treatment groups observed but not attributed to treatment.

ORGAN WEIGHTS: No changes in main dose groups: In recovery group: High dose group absolute and relative liver weight increase (15 and 8% respectively, males only) and relative testes and female lung weight increase (both ~10%). These changes were not thought to be treatment related.
Dose descriptor:
NOAEL
Effect level:
14 ppm
Sex:
male/female
Basis for effect level:
other: This is the maximum concentration that can be tested (effective saturated vapour pressure for a dynamic atmosphere)
Critical effects observed:
no

Measured concentrations in atmosphere.

 Test group  Measured concentration (ppm)  Target concentration (ppm)
 Low 2.0 +/- 0.11   2
Mid  6.0 +/- 0.92  6
 High  14.0 +/- 0.53  14

 

Aerosol particles were not detected in the inhalation atmosphere.

Conclusions:
The organ weight changes in the high dose animals were not deemed biologically significant as they were sex specific and not seen in the main study animals (ie only seen in the recovery group animals.)
Executive summary:

In a 90 day guideline and GLP inhalation study rats were exposed in 3 dose groups up to the maximum saturated vapour pressure of 2 -(2 -butoxyethoxyethanol). Satellite recovery groups were also included for all 3 dose groups and the controls. No adverse effects were seen in any dose group. The NOAEL was therefore 14ppm (94mg/m3), the maximum concentration tested.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
, 5 week exposure period
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, MI)
- Age at study initiation: 7 weeks
- Housing: stainless steel cages, wire bottomed, 2 per cage
- Diet: Purina lab chow, ad libitum except during exposure
- Water ad libitum except during exposure.
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12/11/79 To:
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 157l stainless steel/glass chambers. Vapour generated using counter current liquid/air exchange through a perforated plate distillation column. Outflow air then diluted to required concentration (used neat for highest dose.)
- Temperature, humidity, pressure in air chamber: 21C, 50% RH
- Air flow rate: 25l/min

TEST ATMOSPHERE
- Brief description of analytical method used: Pair of GC with FID. Low and mid concentrations checked every 30 mins, high dose monitored continuoously. Calibration standard run daily.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pair of GC with FID. Low and mid concentrations checked every 30 mins, high dose monitored continuoously. Calibration standard run daily.
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
2, 6 and 18 ppm (0.013, 0.039, 0.117mg/l)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.09(SD=0.28), 5.69(SD=0.78), 18.5 (SD=2.6)ppm
Basis:
analytical conc.
No. of animals per sex per dose:
15
Control animals:
other: no data specified
Details on study design:
- Dose selection rationale: Highest dose is saturated vapour pressure.
- Rationale for selecting satellite groups: 5 animals/sex/dose sacrificed after 3 weeks to examine osmotic red blood cell fragility (taken under methoxyflurane anesthesia.)
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: signs of toxicity, changes in appearance and demeanor.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 6, 9, 14, 19

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at end of study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 17th day (males), 20th day (females) using orbital sinus puncture then at end of study.
- Anaesthetic used for blood collection: Yes for final (methoxyflurane). No data for interim
- Animals fasted: Yes for final, no for interim
- How many animals: 10
- Parameters checked: RBC, Hgb, PCV, platelets, total and differential WBC, MCV, MCH, MCHC
- Other: satellite group of 5 animals sacrificed after 3 weeks to check for osmotic red blood cell fragility.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of study
- Animals fasted: Yes
- How many animals: 10
- Parameters checked: BUN, ALP, GPT, glucose, albumin, total protein and bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: 17th day (males), 20th day (females) then at end of study.
- Metabolism cages used for collection of urine: No data
- Animals fasted: YYes for final, no for interim
- Parameters checked: pH, glucose, ketones, bilirubin, urobilinogen, occult blood, protein, density.


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. As for histopathology.
HISTOPATHOLOGY: Yes : Liver, lymph nodes, ovary, oviducts, uterus, seminal vesicles, pancreas, cecum, skin, spleen, heart, kidneys, thymus, nasal turbinates, pituitary, adrenals, stomach, small and large intestine, epididymides, testes, prostate, thyroid, trachea, eyes, lungs (distended), brain, spinal cord, peripheral nerve, bone and bone marrow, coagulating glands, urinary bladder, skeletal muscle, salivary glands, mediastinal tissue, aorta, esophagus, parathyroid, mammary tissue, tongue, mesenteric tissue, lacrimal glands, larynx, ear. All tissues examined from control and top dose animals plus target organs from mid dose groups.
Other examinations:
none.
Statistics:
Barlett's test (p<0.01), ANOVA (p<0.1) and Dunnett's test or Wilcoxon's test (p<0.05): Haematology, body and organ weights and ratios.
ANOVA and Dunnett's test: Urine density and clinical chemistry data
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:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Some urethral wetness from 13 weeks onwards in females, incidence and severity higher in dose groups than controls. Toxicological significance of this uncertain. No other effects observed

HAEMATOLOGY: The red cell fragility tests showed no differences between control and exposure groups. The white blood cell counts of the mid and high dose males were significantly elevated (+15%) but the dose response was not smooth and the values were within historical ranges and were therefore considered not to be of toxicological significance.

CLINICAL CHEMISTRY: A statistically significant rise in ALP levels in high dose males (+10%) and a decrease in GPT levels in mid dose males was not associated with gross or histopathological changes to the liver and therefore deemed not to be of toxicological significance. The GPT changes also showed no dose reponse relationship.

ORGAN WEIGHTS: Relative liver weights statistically significantly decreased in mid and high dose group males (6&7% respectively). Changes not deemed of toxicological significance as not associated with changed histology. Relative liver weights increased in mid and high dose group females (only significant at high dose where change was still only +6%).

GROSS PATHOLOGY: Paleness in livers of 3/10 females. No other changes.

HISTOPATHOLOGY: NON-NEOPLASTIC: Hepatocyte vacuolisation consistent with fatty change occured in ALL females, including controls, although there was a progression from very slight to slight degree from the control to high dose animals (see table 1). No other significant observations noted nor any significant effects in males
Dose descriptor:
NOAEC
Effect level:
18 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no

Changes in males not deemed of toxicological significance as not associated with changed histology. However, since change in females was associated with histological change, this was deemed significant. However, it is difficult to postulate a toxic effect which would cause a liver increase in one sex and a decrease in the other. The authors postulated that the changes in females could be indicative of a minimal degreee of liver toxicity but could equally be secondary adaptive physiological responses not directly linked to treatment.

Conclusions:
The only effect of possible significance was a minimal increase in liver weight (+6%) associated with slight gross and histopathological changes (slight vacuolisation consistent with fatty change). This was also seen in all control, low and mid dose animals, though severity increased from very slight to slight. In the absence of further differences, these changes in the liver were considered adaptive.
Executive summary:

In a 5 week GLP inhalation study rats were exposed to a concentrations of 2 -(2 -butoxyethoxyethanol) up to and including the saturated vapour pressure. The only effect of note which was both statistically significant and biologically plausible was a slight increase (+6%) in liver weight in high dose females. Vacuolisation also occurred in the livers of these female animals, but this change was also seen in all control, low and mid dose animals. In the absence of further differences, the change in liver weight was considered adaptive. Additionally, inhalation exposure for 90-days did not lead to changes in female livers. The NOAEC was set to 18 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
325 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: TSCA: 40CFR 798.2250 as amended by 40CFR 799.1560
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (reported as 'CD' rats)
- Age at study initiation: 6 wks
- Housing: individual in suspended stainless steel cages, except during mating and lactation period. During latter, solid steel pan fitted and hardwood shaving bedding added.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (ad libitum): Certified Purina lab chow #5002 mash diet
- Water (ad libitum): tap
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 8 - 77
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: back
- % coverage: 3x3cm
- Type of wrap if used: polyethylene (PE) patch held in place by adhesive bandage wrapped around trunk of animal. Gauze pad added beneath PE patch after start of study
- Time intervals for shavings or clipplings:


REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped only.
- Time after start of exposure: 6hrs


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2ml/kg
- Concentration (if solution): neat
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analysis of diluted dosing solutions showed that 10% dose within 99.5(+/-3.1)% and 30% dose within 99.8(+/-2.6)%.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 200, 600 or 2000 mg/kg bw/day, applied as 10, 30 and 100% solution in water to give constant application volume per unit body weight.
Basis:
nominal per unit area
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Rationale for animal assignment (if not random): randomised but to keep mean body weights of control and treatment groups equal.
- other: Post-exposure period: none
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily checks for morbidity/mortality and overt toxic effects

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily qualitative analysis at treatment (dosing) time and after wrapping removed.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-study and near end study

HAEMATOLOGY:
- Time schedule for collection of blood: Pre-study and at 4 and 13 weeks. Collected by venipuncture of orbital sinus.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight and not dosed until after blood collection.
- Parameters examined: Hb, Hct, RBC, MCV, MCH, MCHC, total and differential WBC
- How many animals: no data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-study and at 4 and 13 weeks. Collected by venipuncture of orbital sinus.
- Animals fasted: Yes, overnight
- How many animals: No data
- Parameters examined: ALP, AST, ALT, BUN, glucose, total protein, albumin, globulin, creatinine, bilirubin, Na, K, Cl, Ca, inorganic P.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Pre-study and at 4 and 13 weeks
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: appearance, colour, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, 16hr volume, microscopic analysis.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: During a 14 day period near the end of the study, daily vaginal smears were collected to dermine if normal oestrus cycle was occuring.
Sacrifice and pathology:
Sacrifice by ether anesthesia.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, including adrenals, brain, kidneys, pituitary, prostate, testes, epididymides, seminal vesicles, liver, ovaries and spleen. All weighed and fixed. Other tissues preserved. High dose and control animal tissues sectioned for histology.
Other examinations:
none
Statistics:
Body weights, feed consumption, organ weights using Bartlett's test to determine if variances equal. If variances equal, parametric procedures used (ANOVA followed by Dunnett's test if appropriate). Alternative non-parametric procedures were Kruskal-Wallis test follwed by Dunn's summed rank test if appropriate. Tests for trend using regression analysis and Jonckheere's test. Walsh's test used to determine equality of means. Comparisons made at 1 and 5% signficance levels (two sided.)
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
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 observations related to treatment.

URINALYSIS: Occult blood was noted in females treated at 600 or 2000  mg/kg but there was no evidence of urinary casts or significant numbers of erthyrocytes.

OTHER FINDINGS: IRRITATION: Concentration dependent increase in irritation worse in females. In females it was evident (erythema) from week 1 at the high dose and week 8 at the mid and high dose, with increasing numbers of animals affected with time. In males, it was only seen at the end of the study and and never effected more than 50% of animals. In males the severity was slight or very slight whilst in females there was necrosis and eschar in some animals at high doses.
Dose descriptor:
NOAEL
Effect level:
< 200 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin irritation
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: all other effects
Critical effects observed:
no

Females appeared to be more susceptible than males to concentration-dependent irritation at the application site. Effects at the highest dosage were desquamation,  atonia, eschar and necrosis.

Conclusions:
The only effect of note was dermal irritation at the site of repeated application which occured at all doses, albeit very slight at the low dose and only in males at towards the end of the study. Irritancy was more marked in females than males and produced some necrosis at the highest dose. There were no other adverse findings noted.
Executive summary:

In a well conducted study designed to assess the sub-chronic and reproductive toxicity of 2 -(2 -butoxyethoxy)ethanol to rats, the test substance was administered by the dermal route for 13 weeks to the maximum practical concentration attainable of 2ml/kg. The only effect of note was dermal irritation at the site of repeated application which occured at all doses, albeit very slight at the low dose and only in males at towards the end of the study. Irritancy was more marked in females than males and produced some necrosis at the highest dose. There were no other adverse findings noted.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

There is no sub-chronic or chronic study available for 2-(2-hexyloxyethoxy)ethanol (DEGHE). A Combined Repeated Dose Toxicity Study oral with the Reproduction/Developmental Toxicity Screening Test (OECD 422) was conducted with DEGHE (Dow, 2004). This study differs from a 90 d study according to OECD guidelines in the level of detail in the study design and in the group size. Although the exposure duration of an OECD 422 may be similar for female animals (54 days), it is shorter for the male animals and the level of information derived from the study is different to that from a 90 d study. To cover the endpoint "sub-chronic toxicity", a read-across was conducted to the related analogues ethylene glycol hexyl ether (EGHE) and diethylene glycol butyl ether (DEGBE). A read across justification document has been attached to the corresponding enpoints and to chapter 13.

Data generated with DEGHE

Dietary administration of 100, 300, or 1000mg/kg DEGHE to CD rats in a combined repeated dose / Reproduction toxicity screening test according to OECD guideline 422 resulted in slight treatment-related decreases in body weights of males and females given 1000 mg/kg/day(DOW, 2004). Decreased maternal body weights persisted throughout the gestation and lactation phases of the study. Females given 1000 mg/kg/day had treatment-related increases in serum ALT and ALP activities, along with increases in relative liver weight. These effects corresponded with treatment-related histopathologic changes in the liver that consisted of very slight panlobular hepatocyte hypertrophy. Males given 1000 mg/kg/day had treatment-related increases in relative liver weight, which corresponded with the histological alteration of very slight periportal hepatocyte hypertrophy. At 1000 mg/kg/day, other findings of lesser significance included slightly increased blood urea nitrogen and decreased absolute thymus weights in females, decreased urine pH and increased relative kidney weights in males. Additional histopathologic findings of minor toxicological significance at 1000 mg/kg/day included very slight diffuse acinar hypertrophy of the submandibular salivary gland in males, and very slight atrophy of the mesenteric adipose tissue in females. There were no adverse effects of DEGHE on neurological function.

Based on these results, the no-observed-effect level (NOEL) for general toxicity was 300 mg/kg/day.

 

Data generated with DEGBE

Sub-acute inhalation toxicity[SW1] was assessed in an older study comparable to OECD 412(DOW, 1981). 15 Fischer 344 rats per sex and dose were exposed for 5 weeks to 2, 6, and 18 ppm DEGBE vapour via whole body inhalation. Observations included body and organ weights, ophthalmoscopic examinations, haematology, clinical chemistry, urinalysis, gross, and histopathology. The only effect of note which was both statistically significant and biologically plausible was a slight increase (+6%) in liver weight in high dose females. Vacuolisation also occurred in the livers of these female animals, but this change was also seen in all control, low and mid dose animals. In the absence of further differences, the change in liver weight was considered adaptive. Additionally, inhalation exposure for 90-days did not lead to changes in female livers. The NOAEC was set to 18 ppm.

In a well-documented study similar to OECD 407, ten male rats per group received 891, 1781, or 3564mg/kg DEGBE via gavage on five days each week for six weeks(Eastman Kodak, 1982). Body weight, clinical signs, haematology, and clinical chemistry was assessed. A histopathologic examinations was performed. Six high dose animals died, four of these as a result of intubation errors. The total number of red blood cells, haemoglobin and mean corpuscular haemoglobin concentration were decreased in the mid and high dose animals, while the mean corpuscular volume and the mean corpuscular haemoglobin were increased. Liver and spleen weights were increased at the two highest doses, which was accompanied by read pulp hypocellularity in the four surviving high dose animals. There were no histopathological changes in the liver. There were discrepancies in the reported body weights. As a conservative approach, it is assumed that terminal body weight was reduced in all dose groups. The NOAEL is therefore below 871 mg/kg.

In a sub-chronic study according to OECD 413 and GLP, 10 Wistar rats per sex and dose were exposed to 2, 6, and 14 ppm DEGBE vapour(BASF AG, 1992)via whole body inhalation. Satellite recovery groups were also included for all 3 dose groups and the controls. No adverse effects were seen in any dose group. Liver weights were increased in the high dose recovery group males only. In the absence of histopathological changes and no effect on liver weight in all other groups or females, this change was not considered adverse. The NOAEL was therefore set to 14ppm (94mg/m3), the maximum dose tested.

A 90 day sub-chronic OECD408 guideline and GLP drinking water study was carried out using Fischer 344 rats using doses of 50, 250 and 1000mg/kg/day of DEGBE(Johnson, et al., 2005). End points studied included cage side and detailed clinical observations, body weight, food and water consumption, ophthalmic observations, haematology, clinical chemistry, urine analysis and neurobehavioural observations (functional observation battery). Sperm analysis was also performed. Multiple, albeit mild, effects were seen in body and organ weights, water and food consumption and in the haematology, clinical chemistry and urine analysis the high dose group. In the histopathology, the only finding attributed to treatment was in female livers in the high dose groups where slight or very slight lesions were recorded (foci of aggregates of macrophages/histiocytes, which are common in F344 rats and were observed in greater numbers in the high dose females). Very slight hypertrophy of periportal hepatocytes was also seen in 6 high dose females. No treatment related histopathological effects were found in the bone marrow or spleen in any cohort. The only treatment related effect seen in the mid dose group were equivocal changes (decreases of around 2 -3%) in erythron (RBC count, Hgb and Hct) that were statistically significant to unusually high concurrent controls but within historical control ranges. The mid dose level of 250mg/kg/day was therefore considered the no adverse effect level.

In a well conducted study designed to assess the sub-chronic and reproductive toxicity of DEGBE to SD male and female rats, the test substance was administered by the dermal route for 13 weeks at doses of 200, 600, 2000mg/kgbw/day (Auletta, et al., 1993). The top dose used was the maximum practical concentration attainable. The lower concentrations were diluted in water to give a constant application volume but the top dose was applied neat. 10 animals were used per sex per dose. The only significant effect of note was dermal irritation at the site of repeated application which occurred at all doses, albeit very slight at the low dose and only in males towards the end of the study. Irritancy was more marked in females than males and produced some necrosis at the highest dose. There were no other adverse findings noted. The NOAEL for local skin irritation was <200mg/kgbw/day. The NOAEL for all other effects was >2000mg/kgbw/day.

 

Data generated with EGHE

In a 90-day sub-chronic GLP inhalation study similar to OECD 413, four groups, each consisting of 20 Fischer 344 rats per sex, were exposed for 6 hours per day, 5 days per week, for 14 weeks to the vapours of ethylene glycol hexyl ether at target concentrations of 0 (control), 20, 40, or 85 ppm(DOW, 1985). Exposure was followed by a four week recovery period for half the animals. Actual mean concentrations obtained for this study were 20, 41, and 71 ppm EGHE. Examinations included regular assessments of clinical signs, body weight, food consumption, and water consumption. Prior to sacrifice, blood and urine samples were collected and analysed.Gross examination and histopathology was performed. There were no histopathological changes caused by exposure to EGHE. No effects on red blood cells or histologic changes in the liver or kidney were noted at concentrations up to and including the highest concentration tested (71 ppm or 425 mg/m3). Body weights were significantly decreased for high and mid dose animals. Decreases in body weight and increases in female liver weights observed at 41ppm were considered to be adaptive (and not adverse) since there were no correlative changes in histopathology or serum chemistry. Liver weights were increased in a dose dependent manner and stayed elevated in the 71ppm group at the end of the recovery period. Decreases in transaminases (AST and ALT) and sorbitol dehydrogenase (SDH), and increases in alkaline phosphatase (ALP) were observed at the end of the exposure period for female rats exposed to 71 ppm. This change was not seen after the recovery period. However, there was a statistically significant increase in gamma-glutamyl -transferase for the 71 ppm females at the end of the recovery period. The changes are difficult to interpret since levels of 3 out of 4 enzymes were decreased and only 1 out of 4 was increased. Based on these data, 41 ppm (245 mg/m3) is considered to be NOAEC.

Justification for classification or non-classification

Systemic effects were generic (body weight decrease, liver effects) and occured at high concentrations only. None of the studies presented above gives any indication that DEGHE causes specific target organ toxicity. Consequently, clasification for this endpoint is not required according to (CLP) Regulation (EC) No. 1272/2008.