Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an oral reproductive toxicity study in the OECD 422 design, groups of 12 male and 12 female CD rats were administered the test subatcne via the diet at concentrations supplying 0, 100, 300 and 1000 mg/kg/day for at least 33 days. The NOAEL for general toxicity was found to be 300 mg/kg/day in male and female rats. The NOAEL for reproductive and neurological effects was 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
Limit test:
no
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
- 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 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 of 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
Details on 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.
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
Details on study schedule:
- Age at mating of the mated animals in the study: approx. 10 weeks
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male and 12 female rats
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: limit dose of 1000 mg/kg was chosen as high-dose level based on 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 NOEL.
- Rationale for animal assignment: Animals were stratified by body weight and then randomly assigned to treatment groups using a computer progam designed to increase the probability of uniform mean weights and standard deviations at the start of the study.
- Post-exposure period: none
Positive control:
not required
Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
not investigated
Sperm parameters (parental animals):
not investigated
Litter observations:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of delivery was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Date of parturition,
- litter size on the day of parturition (LD 0),
- clinical observations and the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4 .
- Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see animal observations).
- Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects and then were discarded.

Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
Pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead were examined to the extent possible and discarded.


Statistics:
Accepted methods documented in detail for different data points
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows :
Female mating index =(No . females with evidence of mating/No . paired) x 100
Male mating index =(No . males with evidence of mating/No . paired) x 100
Female conception index =(No . females with evidence of pregnancy/No . mated) x 100
Male conception index =(No. males siring a litter/No . mated) x 10 0
Female fertility index =(No . females with evidence of pregnancy/No . paired) x 100
Male fertility index =(No . males siring a litter/No . paired) x 100
Gestation index =(No . females delivering a viable litter/No . females delivering a litter) x 100
Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss =(No . implants - No . viable offspring)/(No . implants) x 100

Offspring viability indices:
Day 1 or 4 pup survival index =(No . viable pups on day 1 or 4/No . born live) x 100
Clinical signs:
effects observed, non-treatment-related
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 during 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
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects at any dose level on mating, conception, fertility and gestation indices, time to mating, gestation length, post-implantation loss, pup survival or pup sex ratio.








HISTOPATHOLOGY (PARENTAL ANIMALS)


OTHER FINDINGS (PARENTAL ANIMALS)
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related or statistically significant findings . All animals survived until termination.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Slight treatment-related decreases in body weights of males and females given 1000 mg/kg/day. Decreased maternal body weights persisted throughout the gestation and lactation phases of the study.
Decreased feed consumption in male and female animals of the 1000 mg/kg/day dose groups during pre-breeding phase. Not measured during breeding phase. Decreased feed consumption in females of the 1000 mg/kg/day dose group during gestation. No differences in feed consumption in any of the dose groups compared to respective controls during lacation period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
actual test substance intake not reported, only feed consumption data (see above).


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
no adverse effects


ORGAN WEIGHTS (PARENTAL ANIMALS)
Males and females given 1000 mg/kg/day showed treatment-related increases in relative liver weight


GROSS PATHOLOGY (PARENTAL ANIMALS)
no effects observed


HISTOPATHOLOGY (PARENTAL ANIMALS)
very slight panlobular hepatocyte hypertrophy found in females given 1000 mg/kg/day,
very slight periportal hepatocyte hypertrophy in males given 1000 mg/kg/day


OTHER FINDINGS (PARENTAL ANIMALS)
Females given 1000 mg/kg/day had treatment-related increases in serum ALT and ALP activities.
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.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: No adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
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
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
no treatment-related effects
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no treatment-related effects
Gross pathological findings:
no effects observed
Description (incidence and severity):
no treatment-related effects
VIABILITY (OFFSPRING)
no treatment related effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Sex:
male/female
Remarks on result:
other: no adverse effects observed
Reproductive effects observed:
no

Final Body and Organ Weight Effects

MALES Dose (mg/kg/day)         
Parameter 0 100 300 1000
Final Body Weight (g) 393.5 385.7 391.4 371.9
Relative Kidneys (g/100g bw) 0.723 0.749 0.762 0.816*
Relative Liver (g/100g bw) 2.968 3.096 3.166 3.413*
Relative Brain (g/100g bw) 0.523 0.543 0.528 0.559*
Absolute seminal vesicles with coagulating glands (g) 1.735 1.737 1.613 1.424*

FEMALES Dose (mg/kg/day)         
Parameter 0 100 300 1000
Final Body Weight (g) 278.6 270.9 268.6 254.5*
Absolute Thymus (g) 0.255 0.239 0.232 0.195*
Relative Liver (g/100g bw) 3.748 3.815 3.89 4.594 $
Absolute Heart (g) 0.974 0.935 0.935 0.863 *
Absolute Adrenal (g) 0.094 0.081* 0.086 0.081 *
Absolute Ovaries (g) 0.150 0.126* 0.131 0.135

*Statistically different from control mean by Dunnett's test, Alpha = 0 .05. •

$ Statistically different from control mean by Wilcoxon's test, Alpha = 0 .05 .

Bold type indicates the effects judged to be treatment related .

Italics type indicates the effects that were reflective of the treatment-related decrease in final body

weights

Statistically Identified Clinical Chemistry Parameters

FEMALES Dose (mg/kg/day)         
Parameter (mean values) 0 100 300 1000
Urea nitrogen (mg/dl) 18 20 18 22*
ALT (U/L) 60 62 67 80*
ALP (U/L) 83 87 89 121*

*Statistically different from control mean by Dunnett's test, Alpha = 0 .05 .

Bold type indicates the effects judged to be treatment related .

Treatment related Histopathological Effects

Sex

Males          Females         
Dose (mg/kg/day) 0 100 300 1000 0 100 300 1000
Liver (number examined) 12 12 12 12 12 12 12 12
Hypertrophy, hepatocyte, panlobularvery slight 0 0 0 0 0 0 0 12
Hypertrophy, hepatocyte, periportalvery slight 0 0 0 12 0 0 0 0
Submandibular salivary gland (number examined) 12 12 12 12 12 0 0 12
Hypertrophy, acinar, diffusevery slight 2 2 2 9 0 0 0 0
Mesenteric tissue (number examined) 12 0 0 12 12 12 12 12
Atrophy, adipose tissueVery slight 0 0 0 0 3 3 1 8

Bold indicates effects were considered to be treatment related .

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), groups of 12 male and 12 female CD rats were administered the test substance via the diet at concentrations supplying 0, 100, 300 and 1000 mg/kg/day. Females were dosed daily for two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and through post-partum day 4. Females were necropsied on post-partum day 5. The males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test day 34). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, post-mortem examinations included a gross necropsy of the adults with collection of organ weights and extensive histopathologic examination of tissues.Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Dietary administration of the test substance to CD rats resulted in slight treatment-related decreases in body weights of males and females given 1000 mg/kg/day. 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. No adverse effects of the test substance on neurological function were found.

There were no treatment-related effects at any dose level on mating, conception, fertility and gestation indices, time to mating, gestation length, post-implantation loss, pup survival or pup sex ratio. In addition, there were no adverse gross or histopathological findings in the reproductive organs of males or females at the highest exposure level, despite the presence of treatment-related maternal toxicity at this dose.

Based on the results of this test, NOAELfor general toxicity was 300 mg/kg/day. The NOAEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.

There were also no effects on reproductive parameters in three one-generation studies and one study to assess the estrous cycle in rat with the read across substance DEGBE up to 2000mg/kg after dermal exposure and up to 1000mg/kg after oral (gavage or drinking water exposure).


Effects on developmental toxicity

Description of key information

OECD 414, rats, vapour: NOAEL = 500 mg/m³ (highest dose tested), Dow 1987, RA substance EGHE

OECD 414, rats, diet: NOAEL = 633 mg/kg (highest dose tested), Ema 1988, RA substance DEGBE

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study receipt: males (69 days upon arrival) and females (62 days upon arrival)
- Weight at study initiation: not specified in the report
- Fasting period before study: none
- Housing: mating - (1:1, male:female), post-mating - females housed individually
- Diet (e.g. ad libitum): Certified Ground Rodent Chow provided ad libitum, except during exposure
- Water (e.g. ad libitum): Municipal water provided ad libitum, except during exposure
- Acclimation period: Animals were quarantined for two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-28 °C
- Humidity (%): 50-61 %
- Air changes (per hr): standard conditions
- Photoperiod (hrs dark / hrs light): 12 hours (light:dark cycle)
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4 chambers made of glass and stainless steel
- Method of holding animals in test chamber: in cages
- Source and rate of air: ambient
- Method of conditioning air: not specified in the report
- Temperature, humidity, pressure in air chamber: mean temperatures - 25.1-26.5 °C, mean RH - 52.9-56.5%
- Air flow rate: 1000 liters/minute
- Air change rate: 14 air changes/hour
- Treatment of exhaust air: Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack

TEST ATMOSPHERE
- Brief description of analytical method used: A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was used to monitor the hexyl CELLOSOLVE vapor concentrations in the chambers. The GC column was a 10-feet x 1/8 inch (i.d.) stainless steel column packed with 20% SP-2100 on 80/100 mesh Supelcoport (Supelco, Inc., Bellefonte, PA). Calibration of the gas chromatograph was done with dynamically generated gas standards of hexyl CELL0S0LVE prepared by syringe injection of test material into Tedlar (DuPont) gas bags. The series of standards encompassed the entire range of vapor concentrations generated in the exposure chambers. A linear calibration curve was obtained when areas (integration counts) were plotted versus the gas equivalent concentrations of the standards. Each chamber atmosphere was analyzed for hexyl CELLOSOLVE approximately once every 30 minutes during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were also calculated for each chamber.
- Samples taken from breathing zone: not specified in the report
- Liquid hexyl CELLOSOLVE® was metered from a piston pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid (33-52°C). The resultant vapor was carried into the chamber by passage of conditioned air through the evaporator. Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack.
The four chambers employed in this study were rectangular in shape, constructed of glass and stainless steel (Wahmann Manufacturing Company, Timonium, MD), and measured approximately 2.1 m x 1 m x 2.1 m (height). Total volume in each chamber was approximately 4320 L. An orifice plate was positioned in the exhaust duct of the chamber and was connected to a Dwyer Magnehelic Pressure Gauge.
Airflow in each chamber was approximately 1000 L/minute (14 air changes per hour) with a t99 (theoretically-derived time required for the chamber to reach 99% of the equilibrium concentration) of approximately 20 minutes.
The chambers were illuminated with artificial room light. Chamber temperature, relative humidity and air flow rate were recorded at least five (5) times during each exposure. Within each chamber, the animal cages were rotated daily to compensate for any possible, but undetected, variation in chamber exposure conditions.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
target concentrations - 0, 20, 40 and 85 ppm
analytical concentrations - 0, 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm
nominal concentrations - 0, 26.1 ± 1.84, 39.2 ± 1.80 and 91.0 ± 6.28 ppm
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1 (male:female)
- Length of cohabitation: 5 days
- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [no]
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
6 hours/day, gd 6 to gd 15
Frequency of treatment:
daily from gd 6 to gd 15
Duration of test:
9 days
Dose / conc.:
0 ppm
Remarks:
analytical concentrations - 0 ppm, nominal concentrations - 0 ppm
Dose / conc.:
20 ppm
Remarks:
analytical concentrations - 20.8 ± 0.90 ppm, nominal concentrations - 26.1 ± 1.84 ppm
Dose / conc.:
40 ppm
Remarks:
analytical concentrations - 41.1 ± 1.77 ppm, nominal concentrations - 39.2 ± 1.80 ppm
Dose / conc.:
85 ppm
Remarks:
analytical concentrations - 79.2 ± 10.80 ppm, nominal concentrations - 91.0 ± 6.28 ppm
No. of animals per sex per dose:
25 plug positive females/group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on range-fiding study
- Rationale for animal assignment: random assignment
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from gd 0 to gd 21

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gd 0, 6, 9, 12, 15 and 21

FOOD CONSUMPTION AND WATER CONSUMPTION: Yes
- Time schedule for examinations: at intervals gd 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all organs

OTHER: Hematological examination
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: percent live and dead fetuses
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter] - fetuses examined for soft-tissue examinations
Statistics:
The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables (e.g., maternal body weights, liver weights, etc.) were intercompared for the three exposure groups and a control group for each species by use of Levene's test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test.
Nonparametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Indices:
Pre-and post-implantation loss, percent live fetuses, sex ratio
Historical control data:
not applicable
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment related clinical signs of toxicity which included urine stains on fur, lacrimation, ocular wetness and encrustation were observed only at 85 ppm during the exposure period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were significant decreases in maternal body weight and weight gain at both the 40 and 85 ppm dose group during the exposure period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were significant decreases in food consumption at 85 ppm dose group during the exposure period.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased at the 85 ppm dose group during the exposure period.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in the hematological examination
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes on maternal organ weights, gravid uterine weight, gestational body weight and absolute/relative liver weight
Gross pathological findings:
no effects observed
Description (incidence and severity):
At scheduled necropsy, there were no treatment related findings at maternal gross examination.
Details on maternal toxic effects:

.
Key result
Dose descriptor:
NOEC
Effect level:
20 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no effects of exposure on fetal body weight
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects of exposure on sex ratio
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The incidence of fetal malformations and variations were comparable between the treatment groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of fetal malformations and variations were comparable between the treatment groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of fetal malformations and variations were comparable between the treatment groups
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of exposure on gestational parameters including number of corpora lutea per dam, number of total, non-viable or live implantations per litter, pre- and post-implantation loss, sex ratio and fetal body weight. The incidence of fetal malformations and variations were comparable between the treatment groups.
Key result
Dose descriptor:
NOAEC
Effect level:
85 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Exposure to ethylene glycol hexyl ether vapor during organogenesis in Fischer 344 rats resulted in transient maternal toxicity at 85 and 40 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 20 ppm for rats and the developmental toxicity NOEL was at the ambient temperature generated vapor concentration of about 85 ppm.
Executive summary:

Timed-pregnant Fischer 344 rats, 25 per group, were exposed to ethylene glycol hexyl ether vapor for six hours/day on gestational days (gd) 6 through 15 for rats at target concentrations of 0, 20, 40 or 85 ppm (analytical concentrations of 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm for the 20, 40 and 85 ppm groups, respectively). Eighty-five ppm was close to the saturated vapor concentration of the material at 20°C. Maternal body weights were measured on gd 0, 6, 9, 12, 15 and 21 (rats). Food and water consumption was measured, for rats only, for three-day intervals throughout gestation. At scheduled sacrifice on gd 21 (rats), maternal body weight, gravid uterine weight, and liver weight were taken. Maternal blood samples were taken examined for hematologic changes including differential leukocyte counts. Ovarian corpora lutea of pregnancy were counted and all uterine implantation sites were identified and recorded: resorptions (early or late), dead fetuses and live fetuses.All live fetuses were examined for external malformations, including cleft palate, and variations. About 50% of the rat fetuses in each litter were examined for visceral malformations and variations, 50% of the fetuses in each litter were examined for craniofacial defects and the other 50% (intact fetuses) were examined for skeletal malformations and variations.

In rats, maternal toxicity was observed at 40 and 85 ppm, including transient reductions in maternal body weight and weight gain during the exposure period and clinical signs of toxicity at 85 ppm, and reduced weight gain during the exposure period at 40 ppm. Maternal food consumption was reduced at 85 ppm during the exposure period (and increased in the postexposure period). Water consumption was increased at 85 ppm during and after the exposure period. There were no treatment-related clinical signs, gross necropsy observations or hematologic changes. Gestational parameters, including corpora lutea, total, nonviable or live implantations per litter, pre- or postimplantation loss, sex ratio or fetal body weights (males, females or total) per litter, were unaffected by exposures. There were no effects of treatment on the incidence of malformations or variations when examined by individual finding, findings by category or total findings.

Exposure to the test substance vapor during organogenesis in Fischer 344 rats resulted in transient maternal toxicity at 85 and 40 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 20 ppm for rats and the NOEL for developmental toxicity was 85 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
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 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
dosing from GD0-20. 25% of each dose animals allowed to deliver naturally.
Principles of method if other than guideline:
25% of the animals from each dose were allowed to deliver and animals weaned effectively creating a satellite group to assess postnatal toxicity to offspring. Treatment period longer than normal.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks
- Diet: ad libitum Basal diet CE-2 ex Clea, Japan
- Water: tap, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 50-60
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): basal diet ex Clea, Tokyo
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 0 - 20 of gestation.
Frequency of treatment:
continous (feed)
Duration of test:
to GD20
Remarks:
Doses / Concentrations:
0.04, 0.2, 1% in diet (equivalent to 25, 115 and 633 mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes
Details on study design:
Duration of test: 75% of dams killed on day 20 of gestation and the remainder allowed to deliver spontaneously.
- Dose selection rationale: Range finder experiment with dietary doses of 0.2, 1, 5, 10% in diet. 5 and 10% in diet produced marked body weight and food consumption reduction therefore 1% selected as test dose maximum for main study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, daily.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: No

OTHER: Placental weight.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations (pre and post implantation losses): Yes
- Number resorptions: Yes
- Other: live and dead fetuses, sexed and weighed
Fetal examinations:
'Litter' is only 75% of animals per dose as 5/6 were per dose were allowed to litter for a toxicity to reproduction satellite group;

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (fixed in Bouins solution)
- Skeletal examinations: Yes: half per litter (stained with Alizarin red S)
- Head examinations: No
Statistics:
Litter used as unit. Student's t test for body weight (maternal and fetus), tissue weights, food consumption, corpora lutea, implantations, live fetuses, live newborn per litter, degree of ossification and gestation time. Wilcoxon's rank sum, Chi-squared test with Yates' correction or Fisher's exact test for: pre/post implantation losses, all skeletal defects, live birth index, survival rates, sex ratio.
Indices:
not reported
Historical control data:
not reported
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight gain significantly lower than control group at all doses (12-18%) but with no dose response relationship. No other significant effects were noted
Dose descriptor:
NOAEL
Effect level:
633 mg/kg bw/day
Basis for effect level:
other: no adverse effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on any implantation parameters, corpora lutea, live fetus numbers, sex ratios, fetal or placental body weight. No external or internal anomalies were found. One fetus showed skeletal defects (fused sternebrae) in each of the mid and high dose groups. Skeletal variations seen in all dose groups; high dose group rate was higher than control but not statistically significant. Delayed sternebrae ossification was seen in all dose groups with some evidence for a dose response but this was not statistically significant from controls. The degree of ossification of the low dose group was significantly lower than controls but not from the other dose groups.
Dose descriptor:
NOAEL
Effect level:
633 mg/kg bw/day
Basis for effect level:
other: no adverse effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Substance intakes are shown in the section on doses. Skeletal findings are regarded as spontaneous as all are reported to occur historically in control rats (Kameyama, Cong Anom, 20, 25, 1980; Palmer AK, Methods in Perinatal Toxicology, ed Merker, 52, 1977)

Satellite group: There was no significant effect on any of the parameters measured. It should however be borne in mind that the statistical power of this part of the study is low with only 5 or 6 animals per dose group. There was slight evidence for a fall off in survival rate, number of implantation remnants per litter and live newborns per litter but this was far from clear and levels in the low dose group were higher than controls. The only sound conclusion that can be drawn is those of the study author's that there is no evidence for any adverse effects.

Conclusions:
There was no sigificant evidence for developmental toxicity. Maternal and offspring findings were either not biologically significant, or in the case of the single finding that was biologically plausible, not statistically significant.
Executive summary:

In a study that conformed to the basic requirements of the relevant OECD guideline, 2 -(2 -butoxyethoxy)ethanol produced no significant evidence of developmental toxicity when fed to rats in their diet at doses up to 633mg/kg bw/day during the whole gestation period (GD0 -20). A small satellite group which looked at residual effects in pups after birth for up to 10 weeks also failed to show any effects on the parameters assessed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
633 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Species:
rat
Additional information

There are no data available for DEGHE. To cover this endpoint, 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 EGHE

In a teratogenicity study comparable to OECD guideline 414, 25 rats per group inhaled EGHE vapours from gestation day 6 to 15 for 6h each day(DOW, 1987). The concentrations used in the whole body exposures were 20, 40, and 85ppm. The top dose was the highest vapour concentration, which could be achieved.

Body weight, food consumption, and clinical signs were regularly monitored in maternal animals. After sacrifice, all organs and haematological parameters were examined in addition to the number of corpora lutea, number of implantations, resorptions, and live versus dead foetuses. Gross examinations were performed for all foetuses per litter, while half of the foetuses were used for either soft tissue and head or skeletal examinations.

Treatment related clinical signs (urine stains on fur, lacrimation, ocular wetness and encrustation) were observed during the exposure period to 85ppm. There were significant decreases in maternal body weight and weight gain at both the 40 and 85 ppm dose groups and food consumption at 85 ppm dose group. Water consumption was increased at the 85 ppm. At scheduled necropsy, there were no treatment related findings at maternal gross examination. There were no changes in the haematological parameters, on maternal organ weights, gravid uterine weight, and gestational body weight. There were no effects of exposure on gestational parameters including number of corpora lutea per dam, number of total, non-viable or live implantations per litter, pre- and post-implantation loss, sex ratio and fetal body weight. The incidence of fetal malformations and variations were comparable between the treatment groups. The NOEL for maternal toxicity was 20ppm, the NOAEL for teratogenicity was 85ppm (app. 0.5mg/l).

Data generated with DEGBE

In a well-documented study performed similar to OECD guideline 414, 20 rats per group were exposed via feed from gestation day 0 to 20(Ema, 1988). 25% of the animals from each dose were allowed to deliver naturally. Concentrations of 0.04, 0.2, and 1% correspond to daily doses of about 25, 115, and 633mg/kg b.w. The maximum dose was selected based on a pre-study with dietary doses of 0.2, 1, 5, and 10% in diet. 5 and 10% produced marked body weight and food consumption reduction therefore 1% selected as test dose maximum for the main study.

Animals were monitored daily for clinical signs, body weight changes, and food intake. Post-mortem examinations included assessment of the number of corpora lutea, number of implantations, number of resorptions, number, sex, and weight of live and dead foetuses, and calculation of pre- and post-implantation loss. External examinations were performed on all foetuses. Half of the foetuses each were assessed either for soft tissue or skeletal alterations.

The only effect on the maternal animals was a lower body weight gain (-12 - -18%) in all doses, but without dose response relationship. There were no effects on any implantation parameters, corpora lutea, live fetus numbers, sex ratios, and fetal or placental body weight. No external or internal anomalies were found. One fetus each showed skeletal defects (fused sternebrae) in the mid and high dose groups. Skeletal variations occurred in all dose groups. The rate was slightly higher in the high dose group compared to control animals, but the difference was not statistically significant. The degree of ossification of the low dose group was significantly lower than controls but this effect was not seen in any of the other dose groups. Consequently, the NOAEL for maternal, developmental, and feto-toxicity was 633 mg/kg.

Additional information

Teratogenicity studies according to OECD 414 have been performed also in rabbits with DEGBE and EGHE. No developmental or embryotoxic effects were observed.

Justification for classification or non-classification

In all studies either with DEGHE or the read across substances, no effect on fertility or developmental toxicity was observed. Consequently, classification is not warranted according to CLP Regulation (EC) No. 1272/2008.