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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study
Reference
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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%.

Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 function / performance (P0)

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.

Details on results (P0)









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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
no treatment related effects

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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 .

Applicant's summary and conclusion