2-(2-phenoxyethoxy)ethanol

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: OECD 422 screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 422 and EPA OPPTS 870.3650 and in accordance with the Principles of Good Laboratory Practice (GLP).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-phenoxyethoxy)ethanol

EC Number:

203-227-5

EC Name:

2-(2-phenoxyethoxy)ethanol

Cas Number:

104-68-7

Molecular formula:

C10H14O3

IUPAC Name:

2-(2-phenoxyethoxy)ethan-1-ol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Diethylene Glycol Mono Phenyl Ether
- Physical state: liquid
- Analytical purity: 99.92%
- Impurities (identity and concentrations): no information available
- Composition of test material, percentage of components: The purity of the test material was determined to be 99.92%, after correcting for water, by gas chromatography with identification by proton nuclear magnetic resonance and gas chromatography mass spectrometry.
- Lot/batch No.: Lot # 201103261-7-18

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: approximately 8 weeks at initiation of treatment
- Weight at study initiation:
- Fasting period before study: not applicable
- Housing: housed singly in solid stainless steel cages
- Diet (ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form.
- Water: Municipal water was provided ad libitum
- Acclimation period: Animals were acclimated to the laboratory for at least one week prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for two weeks prior to breeding of the P1 adults. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data. Subsequently, the concentrates of the test material in the diets were calculated from the most recent body weight 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. Following breeding, the male diet was prepared weekly until necropsy. During gestation and lactation, females from each dose group were provided with the appropriate dietary concentration of diethylene glycol mono phenyl ether given during breeding.

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: 2 weeks of cohabitation
- Proof of pregnancy: [vaginal plug/sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility was not done.
- After successful mating each pregnant female was caged: The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Confirmation and Homogeneity
Analyses of all dose levels, plus control and premix, were determined from the first mix of the main study prior to the start of dosing. Representative samples of the test diets at the lowest and highest concentration were evaluated for homogeneity concurrent with the dose confirmation analysis. The method used for analyzing the test material in the diet was liquid chromatography-mass spectrometry (LC-MS).
Stability
Diethylene glycol mono phenyl ether was shown to be stable in rodent feed for 73 days at a concentration of 10% (w/w) and 63 days at a concentration ranging from 0.005 to 0.5% (Malowinski and Fiting, 2012). Analysis was performed by high performance liquid chromatography with positive ion electrospray ionization and tandem mass spectrometry detection operating in the multiple reaction monitoring mode (HPLC/ESI-MS/MS). Test diets for the current study were prepared and used within these stability limits.
Analyses of all test diets from the first mix of the main study revealed mean concentrations ranging from 97.6 to 102.1% of targeted concentrations. Analyses of the low-dose female and high-dose male diet indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 0.9%.

Duration of treatment / exposure:

Males were fed the test diets for two weeks prior to breeding and continuing throughout breeding (two weeks) for a treatment period of 34 days. The males were necropsied on test day 35. Females were fed the test diets for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Females were necropsied on post-partum day 5.

Frequency of treatment:

continuous exposure

Details on study schedule:

Males were fed the test diets for two weeks prior to breeding and continuing throughout breeding (two weeks) for a treatment period of 34 days. The males were necropsied on test day 35. Females were fed the test diets for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Females were necropsied on post-partum day 5.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered diethylene mono phenyl ether via the diet at concentrations supplying 0, 100, 300, and 1000 mg/kg/day.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Due to the lack of additional relevant previous toxicity information on DiEPh, a preliminary range-finding study was performed to aid in dose level selection for the main study. This range-finding study also included a characterization of the metabolism of DiEPh and toxicokinetics of DiEPh and relevant metabolites. In the range-finding study, five Crl:CD(SD) rats/sex were administered DiEPh via diet for 14 days at dose levels of 0, 250, 500, or 1000 mg/kg/day. All animals survived to the scheduled termination and had no treatment-related clinical observations. Although there were some minor differences in body weight and feed consumption for animals, the effects were slight and supported use of 1000 mg/kg/day as the high dose level for the definitive study. Organ weight changes were limited to higher relative liver weights in males given 1000 mg/kg/day. There were no treatment-related gross observations recorded at necropsy.

- Other: Qualitative Metabolic Biomarker Identification - A series of analytical screening experiments were conducted to determine the relevant biomarkers in rat plasma and urine following dietary administration of DiEPh at dose levels of 0, 250, 500, or 1000 mg/kg/day. The proposed metabolic scheme of DiEPh is presented in Text Figure 1 which shows the likely metabolic pathways and related biomarkers that were screened. These target analytes included: ethylene glycol (EG), diethylene glycol (DEG), phenoxy ethanol (PE), phenol, hydroxylethoxy acetic acid (HEAA), diglycolic acid (DGA), glycolic acid (GA), and oxalic acid (OA), diethylene glycol mono phenyl ether (DiEPh), phenoxyethoxy acetic (PEAA), and phenoxy acetic acid (PAA). These screening experiments identified the relevant biomarkers as DiEPh, PEAA, and PAA (Appendix F). The levels of the other potential biomarkers were low relative to both background PEAA or PAA levels. Therefore, no definitive quantitative data was generated for EG, DEG, PE, HEAA, DGA, GA, or OA.

Quantitative Toxicokinetic Assessment - DiEPh was not present at levels above the lower limit of quantitation (LLQ) in most of the blood samples (Appendix Tables 10 and 11). The two metabolites, PEAA and PAA, were present at higher levels in the blood. PEAA was present well above the limit of quantitation and in nearly all of the blood samples from exposed animals (Appendix Table 12), while PAA was present in 50 percent of the blood samples. For this reason, systemic exposure (AUC24h) and elimination half-life (t½) values could only be calculated for PEAA. PEAA systemic exposure (AUC24h) values were dose-proportional in both males and females (Appendix Figure 1). Half-life values based on PEAA blood levels were approximately 7.4 hours, on average, and ranged 3.9-15.6 hours.
DiEPh, PEAA, and PAA all were present at concentrations above the LLQ in all urine samples from treated animals. On average, only 1.3 percent of the dose was excreted as DiEPh in the 24 hour urine, whereas 39.3 percent of the dose was excreted as PEAA and 4.3 percent of the dose was excreted as PAA. Urine levels of all three analytes were dose-proportional. These data suggest DiEPh was rapidly metabolized at all dose levels. A major portion of the dose was metabolized to PEAA (≥ 39.3% of the administered dose, 87.5% of the recovered dose). A smaller portion was metabolized to PAA (≥ 4.3% of the administered dose, 9.6% of the recovered dose). There was no apparent saturation of absorption, distribution or elimination at any dose level. Thus, DiEPh exhibits linear kinetics up to, and including, 1000 mg/kg/day.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 gestation days (GD) 0, 7, 14, and 20. Females that delivered litters were weighed on lactation days (LD) 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

CLINICAL PATHOLOGY: Animals were fasted prior to blood collection and blood samples were obtained from the orbital sinus following anaesthesia with O2/CO2 at the scheduled necropsy

- Haematology: Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
Assays - Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), Coagulation - Prothrombin time.

- Clinical chemistry: Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana). Enzyme Activities of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Concentrations of: Albumin (ALB), Albumin/Globulin Ratio (A/G) - calculated, Cholesterol (CHOL), Creatinine (CREA), Electrolytes (NA, K, PHOS, CL and CA), Globulin (GLOB) - calculated, Glucose (GLUC),
Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), Urea nitrogen (UN)

-Urinalysis: Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water was available during this procedure. Assays - Color, appearance, specific gravity (refractometer). Semiquantitative analysis of the following was conducted using Siemens Multistix Reagent Strips on the Clinitek Advantus Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York): pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen and microscopic examination of urine sediment (pooled samples).

OTHER: The functional tests (sensory evaluation, rectal temperature, grip performance, and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4.

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

not evaluated

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 parturition 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. The following information was recorded on each litter: the 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):

SACRIFICE
- Male animals: All surviving animals after at least 4 weeks of exposure
- Maternal animals: All surviving animals on lactation day 5 or at least 24 days after the end of the mating period for females not producing a litter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The tissues indicated below were prepared for microscopic examination on all control and high dose animals -
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, caecum, 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 tissues/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, pituitary and jejunum.

Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (liver) and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.

ORGAN WEIGHTS: Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.

Postmortem examinations (offspring):

All 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 or which were euthanized in moribund condition were examined to the extent possible and discarded.

Statistics:

Standard statistical methods were employed

Reproductive indices:

Female mating index, male mating index, female conception index, male conception index, female fertility index, male fertility index, gestation index

Offspring viability indices:

gestation survival index, post-implantation loss, day 1 or 4 pup survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dietary administration of 1000 mg/kg/day DiEPh resulted in treatment-related slight decreases in feed consumption, body weight and body weight gain during gestation and slightly decreased body weights during lactation, relative to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Dietary administration of 1000 mg/kg/day DiEPh resulted in treatment-related slight decreases in feed consumption, body weight and body weight gain during gestation and slightly decreased body weights during lactation, relative to controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 1000 mg/kg/day had treatment-related very slight hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobular and midzonal hepatocytes and this was deemed to be a non-adverse adaptive effect.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) - There were no effects attributable to treatment in both the sexes.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) - There were no treatment-related effects or statistically-significant differences in body weights for males at any exposure level tested throughout the duration of the study. In females, there were no effects related to treatment on body weights during the pre-breeding phase at any exposure level tested. Although not statistically identified, females given 1000 mg/kg/day had slight treatment-related decreases in gestation body weight and/or body weight gain relative to controls during the gestation phase (up to 4.5 and 14.5%, respectively. The female body weight effects were concomitant with decreased feed consumption. Persisting from the gestation phase, females given 1000 mg/kg/day had slight decreases (up to 4.4%) in lactation body weights relative to controls. There were no effects related to treatment on lactation body weight gains in females given 1000 mg/kg/day nor on gestation or lactation body weight or body weight gain in females given 300 or 100 mg/kg/day.
Feed consumption in males was similar to controls in all exposure levels throughout the study and in females during the pre-breeding period. Consistent with the body weight effects during gestation, there was a slight treatment-related decrease (8.3%) in feed consumption in the 1000 mg/kg/day group between GD 7-14. Lactation feed consumption in the 1000 ppm group was similar to controls. There were no effects related to treatment on feed consumption during gestation or lactation for females in the 300 or 100 mg/kg/day dose groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) - Time-weighted average doses of diethylene glycol mono phenyl ether were 0, 102, 311, or 1054 mg/kg/day for males, and 0, 100, 308, or 1015 mg/kg/day for females during pre-breeding. During the gestation and lactation phases, respective time-weighted average doses of 0, 116, 337, or 1118 mg/kg/day and 0, 179, 511, or 1737 mg/kg/day were obtained for females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) - There were no effects related to treatment at any exposure level on mating, conception, fertility, gestation indices, time to mating, gestation length, postimplantation loss, pup survival, or pup sex ratio.
Percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) was higher in the 1000 mg/kg/day group relative to controls. Although the percent postimplantation loss was slightly outside of the historical control range, it was considered spurious and not attributable to treatment as this difference was not statistically significant and was primarily driven by two litters (5 of 16 and 4 of 14 resorptions). The gestation survival index in females given 1000 mg/kg/day was slightly lower than controls, however, this finding was deemed spurious and unrelated to treatment as the value was within historical control range and did not reach statistical significance.

ORGAN WEIGHTS (PARENTAL ANIMALS) - There were no treatment-related effects on final body weights of males or females at any dose level. Males and females given 1000 mg/kg/day had treatment-related statistically-significant higher relative liver weights (9.0 and 11.9% higher than controls, respectively). The absolute liver weights of males and females given 1000 mg/kg/day were also higher than controls (although not statistically significant), and were also interpreted to be treatment-related. The higher liver weights corresponded to the histopathologic observation of very slight hypertrophy (with altered tinctorial properties) of centrilobular and midzonal hepatocytes in males and females given 1000 mg/kg/day.
Females given 1000 mg/kg/day had lower absolute (statistically significant) and relative thymus weights. The lower thymus weights were interpreted to be treatment- related because they were below the historical control ranges, and most of the individual females given 1000 mg/kg/day had absolute and thymus weights that were below the concurrent control mean values for thymus weights. There was no histopathologic correlate for the lower thymus weights. Therefore, the lower thymus weights were interpreted to be non-adverse.
Females given 1000 mg/kg/day had statistically significant higher relative kidney, absolute thyroid and relative thyroid weights. The absolute kidney weight of females given 1000 mg/kg/day was also higher than controls but did not reach statistical significance. The higher kidney and thyroid weights were interpreted to be unrelated to treatment because the values were within or below historical control ranges, and there were no histopathology correlates

GROSS PATHOLOGY (PARENTAL ANIMALS) - There were no treatment related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure of DiEPh to rats.

HISTOPATHOLOGY (PARENTAL ANIMALS) - Males and females given 1000 mg/kg/day had treatment-related very slight hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobular and midzonal hepatocytes (Text Table 13). There were no treatment-related degenerative or necrotic alterations associated with the hypertrophy of hepatocytes. Hepatocellular hypertrophy was interpreted to be a non-adverse adaptive effect, likely due to the induction of hepatic enzymes necessary to metabolize DiEPh. Males and females given 1000 mg/kg/day had treatment-related lower incidences of hepatocellular vacuolization (consistent with fatty change). The cause of this alteration was not determined, but since there was a decrease in the incidence of hepatocellular vacuolization relative to controls, this finding was interpreted to be a non-adverse effect. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with dietary administration of DiEPh.

OTHER FINDINGS (PARENTAL ANIMALS) -
FUNCTIONAL TESTS -
SENSORY EVALUATION - Examinations performed on males and females at termination revealed no findings related to treatment. There were three observations in males that were statistically identified when compared to controls (alpha = 0.05). Two occurred under baseline conditions (0 vs.1000 mg/kg/day, response to sharp noise moderate, p = 0.0285; 0 vs. 100 response to tail pinch minimal, p = 0.0285), and one following treatment (0 vs. 1000 response to tail pinch pronounced, p = 0.0120). None of these statistically-significant observations were considered treatment-related due to their presence under baseline conditions, their lack of a dose response, and/or their lack of concurrence among the other severities within the observation.
RECTAL TEMPERATURE - There were no effects related to treatment on rectal temperature in males (p = 0.6210) or in females (p = 0.7627).
GRIP PERFORMANCE - There were no effects related to treatment on hindlimb grip performance either in males (p = 0.3471) or females (p = 0.6459). Similarly, there were no treatment-related effects on forelimb grip performance in males (p = 0.1286). In females, the treatment-by-baseline interaction was statistically significant, thereby violating the homogeneity of slopes assumption of the analysis of covariance. As per protocol, the analysis of covariance was replaced by a repeated measures ANOVA in females. There were no effects on forelimb grip performance in females (p = 0.8417).
MOTOR ACTIVITY - There were no effects related to treatment in motor activity. Treatment did not affect motor activity total counts (treatment x time interaction) in males (p = 0.0719). Similarly, the distribution of the motor activity counts within session (treatment x time x epoch interaction) was not affected by treatment in males (p = 0.1847). For females, both the treatment x time interaction (p = 0.0356) and the treatment x time x epoch interaction (p = 0.0179) were significant. The results of the subsequent linear contrasts for the treatment x time interaction are as follows: control vs. low x time (p = 0.1185), control vs. mid x time (p = 0.2287), and control vs. high x time (p = 0.3870). The results of the subsequent linear contrasts for the treatment x time x epoch interaction are as follows: control vs. low x time (p = 0.5569), control vs. mid x time (p = 0.9300), and control vs. high x time (p = 0.1105). The linear contrasts (control vs. treatment group) for both interactions indicated that the double and the triple interaction were not statistically significant (α = 0.02). Furthermore, the relative p values for each of the linear contrasts were not grossly different and did not tend to suggest any dose relationship.

CLINICAL PATHOLOGY -
HEMATOLOGY - There were no treatment-related hematologic effects in males or females at any dose level. Females given 100 mg/kg/day had a statistically-significant lower platelet count that was interpreted to be unrelated to treatment due to the lack of a dose response and there were no treatment-related effects on prothrombin times for males and females at any dose level.
CLINICAL CHEMISTRY - Males and females given 1000 mg/kg/day had statistically-significant higher serum urea nitrogen concentrations and males given 1000 mg/kg/day had a statistically-identified lower chloride concentration. The lower chloride in males given 1000 mg/kg/day was interpreted to be unrelated to treatment because the value was within the historical control range. Although the elevated urea nitrogen concentrations in males and females given 1000 mg/kg/day were within or slightly higher than the historical control range, several individual animals at this dose from both sexes had urea nitrogen concentrations that were higher than the concurrent control group range of values. Therefore, the higher urea nitrogen concentrations in males and females given 1000 mg/kg/day were interpreted to be a treatment-related effect. This effect was considered to be non-adverse because there were no corresponding histopathologic alterations in the urinary tract, and no evidence of other predisposing conditions such as dehydration or increased protein catabolism.
URINALYSIS - There were no treatment-related effects on urinalysis parameters for males at any dose level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

300 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on overall effects (equivalent to time weighted average dose of 311 mg/kg/day for males and ranged between 308-511 for females)

2

Key result

Dose descriptor:

NOEL

Remarks:

for reproductive and neurological effects

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on overall effects (equivalent to time-weighted average dose of 1054 for males and ranged between 1054-1118 for females)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no visible external morphologic alterations noted in any of the offspring.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: There were no treatment-related effects of DiEPh on prenatal neonatal growth and survival of the offspring.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the no-observed-effect level (NOEL) for general toxicity was 300 mg/kg/day and the NOEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.

Executive summary:

The purpose of this study was to evaluate the potential effects of diethylene glycol mono phenyl ether (DiEPh) on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and survival of the offspring, following dietary administration (conducted as per OECD TG 422 and in accordance with GLP).

Groups of 12 male and 12 female Crl:CD(SD) rats were administered DiEPh via the diet at targeted doses 0, 100, 300, or 1000 mg/kg/day which corresponded to time-weighted average doses for males of 0, 102, 311, or 1054 mg/kg/day and ranged from 0, 100-179, 308-511, or 1054-1118) mg/kg/day for females during the various study phases. Males were fed the test diets for two weeks prior to breeding and continuing throughout breeding (two weeks) for a treatment period of 34 days. The males were necropsied on test day 35. Females were fed the test diets for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Females were necropsied on post-partum day 5. 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 1000 mg/kg/day DiEPh resulted in treatment-related slight decreases in feed consumption, body weight and body weight gain during gestation and slightly decreased body weights during lactation, relative to controls. There were no effects on body weight, body weight, body weight gain, or feed consumption in males or females in the 100 or 300 mg/kg/day dose groups. The only treatment-related effect on clinical pathology parameters was a slightly higher serum urea nitrogen concentration in males and females given 1000 mg/kg/day. This effect was considered to be non-adverse because there were no corresponding histopathologic alterations in the urinary tract, and no evidence of other predisposing conditions such as dehydration or increased protein catabolism.

Treatment-related very slight hypertrophy of centrilobular and midzonal hepatocytes occurred in males and females given 1000 mg/kg/day. The hypertrophy corresponded with higher absolute and relative liver weights at this dose level in both sexes. These changes were considered to be an adaptive response associated with increased hepatic metabolism of diethylene glycol mono phenyl ether. Males and females given 1000 mg/kg/day had lower incidences of hepatocellular vacuolization (consistent with fatty change), which was interpreted to be a non-adverse effect of treatment. Females given 1000 mg/kg/day had treatment-related lower absolute and relative thymus weights, which were interpreted to be non-adverse because there were no corresponding histopathologic observations.

There were no treatment-related effects of DiEPh on neurological or reproductive function, or prenatal neonatal growth and survival of the offspring.

Based on these results, the no-observed-effect level (NOEL) for general toxicity was 300 mg/kg/day. The NOEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.