Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two studies are available for this endpoint. On OECD 422 combined repeated dose/reproductive toxicity screen on the substance being registered (Di-EPh) and a 2-generation reproductive toxicity study using the read across analogue, EPh.

Link to relevant study records
Reference
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).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3650
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals 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.)
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.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in diet
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
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)
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)
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
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.
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.
Reproductive effects observed:
not specified

None

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.

 

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

Data on Di-EPh:

In a well conducted OECD 422 screening study in rats there were no effects on any reproductive parameter assessed at any dose level (top dose, 1000 mg/kg bw/day).

Supporting data from EPh:

A multi-generation reproduction study evaluating 2-phenoxyethanol according to the Reproductive Assessment by Continuous Breeding Protocol (NTP, 1984) was conducted by the National Toxicology Program and reported in a peer-reviewed journal (Heindel et al, 1990). In this study, male and female mice were given 2-phenoxyethanol in feed at dose levels of approximately 0, 375, 1875 or 3,700 mg/kg bw/day. The oral feeding with 2-phenoxyethanol resulted in loss of body weights (-2%) of high-dose males compared to controls, while female body weights were unaffected. There were also no significant differences in feed consumption. Liver weights were increased at the highest-dose level. 2-Phenoxyethanol had no effect on fertility. However, the number of litters/pair, the litter size, proportion of pups born alive and pup body weights were decreased at the highest dose. The only effect observed at the mid-dose level consisted of an equivocal lower pup body weight. There were no effects on any of these parameters at the low-dose level. A crossover mating trial demonstrated no effects on mating or fertility, however, pup body weights of females given 3,700 mg/kg bw/day of the test substance were lower than the controls. In summary, there was evidence of reproductive toxicity only at very high doses (almost 4x the current recommended limit dose for this assay). At the mid dose of 1875 there was an equivocal effect on pup bodyweight only. Based on these minimal findings, it is likely that the actual NOEL for reproductive effects is >1000 mg/kg bw/day.

The available data on both EPh and Di-EPh indicate an absence of reproductive toxicity in two species at doses lower than 1000 mg/kg bw/day.


Short description of key information:
2 studies are available for this endpoint. On OECD 422 combined repeated dose/reproductive toxicity screen on the substance being registered (Di-EPh) and a 2-generation reproductive toxicity study using the read across analogue, EPh.

Justification for selection of Effect on fertility via oral route:
K1 reliable OECD guideline study

Effects on developmental toxicity

Description of key information
No developmental toxicity studies are available for Di-EPh. The results of the OECD 422 screening study can be used to support this endpoint, however the developmental toxicity data from rats and rabbits using EPh priovide the basis for the developmental toxicity characterisation.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Sep 2005 - 07 Oct 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP
Justification for type of information:
Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Experimental Toxicology and Ecology BASF Aktiengesellschaft
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH [former: Charles River Laboratories, Germany]
- Age at study initiation: 11 to 13 weeks
- Weight at study initiation: 144.9 – 185.3 g
- Housing: Rats were housed singly from day 0-20 p.c. in type DK III stainless steel wire mesh cages.
- Diet (ad libitum): Ground Kliba maintenance diet mouse/rat "GLP"
- Water (ad libitum): tap water
- Acclimation period: 6 days


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


IN-LIFE DATES: 1st cohort From: 27 Sep 2005 To: 17 Oct 2005; 2nd cohort From: 28 Sep 2005 To: 18 Oct 2005
Route of administration:
oral: gavage
Vehicle:
other: tap water and a few drops Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance emulsions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification. For the preparation of the emulsions, appropriate amounts of the test substance were weighed into calibrated beakers and were topped up with tap water and a few drops Tween 80 (as emulsifier). Afterwards they were intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the emulsions homogeneous during the procedure of dosing the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in demineralized water for a period of at least 96 hours at room temperature were carried out before the study was initiated (in a similar batch). Samples of the test substance emulsions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the first concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (100 and 1,000 mg/kg body weight/day). Three samples (one from the top, middle and bottom in each case) were taken for each of these
concentrations from the beaker with a magnetic stirrer running.
Details on mating procedure:
The animals were paired by the breeder and supplied on day 0 post coitum (= detection of vaginal plug / sperm).
Duration of treatment / exposure:
gestation day 6-19 (post mating)
Frequency of treatment:
once daily
Duration of test:
28 days
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Animals were checked from day 0 to 20 p.c. at least once daily for clinical symptoms whereas check for mortality was conducted twice a day on working days, and once a day during weekend or public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 post mating (p.c.).


FOOD CONSUMPTION was recorded on days 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c..


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

The scope of examinations exceeded the respective test guideline requirements. It was enhanced by clinical pathology examinations (hematology and clinical chemistry). For this purpose, blood samples were collected from the retroorbital venus plexus at study termination.
Hematology
The following parameters were determined in blood:
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobin
• mean corpuscular hemoglobin concentration
• platelets
• differential blood count
• reticulocytes

• prothrombin time (Hepato Quick's test)


Clinical chemistry
An automatic analyzer was used to examine following clinical chemical parameters:
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatase
• γ-glutamyltransferase
• sodium
• potassium
• chloride
• inorganic phosphate
• calcium
• urea
• creatinine
• glucose
• total bilirubin
• total protein
• albumin
• globulins
• triglycerides
• cholesterol
• magnesium


Ovaries and uterine content:
At study termination and following blood samples collection, the animals were sacrificed. The sacrificed animals as well as the one animal of the 1,000mg/kg bw/day group that was prematurely killed because of moribund state were subjected to necropsy and gross pathology, and the ovaries and uterus were removed for following examination:

Gravid uterine weight (excepted for the prematurely sacrificed animal)
Number of corpora lutea
Number of implantations (implantation sites, live/dead implantations)
The conception rates, as well as the pre- and post-implantation losses, were calculated.
Fetal examinations:
Litter size, number of dead fetuses, fetal weight, sex ratio, external alterations. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded.

Following sacrifice, 50% of the fetuses were prepared for the purpose of skeletal examination.

The remaining half of the sacrificed fetuses was prepared for the purpose of visceral examination.
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Pairwise comparison of each dose group with the control group using FISHER'S EXACT-test (one-sided) for the hypothesis of equal proportions.
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Non-parametric one-way analysis using KRUSKAL-WALLIS-test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Test group 3 (1,000 mg/kg b.w./day):
• one high dose female was sacrificed in moribund condition, preterminally, lateral position, unsteady gait, vaginal hemorrhage, salivation and respiratory sounds were observed
• all dams at least once showed unsteady gait (from day 6 p.c. onwards) and transient salivation (from day 8 p.c. onwards), although not all animals exhibited these findings on any one day, those findings persisted for a short time (up to 3.5 hours) after the dosing
• individual dams were found in lateral position shortly after treatment (5/25), had vaginal hemorrhage (2/25) and/or urine smeared fur (2/25) on several occasions during dosing
• mean food consumption was statistically significantly reduced (at maximum 10 % below control) on days 6 - 13 p.c., if calculated for the entire treatment period (days 6 - 19 p.c.), the food intake was about 6 % below control
• mean body weights were slightly, but statistically significantly below control on days 8 – 20 p.c., on day 20 p.c. mean body weight was about 4 % below control
• statistically significantly lower body weight gain was noted after initiation of treatment (days 6 - 8 p.c., about 44 % below control), average weight gain during entire treatment period (days 6 - 19 p.c.) was statistically significantly below control (about 12 %)
• net maternal body weight was about 4 % below control, net maternal body weight gain was about 14 % below control

No test substance-related adverse effects on dams in test group 1 (100 mg/kg bw/day) and test group 2 (300 mg/kg bw/day).
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Gestational parameters:
At terminal sacrifice 22 - 25 females/group had implantation sites.
2-phenoxyethanol had no influence on gestational parameters up to and including the limit dose of 1,000 mg/kg bw/day.

Developmental toxicity:
2-phenoxyethanol induced no signs of developmental toxicity.

Fetal external, soft tissue and skeletal observations:
The external and skeletal malformations in one individual fetus from control and in 2 low dose group fetuses (0 and 100 mg/kg bw/day),


Fetal external, soft tissue and skeletal observations:
The external and skeletal malformations in one individual fetus from control and in 2 low dose group fetuses (0 and 100 mg/kg bw/day), which were related to the head/skull and sternebra, are considered to be spontaneous findings.
No soft tissue malformations were recorded at all. If all the different types of malformations are summarized, in total one out of 210 examined control fetuses [= 0.5 %] in one out of 25 litters [= 4.0 %], 2 out of 206 low dose fetuses [= 1.0 %] in 2 out of 25 litters [= 8.0 %], none of the 204 mid dose fetuses (from 23 litters) and none of the 180 high dose fetuses (from 22 litters) showed malformations.
The mean percentages of affected fetuses/litter with total malformations amounted to 0.4, 1.0, 0.0, and 0.0 % at 0, 100, 300 or 1,000 mg/kg bw/day respectively. These low, not dose-related incidences do not suggest any relationship to the test substance.
External variations did not occur in any of the fetuses in this study. Soft tissue variations, in the form of dilated renal pelvis or short innominate occurred in all test groups including the controls without any relation to dosing. The skeletal variations consisted primarily of small disturbances or transient delays of ossification. Most of the skeletal variations were equally distributed among the different test groups including controls, if normal biological variation is taken into account. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a relation to dosing, were not considered to be of any toxicological relevance and/or can be found at a comparable frequency in the historical control data. If all variations are summarized, in total 119 of the 210 examined control fetuses [= 57 %] in all 25 litters [= 100 %], 107 of the 206examined low dose fetuses [= 52 %] in all 25 litters [= 100 %], 110 out of 204 mid dose fetuses [= 54 %] in all 23 litters [= 100 %] and 104 out of 180 high dose fetuses [= 58 %] in all 22 litters [= 100 %] showed variations. The mean percentages of affected fetuses/litter with total variations amounted to 57.2, 52.3, 54.0, and 58.1 % at 0; 100; 300 or 1,000 mg/kg bw/day, respectively. These overall incidences do not suggest a treatment-relationship, but reflect the usual biological variation inherent in the strain of rats used for this experiment. A spontaneous origin is assumed for the unclassified external and the unclassified cartilage observation, that were recorded for very few fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 or 1,000 mg/kg bw/day). Distribution and type of these findings (i.e. blood coagulum around placenta, bipartite processus xiphoideus, notched cartilage between basisphenoid and basioccipital and notched manubrium) do not suggest any relation to treatment. Thus, the oral administration of phenoxyethanol to pregnant Wistar rats had no effect on development and morphology of offspring at any of the dose levels tested (100, 300 and 1,000 mg/kg bw/day).

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: the oral administration of phenoxyethanol to pregnant Wistar rats had no effect on development and morphology of offspring at any of the dose levels tested (100, 300 and 1,000 mg/kg bw/day).
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified

Hematology and serum enzyme examinations revealed no treatment-related changes. Clinical chemistry investigations showed decreased concentrations in total bilirubin, total protein, albumin and globulin as well as increased triglyceride values in the serum of the high dose animals (1,000 mg/kg bw/day). Similar effects on clinical chemistry parameters, with somewhat lower severity, were noted in the mid dose animals (300 mg/kg bw/day). All of these findings were rather related to adaptive metabolic changes, than indicative of particular organ toxicity. Although these effects were likely to be caused by the test material, they were not considered as adverse per se.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was comparable to a guideline study with acceptable restrictions
Justification for type of information:
Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
test substance was applied dermal; gravid uterine weight was not determined
GLP compliance:
yes
Remarks:
Mammalian and Environmental Toxicology Research Laboratory
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton-Dutchland, Inc. (Denver)
- Weight at study initiation: 3.5-4.5 kg
- Housing: Animals were housed in wire-bottom cages.
- Diet (ad libitum): Certified Laboratory Rabbit Chow #5322, Ralston Purina Company, St. Louis, MO
- Water (ad libitum): municipal tap water
- Acclimation period: at least two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 54-80
- Photoperiod (hrs dark / hrs light): 12

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
artificial insemination according to Gibson et al., 1966
Duration of treatment / exposure:
days 6-18 of gestation
Frequency of treatment:
once daily
Duration of test:
28 days
No. of animals per sex per dose:
25
Control animals:
other: distilled water
Details on study design:
Control animals were treated with distilled water at a targeted volume of 0.91 ml/kg body weight. The targeted dose volume of undiluted 2-phenoxyethanol (specific gravity = 1.1) was 0.27, 0.55 and 0.91 ml/kg for the targeted dose levels. The highest dose of 1000 mg/kg/day was the maximum amount of liquid which could be applied to the clipped area on the animal´s back without excessive run off at the time of application or subsequent loss from absorption through he gauze liner into the cloth.
The methods of application and occlusion of the application site were similar to those developed by Quellette et al., 1983. A section (10x15 cm) on the back of each rabbit was clipped free of hair with electric clippers on day zero of gestation. On day 6 of gestation, an occlusive bandage of absorbent gauze, non-absorbent cotton and cloth held in place with adhesive tape was placed over the dosing area on the back. Rabbits remained in these bandages throughout the dosing period. The bandages were loosened in order to apply the test material each day and then retaped with adhesive tape. Bandages were replaced as needed during the treatment if they became loose or damaged due to the animal´s movement. On day 19 of gestation, the bandages were removed and the area of application wiped to remove any residue of test material.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded daily throughout the treatment period (day 6 to 18 of gestation) and thereafter on days 19 and 28 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28

Animals were observed daily throughout the experimental period for indications of toxicity. Animals found dead or moribund were submitted to gross pathological examination. Statistical analysis of body weight and body weight gain were performed using data recorded on gestation days 6, 9, 12, 15, 19 and 28. In order to assess hematologic effects observed in dead and moribund animals, blood was collected from an ear vein from approximately 10 animals per dose group (0, 300 and 600 mg/kg b.w./day) on day 19 of gestation for the following measurements: packed cell volume (PCV), hemoglobin (HgB), erythrocyte count (RBC), total leukocyte count (WBC), red blood cell indices (MCV, MCM, MCHC), reticulocyte count, red cell osmotic fragility, WBC differential counts and platelet count. Urine was collected at the time of necropsy from the bladders of two moribund rabbits via aspiration for urinalysis. Maternal liver weights were recorded and sections of skin from the application site were collected from all animals at caesarean section. In addition, liver, kidney, spleen, bone marrow (sternum and vertebra) and urinary bladder were collected at either caesarean section or necropsy.





Ovaries and uterine content:
Following caesarean section, the number of corpora lutea and the number and position of implantations, resorptions and live or dead fetuses were recorded. The uteri of apparently non pregnant females were stained and examined for evidence of early implantation sites.
Fetal examinations:
Fetal examinations included litter size, number of dead fetuses, foetuses weights, crown-rump length measurement, sex determination and external alterations.

All fetuses were preserved and examined for skeletal alterations.

The number of fetuses (and litters) externally examined from the control, 300, 600 and 1000 mg/kg bw/day/ groups were 128 (17), 142 (20), 136 (15) and 49 (5), respectively; one half of each litter was examined for evidence of visceral alterations.
Statistics:
Statistical assessment: Maternal and fetal body weights, absolute and relative organ weights, hematologic variables and fetal length data were analyzed using a parametric or nonparametric analysis of variance followed by a Dunnett's test or the Wilcoxon rank sum test with Bonferroni's correction where appropriate. Preimplantation loss, resorptions, and fetal alterations were analyzed by a censored Wilcoxon test with Bonferroni's correction. Corpora lutea, implants and litter size were analyzed with a nonparametric analysis of variance followed by the Wilcoxon rank sum test with Bonferroni's correction. Pregnancy rates were analyzed by the Fisher's exact probability test and fetal sex ratios were analyzed by a binomial distribution test. The critical value for statistical significance was p<0.05.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Four rabbits in the 600 mg/kg bw/day group and 3 in the 1000 mg/kg bw/day group had darkened areas of skin at the application site. Staining in the perineal region or dark colored urine was noted in several animals in these groups. Nine rabbits in the 1000 mg/kg bw/day and five in the 600 mg/kg bw/day groups died or were killed moribund. Most deaths occurred between gestation days 11 and 18. Most of these animals had dark colored urine in the bladder, jaundice and dark kidneys. Gross findings were consistent with hemolysis. In moribund animals for which hematological parameters were evaluated, red blood cell and platelet counts were severely depressed, reticulocytes were elevated, and red cell fragility was increased. There were no intact red blood cells in the urine sediment. Blood from animals that survived treatment with 600 or 1000 mg/kg bw/day appeared normal. The specific cause of death for 3 animals (2 at 600 mg/kg bw/day and one at 1000 mg/kg bw/day) could not be determined. With the exception of 5 animals that had already survived to day 28 due to staggered initiation, all remaining animals of the 1000 mg/kg bw/day group that survived to day 18 were killed on this day for humane reasons with no further observations. Animals in the 600 (N = 20) and 1000 mg/kg bw/day (N = 5) groups that survived to day 28 of gestation had no evidence of treatment-related effects. No signs of maternal toxicity were seen at 300 mg/kg bw/day. No differences in body weight gains or absolute or relative liver weights were observed between treated rabbits or controls.
Dose descriptor:
LOAEL
Effect level:
ca. 600 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
> 600 mg/kg bw/day (nominal)
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gestational parameters and developmental toxicity:
Treatment with 300 or 600 mg/kg bw/day had no adverse effect on the pregnancy rate, the number of corpora lutea, implantations, resorbed implantations, or live fetuses per litter or fetal body measurements.
Teratogenicity:
Treatment with up to 600 mg/kg bw/day had no effect on the incidence or type of external, visceral or skeletal malformations. Single occurrences of hemivertebrae and clinodactyly were observed among litters of dosed animals. The low incidences of these malformations are considered to be of sporadic occurrence and not indicative of a treatment-related effect. Fetuses from the 5 animals treated with 1000 mg/kg bw/day that survived to day 28also did not exhibit external, visceral or skeletal alterations (No statistical evaluations were performed on these data).
Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment with 300 or 600 mg/kg bw/day had no adverse effect on the pregnancy rate, the number of corpora lutea, implantations, resorbed implantations, or live fetuses per litter or fetal body measurements.
Remarks on result:
other: Treatment with up to 600 mg/kg bw/day had no effect on the incidence or type of external, visceral or skeletal malformations.
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Additional information

Data on Di-EPh

In a well conducted OECD 422 screening study in rats there were no effects on any reproductive parameter assessed at any dose level (top dose, 1000 mg/kg bw/day).

Data on EPh

In prenatal developmental toxicity studies, no effects on the developing foetus were seen in rats and rabbits. In rats, oral administration of 2-phenoxyethanol elicited distinct signs of maternal toxicity (decreased bodyweight gain and reduced feed consumption) at a dose level of 1,000 mg/kg bw/day (BASF AG, 2006). The test compound had no influence on gestational parameters and induced no signs of developmental toxicity up to and including the highest test dose of 1,000 mg/kg bw/day. Therefore, the NOAEL for prenatal developmental toxicity was 1000 mg/kg bw/day.

In rabbits, dermal administration of 300, 600 and 1000 mg/kg bw/day resulted in intravascular red blood cell haemolysis and death of some dams (Dow Chemical USA, 1985 and 1987) of the mid and high dose groups. In the high dose group the survival rate of the dams was too low to definitively assess the developmental toxicity potential of EPh. However in this group the fetuses from animals which survived to day 28 did not exhibit external, visceral or skeletal alterations. No developmental toxicity was observed in the mid and low dose groups. The NOAEL for teratogenicity and embryotoxicity was >600 mg/kg bw/day and for maternal toxicity was 300 mg/kg bw/day.

The read across argumentation appended to this dossier in section 13 provides the justification for the use of read across to address this endpoint, and based on that justification it is considered justified to conclude that Di-EPH would not be a developmental toxicant based on the absence of developmental toxicity observed with EPh.


Justification for selection of Effect on developmental toxicity: via oral route:
Reliable developmental toxicity study on the analogue substance used for read across

Justification for classification or non-classification

Di-EPh does not meet the criteria for classification as a reproductive or developmental toxicant.