[(butoxymethylethoxy)methylethoxy]propan-1-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A two-generation inhalation reproduction study in Sprague-Dawley rats is available. The study was conducted under GLP and according to OECD guideline 416. Supporting data is available through a summary report on a continuous breeding study in mice. Both studies were conducted with the surrogate substance propylene glycol methyl ether (PGME). In addition, a GLP study (oral gavage in rats) according to OECD guideline 421 is available for dipropylene glycol butyl ether (DPnB).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

08/2005-06/2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD guideline 422

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: 8 weeks
- Weight at study initiation: (P) Males: ca. 246 g; Females: ca. 194 g;
- Housing: singly in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material from approximately GD 19 until completion of lactation.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

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

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test material was administered in a 0.5% methylcellulose vehicle, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose suspensions were prepared periodically throughout the study period based upon stability.
Stability of the test material in the vehicle was determined prior to the start of the study at concentrations of 0.250, 2.50, and 150 mg/ml. Dose suspensions for the current study were prepared and used within these stability limits.

Details on mating procedure:

Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If a breeding male died, a substitute partner (from the same dose group) that had already completed mating was provided. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The low- and high-dose suspensions from the first mix of the main study were analyzed to confirm homogeneous distribution of the test material prior to the start of the study using gas chromatography with flame ionization detection with external standards. Analysis of all dosing suspensions from the first mix of the main study were initiated prior to the start of dosing and were conducted concomitant with the homogeneity analysis.

Duration of treatment / exposure:

Male rats were dosed for 14 days prior to mating and continuing throughout the mating for 28 days. Females were dosed for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4.

Frequency of treatment:

daily

Details on study schedule:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered the test material daily, by gavage, at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29). Effects on general toxicity, gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with histopathologic examination of tissues. The key study parameters and study schedule are presented in Table 1. Gavage dosing for both males and females began on October 05, 2005. The adult males were necropsied on November 02, 2005. The adult females were necropsied on November 14, 2005 to November 28, 2005. In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed. Pups were euthanized on PND 4.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The high-dose level was based upon data obtained from a preliminary range-finding study and was expected to induce some toxic effects, but not
death or obvious suffering. In addition, the high-dose of 1000 mg/kg/day represented a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3550, Reproduction/Developmental Screening Test). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a NOEL.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Animals were examined for abnormalities such as, but not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation,
tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Any animals found dead were necropsied as soon as practical. Moribund animals not expected to survive until the next observation period were humanely euthanized and necropsied as soon as practical.

BODY WEIGHT: Yes
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly until termination. Body weight analyses were conducted for the following days: GD 0, 7, 14, 20, and LD 1 and 4. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Feed consumption was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co- housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After
parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.

Sperm parameters (parental animals):

The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Sciffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules.

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 partur ition 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: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on LD 0, 1, and 4, 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 Daily In-Life Observations). In addition, pup clinical observations were recorded on each litter on PND 0 through 4. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and then discarded.

Postmortem examinations (parental animals):

Adult males (fasted) were submitted for necropsy after at least four weeks (actual: TD 29) of exposure. Adult females (fasted) were terminated on LD 5-7, or at least 24 days after the end of the mating period for females not producing a litter. The animals were anesthetized by the inhalation of CO2 and weighed. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and were examined for the presence and number of implantation sites. After evaluation, uteri was gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the epididymides, kidneys, liver, and testes were recorded, and organ:body weight ratios calculated. Representative samples of tissues listed in Table 2 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides which were fixed in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues. During routine working hours, any animal found dead or euthanized prior to the scheduled necropsy was necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were
followed for these animals except that terminal body and organ weights were not recorded and the testes and epididymides were preserved in neutral, phosphatebuffered 10% formalin.

Histologic examination were conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups (low and mid-dose) was limited to liver, kidneys, 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. Kidneys from selected male rats were also stained with periodic acid Schiff and Mallory’s Heidenhain stain for the evaluation of hyaline droplets. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Sciffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Selected histopathologic findings were graded to reflect the severity of specific
lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions s a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tis sue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening. A complete set of tissues (listed in Table 2) was examined from rats found dead or moribund. Histological examination was conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin.

Postmortem examinations (offspring):

All pups surviving to PND 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead were examined to the extent possible and discarded.

Statistics:

See any other infromation on materials and methods.

Reproductive indices:

Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100

Offspring viability indices:

Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Examinations performed on all animals pre-exposure revealed that all animals were in good health. Two high-dose males died or were euthanized prior to the scheduled necropsy due to apparent test material aspiration associated with the dosing procedure. Male 7861 appeared to have refluxed test material and was observed with noisy respiration on TD 11. On TD 12, this rat exhibited excess salivation and labored respiration and was found dead the following morning (TD 13). Male 7870 was submitted to necropsy on TD 24 prior to dosing due to labored respiration (mouth breathing). Following microscopic examination, both animals had significant inflammation of the lungs consistent with inadvertent aspiration of test material during or following the oral gavage dosing procedure. All remaining animals survived to the scheduled termination of the study.
Transient, post-dosing salivation (recorded as clear perioral soiling) was noted sporadically in several high-dose males and females. The fact that
this salivation was observed immediately following dosing suggested a local response, perhaps to the taste of the test material. In addition,
transient (1-2 hours post-dosing), subtle incoordinated gait was observed only on the first day of dosing in three high-dose females. Incoordinated gait was not seen again for the remainder of the study. The incoordination presented itself as a slight stumbling behavior that affected both the fore- and hindlimbs when the rats jumped from their feed crock or were attempting to walk. This finding was consistent with effects noted in previously conducted repeated dose toxicity studies with propylene oxide based glycol ethers. All other observations were incidental bearing no relationship to treatment. There were no notable observations made during the cageside observations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No significant differences in body weights were observed for males at any dose level. Similarly, there were no treatment-related differences in the body weights or body weight gains of females at any dose level tested during the pre- mating, gestation, or lactation periods.

FOOD INTAKE (PARENTAL ANIMALS)
There were no significant differences in the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects at any dose level on reproductive indices, time to mating, gestation length, postimplantation loss, pup survival, or pup sex ratio. Post implantation loss of females given 300 and 1000 mg/kg/day was numerically higher, but not significantly different from controls. The differences were not dose related, and were most likely due to normal variability, which is common for postimplantation loss values from studies with small group sizes. This is evident from examination of recent historical control data. The 300 and 1000 mg/kg/day group values for postimplantation loss (8.85 and 4.76%, respectively) were well within the range of recent historical values. In addition, there were no adverse gross or histopathological findings in the reproductive organs of males or females at these dose levels. Therefore, these differences were considered to be spurious and unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no treatmentrelated effects on final body weights of males or females at any dose level. Males given 300 or 1000 mg/kg/day and females given 1000 mg/kg/day had treatmentrelated, statistically identified, higher mean absolute and relative liver weights. Males given 100 mg/kg/day had slightly increased absolute and relative liver weights. Although the absolute weights were statistically identified, these minimal absolute and relative weight changes were within the historical control range, and thus, were not considered to be treatment-related. The higher liver weights corresponded with centrilobular hepatocellular hypertrophy seen at all dose levels in males, and in highdose females. Treatment-related increases in absolute and relative kidney weights occurred in males and females given 1000 mg/kg/day. Histopathological evidence of hyaline droplet formation was noted in the kidneys of the 1000 mg/kg/day males, as well as in 300 mg/kg/day males. However, there was no histopathological correlate to the increased kidney weights of the high-dose females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related gross pathologic observations of males and females at any dose level.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males given 100, 300, or 1000 mg/kg/day and females given 300 or 1000 mg/kg/day had treatment-related increases, relative to controls, in the incidences of very slight to slight hepatocellular hypertrophy in the liver, relative to controls. The hepatocellular hypertrophy was characterized by an increase in the size of hepatocytes in a centrilobular/midzonal distribution. Hepatocellular hypertrophy was believed to be an adaptive effect, due to the induction of hepatic enzymes likely necessary to metabolize DPnB.
Male rats given 300 or 1000 mg/kg/day had treatment-related hyaline droplet formation in the renal proximal tubules (Text Table 8). The hyaline droplets stained PAS negative and positive with Mallory’s Heidenhain stain. These results were consistent with, but not diagnostic for, alpha 2m globulin. The occurrence of increased levels of this protein within the proximal convoluted tubular epithelial cells of male rats has been shown to cause tubular degeneration. However, the re were no treatment-related degenerative effects in the renal tubules at any dose level for this study. Alpha 2m globulin nephropathy in male rats has been considered not relevant for human risk assessment, as humans do not develop the nephropathy due to quantitative and qualitative differences in this protein.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: overall reproductive effects

Remarks on result:

other: Generation: overall (migrated information)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: overall systemic effects

Remarks on result:

other: Generation: overall (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
no treatment-related effects observed

CLINICAL SIGNS (OFFSPRING)
Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. One low dose pup exhibited agnathia, a malformation comprised of a missing jaw. Agnathia was not observed in pups at the higher doses and, therefore, was considered incidental and not reated to exposure to DPnB.

BODY WEIGHT (OFFSPRING)
There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Reproductive effects observed:

not specified

Conclusions:

The no-observed-effect level (NOEL) for reproductive effects was 1000 mg/kg/day, the highest dose tested.

Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered dipropylene glycol n-butyl ether (DPnB) daily, by gavage at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29). Effects on reproductive function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. On the first day of dosing with 1000 mg/kg/day of DPnB, three females exhibited a transient, subtle, incoordinated gait which resolved within 1-2 hours after dosing and was not seen again for the remainder of the study. The only other treatment-related clinical observation was transient, post-dosing salivation (clear perioral soiling) noted sporadically in several high-dose males and females. This salivation was considered to be a local response to the taste of the test material, rather than evidence of toxicity. Treatment-related increases in the incidence of hepatocellular hypertrophy occurred in males of all dose groups, and in females given 300 or 1000 mg/kg/day DPnB. The hypertrophy corresponded with increased liver weights in the 1000 mg/kg/day males and females, and 300 mg/kg/day males (absolute and relative weights affected). These changes were considered to be an adaptive response associated with increased hepatic metabolism of DPnB. Treatment-related increases in absolute and relative kidney weights also were found in males and females given 1000 mg/kg/day. In addition, hyaline droplet formation in the proximal renal tubules was observed in males given 300 or 1000 mg/kg/day. Females given 1000 mg/kg/day had treatment-related higher mean absolute and relative kidney weights, but a histopathologic correlate to the higher kidney weights was lacking. There were no treatment-related effects on any reproductive parameters. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 100 mg/kg/day, based on very slight to slight hepatocellular hypertrophy with no corresponding increases in liver weights in low-dose males. The no-observed-effect level (NOEL) for reproductive effects was 1000 mg/kg/day, the highest dose tested.

Reference 2

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

06/1995-02/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-report according to OECD guideline 416

Justification for type of information:

Please refer to category document.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY
- Age at study initiation: 6 weeks
- Weight at study initiation: (P) Males: g; Females: 120-121 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: none
- Housing: singly in wire mesh stainless steel cages
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): ad libitum except during inhalation exposure
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 40-60%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:14.5m3 (2.4 m wide x 2.4 m high x 2.4 m deep with pyramidal top)
- Method of conditioning air: the various concentrations of PGME were generated using a glass J-tube method. Liquid PGME was metered into a glass J-tube assembly through which a preheated stream of approximately 90 liters per minute of compressed air was passed to vaporize the test material. The compressed air was heated to the minimum extend necessary to facilitate complete vaporization of the test material (approximately 65°C for the 300 ppm chamber, 115°C for the 1000 ppm chamber and 150-170°C for the 3000 ppm chamber). The compressed air and PGME vapors were diluted and mixed with room temperature air to the desired concentration at a flow rate of 2900 liters per minute into whole-body inhalation chambers.
- Temperature, humidity, pressure in air chamber: 22°C, 40-60%, The chambers were operated at a slightly negative pressure relative to the surrounding area.
- Air flow rate: 2900 liters per min
- Air change rate: 12 changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

Details on mating procedure:

Breeding of the P1 and P2 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same exposure group (1:1 mating) until pregnancy occurred or two weeks had elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal plug was observed in situ
was considered day 0 of gestation. Sperm and plug positive females were then removed from the male’s cage and placed back into wire mesh, stainless steel cages. If mating did not occur during the two weeks, the animals were separated without further opportunity for mating. For the P2 mating, cohabitation of male and female litter mates was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of PGME in the breathing zone of the animals in each chamber was measured at least once per hour using a MIRAN 1A infrared spectrophotometer (Foxboro Analytical, Norwalk, CT) at a wavelength of 10.2 microns. The spectrophotometer was calibrated using standards having a known PGME vapor concentration contained in 90 liter SARANÒ or Tedlar film gas bags prior to the first exposure and approximately monthly thereafter. Daily checks of the spectrophotometer were performed prior to each exposure period using a single PGME standard concentration. In addition, the amount of PGME used each day was recorded and the nominal concentration (amount of PGME used / total chamber airflow) of PGME was calculated. Prior to the start of the study, each of the chambers to be used were checked to ensure that a uniform distribution of vapors occurred within the animal’s breathing zone.

Duration of treatment / exposure:

Exposure period: Before mating, through gestation, and post-birth.
Premating exposure period (males): 5 days/week prior to mating; 7 days/week post mating
Premating exposure period (females): 5 days/week prior to mating; 7 days/week post mating

Frequency of treatment:

6 hr/day

Details on study schedule:

Groups of 30 male and 30 female rats were exposed to 0, 300, 1000 or 3000 ppm PGME via inhalation, for 6 hours/day, 5 days/week prior to mating, and 6 hours/day, 7 days/week during mating, gestation and lactation. Treatment of the first generation parental (P1) rats began at approximately 6 weeks of age. After approximately 10 weeks of exposure (5 days/week, excluding holidays), P1 rats were mated (one male to one female of the respective treatment group) to produce the F1a litters. To aid in the interpretation of F1a litter weight data which indicated non dose-related decreased litter weights at 300 and 1000 ppm PGME (statistically identified for 300 ppm PGME day 14 females only) and to confirm significant findings noted for the 3000 ppm litters, the P1 adults were mated a second time to produce an F1b litter. Mating of the P1 adults for the second time
commenced approximately one week following weaning (3 weeks of age) of the last F1a litter. Initially, 30 males and 30 females from each treatment group were randomly selected from the F1a litters and assigned to treatment groups to become the second generation parents (P2). However, soon after exposure of the F1a weanlings commenced (1-3 days beginning on postnatal day 22) it became apparent that the weanlings in all dose groups were too small to be singly housed in wire mesh inhalation cages and to go without feed during the exposure period. Therefore, exposures were stopped. Weanlings in all dose groups appeared lethargic/weak following exposures and did not appear interested in feed or water. Given the limitations of starting the F1a litters at such a young age, and space limitations within the exposure chambers which precluded the simultaneous maintenance of the P1 and P2 generation animals, the decision was made to terminate the F1a weanlings and to choose the second generation parents from the F1b litters. Following weaning (3 weeks of age) of the F1b litters, 30 males and 30 females from each treatment group were randomly selected to become the P2 generation. To avoid the aforementioned problems with the F1a litters, exposure of the F1b weanlings/P2 adults began on their respective postnatal day 28. After approximately 10 weeks of treatment, the P2 adults were bred to produce the F2 litters. Exposures of P1 and P2 adults rats to PGME continued until the adults were sent to necropsy. All rats were housed continuously in exposure chambers following the initial exposure to PGME, except during late gestation and throughout the lactation period, when female rats were housed outside of the exposure chambers in nesting boxes. Maternal rats were not exposed to PGME after day 20 of gestation through the fourth day postpartum, in order to allow for parturition and initiation of lactation. During the lactation period, pups were not placed in the exposure chambers, but remained in the nesting boxes separated from the dam for
approximately 6 hours/day on lactation days 5 through 21.

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Rats were exposed to target concentrations of 0, 300, 1000 or 3000 ppm PGME. These concentrations corresponded to oral equivalent doses of approximately 0, 396, 1325 and 3974 mg/kg/day assuming ventilation rates of 1 l/min/kg and 100 percent absorption. The
calculated middle and high dose oral equivalents exceeded the 1 g/kg/day oral limit dose as defined by both the EPA (EPA, 1985) and OECD (OECD, 1981). The chosen concentrations were selected by the sponsor and based upon the results of the inhalation toxicity studies conducted previously.
- Rationale for animal assignment (if not random): random by body weight

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Each rat on study was observed twice daily (a.m. and p.m.) for mortality, morbidity and moribundity as well as availability of feed and water. In addition, changes in behavior or demeanor and indications of overt toxicity were evaluated during the a.m. or p.m. observation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A thorough clinical examination was conducted on all animals prior to the start of the study and weekly thereafter. This examination included evaluations of the skin and fur, mucous membranes, respiration, nervous system and behavior pattern. All adult rats found dead or in moribund condition were submitted for a gross pathologic examination. Adult rats found dead after normal working hours were refrigerated until a necropsy could be performed. All pups found dead, or pups that were euthanized in moribund condition, were examined to the extent possible for defects and/or cause of death and preserved in neutral, phosphate-buffered 10% formalin. Cannibalized pups were examined to the extent possible and discarded.

BODY WEIGHT: Yes
- Time schedule for examinations: All P1 animals had body weights recorded weekly during the 10-week pre-breeding treatment period, beginning on or before the first week of the study. Body weights for males were recorded weekly throughout the course of the study. Sperm and plug positive females were weighed on days 0, 7, 14 and 21 of gestation. Females that delivered litters were weighed on days 1, 4, 7, 14, and 21 of lactation. A similar schedule was followed for the P2 generation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Feed consumption was not measured in this study

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily by vaginal lavage (Cooper et al., 1993) for all P1 and P2 females starting three weeks prior to the F1a and F2 matings only, and continued throughout cohabitation.

Sperm parameters (parental animals):

At termination, samples of sperm from the right distal cauda epididymis from the first ten P1 and P2 males sacrificed in each exposure group were collected for evaluation of sperm motility. Sperm motility was determined with the use of the Hamilton-Thorn (HTM) Intergrated Visual Optical System (IVOS) motility analyzer (Hamilton-Thorn Research, Beverly, Massachusetts). Images of all samples for motility analyses were captured on an
optical disk and kept as raw data. The entire left cauda epididymis was weighed and then minced in saline to enumerate the total number of sperm (cauda reserves). Sperm counts were performed manually using a hemocytometer. Additionally, slides were prepared from sperm samples obtained from the left cauda epididymis for possible morphological evaluation and saved, but were not evaluated as no effects were noted on sperm counts or
motility.

Litter observations:

All litters were examined as soon as possible after delivery. The following parameters were recorded for each litter: total litter size on the day of parturition (day 0), the number of live and dead pups on days 0, 1, 4, 7, 14, and 21 postpartum, and the sex and the weight of each pup on days 1, 4, 7, 14, and 21 of lactation. Any visible physical abnormalities or demeanor changes in the neonates were recorded during the lactation period.

Postmortem examinations (parental animals):

A complete necropsy of all P1 and P2 adults was conducted by a team of trained individuals, including and directly supervised by a veterinary pathologist. The scheduled necropsy was performed after the last litter of the respective generation had been weaned, with the following exception: P1 adult males were necropsied early as it was determined that the age of the P1 adult population at the completion of the F1b lactation phase would have precluded any additional breeding of these adults. The rats were fasted overnight, anesthetized with methoxyflurane and euthanized by decapitation. The eyes were visually examined in situ through a moistened glass slide gently pressed against the cornea. Tissues routinely collected (Table 3) were saved from these rats and preserved in neutral, phosphate-buffered 10% formalin, with the following exceptions: testes and epididymides were preserved in Bouin's fixative. The lungs were infused with formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Moribund rats and those dying spontaneously were necropsied in a similar manner. However, body and organ weights were not recorded.

The following organs of the first ten P1 and P2 parental animals sacrificed were weighed: ovaries or testes, left epididymis (total and cauda), seminal vesicles (with coagulating glands and their fluids), prostate, brain, liver, kidneys, lungs, adrenal glands, spleen, and thymus, and the organ-to-body weight ratios calculated.

Histologic examination of potential target organs and reproductive tissues was performed on the control and high concentration groups. Examination of tissues from the low and middle groups was limited to those tissues which demonstrated treatment-related histologic changes at the high concentration; those tissues were the liver and ovaries. Grading for atrophic ovaries was based primarily on the number of recognizable corpora lutea of any stage. In near optimal sections the grades were as follows: Very Slight, 16- 25 corpora lutea; Slight, 6-15; and Moderate, £5. If sections were less than complete, the presence of large cystic follicles and the density of corpora lutea were also considered. A complete set of histologic slides from all tissues listed in Table 3, was prepared from all rats that died spontaneously or were euthanized in a moribund condition, and were examined in an attempt to determine cause of death.

Postmortem examinations (offspring):

One pup/sex/exposure concentration from the first ten F1 and F2 litters to be weaned was given a complete necropsy by a team of trained individuals including and under the direct supervision of a veterinary pathologist. In order to control for variation in body and organ weight, pups selected for a complete necropsy were euthanized at the same age (postnatal day 22). Pups were anesthetized with methoxyflurane and euthanized by decapitation. Terminal body weights were recorded. Gross pathologic examination and preservation of tissue samples (Table 3) was performed as described above for adults.

For the F1 and F2 pups that were examined macroscopically (one/sex of the first ten litters/concentration weaned), the following organs were weighed: ovaries or testes, brain, heart, liver, kidneys, adrenal glands, spleen and thymus. Organ to body weight ratios were calculated.

Organs that demonstrated treatment-related effects (decreased absolute or relative weight) in weanlings chosen for necropsy were examined microscopically in the control and high concentration groups and included the liver, spleen, thymus and testes. These tissues were chosen for histologic examination because their absolute and/or relative organ weights were depressed (at least one statistically significantly, the other with the same downward difference) in one or more generations of high concentration weanlings. Examination of tissues from the low and middle groups was not conducted as the morphologic changes observed among the high concentration weanlings were clearly related to severe body weight depressions, and because these organ weight changes were not statistically significant at the lower exposure concentrations.

Statistics:

Body weights, gestation/lactation body weight gains, organ weights, sperm count per gram of cauda epididymis and percent motile sperm were first evaluated by Bartlett’s test for equality of variances. Based upon the outcome of Bartlett’s test, either a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, a Dunnett’s test or the Wilcoxon Rank-Sum test with Bonferroni’s correction was performed. Gestation length, average time to mating, litter size, age at vaginal opening and age at preputial separation were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni’s correction was performed. Statistical outliers were identified by the method of Grubbs (1969), but were only excluded from analysis for documented, scientifically sound reasons. Fertility indices were analyzed by the Fisher exact probability test and Bonferroni’s correction was used for multiple testing of groups in comparison to a single control. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates were analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974).

Reproductive indices:

see statistics

Offspring viability indices:

see statistics

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 3000 ppm PGME, sedation as evidenced by incoordination and decreased activity which resolved by the next exposure day was observed in both
sexes immediately following the first exposure to PGME and continued to be observed through test day 32 in males, and test day 23 in females. Sedation was not observed in males or females exposed to 300 or 1000 ppm PGME at any time during the study. No other treatment-related observations regarding behavior or demeanor were observed in P1 adults males at any exposure concentration during the entire study or in P1 adult females at any exposure concentration during the pre-mating, gestation or lactation periods. Six P1 adult females were found dead prior to the scheduled necropsy. Cause of death and other relevant information is summarized in Text Table 1. Detailed gross and histopathologic observations for these animals may be found in the individual animal pathology data, starting with Appendix Table 58. The deaths of these rats were not considered treatment-related. All other rats survived to the scheduled termination.
P2 adults and F2 litters: A single 1000 ppm male, 95B0860, died early on test day 72. No prior clinical observations were made on this male. The only gross observation made for this male was congested viscera. Additionally, inflammation of the heart and atrophy of the pancreas was noted during histopathologic examination. The cause of death for this male was not determined. All other P2 adults survived the test period and were necropsied on
the scheduled date of termination. Similar to the P1 adults, P2 male and female rats exposed to 3000 ppm PGME exhibited sedation, initially evidenced as incoordination following their first exposure to PGME (postnatal day 28). Incoordination was resolved within approximately one week of initial exposure to PGME vapors followed by approximately one week of decreased activity observed immediately following daily exposures. Incoordination and sedation observed during and post-exposure resolved by the next exposure day during this interval. Sedation was not observed in males or females
exposed to 300 or 1000 ppm PGME at any time during the study. The only other treatment-related observation noted was vaginal bleeding, which was noted among five high dose females late in the study (gestation/lactation phase). In three of the five cases the vaginal bleeding was associated with the pending delivery of a litter. In the other two cases the bleeding stopped, however, no litters were delivered. Of these two females, only one had histopathologic indications of a prior pregnancy (pigment laden macrophages in the uterus). No other treatment-related observations regarding behavior or demeanor were observed in P2 adults males at any exposure concentration during the entire study or in P2 adult females at any exposure concentration during the pre-mating, gestation or lactation periods.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weight data for P1 males and females, including the F1a/F1b gestation and lactation periods (females only) and interim body weight data collected between the F1a and F1b breeding (females only), are presented in Tables 10 through 19. Body weights of 3000 ppm PGME males were significantly decreased relative to control values beginning on test day 7 and remained decreased throughout the study, with statistical significance achieved on days 7, 14, and 77-168. Body weights of males exposed to 300 or 1000 ppm PGME were not significantly different from controls at any time during the study. Pre-mating body weights of 3000 ppm PGME females were significantly decreased beginning on test day 7 and remained decreased throughout the pre-mating period. In fact, mean body weight in the high concentration group was approximately 10% lower than control values by the end of the pre-mating period. Body weights of high concentration dams remained significantly decreased throughout most of the F1a and F1b gestation periods, and the first one to two weeks of the F1a and F1b lactation periods. Body weight gains of 3000 ppm PGME females were not affected during the F1a or F1b gestation phases or the respective first week of lactation. Compensatory increases in body weight gains relative to controls were noted during the last two weeks of lactation (F1a and F1b) for the 3000 ppm PGME dams. Dams exposed to 1000 ppm PGME during the pre-mating phase exhibited slight decreases in body weight, with the decreases statistically identified only on test days 7, 14 and 63. No significant effects on body weight were observed among 300 ppm females during the pre-mating period. Additionally, no significant differences in F1a or F1b gestation or lactation body weights or body weight gains were noted for 300 or 1000 ppm females relative to controls.
Mean body weights of P2 males and females, including the F2 gestation and lactation period (females only) are presented in Tables 20 through 25. Body weights of 3000 ppm PGME males were significantly decreased (20%) relative to controls at the beginning of the P2 exposures and remained decreased throughout the study (9% decrease on day 70, the end of the pre-mating period). Body weights of the P2 males exposed to 300 or 1000 ppm PGME were not affected at any time during the study. Pre-mating body weights of 3000 ppm PGME females were significantly decreased (21%) at the beginning of the P2 exposures and remained decreased throughout the pre-mating period (16% decrease on day 70, the end of the pre-mating period). Body weights of high concentration dams were also significantly decreased throughout the F2 gestation period, and the first one to two weeks of the F2 lactation period. Body weight gains of 3000 ppm PGME females were significantly decreased on gestation days 14-21, resulting in a statistically identified overall decrease on gestation days 0-21 as well. Compensatory increases in body weight gains relative to controls were statistically identified during lactation days 7-14 and 14-21, resulting in an overall increase in body weight gain for the entire lactation period (days 1-21) for the 3000 ppm PGME dams. Dams exposed to 1000 ppm PGME during the pre-mating period exhibited slight decreases in body weight, relative to control values, with the decreases statistically identified only on test days 63 and 70. No significant effects on body weight were observed among 300 ppm PGME females during the pre-mating period. Additionally, no significant differences in P2/F2 gestation or lactation body weights or body weight gains were noted for 300 or 1000 ppm PGME females relative to controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cyclicity data collected for ten P1 females per exposure concentration are presented in Table 26. Although not statistically identified, dams exposed to 3000 ppm PGME had a slight increase in the mean number of days per cycle as well as a resultant decrease in the mean number of estrous cycles observed per dam prior to placement with males for the F1a mating. Additionally, cycling slides obtained for four out of ten high exposure females were noted as exhibiting atypical cellularity. No effects on mean number of days per cycle or number of cycles per dam were noted for 300 or 1000 ppm dams. Estrous cycle length and normality were not evaluated prior to the F1b mating.
P2 estrous cyclicity data collected for ten P2 females per exposure concentration are summarized in Table 27. Dams exposed to 3000 ppm PGME had a statistically significant increase in the mean number of days per cycle as well as a statistically significant decrease in the mean number of estrous cycles observed per dam prior to placement with males for the F2 mating. This was consistent with the slight trend for increased cycle length and decreased number of cycles noted in P1 females for the F1a mating. Also, as for the P1 females, atypical cellularity was noted among the cycling slides for four high exposure females. No effects on mean number of days per cycle or number of cycles per dam were noted for 300 or 1000 ppm PGME dams.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Summaries of mean sperm counts and sperm motility (percent motile and progressively motile sperm) observed for ten P1 males per exposure concentration: No treatment-related differences in sperm counts or motility were observed for any exposure concentration tested. A significant increase in the number of progressively motile sperm obtained from P1 3000 ppm PGME males was considered spurious as a similar effect was not observed in the subsequent P2 generation.
Summaries of mean sperm counts and sperm motility (percent motile and progressively motile sperm) observed for ten P2 males per exposure concentration: No treatment-related differences in sperm counts or motility were observed for any exposure concentration tested.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related effects were observed on male or female P1 adult reproductive indices, gestation survival index, pup sex ratios, gestation length, or time to mating at any exposure concentration for the F1a mating/litters. Survival of pups from 3000 ppm PGME litters was significantly decreased relative to controls on lactation days 1 and 4. Survival of 3000 ppm PGME pups was also decreased, although not statistically identified, on lactation days 14 and 21. No effects on survival were observed for 300 or 1000 ppm PGME F1a litters at any time.
Reproductive indices and pup survival for P2 adults and their F2 litters: At 3000 ppm PGME, P2 male and female fertility was lower (not statistically
identified) than controls and recent historical control ranges (Table 31), while male and female conception rates were identified as significantly lower than controls, also falling outside the range of recent historical control values. No effects were observed on the P2 male or female mating index, gestation survival, pup sex ratios, gestation length, or time to mating. However, survival of 3000 ppm PGME F2 pups was significantly decreased relative to controls on lactation days 1 and 4. No treatment-related effects were observed on P2/F2 male or female reproductive indices, gestation survival, pup survival indices, pup sex ratios, gestation length, or time to mating at 300 or 1000 ppm PGME.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weight data (absolute and relative) for P1 males and females: Males exposed to 3000 ppm PGME exhibited statistically significant increases in relative testes, brain and kidney weights as well as a decrease in absolute and relative thymus weight. These effects were considered likely secondary to the significant decrease in mean P1 male body weight at this exposure concentration (probable decreased feed intake and resultant nutritional stress). No other significant effects on any other organ weights were observed among P1 3000 ppm PGME males. There were no treatment-related effects on terminal body weights or organ weights of males exposed to 300 or 1000 ppm PGME. P1 females exposed to 3000 ppm PGME had significantly higher relative adrenal, liver, and lung weights, when compared to controls, also primarily because of significantly lower body weights, however the interpretation of the liver weights is more complicated and will be discussed. No significant differences were observed for organ weights of P1 300 or 1000 ppm PGME females relative to controls.
Organ weight data (absolute and relative) for P2 males and females: P2 males exposed to 3000 ppm PGME exhibited statistically significant increases in relative liver, lung and seminal vesicle weights. Trends were similar, but not significant in P1 3000 ppm males, and again are interpreted primarily as the result of decreased body weight. No other significant effects on any other organ weights were observed among P2 3000 ppm PGME males. There were no treatmentrelated differences in organ weights of P2 males exposed to 300 or 1000 ppm PGME. P2 females exposed to 3000 ppm PGME had significantly decreased brain (absolute) and ovary (absolute and relative) weights relative to controls. The brain weights of the highexposure
P2 females will be discussed in the context of the entire study. The decreased ovary weights noted at 3000 ppm PGME appeared consistent with an increased incidence of ovarian atrophy observed histologically at this concentration. No other significant differences in any other organ weights were noted for 3000 ppm P2 females relative to controls. There were no treatment-related differences in organ weights of P2 females exposed to 300 or 1000 ppm PGME.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related gross pathologic changes were observed among the P1 and P2 adults at any exposure concentration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
An increased incidence of histologic ovarian atrophy and decreased ovarian weight was observed among the 3000 ppm PGME P1 females and was interpreted to be the result of non-specific toxicity and nutritional stress (see Discussion). Histologically, typical atrophic ovaries had fewer, or no, corpora lutea, and multiple large cystic and atretic follicles. There was no apparent increase in follicular atresia (identified by apoptotic granulosa cells) among developing follicles. Primordial and all subsequent stages in follicular maturation were evaluated qualitatively; they appeared to be present in normal numbers and were of normal appearance.
Similar to the P1 adults, an increased incidence of histologic ovarian atrophy associated with decreased ovarian weight was observed among the P2 3000 ppm PGME females and likewise was interpreted to be the result of non-specific toxicity and nutritional stress (see Discussion). Similar to the P1 females, typical P2 atrophic ovaries had fewer, or no, corpora lutea, and multiple large cystic and atretic follicles and there was no apparent increase in follicular atresia (identified by apoptotic granulosa cells) among developing follicles. Primordial cells and all subsequent cell stages in follicular maturation appeared to be present in normal numbers and were of normal appearance.

Dose descriptor:

NOAEL

Effect level:

300 ppm

Sex:

male/female

Basis for effect level:

other: sedation as evidenced by incoordination and decreased activity which resolved by the next exposure day was observed in both sexes immediately following the first exposure to PGME

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING)
No treatment-related effect observed.

CLINICAL SIGNS (OFFSPRING)
No treatment-related effect observed.

BODY WEIGHT (OFFSPRING)
Significant body weight decreases observed among the 3000 ppm PGME weanlings relative to controls.

SEXUAL MATURATION (OFFSPRING)
No treatment-related effect observed.

ORGAN WEIGHTS (OFFSPRING)
Mean absolute and relative organ weights and terminal body weights were collected for ten males and females per exposure concentration (F1a and F1b). Several organ weights changes (absolute or relative) were noted for male and female weanlings from F1a 3000 ppm PGME litters. F1a 3000 ppm PGME males had statistically identified decreases in absolute kidney, liver, spleen, and testes weights, while high concentration F1a females exhibited decreased absolute heart, and thymus weights and increased relative brain and kidneys weights. All of the differences identified at 3000 ppm PGME apeared to be secondary to significant decreases in male and female pup body weights at the time of necropsy/weaning (see Table 34) and were entirely consistent with nutritional stress. No significant organ weight changes were noted for F1a 300 or 1000 ppm PGME male or female weanlings. High concentration F1b male and female weanlings had significantly decreased absolute brain weights (males and females) and increased relative heart weights (males only). As with the F1a weanlings, these changes were likely due to the significant body weight decreases observed among the 3000 ppm PGME weanlings relative to controls. F1b males from 1000 ppm PGME litters were noted as having statistically identified decreases in absolute brain weight. This change was not considered toxicologically significant as relative brain weight was not affected and similar decreases were not observed among the F1a or F2 3000 ppm PGME males. A statistically significant increase in relative heart weight in 300 ppm PGME F1b males and females was considered spurious and unrelated to treatment as no dose-response was observed and similar increases were not observed in the F1a or F2 weanlings. Similarly, a significant increase in relative adrenal weight observed for 300 ppm F1b females was not consistent with data generated for the F1a or F2 generations, nor was a dose response observed. No significant organ weight changes were identified for 1000 F1b ppm PGME females.
F2 males at 3000 ppm PGME exhibited significant weight changes (absolute and/or relative) for all organs weighed and included the following: adrenals, brain, heart, kidneys, liver, spleen, testes, and thymus. F2 3000 ppm PGME females exhibited similar changes, however, only absolute brain and spleen weights were statistically identified. As with the 3000 ppm PGME F1a and F1b weanlings, organ weight changes noted for the F2 3000 ppm PGME males and females were likely secondary to significant body weight decrements (see Table 37) relative to control values and nutritional stress. A statistically significant decrease in relative thymus weight was identified for F2 300 ppm PGME males. As this effect was not dose-related and was not observed among the F1a or F1b 300 or 1000 ppm PGME males, it was considered a spurious finding. No other significant organ weight changes were noted for 300 or 1000 ppm PGME F2 males or females.

GROSS PATHOLOGY (OFFSPRING)
There were no gross lesions identified at the F1a or F1b necropsy that were associated with PGME exposure. There were no treatmentrelated gross lesions identified in the F2 males or females at any exposure concentration.

HISTOPATHOLOGY (OFFSPRING)
Histologically, the livers of F1a and F1b 3000 ppm PGME weanlings often lacked the normal degree of glycogen vacuolation; glycogen depletion was diagnosed and recorded. This finding, along with an increase in thymic single cell necrosis, is consistent with nutritional stress. Although spleen weights were in general depressed, no particular cell compartment was identified as contributing disproportionately, and no necrosis was observed, so no histopathologic correlate was recorded. Similarly, testicular weights for the F1a and F1b 3000 ppm PGME males were decreased, however, they were also histologically normal for their slightly earlier stage of development. Also, it should be recalled that the high-exposure P2 males, selected from the F1b pups, demonstrated normal testicular weights and histopathology, as well as sperm count and motility.
Changes similar to those observed in the F1a and F1b weanlings were noted histologically in the livers and thymus of F2 3000 ppm PGME weanlings suggesting some degree of nutritional stress. Despite weight changes observed, no histopathologic observations were noted in the spleens of 3000 ppm PGME F2 males or females. The testes of 3000 ppm PGME F2 males were also histologically normal, despite decreases noted in weight relative to controls.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: Highest concentration tested (3000ppm: significant body weight decreases; several organ weights changes

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: Highest concentration tested (3000ppm: significant body weight decreases; several organ weights changes

Reproductive effects observed:

not specified

At 3000 ppm, toxicity in the P1 and P2 adults was marked, as
evidenced by sedation during and after exposure for several
weeks, and mean body weights which were as much as 21% lower
than controls. This marked parental toxicity was accompanied
by lengthened estrous cycles, decreased fertility, decreased
ovary weights, reduced pup survival and litter size, slight
delays in puberty onset, and histologic changes in the liver
and thymus of the F1 and F2 offspring.  At 3000 ppm, there
was an increase in histologic ovarian atrophy in P1 and P2
females, and at 1000 ppm, there was a decrease in pre-mating
body weight in the P1 and P2 females. No treatment-related
differences in sperm counts or motility were observed among
the P1 or P2 males.

Conclusions:

The NOAEL for paternal toxicity is 300 ppm and for offspring toxicity is 1000 ppm. Effects appear secondary to parental weight loss.

Executive summary:

The objective of this two-generation inhalation reproduction study was to evaluate the effects of propylene glycol monomethyl ether (PGME) on the reproductive capability and neonatal growth and survival of rats. Groups of 30 male and 30 female Sprague-Dawley rats, approximately 6 weeks of age, were exposed to 0, 300, 1000 or 3000 ppm PGME via inhalation, for 6 hours/day, 5 days/week prior to mating and 6 hours/day, 7 days/week during mating, gestation and lactation for two generations. Inhalation exposure of adult male and female rats to 1000 (females only) and 3000 (males and females) ppm PGME resulted in dose-related parental effects. Toxicity in 3000 ppm PGME P1 and P2 males and females was evidenced primarily as an increased incidence of sedation for several weeks early in the exposure regimen and significant decreases in body weights, which achieved decrements of as much as 20 and 21% relative to controls, respectively. Decreased body weights in the P1 and P2 high concentration females generally persisted throughout the pre-breeding, gestation and lactation phases of the study. Additional effects noted among P1 and P2 adult females exposed to 3000 ppm PGME included lengthened estrous cycles, decreased fertility, decreased ovary weights and an increased incidence of histologic ovarian atrophy. The effects on fertility, estrous cyclicity and ovarian weight/histology appeared to be interrelated and associated with the significant decreases in 3000 ppm PGME female body weights and general toxicity/nutritional stress throughout the test period. No treatment-related differences in sperm counts or motility were observed among P1 or P2 adult males. Neonatal effects observed at 3000 ppm PGME consisted of decreased pup body weights, reduced pup survival and litter size, increased time to vaginal opening or preputial separation, and histopathologic observations in the liver and thymus of weanling rats. These neonatal effects also were considered secondary to maternal toxicity, particularly with respect to the compromised nutritional status of the maternal animals of the 3000 ppm PGME group. In the 1000 ppm PGME group, mild parental toxicity was evidenced by slightly decreased pre-mating body weights among P1 and P2 females, but was not accompanied by any statistically significant effects on parental reproduction or neonatal survival, growth or development. There were no treatment-related parental or neonatal effects related to exposure of rats to 300 ppm PGME. In conclusion, the no-observed-effect-level (NOEL) for fertility and reproductive effects in this two-generation inhalation reproduction study was 1000 ppm PGME.

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

11 632 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity test has been performed with TPGBE.

In the absence of reproductive toxicity data on TPGBE, data available for structurally related propylene glycol methyl ether (PGME) and dipropylene glycol n-butyl ether (DPGBE) are used to predict the reproductive toxicity potential of TPGBE. PGME, DPGBE and TPGBE are closely related in molecular structure and physicochemical properties and thus, the potential for toxicological effects. No major differences in the toxicological profile have been observed between propylene glycol ethers e.g. mono-, di- and tri-propylene glycol methyl ethers and mono-, di- and tri-propylene glycol n-butyl ethers). This is supported by repeated dose toxicity data on TPGBE (absence of effects on reproductive organs).

The justification for using data on structural related substances is provided in the category document attached to Section 13 of the IUCLID dossier.

Repeated dose toxicity data on TPGBE:

In a 90-day drinking water study, effects related to TPGBE occurred in the liver and kidneys of male rats given 1000 mg/kg/day and only in liver of females given 1000 mg/kg/day. Liver effects consisted of an increase in size of hepatocytes in centribular region of hepatic lobule and were accompanied by altered staining of the cytoplasm. These effects were accompanied by an increase in liver weight of male and female rats. This alteration was not accompanied by evidence of hepatocellular degeneration and was interpreted to be an adaptive change, possibly associated with the metabolism of TPGBE. A slight increase in the severity of renal tubular degeneration occurred in the kidneys of three of ten male rats given 1000 mg/kg/day, as compared to the control male rats. The kidney effects were only observed in male rats and findings with other propylene glycol ethers (e. g. propylene glycol methyl ether) suggest that these effects are related to alpha 2µ-globulin nephropathy. Alterations were not observed in sections of bone marrow or spleen that correlated with the hematologic differences statistically identified in male and female rats given 1000 mg/kg/day. Therefore, these effects are not considered to be relevant for human since it is assumed to be due to a male rat specific mechanisms. Results from this repeated dose study indicate no toxicity to the testes (no reduction in testicular weight), no damage to the sperm or sperm-producing cells, and no damage to the epididymis or seminiferous tubules. Likewise, no damage to female reproductive organs was found. Based on results of this study the NOAEL for male and female rats was 1000 mg/kg/day.

Data available for PGME:

The objective of this two-generation inhalation reproduction study was to evaluate the effects of propylene glycol monomethyl ether (PGME) on the reproductive capability and neonatal growth and survival of rats. Groups of 30 male and 30 female Sprague-Dawley rats were exposed to 0, 300, 1000 or 3000 ppm PGME via inhalation, for 6 hours/day, 5 days/week prior to mating and 6 hours/day, 7 days/week during mating, gestation and lactation for two generations. Inhalation exposure of adult male and female rats to 1000 (females only) and 3000 (males and females) ppm PGME resulted in dose-related parental effects. Toxicity in 3000 ppm PGME P1 and P2 males and females was evidenced primarily as an increased incidence of sedation for several weeks early in the exposure regimen and significant decreases in body weights, which achieved decrements of as much as 20 and 21% relative to controls, respectively. Decreased body weights in the P1 and P2 high concentration females generally persisted throughout the pre-breeding, gestation and lactation phases of the study. Additional effects noted among P1 and P2 adult females exposed to 3000 ppm PGME included lengthened estrous cycles, decreased fertility, decreased ovary weights and an increased incidence of histologic ovarian atrophy. The effects on fertility, estrous cyclicity and ovarian weight/histology appeared to be interrelated and associated with the significant decreases in 3000 ppm PGME female body weights and general toxicity/nutritional stress throughout the test period. No treatment-related differences in sperm counts or motility were observed among P1 or P2 adult males. Neonatal effects observed at 3000 ppm PGME consisted of decreased pup body weights, reduced pup survival and litter size, increased time to vaginal opening or preputial separation, and histopathologic observations in the liver and thymus of weanling rats. These neonatal effects also were considered secondary to maternal toxicity, particularly with respect to the compromised nutritional status of the maternal animals of the 3000 ppm PGME group. In the 1000 ppm PGME group, mild parental toxicity was evidenced by slightly decreased pre-mating body weights among P1 and P2 females, but was not accompanied by any statistically significant effects on parental reproduction or neonatal survival, growth or development. There were no treatment-related parental or neonatal effects related to exposure of rats to 300 ppm PGME. In conclusion, the no-observed-effect-level (NOEL) for fertility and reproductive effects in this two-generation inhalation reproduction study was 1000 ppm PGME.

Data available for DPGBE:

In an OECD 421 study, groups of 12 male and 12 female Crl:CD(SD) rats were administered dipropylene glycol n-butyl ether (DPGBE) daily, by gavage at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29). On the first day of dosing with 1000 mg/kg/day of DPGBE, three females exhibited a transient, subtle, incoordinated gait which resolved within 1-2 hours after dosing and was not seen again for the remainder of the study. The only other treatment-related clinical observation was transient, post-dosing salivation (clear perioral soiling) noted sporadically in several high-dose males and females. This salivation was considered to be a local response to the taste of the test material, rather than evidence of toxicity. Treatment-related increases in the incidence of hepatocellular hypertrophy occurred in males of all dose groups, and in females given 300 or 1000 mg/kg/day DPGBE. The hypertrophy corresponded with increased liver weights in the 1000 mg/kg/day males and females, and 300 mg/kg/day males (absolute and relative weights affected). These changes were considered to be an adaptive response associated with increased hepatic metabolism of DPGBE. Treatment-related increases in absolute and relative kidney weights also were found in males and females given 1000 mg/kg/day. In addition, hyaline droplet formation in the proximal renal tubules was observed in males given 300 or 1000 mg/kg/day. Females given 1000 mg/kg/day had treatment-related higher mean absolute and relative kidney weights, but a histopathologic correlate to the higher kidney weights was lacking. There were no treatment-related effects on any reproductive parameters and no adverse gross or histopathological findings in the reproductive organs of males or females. The no-observed-effect level (NOEL) for reproductive effects was 1000 mg/kg/day, the highest dose tested. 

Summary:

Although there are no reproductive toxicity data available for TPGBE, there are sufficient data available for structurally related substances to make a conclusion about the reproductive toxicity of TPGBE to support classification and risk assessment.

Repeated dose toxicity data on TPGBE do not indicate that the reproductive organs are a target following oral exposure. The no-observed-effect-level (NOEL) for fertility and reproductive effects in the two-generation inhalation reproduction study on PGME was 1000 ppm. The NOAEL for paternal toxicity is 300 ppm and for offspring toxicity is 1000 ppm.  Effects appear secondary to parental weight loss. The reproductive NOEL from the OECD 421 oral screening study on DPGBE was 1000 mg/kg/day. There were no treatment-related effects on any reproductive parameters observed in this study. Based on the parameters covered in these studies the overall assessment indicates there would be no reproductive effects expected, in the absence of general toxicity, for TPGBE.

Effects on developmental toxicity

Description of key information

No teratology studies are available for tripropylene glycol butyl ether. Therefore, available data on structural analogues propylene glycol butyl ether (PnB) and dipropylene glycol butyl ether (DPnB) is used for read-across.
For PnB an OECD 414 dermal developmental toxicity study in rats is available. This data is supported by peer-reviewed published data on dermal developmental toxicity in rabbits. For DPnB an OECD 414 dermal developmental toxicity study in rats is available.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

08/1987-10/1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD guideline 414.

Justification for type of information:

Please refer to category document.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

only 2 dose levels

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann Versuchstierzucht GmbH & Co. KG, Borchen, Germany
- Age at study initiation: 13 weeks (females) & 14 weeks (males)
- Weight at study initiation: 182 to 218 g (females)
- Fasting period before study: none
- Housing: five females per cage and males individually housed in suspended steel cages fitted with wire-mesh floors and fronts
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ca. one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): at least 40%
- Air changes (per hr): 8-10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

propylene glycol

Details on exposure:

TEST SITE
- Area of exposure: shaved back skin
- coverage: 20cm2
- Type of wrap if used: no wrap (uncovered)
- Time intervals for shavings or clipplings: no data

REMOVAL OF TEST SUBSTANCE
- Washing (if done): prior to daily application the treated skin was cleaned with lukewarm water moistened sponge
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.5 ml/kg body weight
- Concentration (if solution): ratios of 40:60 and 12:60 PnB in propylene glycol
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on preliminary dermal irritation study and the pilot dermal embryotoxicity study it was decided to apply the test substance in a solution with propylene glycol
- Amount(s) applied (volume or weight with unit): 1.5 ml/kg body weight

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (neck collars)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from each test solution and stored at 4°C until analysis. Samples were diluted with acetone and analyzed by gas chromatography using a flame ionization detector.
For gas chromatographic conditions see attachment provided below.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight untill detection of sperm cells in the female (max. 4 days)
- Males found to have mated were assigned to each group in rotation
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 to 16 of pregnancy

Frequency of treatment:

daily

Duration of test:

untill day 21 of pregnancy

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a preliminary dermal toxicity study showing that PnB was well-tolerated by pregnant female rats applied dermally at levels up to 1.0 ml/kg bw/day
- Rationale for animal assignment: random

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: general condition and behaviour of the animals was checked daily and local skin reactions, if any, were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: animals were individually weighed on day 0, day 6, day 16 and day 21 of pregnancy

FOOD CONSUMPTION : Yes
-quatitiy of food consumed by each animal of each group was determined during the following periods: day 0 to day 6 and day 6 to day 16 and day 16 to day 21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: both ovaries, uterus, kidneys and the liver

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

For the statistical analyses of differences in degree of ossification between the test and control groups Student's t-test was applied to transformed ossification values expressed in degrees.
Statistical analysis of differences in body weight, food consumption, organ weights, litter data, foetus weights and lengths and placenta weights was carried out by applying analysis of (co)-variance, with body weights on day 0 as the covariable, followed by Dunnet's multiple comparison test, wherea skeletal and visceral anomalies were evaluated by the Chi-square test.

Indices:

Percentage of pre-implantation loss (PRIL) was calculated for each litter according to the formula below.

PRIL=100%*(number of corpora lutea-number of implantation sites/number of corpora lutea)

Percentage of post-implantation loss (POIL) was calculated for each litter according to the formula below.

POIL=100%*(number of implantation sites-number of live pubs/number of implantation sites)

Degree of ossification (DgO) of foetus skeletons was calculated for each litter according to the formula below.

DgO=number of bones without ossification (or with incomplete ossification)/number of bones examined

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Minor skin reaction were observed. However, these were not considered of importance to the study. There were no statistical differences in body weight and food intake, in the weight of ovaries, uterus, kidneys and liver between control- and test groups.

Dose descriptor:

NOAEL

Effect level:

880 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

880 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No compound related visceral and skeletal malformations, anomalies or variants were observed at any dose level.

Dose descriptor:

NOAEL

Effect level:

880 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: overall effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Propylene glycol n-butyl ether did not induce developmental effects up to 1.0 ml/kg bw/day (highest dose level tested).

Executive summary:

Three groups of 25 mated Wistar rats recieved dermal applications ofg 0, 0.3 and 1.0 ml/kg bw/day propylene glycol n-butyl ether (DOWANOL PnB) from day 6 -16 of pregnancy. The control group was treated with 1.5 ml propylene glycol/kg bw/day. The PnB in the test groups was provided as two mixtures in propylene glycol at the ratios 12:60 and 40:60. Because of the non-occluded applications, collars were used to prevent oral uptake. The study was conducted according to OECD guideline 414.

No mortality, and no abnormalities in condition and behaviour occurred. Minor skin reactions were observed in the PnB. These were not considered of importance for the study. There were no statistical differences in body weight and food intake, in the weight of ovaries, uterus, kidneys and liver between control- and test groups.

Maternal performance was comparable in all groups. no compound related visceral and skeletal malformations, anomalies or variants were observed, that could be related to the treatment with PnB.

The limit concentration of 1.0 ml/kg bw/day produced minor local skin reactions, but no further toxicity in the dams. Daile dermal applications of 0.3 or 1.0 ml/kg bw/day from day 6 through 16 of pregnancy did not induce embryo/foeto toxicity nor teratogenic effects.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The data quality from this study is considered acceptable. The report included documentation for methods and results and is equivalent to OECD Guideline 414 "Teratogenicity" (was not specified in this peer-reviewed paper, but the reported methodology satisfies the criteria described in these guidelines).

Justification for type of information:

Please refer to category document.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research animals (Denver, PA)
- Age at study initiation: 6 months
- Weight at study initiation: Females: Group 0 controls, 3.89 ± 0.25 kg; Group 2 (10 mg/kg), 3.83 ± 0.36 kg: Group 3 (40 mg/kg), 3.92 ± 0.26 kg, Group 4 (100 mg/kg), 3.75 ± 0.22 kg.
- Fasting period before study: none
- Housing: Individually in stainless steel cages with wire-mesh bottoms.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 32 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 22 °C
- Humidity (%): 50-72%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

water

Details on exposure:

Dilutions of PnB in water resulted in applied volumes of 2.0 ml test solution per kg body weight. Treatment solutions were applied to the shaved dorsal trunk (area of 10 x 20 cm) of each rabbit for a period of 6 hours per day, whereafter residual material was removed with warm water. Rabbits wore neck collars to prevent grooming and ingestion of test material during the daily 6-hour exposure periods. Solutions were applied unoccluded since the low vapor pressure of PnB was considered to preclude evaporative loss.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: artificial insemination, the day of insemination was considered day 0 of pregnancy

Duration of treatment / exposure:

day 7 through day 18 of presumed gestation

Frequency of treatment:

daily

Duration of test:

until day 29 of gestation

No. of animals per sex per dose:

17 in the control group and 19 in each of the test groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on solubility of butoxypropanol in water and the results of a pilot study.

Maternal examinations:

Rabbits were observed for clinical signs of toxicity, abortion, delivery, and skin reactions several times per day over the exposure and post-exposure periods. Individual body weights and feed consumption were recorded daily during pregnancy. At sacrifice (day 29 of pregnancy), all animals were subjected to necropsy and examined for gross abnormalities.

Ovaries and uterine content:

The ovaries, uterus, kidneys, and livers were removed and weighed. The number of corpora lutea were counted in each ovary. Early and late resorptions and live and dead fetuses were counted. Implantation sites in both uterine horns were counted and the empty uterus weighed.

Fetal examinations:

Fetuses were removed from the uterus, weighed, lengths recorded, and examined for gender and external and internal gross abnormalities, according to the method of Staples. All the fetuses from each litter were eviscerated, skinned and stripped of most subcutaneous tissue, then fixed in 96% ethanol. These fetuses were then stained with Alizarin Red S for examination for skeletal anomalies. Percentages of pre- and post-implantation loss were calculated as was the degree of ossification for each fetus. Soft tissue and skeletal anomalies or abnormalities were recorded

Statistics:

Maternal clinical sign data and necropsy observation data were analyzed using Fisher's Exact Test. maternal bodey weight and feed consumption data, fetal body weights, litter averages (calculated as a percentage basis), anomaly average data, Caesarean-sectioning data. and fetal ossification site data were analyzed using Bartlett's Test of Homogeniety of Variances and either the analysis of variance or the Mann-Whitney U test. If Bartlett's Test was significant, then the Mann-Whitney U test was used to analyze the data by testing each group individually against the control group. If Bartlett's Test was not significant, then the one-way analysis of variance was used to analyze the data. If the analysis of variance was significant, then Dunnett's test was used to analyze the data. Proportion data for Cesarean-sectioning and fetal observations were analyzed using the Fisher's Exact test; observations for the late resorptions were excluded from statistical analyses.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Erythema of the skin at the site of application occurred at a greater incidence and severity in does from the high exposure group.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: developmental toxicity overall effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

There were no statistically significant differences between test and control groups for maternal body weight gain, feed consumption, number of corpora lutea per ovary, implantations, live fetuses, early and late resorptions, fetal body weights, gender, or gross external changes. There were no visceral or skeletal fetal alterations at any dose tested. No signs of maternal toxicity were observed; however, mild erythema occurred at the site of application at the 100 mg/kg/day dose level. The developmental no-effect level was greater than 100 mg/kg/day.

Executive summary:

The developmental toxicity (embryofetotoxicity or teratogenicity) and maternal toxicity of butoxypropanol, a propylen glycol ether, was evaluated in New Zealand White rabbits. Dosages of 0 (water), 10, 40 or 100 mg/kg/day, selected on the basis of the water solubility of butoxypropanol and the results of a pilot study, were administered percutaneously to groups of artificially inseminated (Day )) female rabbits on Days 7 through 18 of gestation. The dosage volume was 2 ml/kg. The control group contained 17 does and each test group contained 19 does. Does were observed for signs of test substance effect on abortion or premature delivery, body weight, and feed consumption. On Day 29, they were euthanized and examined for pregnancy, number and placement of implantations, early and late resorptions, and live fetuses. Pregnancy occurred in 15 control does (88%) and in 16 does in each test group (84%). There were no statistically significant differences between test and control groups for maternal body weight gain, feed consumption, number of corpora lutea per ovary, implantations, live fetuses, early and late resorptions, fetal body weights, gender, or gross external changes. There were no visceral or skeletal fetal alterations at any dose tested. No signs of maternal toxicity were observed; however, mild erythema occurred at the site of application at the 100 mg/kg/day dose level. The developmental no-effect level was greater than 100 mg/kg/day.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

880 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

No developmental toxicity studies have been performed with TPGBE.

In the absence of developmental toxicity data on TPGBE, data available for structurally related propylene glycol butyl ether (PGBE) and dipropylene glycol n-butyl ether (DPGBE) are used to predict the develpmental toxicity potential of TPGBE. PGBE, DPGBE and TPGBE are closely related in molecular structure and physicochemical properties and thus, the potential for toxicological effects. No major differences in the toxicological profile have been observed between propylene glycol ethers e.g. mono-, di- and tri-propylene glycol n-butyl ethers).

The justification for using data on structural related substances is provided in the category document attached to Section 13 of the IUCLID dossier.

Data available for PGBE:

In a study conducted in accordance to the OECD guideline 414, three groups of 25 mated Wistar rats received dermal applications of 0, 0.3 and 1.0 ml/kg bw/day propylene glycol n-butyl ether (PGBE) from day 6 -16 of pregnancy. The control group was treated with 1.5 ml propylene glycol/kg bw/day. The PGBE in the test groups was provided as two mixtures in propylene glycol at the ratios 12:60 and 40:60. Because of the non-occluded applications, collars were used to prevent oral uptake. No mortality, and no abnormalities in condition and behaviour occurred. Minor skin reactions were observed in the PGBE treated groups. There were no statistical differences in body weight and food intake, in the weight of ovaries, uterus, kidneys and liver between control- and test groups. Maternal performance was comparable in all groups. There were no compound related visceral and skeletal malformations, anomalies or variants observed that could be related to the treatment with PGBE. The limit concentration of 1.0 ml/kg bw/day produced minor local skin reactions, but no further toxicity in the dams. Daily dermal applications of 0.3 or 1.0 ml/kg bw/day from day 6 through 16 of pregnancy did not induce embryo/foeto toxicity nor teratogenic effects. The NOAEL for developmental toxicity was greater than 1.0 ml/kg bw /day (880 mg/kg bw/day), the highest dose tested.

The developmental toxicity (embryofetotoxicity or teratogenicity) and maternal toxicity of propylene glycol n-butyl ether (PGBE) was evaluated in New Zealand White rabbits. Dosages of 0 (water), 10, 40 or 100 mg/kg/day, selected on the basis of the water solubility of PGBE and the results of a pilot study, were administered percutaneously to groups of artificially inseminated female rabbits on Days 7 through 18 of gestation. Pregnancy occurred in 15 control does (88%) and in 16 does in each test group (84%). There were no statistically significant differences between test and control groups for maternal body weight gain, feed consumption, number of corpora lutea per ovary, implantations, live fetuses, early and late resorptions, fetal body weights, gender, or gross external changes. There were no visceral or skeletal fetal alterations at any dose tested. No signs of maternal toxicity were observed; however, mild erythema occurred at the site of application at the 100 mg/kg/day dose level. The developmental no-effect level was greater than 100 mg/kg/day, the highest dose on the study.

Data available for DPGBE:

Dipropylene glycol n-butyl ether (DPGBE) was applied daily to the skin of pregnant rats on gestation days 6 through 15. DPGBE was applied to the clipped skin of two groups of Wistar rats (>20/sex/dose level) at various dilutions in propylene glycol (PG) equivalent to doses of 0 (PG-only; 1.5 ml/kg bw/day), 0.3 or 1.0 ml DPGBE/kg bw/day. These doses equate to 0, 273, or 910 mg DPGBE/kg bw/day. Rats wore neck collars to prevent grooming and ingestion of test material. Solutions were applied unoccluded since the low vapor pressure of DPGBE and PG precluded evaporative loss. Slight skin reactions were found in the dams from all treatment groups and thus were not considered to be treatment related. No maternal toxicity was found: clinical signs and organ or body weights did not differ between treatment and controls groups. No deaths occurred in any groups over the course of the study. Fecundity was comparable among groups. No embryo- or fetotoxicity was evident since pre- and post-implantation loss, number of viable fetuses, and fetal weights and lengths were comparable between treatment and control groups. DPGBE did not cause developmental toxicity in skeletal or soft tissue. Skeletal malformations were observed only in the control group and skeletal variants were observed in all dose groups. The high dose group did exhibit a slight increase (not statistically significant) in the incidence of supernumerary rudimentary thoracic ribs when compared to controls. However, this finding was not considered biologically significant since the incidence was within normal limits for this species. In conclusion, DPGBE is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0 ml/kg bw/day during organogenesis (days 6 - 15). The NOAEL for maternal toxicity, embryo- or fetal toxicity, or developmental toxicity is 1.0 ml/kg bw/day (910 mg/kg bw/day), the highest dose level on the study.

Summary:

Although there are no developmental toxicity data available for TPGBE, there are sufficient data available for structurally related substances to make a conclusion about the developmental toxicity of TPGBE to support classification and risk assessment.

PGBE is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0 ml/kg bw/day (880 mg/kg bw/day) during organogenesis (days 6 - 15). In rabbits, PGBE did not cause maternal toxicity, embryo- or fetal toxicity, or developmental abnormalities in fetuses at any dose level; therefore the NOAEL was > 100 mg/kg. DPGBE is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0 ml/kg bw/day during organogenesis (days 6 - 15). The NOAEL for maternal toxicity, embryo- or fetal toxicity, or developmental toxicity is 1.0 ml/kg-d (910 mg/kg-d) which is the highest dose tested. Based on the parameters covered in these studies the overall assessment indicates there would be no developmental effects expected for TPGBE.

Justification for classification or non-classification

No fertility and teratology studies are available for tripropylene glycol butyl ether (TPnB). Since TPnB has undergone repeated dose toxicity testing at substantial doses with extensive histopathology, definitive conclusions can be drawn regarding damage to reproductive organs. Results from these repeated dose tests indicate that TPnB did not cause toxicity to the testes. Specifically, no reduction in testicular weight, no damage to the sperm or sperm-producing cells, and no damage to the epididymis or seminiferous tubules were reported. Likewise, no damage to female reproductive organs was found. In addition read-across data from other p-series glycol ethers (2-generation reprotox study on PGME and an OECD 421 study on DPnB as well as teratology studies in rats and rabbits for PnB and DPnB) is available to address these endpoints. No treatment-related effects on reproductive parameters or developmental effects were observed with PGME, PnB or DPnB. Therefore, tripropylene glycol n-butyl ether should not be classified for reproductive toxicity or developmental effects.