Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An oral gavage reproductive/developmental toxicity screening test in rats is available. The study was conducted under GLP and equivalent to OECD guideline 421. Supporting data are available through read-across from a 2-generation reproductive toxicity study on propylene glycol methyl ether (PGME) via inhalation in rats.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 22, 2005 - June 23, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 421
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: approximately 8 wks
- Weight at study initiation: Males: around 265 g; Females: around 194 g
- Fasting period before study: not described
- Housing: two-three per cage in stainless steel cages before dosing, and one per cage except during breeding and during littering phases of the study after dosing. 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.
- Use of restrainers for preventing ingestion (if dermal): as described above
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


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


IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
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.

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.

Dose Selection:
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 (1000 mg/kg bw/day). 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.


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:
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:
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).
Frequency of treatment:
Females and males were dosed once daily.
Details on study schedule:
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).
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
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.

Text Table 1. Dose Levels
Dose Levels No. of Adult Rats/Sex/Dose Level
(mg/kg/day)
0 12
100 12
300 12
1000 12
Total Number Adults: 96
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least twice daily for parental rats. Cage-side examinations were conducted on dams and their litters, at least twice daily.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations:
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 (if feeding study):
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. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)


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

OTHER: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.
Oestrous cyclicity (parental animals):
n/a
Sperm parameters (parental animals):
n/a
Litter observations:
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 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 of the tissues indicated in Table 2 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.
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:
Various endpoints were analyzed using statistical models, including body weights, feed consumption, organ weights, etc.
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 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
Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Offspring viability indices:
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
Clinical signs:
no effects observed
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
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.
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no reproductive effects observed
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Litter 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.

There were no treatment-related effects on litter size or pup body weights at any dose level tested.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on litter size or pup body weights at any dose level tested.
Reproductive effects observed:
not specified

none

Conclusions:
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. According to EU classification and labeling criteria, DPnB is not classified as a reproductive toxicant and is not required to be labeled.
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. According to EU classification and labeling criteria, DPnB is not classified as a reproductive toxicant and is not required to be labeled.

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-study according to OECD guideline 416
Justification for type of information:
Please refer to category document.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
Gross pathological findings:
no effects observed
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 parental 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
Quality of whole database:
acceptable
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
7 783 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the absence of a full reproductive toxicity study on DPGBE, data available for the structurally related propylene glycol methyl ether (PGME) will be used, along with data from DPGBE, to predict the reproductive toxicity potential of DPGBE. PGME and DPGBE 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).

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

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. 

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.

Summary:

Although there are no full reproductive toxicity studies available for DPGBE, there is sufficient data available for a structurally related substance (PGME) along with reproductive screening data on DPGBE to make a conclusion about the reproductive toxicity of DPGBE to support classification and risk assessment.

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 DPGBE.

 

Effects on developmental toxicity

Description of key information
A dermal embryotoxicty/teratogenicity study in rats is available. This is a GLP study conducted according to OECD 414 (Prenatal Developmental Toxicity Study). In addition there are developmental toxicity studies available for category members, DPnP (dermal, rabbit) and DPM (inhalation rat and rabbit). Justification for using these studies is included in the catgory document attached to the IUCLID.
Link to relevant study records

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1987- June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 414
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar derived SPF-bred albino rats (Bor; WISW, SPF TNO)
Details on test animals and environmental conditions:
Age at dosing:       Approximately 12 weeks (females) and 13 eeks (males) of age.
Source:                Winkelmann Versuchstierzucht GmbH & Co. KG, Borchen, West-Germany.
Acclimation period:  Approximately 1 week.
Average weight (start of study):       Males: not specified Females: 186 - 209 grams.
Assignment to  groups:      Computerized, random number-based  procedure.
Diet:                "Basal Diet" (analysis provided in report)
Access to food:      Available ad libitum.
Access to water:     Available ad libitum.
Method of  Identification:      "V" notches on ears.
Housing:          Prior to mating:  males - individually, females - 5 per group in stainless steel cages with wire-mesh bottoms. 
After mating: housing-type for females not specified.
Environmental Conditions:
Temperature:         22 ± 2°C. Recording frequency not reported.
Humidity:            At least 40%. Range & recording  frequency not reported.
Air changes:         8-10 air changes per hour.
Photoperiod:         12 hr light/12 hr dark.
Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
propylene glycol
Details on exposure:
Dipropylene glycol n-butyl ether (DPnB) was applied daily to the skin of pregnant rats on gestation days 6 through 15 (detection of sperm in vaginal smears was designated day 0).  DPnB 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-day), 0.3 or 1.0 ml DPnB/kg-day.  These doses equate to 0, 273, or 910 mg DPnB/kg-day.  Treatment solutions were applied to the clipped dorsal trunk of each rat over an area of about 20 cm2.  Dilutions of DPnB in PG resulted in applied volumes of 1.5 ml (PG-only), 1.8 ml (1.5 ml PG & 0.3 ml DPnB), or 2.5 ml (1.5 ml PG & 1.0 ml DPnB) test solution per kg body weight.  Rats wore neck collars to prevent grooming and ingestion of test material.  Solutions were applied unoccluded since the low vapor pressure of DPnB and PG precluded evaporative loss. 
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of DPnB was determined in the test solutions prepared on July 20-1987. Samples were taken from each solution and stored at -20 celcius until further analysis. The analysis was carried out at TNO Institute CIVO-Analysis.
Details on mating procedure:
Females were placed overnight with males, e.g. two females with one male. Vaginal smears were taken to determine whether mating occured. If no sperm cells were detected, the female of question was placed with another male again. The day of detection of sperm cells was considered day 0 of pregnancy. This procedure was continued for 4 days to have sufficient number of possibly pregnant animals in each of the groups. Animals found to have mated were assigned to each group in rotation.
Duration of treatment / exposure:
Days 6-15 of gestation (day plug found was Day 0)
Frequency of treatment:
daily
Duration of test:
Treatment from Day 0 to Day 15 of pregnancy. Test until day 21 of pregnancy.
No. of animals per sex per dose:
22 in control, 21 in 273 mg/kg, and 25 in 910 mg/kg dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: until day 21 of pregnancy
Maternal examinations:
Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Individual body weights were recorded on days 0, 6, 16, and 21 of pregnancy and food consumption was monitored over days 0 - 6, 6 - 16, and 16 - 21 of pregnancy.  At sacrifice, all animals were subjected to necropsy and examined for gross abnormalities. 
Ovaries and uterine content:
The number of corpora lutea was counted. 
Fetal examinations:
Fetuses were removed from the uterus, weighed, lengths recorded, and examined for gross abnormalities.  Early and late resorptions and live and dead fetuses were counted. Implantation sites in both uterine horns were counted and the empty uterus weighed.  Half 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.  The remaining fetuses were fixed in Bouin's fluid, transferred to 70% ethanol and sectioned into slices (after Wilson) for soft tissue examination.  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:
For the statistical analysis of difference in degree of ossification between test and control student t-test was applied. Statistical analysis for the 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 weight on day 0 as covairable, followed by Dunnet's multiple comparison test, whereas skeletal and visceral anormalies were evaluated by the Chi-square test.
Indices:
Percentage pre-implantation loss (PRIL)
Percentage post-implantation loss (POIL)
Degree of ossificatin of foetus skeleton (DgO)
Transformed ossification values (DfOt)

Historical control data:
Incidence of spontaneous malformations, anomalies and variants in Albino rats of the Wistar strain (Cpb-WU; Wistar random). TNO report # v82.043/020283
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
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.  
Dose descriptor:
NOAEL
Effect level:
910 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.  DPnB did not cause frank developmental toxicity in skeletal or soft tissue.  Frank skeletal malformations were observed only in the control group (6 fetuses from 2 litters).  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 by the authors of the study since the incidence was within normal limits for these species.
Dose descriptor:
NOAEL
Effect level:
910 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
DPnB is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0 ml/kg-d 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) and a LOAEL was not established.
Executive summary:

Dipropylene glycol n-butyl ether (DPnB) was applied daily to the skin of pregnant rats on gestation days 6 through 15 (detection of sperm in vaginal smears was designated day 0).  DPnB 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-day), 0.3 or 1.0 ml DPnB/kg-day.  These doses equate to 0, 273, or 910 mg DPnB/kg-day.  Treatment solutions were applied to the clipped dorsal trunk of each rat over an area of about 20 cm2.  Dilutions of DPnB in PG resulted in applied volumes of 1.5 ml (PG-only), 1.8 ml (1.5 ml PG & 0.3 ml DPnB), or 2.5 ml (1.5 ml PG & 1.0 ml DPnB) test solution per kg body weight.  Rats wore neck collars to prevent grooming and ingestion of test material.  Solutions were applied unoccluded since the low vapor pressure of DPnB and PG precluded evaporative loss. 

The content of DPnB was determined in the test solutions prepared on July 20-1987. Samples were taken from each solution and stored at -20 celcius until further analysis. The analysis was carried out at TNO Institute CIVO-Analysis.

 

Females were placed overnight with males, e.g. two females with one male. Vaginal smears were taken to determine whether mating occured. If no sperm cells were detected, the female of question was placed with another male again. The day of detection of sperm cells was considered day 0 of pregnancy. This procedure was continued for 4 days to have sufficient number of possibly pregnant animals in each of the groups. Animals found to have mated were assigned to each group in rotation.

 

Pregnant animals were randomly assigned to each groups, with 22 in control, 21 in 273 mg/kg, and 25 in 910 mg/kg dose group. Treatments of the animals were conducted on days 6-15 of gestation (day plug found was Day 0) daily. The doses used were 273 or 910 mg/kg.

 

The experimental design is shown in the table below.

Group  DPnB      Vehicle   DPnB      No/F   Treatment
       Dose      PG Dose   Dose      Dose   Period 
       (ml/kg-d) (ml/kg-d) (mg/kg-d) Group  (days)    
===========================================================
1      0         1.5       0         22     6 thru 15 gest.
2      0.3       1.5       273       21     6 thru 15 gest.
3      1.0       1.5       910       25     6 thru 15 gest.

 

Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Individual body weights were recorded on days 0, 6, 16, and 21 of pregnancy and food consumption was monitored over days 0 - 6, 6 - 16, and 16 - 21 of pregnancy.  At sacrifice, all animals were subjected to necropsy and examined for gross abnormalities. The number of corpora lutea was counted. Fetuses were removed from the uterus, weighed, lengths recorded, and examined for gross abnormalities.  Early and late resorptions and live and dead fetuses were counted. Implantation sites in both uterine horns were counted and the empty uterus weighed.  Half 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.  The remaining fetuses were fixed in Bouin's fluid, transferred to 70% ethanol and sectioned into slices (after ) for soft tissue examination.  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. For the statistical analysis of difference in degree of ossification between test and control student t-test was applied. Statistical analysis for the 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 weight on day 0 as covairable, follwoed by Dunnet's multiple comparison test, whereas skeletal and visceral anormalies were evaluated by the Chi-square test.

Indeces include Percentage pre-implantation loss (PRIL), Percentage post-implantation loss (POIL), Degree of ossificatin of foetus skeleton (DgO) and Transformed ossification values (DfOt). Incidence of spontaneous malformations, anomalies and variants in Albino rats of the Wistar strain (Cpb-WU; Wistar random). TNO report # v82.043/020283.

 

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.  DPnB did not cause frank developmental toxicity in skeletal or soft tissue.  Frank skeletal malformations were observed only in the control group (6 fetuses from 2 litters).  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 by the authors of the study since the incidence was within normal limits for these species.

 

In conclusion, DPnB is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0

 ml/kg-d 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) and a LOAEL was not established.

 

In a pilot study 0, 0.1, 0.3 or 1 ml DPnB was applied dermally to Wistar rats during gestation day 6 till 16. No mortalities were observed. The reproduction and litter data did not reveal any treatment related effect. From this study
it was concluded that DPnB at levels up to 1 ml (910 mg/kg bw) was not embryo/fetotoxic to rats.

This study was identified as key for this toxicity endpoint because of the methods followed (which were comprehensively
documented in the report).  The report included GLP and Quality Assurance statements, signed by the Study Director
and Head of the QA Unit, respectively.  The study report followed OECD Protocol 414: "Teratogenicity" (12 May 1981),
the numbers and type of test animals used and their husbandry conditions were as prescribed in the  guidance. 
Test material characterization was adequate. The amount of test material applied complied with guidance, the length of
the treatment period (organogenesis) was sufficient, and evaluation criteria and statistical methods were typical for
this type assay and adequately recorded.

 

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 414 and OPPTS Guidleines 870.3700 and in accordance with the Principles of GLP.
Justification for type of information:
Please refer to category document.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Minor deviations were observed and reported and these did not negatively impact the quality or integrity of the data nor the outcome of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Minor deviations were observed and reported and these did not negatively impact the quality or integrity of the data nor the outcome of the study.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: 5.5 months old upon receipt
- Weight at study initiation: 3084 g to 4442 g
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Industrial Products Division, Maumee, Ohio). The bedding was changed at least twice each week. Nesting material was not required, as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the
Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research Laboratories, LLC are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
- Diet (e.g. ad libitum): The basal diet (PMI Nutrition International, LLC, Certified Rabbit LabDiet® 5322) was offered 3 times at 25 g on the day of arrival, 3 times at 50 g on the day after arrival and ad libitum on all subsequent days until euthanasia.
- Water (e.g. ad libitum): Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum during the study.
- Acclimation period: not specified in the report

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66 ± 5°F (19 ± 3°C), Actual mean daily temperature ranged from 65.6°F to 66.5°F (18.7°C to 19.2°C)
- Humidity (%): 50 ± 20% relative humidity, Actual mean daily relative humidity ranged from 43.5% to 54.8%
- Air changes (per hr): Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Light timers were calibrated to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: The appropriate volume of the test or control article was applied evenly over the shaved, intact dorsal skin of each animal (approximately 10 cm x 15 cm) using a syringe and stainless steel cannulae (16-gauge).
- % coverage: The area dosed corresponded to approximately 5.3% to 7.2% of the body surface area (cm2).
- Type of wrap if used: A bandage of absorbent gauze was placed over the dosing area followed by a layer of non-absorbant material (polyethylene plastic). These 2 layers were held in place with tape. The treated area and the 2 layers of material were then secured by wrapping the torso with gauze bandaging and secured with Durapore® wrap.
- Time intervals for shavings or clipplings: One day prior to the initiation of dose administration and throughout the study as necessary (at least 1 time each week), a section slightly larger than the dosing area of the scapular and lumbar regions of each rabbit was shaved with Oster® small animal clippers.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Following the 6-hour exposure period each day, the collars and wrappings were removed. The test sites were gently washed with disposable paper towels and deionized water to remove any residual test article, and then gently dried.
- Time after start of exposure: 6 hours/day

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The dosage volumes were 1.1, 0.55, 0.83 and 1.1 mL/kg for the control, 500, 750 and 1000 mg/kg/day groups, respectively.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. The duration of the exposure was 6 hours, during which Elizabethan collars were
applied to each animal to prevent ingestion of the test article and/or wrappings.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
As the test article was administered undiluted, analyses to confirm stability, homogeneity and concentration of the test article were not conducted.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
The control and test article were administered by dermal application for 6 hours, once daily during gestation days 6-28.
Frequency of treatment:
The control and test article were administered by dermal application for 6 hours, once daily during gestation days 6-28.
Duration of test:
The control and test article were administered by dermal application for 6 hours, once daily during gestation days 6-28.
No. of animals per sex per dose:
25 time mated rabbits per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a range finding study, there was no maternal toxicity or embryo/fetal lethality observed at the limit dose of 1000 mg/kg/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Animals were also observed for signs of toxicity approximately 1-2 hours following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual detailed clinical observations were recorded from the day of receipt through gestation day 29 (prior to dose administration during the treatment period).

DERMAL OBSERVATIONS: Yes
- Time schedule: Application sites were examined for erythema, edema, scaling, fissuring and other dermal findings daily within approximately 1 hour (target time between 30 and 60 minutes) after the end of each 6-hour exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 (by supplier), 4 and 6-29 (daily).

FOOD CONSUMPTION: Yes
- Time schedule: Individual food consumption was recorded daily on gestation days 4-29. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals and also for gestation days 6-9, 9-12, 12-15, 15-18, 18-21, 21-25, 25-29 and 6-29.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: rabbits were euthanized on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded.

OTHER: Liver weights were recorded for all does. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The carcass of each female was then discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Standard statistical procedures were used.
Indices:
Postimplantation loss, viable fetuses affected/litter
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All animals survived to the scheduled necropsy. Slight to severe scaling and slight fissuring was noted for a majority of animals in the 1000 mg/kg/day group. The incidence of these findings was highest mid-way through dosing, and decreased in severity and incidence generally throughout the remainder of the study. The control, 500 and 750 mg/kg/day groups also experienced slight scaling, but the incidence was greatly reduced compared to the 1000 mg/kg/day group. Moderate erythema was noted in 1 and 4 females in the 750 and 1000 mg/kg/day groups, respectively. Slight erythema was noted in all treatment groups, with the highest occurrence in the 1000 mg/kg/day group. The incidence of very slight erythema (barely perceptible) was similar between the control and 500 mg/kg/day group; however, this finding was noted up to approximately twice as often in the 750 and 1000 mg/kg/day groups throughout the treatment period. Very slight to slight edema was noted for multiple females in the 500, 750 and 1000 mg/kg/day groups, compared to a single occurrence of very slight edema in the control group; no dose-related trend was evident. The dermal findings in this study were generally considered test article-related, but because the severity and incidence decreased substantially or were absent by the end of the dose administration period, the dermal findings were not considered adverse.

Other than the dermal irritation described above, no test article-related clinical or macroscopic findings were noted at any dose level. Mean maternal body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights and food consumption were unaffected by test article administration.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Intrauterine growth and survival in the test article-treated groups were similar to the control group. There were no test article-related malformations or developmental variations observed in any fetus evaluated in this study.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity overall effects
Abnormalities:
not specified
Developmental effects observed:
not specified

None

Conclusions:
Based on the results of this study, the dermal application of 1000 mg/kg/day (the limit dose based on OPPTS 870.3700 Guidelines) was considered to be the no-observed-adverse-effect level (NOAEL) for maternal systemic toxicity; however, dermal irritation (local toxicity) was transiently observed at all dosage levels. A dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal development when DPnP was administered by dermal exposure to pregnant rabbits.
Executive summary:

The objective of the study was to determine the potential maternal toxicity (local and systemic) and/or prenatal developmental toxicity of the test article, dipropylene glycol n-propyl ether (DPnP), when administered by dermal exposure to pregnant rabbits throughout the period of major organogenesis up to a limit dose of 1000 mg/kg/day and to determine a NOAEL (no-observed-adverse-effect level) for maternal toxicity and developmental toxicity.

All animals survived to the scheduled necropsy. Slight to severe scaling and slight fissuring was noted for a majority of animals in the 1000 mg/kg/day group. The incidence of these findings were highest mid-way through dosing, and decreased in severity and incidence generally throughout the remainder of the study. The control, 500 and 750 mg/kg/day groups also experienced slight scaling, but the incidence was greatly reduced compared to the 1000 mg/kg/day group. Moderate erythema was noted in 1 and 4 females in the 750 and 1000 mg/kg/day groups, respectively. Slight erythema was noted in all treatment groups, with the highest occurrence in the 1000 mg/kg/day group. The incidence of very slight erythema (barely perceptible) was similar between the control and 500 mg/kg/day group; however, this finding was noted up to approximately twice as often in the 750 and 1000 mg/kg/day groups, respectively, throughout the

treatment period. Very slight to slight edema was noted for multiple females in the 500, 750 and 1000 mg/kg/day groups, compared to a single occurrence of very slight edema in the control group; no dose-related trend was evident. No maternal systemic toxicity (clinical observations, body weight, food consumption, macroscopic findings or liver weights) was observed at any dosage level in this study. Dermal irritation (local toxicity) was transiently observed at all dosage levels. Intrauterine growth and survival in the test article-treated groups were similar to the control group. There were no test article-related malformations or developmental variations observed in any fetus evaluated in this study.

Based on the results of this study, the dermal application of 1000 mg/kg/day (the limit dose based on OPPTS 870.3700 Guidelines) was considered to be the no-observed-adverse-effect level (NOAEL) for maternal systemic toxicity; however, dermal irritation (local toxicity) was transiently observed at all dosage levels. A dosage level of 1000 mg/kg/day was considered to be the NOAEL for prenatal development when DPnP was administered by dermal exposure to pregnant rabbits.

Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
910 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable
Additional information

There is a developmental toxicity study in rats which has been performed with DPGBE but not in rabbit.

In the absence of developmental toxicity data on DPGBE in rabbit, data from the structurally related dipropylene glycol methyl ether (DPGME) and dipropylene glycol n-propyl ether (DPGPE), which have undergone developmental toxicity testing in rabbit, can be used along with another developmental rat study with DPGME. DPGME, DPGPE and DPGBE 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).

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 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.

Data available for DPGPE:

The objective of the study was to determine the potential maternal toxicity (local and systemic) and/or prenatal developmental toxicity of the test article, dipropylene glycol n-propyl ether (DPGPE), when administered by dermal exposure to pregnant rabbits throughout the period of major organogenesis up to a limit dose of 1000 mg/kg bw/day. All animals survived to the scheduled necropsy. Dermal irritation, fissuring, scaling, erythema, and/or edema (local toxicity) was observed at all treatment levels. No maternal systemic toxicity (clinical observations, body weight, food consumption, macroscopic findings or liver weights) was observed at any dosage level in this study. Intrauterine growth and survival in the test substance-treated groups were similar to the control group. There were no test substance-related malformations or developmental variations observed in any fetus evaluated in this study. Based on the results of this study, the dermal application of 1000 mg/kg bw/day (the limit dose based on OPPTS 870.3700 Guidelines) was considered to be the no-observed-adverse-effect level (NOAEL) for maternal systemic toxicity; however, dermal irritation (local toxicity) was transiently observed at all dosage levels. A dosage level of 1000 mg/kg bw/day, the highest dose for the study, was considered to be the NOAEL for prenatal development when DPGPE was administered by dermal exposure to pregnant rabbits.

Data available for DPGME:

In a Prenatal Developmental Toxicity study, Fischer 344 rats were exposed via inhalation for 6 hours/day to 0 (control filtered air), 50, 150 and 300 ppm of DPGME vapors on day 6 through 15 of gestation. In-life parameters examined included clinical observations, feed consumption, water consumption and body weight. On day 21 of gestation, all animals were euthanased prior to cesarean section. Maternal liver and gravid uterine weights, the number of corpora lutea, implantations, resorptions and live/dead fetuses were recorded. The fetuses were removed from the uterus, weighed and examined for external, visceral and skeletal alterations. No treatment-related effects were observed on any of the maternal, embryonal and fetal parameters evaluated. In conclusion, inhalation exposure of pregnant rats to concentrations of DPGME as high as 300 ppm on days 6 through day 15 of gestation was not maternally toxic, embryo/fetotoxic or teratogenic. Three hundred ppm was the highest concentration of DPGME that was practically attainable at normal room temperature and pressure.

In a Prenatal Developmental Toxicity study, New Zealand White rabbits were exposed via inhalation for 6 hours/day to 0 (control, filtered air), 50, 150 and 300 ppm of DPGME vapors on days 7 through 19 of gestation. In-life parameters included clinical observations and body weight. On day 28 of gestation, all animals were euthanased prior to cesarean section. All fetuses were then removed from the uterus, weighed and examined for external, visceral and skeletal alterations. No treatment related effects were observed on any of the maternal and embryonal/fetal parameters evaluated at any exposure level. In conclusion, inhalation exposure of pregnant rabbits to concentrations of DPGME as high as 300 ppm on days 7 through 19 of gestation was not maternally toxic, embryo/fetotoxic or teratogenic. Three hundred ppm was the highest concentration of DPGME that was practically attainable at normal room temperature and pressure.

Summary:

There is sufficient data available for DPGBE along with structurally related substances, DPGME and DPGPE, to make a conclusion about the developmental toxicity of DPGBE to support classification and risk assessment.

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-d). In the DPGPE developmental toxicity study, the dermal application of 1000 mg/kg bw/day (the limit dose) was considered to be the no-observed-adverse-effect level (NOAEL) for maternal systemic toxicity; however, dermal irritation (local toxicity) was transiently observed at all dosage levels. A dosage level of 1000 mg/kg bw/day was considered to be the NOAEL for prenatal development when DPGPE was administered by dermal exposure to pregnant rabbits. In the studies using DPGME, no treatment related adverse effects - no maternal toxicity, no embryo-/fetotoxicity and no teratogenicity - were observed in rats or rabbits at the highest attainable concentration of DPGME. The NOAEL for DPGME is at least 300 ppm in both species. Based on the parameters covered in these studies the overall assessment indicates there would be no developmental effects expected for DPGBE.

 

 

Justification for classification or non-classification

Only a reproductive/developmental screening study (OECD 421) has be performed with DPGBE. This study showedno treatment-related effects on any reproductive parameters and no adverse gross or histopathological findings in the reproductive organs of males or females.Further inferences may be drawn from the developmental toxicity study in rats on DPGBE. In addition read-across data from other p-series glycol ethers (2-generation reprotox study on PGME) is available to address the fertility endpoint. No treatment-related effects on reproductive parameters were observed with PGME or DPGBE. Therefore, dipropylene glycol n-butyl ether should not be classified for reproductive toxicity.

DPGBE did not cause any developmental effects in rats. In addition read-across data from other p-series glycol ethers in rats and rabbits (DPGPE – rabbit; DPGME – rat, rabbit) are available to further address the developmental endpoint. No treatment-related effects on developmental parameters were observed with DPGBE, DPGME or DPGPE. Therefore no classification for developmental effects is required.