Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06/1995-02/1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-report according to OECD guideline 416
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
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
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 ppm
Reproductive effects observed:
not specified

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

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

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

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
6 061.35 mg/m³
Species:
rat
Quality of whole database:
Good (Klimisch 2)
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No fertility studies are available for dipropylene glycol methyl ether (DPGME). Therefore, a study on propylene glycol methyl ether (PGME) is used as a surrogate for DPGME.In a 2-generation inhalation reproduction study with propylene glycol monomethyl ether (PGME) no adverse fertility or reproductive effects were observed at 1,000 ppm PGME.The NOAEL for paternal toxicity is 300 ppm and for offspring toxicity is 1000 ppm. Effects appear secondary to parental weight loss.

 

Propylene glycol methyl ether and dipropylene glycol methyl ether are closely related in molecular structure and physicochemical properties and thus, the potential for toxicological effects. They are liquids with similar boiling points, low to moderate volatility, and high water solubility. Increasing boiling point and vapor pressure are consistent with increasing molecular weight. Major metabolic pathways for DPGME include conjugation with glucuronic acid and sulfate; hydrolysis of the methoxy group to form dipropylene glycol; and hydrolysis of the dipropylene glycol backbone of DPGME to form PGME and propylene glycol (see section 7.1.1). The glucuronide and sulfate conjugates of DPGME are essentially non-toxic and rapidly eliminated from the body. DPGME is less volatile and has been shown in comparable studies to be similar to, or less toxic than dipropylene glycol, PGME and propylene glycol, each of which are of low toxicity, themselves. Based upon the similarities in metabolism and modes of action of DPGME and its metabolites, it is highly probable that DPGME will be similar to or less toxic than its metabolites in reproductive toxicity studies.

 

PGME and DPGME have also undergone repeated dose toxicity testing at substantial doses with extensive histopathology, which allows to draw conclusions regarding effects on reproductive organs. Results from these repeated dose tests indicate that neither PGME nor DPGME caused toxicity to the testes. Specifically, no reduction in testicular weight, no damage to the sperm or sperm producing cells, and no damage to the epididymis or seminiferous tubules were reported. Likewise, no damage to female reproductive organs was found.

Further justification for the use of read across is contained in the category document attached at section 13.


Short description of key information:
No specific study is available for dipropylene glycol methyl ether. A two-generation inhalation reproduction study with propylene glycol methyl ether (PGME) in Sprague-Dawley rats is used for read-across. The study was conducted under GLP and according to OECD guideline 416.

Justification for selection of Effect on fertility via oral route:
There is an inhalation (most relevant route of exposure) two generation study (read across from PGME) available

Justification for selection of Effect on fertility via inhalation route:
Read across from PGME - GLP-report according to OECD guideline 416

Justification for selection of Effect on fertility via dermal route:
There is an inhalation (most relevant route of exposure) two generation study (read across from PGME) available

Effects on developmental toxicity

Description of key information
Inhalation teratology studies in rats and rabbits are available for dipropylene glycol methyl ether. Both studies were conducted under GLP and according to guidelines. 
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 818.4 mg/m³
Species:
rabbit
Quality of whole database:
Good (Klimisch 1)
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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 dipropylene glycol methyl ether. The studies in both species are of good quality and reliable without restrictions. The no observed adverse effect level for dipropylene glycol methyl ether is 300 ppm in both species.


Justification for selection of Effect on developmental toxicity: via oral route:
There are inhalation (most relevant route of exposure) developmental studies in rats and rabbits available.

Justification for selection of Effect on developmental toxicity: via inhalation route:
The study was conducted according to EPA TSCA guidelines and in accordance with the Principles of GLP.

Justification for selection of Effect on developmental toxicity: via dermal route:
There are inhalation (most relevant route of exposure) developmental studies in rats and rabbits available.

Justification for classification or non-classification

Fertility studies are not available for dipropylene glycol methyl ether (DPGME). Since DPGME has undergone repeated dose toxicity testing at substantial doses with extensive histopathology, definitive conclusions can be drawn regarding damage to reproductive organs. Results from these repeated dose tests indicate that DPGME did not cause toxicity to the testes. Specifically, no reduction in testicular weight, no damage to the sperm or sperm-producing cells, and no damage to the epididymis or seminiferous tubules were reported. Likewise, no damage to female reproductive organs was found. In addition read-across data from another P-series glycol ether (2-generation reprotox study on propylene glycol methyl ether, PGME) is available to address the fertility endpoint. No treatment-related effects on reproductive parameters were observed with PGME. Therefore, dipropylene glycol methyl ether should not be classified for reproductive toxicity. Two inhalation teratology studies in rats and rabbits conducted with DPGME are available to address the developmental toxicity endpoint. No developmental toxicity was observed with DPGME. Therefore, dipropylene glycol methyl ether should not be classified for developmental toxicity.