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Toxicological information

Toxicity to reproduction

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Administrative data

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
(adopted 1983)
Deviations:
yes
Remarks:
several newer parameters (e .g ., sperm analysis, developmental landmarks, estrous cycling, weanling organ weights) were also evaluated .
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 97.99 - 98.07%
There were no impurities present at greater than 0.1% .

Test animals

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: (P) 6 weeks
- Housing: rats were housed singly in wire mesh, stainless-steel cages during and after inhalation exposure (exceptions were after exposure in late gestation and throughout lactation (pups were weaned at 21 days-of-age), when the females were housed in plastic nesting boxes with ground corn cob bedding)
- Diet: Purina Certified Rodent Chow No. 5002 (Purina Mills Inc., St . Louis, MO) was provided ad libitum except during the 6-h/day exposures, at which time feed was withheld
- Water: drinking water, ad libitum throughout the study
- Acclimation period: ca. 2 weeks

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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Whole-body inhalation exposures were conducted in 14.5 m3 chambers under dynamic airflow conditions (approximately 2900 liters per min). The chambers were operated at a slightly negative pressure relative to the surrounding area. Control rats were placed in a chamber of identical design supplied with air without test material. Test atmospheres containing PGME were generated using the glass J-tube method of Miller et al. (1980). Liquid PGME was metered into the glass J-tube assembly using a fluid-metering pump. Compressed air (up to 100 lpm) was passed through the J-tube simultaneously to volatilize the test material. The air was heated with a heat gun to the minimum extent necessary to facilitate complete vaporization of the test material (approximate temperatures were; 65°C for the 300 ppm chamber, 115°C for the 1000 ppm chamber, and 150-170°C for the 3000 ppm chamber). The PGME vapors were diluted and mixed with room air to achieve the previously indicated total flow rate of approximately 2900 lpm and the desired concentration.
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 until pregnancy occurred or 2 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. If mating did not occur during the 2 weeks, the animals were separated without further opportunity for mating. For the P2 mating, cohabitation of male and female littermates 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 h using a MIRAN 1A infrared spectrophotometer at a wavelength of 10.2 microns. The spectrophotometer was calibrated using standards having a known PGME vapor concentration contained in 90-liter SARAN® 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 animals' breathing zone.
Airflow through each chamber was recorded hourly during the exposure with a factory calibrated Universal Venturi tube. This was coupled to a Setra Differential Pressure Transmitter and an incline manometer, which allowed for visual inspection throughout the exposures. Chamber temperatures and relative humidities were recorded hourly. On one exposure day, chamber temperature, humidity, and airflow values were monitored with a thermometer and hygrometer, respectively, and airflow was monitored with the incline manometers. The sensor in each chamber was calibrated prior to study start and approximately monthly thereafter. The temperature and relative humidity in each chamber were controlled by a system designed to maintain temperature and relative humidity at approximately 22 ± 2°C and 40-60%, respectively.
Duration of treatment / exposure:
10 weeks prior to mating and 7 days/week during mating, gestation and lactation for 2 generations
Frequency of treatment:
6 h/day, 5 days/week for 10 weeks prior to mating, and 6 h/day, 7 days/week during mating, gestation and lactation
Details on study schedule:
After approximately 10 weeks of exposure, Pl rats were mated (one male:one female) to produce the F1a litters. In order to confirm findings noted for the 3000 ppm F1la litters, the P1 adults were mated a second time (1 week alter weaning the last F1a litter) to produce the F1b litters. 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, 1-3 days after exposure of the F1a weanlings commenced on postnatal day 22, it became apparent that the weanlings were too small to tolerate an absence of feed during the 6 hours of inhalation exposure. The F1a weanlings in all dose groups appeared lethargic and/or weak following exposures and did not appear interested in feed or water, even after being removed from the inhalation chambers. Therefore, exposures were temporarily discontinued for several days, until the weanlings could achieve a larger size. Exposures of these F1a weanlings resumed on postnatal day 28 and continued through to evaluation of vaginal opening and preputial separation, after which time the animals were euthanized. Due to this modification involving the F1a litter, parents for the second generation (30 males, 30 females) were randomly chosen from the F1b litters. These litters were weaned on postnatal day 21, but exposure was not initiated until postnatal day 28. After approximately 10 weeks of treatment, the P2 adults were bred to produce the F2 litters. Exposures of Pl and P2 adult 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. Maternal rats were not exposed to PGME from gestation day 20 to lactation day 4, 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 6 h/day on lactation days 5 through 21.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 300, 1000 and 3000 ppm, corresponding to ca. 1.11, 3.69 and 11.07 mg/L (calculated with a conversion factor of 3.69 mg/m3 per ppm)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.00, 302.7 ± 10.16, 1005.7 ± 26.93, and 2996.3 ± 65.47 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 396, 1325, and 3974 mg/kg/day, assuming ventilation rates of 1 liter/min/kg and 100 percent absorption
Basis:
actual ingested
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
- Dose selection rationale: based an a 13-week inhalation study in Fischer-344 rats, which was used for dose selection (Cieszlak et al., unpublished data).

Examinations

Parental animals: Observations and examinations:
All adult rats were observed daily for changes in behavior, demeanor, or overt indications of toxicity. Rats found dead or moribund were submitted for a complete pathologic examination in an effort to determine cause of death. P1 and P2 rats were weighed weekly during the pre-breeding treatment period. Following the pre-breeding period, male body weights continued to be recorded weekly. Females exhibiting evidence of mating were weighed on days 0, 7, 14, and 21 of gestation and those with litters were weighed on days 1, 4, 7, 14, and 21 of lactation. 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 3 weeks prior to the F1a and F2 matings only, and continued throughout cohabitation.
Sperm parameters (parental animals):
Samples of sperm from the right distal cauda epididymis from the first ten Pl and P2 males sacrificed in each exposure group were collected for evalu¬ation of sperm motility. Sperm motility was determined with the use of the Hamilton-Thorne (HTM) Integrated Visual Optical System (IVOS) motility analyzer. 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.
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 parturition was initiated (day 0), the number of live and dead pups on days 0, 1, 4, 7, 14, and 21 postpartum, and the sex and weight of each pup on days 1, 4, 7, 14, and 21 of lactation. To reduce the variation in the growth of the pups, litters with more than eight pups were randomly culled on postpartum day 4 (4 males and 4 females per litter whenever possible). Litters with fewer than 8 pups were not culled. Any visible physical abnormalities or demeanor changes in the neonates were recorded during the lactation period. 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.
Thirty male and thirty female F1a and F1b weanlings per concentration were observed daily for vaginal opening, beginning on postnatal day 25 or male preputial separation beginning on day 35. Body weights of the respective F1b rats were recorded at the time of vaginal opening or preputial separation. Triggered by effects observed on these developmental landmarks in the F1b rats, anogenital distance was measured on postnatal day 4 (after culling) for F2 male and female pups in 10 litters per exposure concentration. Vaginal opening and preputial separation were not evaluated in the F2 offspring, as the pups were sacrificed on postnatal day 22.
Postmortem examinations (parental animals):
All P1 and P2 adults were given a complete gross necropsy examination. 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. Tissue sections from all major organ systems were saved from these rats and preserved in neutral, phosphate-buffered 10% formalin or Bouin's solution (testes and epididymides). 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. Weights of the 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 were determined in the first ten Pl and P2 parental animals sacrificed in each treatment group. Histologic examination of the cervix, coagulating glands, epididymides, kidneys, mammary gland, nasal tissues, ovaries, oviducts, pituitary, prostate, seminal vesicles, testes, thymus, uterus, vagina, and any gross lesions was performed on all rats in the control and high-concentration groups. Examination of tissues from the low and middle groups was limited to the liver and ovaries, as these organs demonstrated treatment-related histologic changes at the high concentration. Examined tissues were embedded in paraffin, sectioned at 6 pm and stained with hematoxylin and eosin. In order to classify the severity of ovarian atrophy, a grading system was developed as follows: normal, > 25 corpora lutea; very slight, 16-25; slight, 6-15; and moderate <5; the severe grade was not used.
Postmortem examinations (offspring):
A complete gross necropsy on one pup/sex/exposure concentration from the first ten F1 and F2 litters was conducted on postnatal day 22. Pups were anesthetized with methoxyflurane and euthanized by decapitation. Terminal body weights were recorded. Gross examination and preservation of tissue samples were performed as described above for adults. The ovaries or testes, brain, heart, liver, kidneys, adrenal glands, spleen and thymus of these pups were weighed. The kidneys, liver, spleen, thymus, and testes were examined microscopically in the control and high concentration groups.
Statistics:
Body weights, gestation/lactation body weight gains, organ weights, sperm count per gram of cauda epididymis, percent motile sperm, pup body weight (litter mean), and anogenital distance (litter mean) were first evaluated by Bartlett's test (a, 0.01) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) was performed (a = 0.10). If the ANOVA was significant, a two-sided Dunnett's test, or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed (a = 0.05). 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 (a = 0.05) 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 (Steel and Torrie, 1960). Survival indices and other incidence data among neonates were analyzed, using the litter as the experimental unit, by the Wilcoxon test (a = 0.05) as modified by Haseman and Hoel (1974). Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type 1 errors) was much greater than the cited a levels would suggest. Thus, the final interpretation of numerical data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were plausible in light of other biologic and pathologic findings.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Mortality
Of the 480 adult rats on test, one control, one 300 ppm, four 1000 ppm and one 3000 ppm group rats were found dead prior to the scheduled necropsy. The causes of death of these rats were generally incidental (e.g., leukemia, peritonitis) and unrelated to treatment with PGME.

Clinical Signs
Exposure to 3000 ppm of PGME resulted in sedation, as evidenced by incoordination and decreased activity, which was observed during the 6-hour exposure period and for various lengths of time after exposure. In all cases the sedation resolved by the next exposure day. This sedation was apparent in P1 adults during the first 3-5 weeks of exposure and in postnatal day 28 rats selected as P2 parents during the first 2 weeks of exposure. Sedation was not observed thereafter in the 3000 ppm rats nor in any rats exposed to 300 or 1000 ppm PGME at any time during the study. No other treatment-related changes in behavior or demeanor were observed in P1 adults at any exposure concentration during any phase of the study. No treatment-related clinical observations or physical alterations were observed for any F1a, F1b, or F2 pups from any of the exposure groups.

Body weights
Body weights of 3000 ppm P1 and P2 males and females were significantly decreased during the pre-mating, gestation, and early lactation periods relative to control body weights. These body weight decreases were especially marked in the P2 generation, with mean body weights as much as 21% lower than controls. Among the 1000 ppm group female rats, body weights were significantly lower than controls during a limited portion of the pre-mating phase, but not during gestation or lactation. No significant effects on body weight were observed in 1000 ppm group males nor in 300 ppm group males or females at any time during the study.

Estrous cyclicity
P1 and P2 females exposed to 3000 ppm PGME exhibited an increase in the mean number of days per estrous cycle as well as a resultant decrease in the mean number of estrous cycles during the period of evaluation. This effect on estrous cyclicity achieved statistical significance only in the P2 females, but was considered to be biologically significant in both the P1 and P2 animals. Also, 4 out of 10 high-exposure females in each of the 2 generations exhibited atypical vaginal cytology (specific stage of cycle was difficult to ascertain). No effects on mean number of days per cycle or number of cycles per dam were noted for the 300- or 1000 ppm group dams.

Reproductive data.
No treatment-related effects were observed on gestation survival index, pup sex ratios, gestation length, or time to mating at any exposure concentration for the F1a, F1b or F2 mating/litters. However, decreases in F1b and F2 male and female conception and fertility indices were observed among the 3000 ppm group animals. These decreases were not always statistically identified, but were usually outside of historical control ranges and were considered treatment-related. Decreased pup survival also was noted during the lactation phase for the F1a, F1b, and F2 litters of the 3000 ppm PGME group. No treatment-related effects were observed on any index of reproduction or fertility, gestation survival (% liveborn pups), pup survival, pup sex ratios, gestation length, or time to mating at 300 or 1000 ppm PGME.
Decreased litter size and decreased pup body weights also were noted in the high-exposure group for the F1a, F1b and F2 litters. The decreased litter sizes were reflective of significant decreases in pup survival during lactation, as mentioned above. No effects on mean litter size were observed for any of the 300 or 1000 ppm PGME group litters at any time during their respective lactation periods.

Developmental markers
Offspring from the 3000 ppm group exhibited statistically significant delays in age at vaginal opening (F1a and F1b) and age at preputial separation (F 1a only). In the females, vaginal opening was delayed by 2.6-2.9 days, while in males, a delay of 3.1 days was observed for preputial separation. Both the male and female F1b rats in the 3000 ppm group weighed significantly less than controls at the time these markers of puberty were achieved. No effects on time to preputial separation or vaginal opening, or on body weight at the time of vaginal opening or preputial separation, were noted for the 300 or 1000 ppm PGME groups. No significant effects were observed on anogenital distance for F2 male or female pups at any exposure concentration tested.

Gross necropsy and histopathology
P1 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, consistent with their decreased body weights (organ weight data not shown). Among P2 males, significant increases in relative liver, lung, and seminal vesicle weights were observed. P1 females from the 3000 ppm group had significantly higher relative adrenal, liver, and lung weights, while P2 females exposed to 3000 ppm PGME had significantly decreased brain (absolute) and ovary (absolute and relative) weights relative to controls. There were no treatment-related differences in organ weights of P1 or P2 males or females exposed to 300 or 1000 ppm PGME.
No treatment-related gross pathologic changes were observed among the P1 or P2 adults at any exposure concentration. Histologically, an increased incidence of ovarian atrophy (consistent with decreased ovarian weight) was observed among the 3000 ppm PGME P1 and P2 females. 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. The severity of ovarian atrophy was classified either as very slight, slight, or moderate. Moderate ovarian atrophy was observed in 8/30 (P1) and 10/30 (P2) females in the 3000 ppm group, as compared to a control incidence of 1/30 (P1) and 0/30 (P2). Among P2 females in the 3000 ppm group, an increased incidence of slight ovarian atrophy also occurred (4/30 vs. 0/30 for controls). However, the incidence of slight or very slight ovarian atrophy was similar among all groups in the P1 generation, probably reflecting an increased background incidence of reproductive senescence of the P1 females (8 months old) vs. the P2 females (5.6 months old). There were no treatment-related histopathological changes noted among the P1 or P2 adults exposed to 300 or 1000 ppm of PGME.

Sperm count and motility
Sperm count and motility was determined at necropsy after completion of the mating period for the adult P1 and P2 males. 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 (exhibiting forward motion) obtained from P1 3000 ppm PGME males was considered spurious and not of toxicological significance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 1.11 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: corresponding to 300 ppm; reduced body weights in higher doses
Remarks on result:
other: Generation: parental toxicity (P1, P2) (migrated information)
Dose descriptor:
NOEL
Effect level:
ca. 3.69 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: reproductive toxicity (F1a, F1b, F2) (migrated information)

Results: F1 generation

Details on results (F1)

Gross necropsy and histopathology
High concentration male and female weanlings generally exhibited decreased absolute weights of the kidneys, liver, spleen, testes, thymus and brain, and increased relative brain weights. Although these changes were not always statistically identified, the trends in these organs appeared fairly consistent across the F1a, F1b and F2 litters. There were no gross lesions identified at the F1la, F1b or F2 weanling necropsy that were associated with PGME exposure. Histologically, the livers of F1a, F1b and F2 3000 ppm PGME weanlings often lacked the normal degree of glycogen vacuolation (depletion) and exhibited thymic single-cell necrosis. No significant treatment-related organ weight, gross pathological, or histopathological changes were identified among the 300 or 1000 ppm PGME weanlings.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

At 3000 ppm, toxicity in the P1 and P2 adults was marked, as evidenced by sedation during and after exposure, 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, and histologic ovarian atrophy in maternal rats. In the offspring from these dams, decreased body weights, reduced sur­vival and litter size, slight delays in puberty onset, and histologic changes in liver and thymus in the F1 and F2 offspring were observed. The nature of the reproductive/neonatal effects and their close individual animal correlation with decreased maternal body weights suggested that these effects were secondary to general toxicity and/or nutritional stress. No such reproductive/neonatal effects were observed at 1000 ppm, a concentration which caused less marked, but significant body weight effects without sedation. There were no treatment-related effects of any kind noted at 300 ppm of PGME. Therefore, the no-observable-effect level (NOEL) for reproductive/neonatal effects was 1000 ppm, and that for parental toxicity was 300 ppm.

Applicant's summary and conclusion