Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 21, 2006 - June 11, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 421 and in accordance with the principles of GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
USEPA OPPTS 870.3550
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity of the test material was 99.6 +/- 0.002% (GC with FID). The isomer ratios were DPnP-2,2 = 83.86%, DPnP-2,1 = 3.76%, DPnP-1,1 = 0.58%, and DPnP-1,2 = 11.80%. Identification was determined by GC/MS and Fournier Transfer infrared.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals: CD (Crl: CD(SD)IGS BR) rats were obtained from Charles River Laboratories, Inc (Portage, MI). They were acclimated to the laboratory at least one week prior to treatment, and were approximately 8 weeks old at the beginning of the study. Animals were housed two to three per cage during acclimation, one per cage after assignment or two per cage (one per sex during breeding) in stainless steel cages. Dams were housed one per cage (with litter) in plastic cages with ground corn cob nesting material from approximately gestation day 19 through lactation, under a 12 hour light/dark photocycle. Room humidity and temperature were maintained at 40-70% and 22 +/- 1 degrees C. Room air was exchanged approximately 12-15 times/hour. Animals were allowed free access to food (LabDiet Certified Rodent Diet #5002, PMI Nutrition International, St. Louis, MO) and municipal water. There were no contaminants in the feed or water that would interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
Groups of 12 male and 12 female CD rats randomly assigned to four treatment groups and were gavaged with 0 (control), 100, 300 and 1000 mg/kg/day test material in 0.5% methylcellulose vehicle (4 ml/kg bw). Females were dosed daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4 (one day prior to necropsy). The males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test Day 29). Dose volumes were adjusted weekly using the most current body weight. Dosing suspensions were prepared periodically throughout the study period based on stability.
Details on mating procedure:
Breeding commenced after approximately 2 weeks of treatment. During breeding, each female was caged with a male from the same group until pregnancy occurred (presence of sperm in vaginal lavage sample evaluated daily or presence of a copulatory plug) or two weeks had elapsed. The day on which pregnancy was detected was considered gestation day 0. The sperm- or plug-positive females were then separated from the males and returned to their home cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The low- and high-dose suspensions from the first mix of the main study were analyzed by GC/FID to confirm homogeneous distribution of the test material prior to the start of the study. The relative standard deviations were 1.1 and 1.7%, respectively, confirming homogeneous distribution. Concentrations in suspensions taken concomitantly with the homogeneity analyses were 101. 7 - 103.2% of targets. Stability of the test material (0.250, 2.50 and 250 mg/ml) in the vehicle was determined prior to study start. The material was stable for 39 days at the tested concentrations.
Duration of treatment / exposure:
Females were dosed daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4. The males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test Day 29)
Frequency of treatment:
once/day
Details on study schedule:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The high-dose level was based upon data obtained from the preliminary range-finding study and was expected to induce some toxic effects, but not
death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a NOEL.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
Cage-side examinations (activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function, altered respiration blue/pale skin and mucous membranes, severe eye injury (rupture), fecal consistency and fecal/urinary quantity were conducted twice daily. All animals were observed for morbidity, mortality and the availability of food and water at least twice daily. Clinical exams (hand-held examination of the animals with an evaluation of the abnormalities in the eyes, urine, feces, GI tract, extremities, movement, posture, reproductive system, respiration, skin/hair coat mucous membranes, general behaviour, injuries or palpable masses/swellings) were conducted once daily. Females were observed for signs of parturition on or about gestation day 20. Females that delivered received clinical examinations on lactation days 0, 1 and 4. Females that failed to mate or deliver were subjected to hand-held evaluations weekly.

All rats were weighed prior to exposure and on the first day of dosing. Male body weights were recorded weekly throughout the study and females were weighed weekly prior to gestation. Presumed pregnant females were weighed on gestation day 0, 7, 14 and 20. Females that delivered litters were weighed on lactation days 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly until termination.

Feed consumption was determined once a week during the pre-breeding phase. It was not determined during breeding. After breeding, it was determined on gestation days 0, 7, 14, and 20 for mated females. After parturition, feed consumption was measured on lactation days 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter of for males after breeding.
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
Weights of the epididymides and testes were calculated. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis.
Litter observations:
Females were observed for signs of parturition beginning on or about gestation day 20. If possible, parturition was observed for signs of difficulty or unusual duration. The day of delivery was recorded as lactation day 0. All litters were examined as soon as possible after delivery. The litter size on lactation day 0, numbers of live and dead pups on postpartum days (PND) 0, 1 and 4, and sex and weight of each pup on lactation days 1 and 4 were recorded. Physical abnormalities or demeanor changes in the neonates were recorded as observed during the lactation period. Pup clinical observations were recorded on each litter on PND 0 through 4. Any pups found dead were sexed and examined grossly for external and visceral defects.
Postmortem examinations (parental animals):
On the day prior to the scheduled necropsy, all males and females in each group were fasted overnight. At necropsy (test day 29 for males or lactation days 5-7 for females), the animals were anesthetized with CO2 and decapitated. Females that did not deliver were terminated and necropsied at least 24 days after the last day of the mating period.

Post-mortem examinations included a gross necropsy of all adults (which included a gross examination of the eyes, brain and pituitary, and external and visceral tissues). The histopathological examination of the testes included a qualitative assessment of states of spermatogenesis. The uteri of all females were stained with a 10% solution of sodium sulfide and the number of implantation sites recorded. Weights of the testes, epdidymides, kidneys and liver were recorded and organ to body weight ratios calculated.

Samples of the cervix, coagulating glands, epididymides, gross lesions, kidneys, liver, mammary gland (females), ovaries, oviducts, pituitary, prostate, seminal vesicles, testes, uterus and vagina were collected and preserved from adults surviving to study termination. These tissues plus the adrenals, aorta, auditory sebaceous glands, bone and bone marrow, brain, cecum, colon, cranial nerve, duodenum, esophagus, eyes, heart, ileum, jejunum, lacrimal/Harderian glands, larynx, lungs, mediastinal lymph node and tissues, mesenteric lymph node and tissues, nasal turbinates/pharynx, oral tissues, pancreas, parathyroid glands, peripheral nerve, rectum, salivary glands, skeletal muscle, skin and subcutis, spinal cord, spleen, stomach, thymus, thyroid gland, tongue, trachea, urinary bladder were collected from rats found moribund. All tissues collected from control and high dose animals and rats found moribund were examined histologically. Liver, kidneys and relevant gross lesions were examined from low and mid dose rats.
Postmortem examinations (offspring):
All pups surviving to PND 4 were euthanized by i.p. administration of sodium pentobarbital and examined for gross external alterations. Any pups found dead were also examined (if possible).
Statistics:
Parental body weights, gestation and lactation body weight gains, litter mean body weights, feed consumption and organ weights were first evaluated by Bartlett’s test for homogeneity. Parametric and nonparametric data were then analyzed by the appropriate analysis of variance (ANOVA). If the ANOVA was significant at alpha = 0.05, a Dunnett’s test (alpha = 0.05) or the Wilcoxon Rank Sum test with Bonferroni’s correction was performed. Feed consumption data were excluded if the feed was spilled or scratched.

Gestation length, average time to mating, and litter size were analyzed using a non-parametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank Sum test with Bonferroni’s correction was performed. Statistical outliers (alpha = 0.02) were identified and excluded from the analysis only for documented, scientifically sound reasons. Mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test, with Bonferroni’s correction. The neonatal sex ratio was analyzed using the binomial distribution test. Genders of pups found dead were included in the ratio. Survival indices, post-implantation loss and other neonatal incidence data were analyzed using the litter as the experimental unit by the censored Wilcoxon test (alpha = 0.05). Females failing to deliver a litter were excluded from the appropriate analyses.
Reproductive indices:
Female and male mating indices, female and male conception indices, female and male fertility indices, gestation index, post-implantation loss, sex ratio
Offspring viability indices:
Gestation survival index, Day 1 or 4 pup survival index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

1000 mg/kg bw: Very slight hepatocellular hypertrophy (11/12 males and 12/12 females), increased absolute (25.6% in males and 20.8% in females) and relative (28.2% in males and 16.5% in females) liver weights, increased absolute (8.1 % in males and 13.5% in females) and relative (10.5 % in males and 9.5% in females) kidney weights, very slight hyaline droplet formation in proximal renal kidney tubules of males (9/12), excess salivation immediately after dosing (all males and majority of females), increased post implantation loss (11.26% vs. 6.47% in control), decreased litter size (14.0 vs. 14.4 live pups/litter in control not significant but considered to be related to treatment). The mean litter size would have been lower (13.4) if it had not been for one animal that had a very large litter (20 pups). Four of 11 (36%) high dose litters had three or more resorptions (maximum in control litters was two). One female had a difficult birth and retained the placenta. One high dose male was terminated on test day 18 due to labored respiration. This animal had changes indicative of aspiration after dosing.

No effects were noted at 300 or 100 mg/kg bw/day.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

1000 mg/kg: Decreased gestation survival (97.5% vs. 98.9% in control), consistent with increased postimplantation loss. No malformations noted.

No effects were noted at 300 or 100 mg/kg bw/day.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the results, the no-observed-effect level (NOEL) for DPnP for parental and reproductive toxicity was 300 mg/kg/day.
Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered Dipropylene Glycol n-Propyl Ether (DPnP) 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 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.

Administration of 1000 mg/kg/day of DPnP resulted in treatment-related parental toxicity in males and females consisting of increases in the incidence of hepatocellular hypertrophy and corresponding increases in absolute and relative liver weights. In addition, absolute and relative kidney weights were increased in males and females at this dose level. Microscopic examination of the kidneys revealed hyaline droplet formation in the proximal renal tubules of males given 1000 mg/kg/day, but there were no treatment-related histopathologic findings in the kidneys of high-dose females. Transient, excess salivation was noted in the majority of high-dose males and females immediately after dosing, but was considered to be a local response and of no toxicological significance.

Accompanying the parental toxicity at 1000 mg/kg/day was a slight, treatment-related increase in post implantation loss, along with a corresponding slight increase in gestation survival and very slight decrease in litter size. One high-dose female also had a difficult birth and retained placentae, although the relationship of this finding to treatment is equivocal. There was no parental or reproductive toxicity at 300 or 100 mg/kg/day.

Based on these results, the no-observed-effect level (NOEL) for parental and reproductive toxicity and was 300 mg/kg/day.