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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 22, 2005 - June 23, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 421
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:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DOWANOL DPnB glycol ether (chemical name: 1-(2-butoxy-1-methylethoxy)-2-propanol)
- Molecular formula (if other than submission substance): C10H22O3
- Molecular weight (if other than submission substance): 190
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: Colorless liquid
- Composition of test material, percentage of components: 99.34% purity
- Lot/batch No.: # SC1755R5K1

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: approximately 8 wks
- Weight at study initiation: Males: around 265 g; Females: around 194 g
- Fasting period before study: not described
- Housing: two-three per cage in stainless steel cages before dosing, and one per cage except during breeding and during littering phases of the study after dosing. During littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material from approximately GD 19 until completion of lactation.
- Use of restrainers for preventing ingestion (if dermal): as described above
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


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


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on exposure:
Groups of 12 male and 12 female Crl:CD(SD) rats were administered the test material daily, by gavage, at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day.

PREPARATION OF DOSING SOLUTIONS:
The test material was administered in a 0.5% methylcellulose vehicle, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose suspensions were prepared periodically throughout the study period based upon stability.

Dose Selection:
The high-dose level was based upon data obtained from a preliminary range-finding study and was expected to induce some toxic effects, but not death or obvious suffering. In addition, the high-dose of 1000 mg/kg/day represented a limit dose (1000 mg/kg bw/day). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a NOEL.


Details on mating procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If a breeding male died, a substitute partner (from the same dose group) that had already completed mating was provided. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of all dosing suspensions from the first mix of the main study were initiated prior to the start of dosing and were conducted concomitant with the homogeneity analysis.
Duration of treatment / exposure:
Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29).
Frequency of treatment:
Females and males were dosed once daily.
Details on study schedule:
Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
Groups of 12 male and 12 female Crl:CD(SD) rats were administered the test material daily, by gavage, at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 29). Effects on general toxicity, gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with histopathologic examination of tissues. The key study parameters and study schedule are presented in Table 1. Gavage dosing for both males and females began on October 05, 2005. The adult males were necropsied on November 02, 2005. The adult females were necropsied on November 14, 2005 to November 28, 2005. In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed. Pups were euthanized on PND 4.

Text Table 1. Dose Levels
Dose Levels No. of Adult Rats/Sex/Dose Level
(mg/kg/day)
0 12
100 12
300 12
1000 12
Total Number Adults: 96
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least twice daily for parental rats. Cage-side examinations were conducted on dams and their litters, at least twice daily.


DETAILED CLINICAL OBSERVATIONS: No


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


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumption was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co- housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)


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

OTHER:Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of partur ition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery.
Oestrous cyclicity (parental animals):
n/a
Sperm parameters (parental animals):
n/a
Litter observations:
All litters were examined as soon as possible after delivery. The following information was recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily In-Life Observations). In addition, pup clinical observations were recorded on each litter on PND 0 through 4. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and then discarded.
Postmortem examinations (parental animals):
Adult males (fasted) were submitted for necropsy after at least four weeks (actual: TD 29) of exposure. Adult females (fasted) were terminated on LD 5-7, or at least 24 days after the end of the mating period for females not producing a litter.

The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and were examined for the presence and number of implantation sites. After evaluation, uteri was gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.

Weights of the epididymides, kidneys, liver, and testes were recorded, and organ:body weight ratios calculated. Representative samples of tissues listed in Table 2 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides which were fixed in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues.

During routine working hours, any animal found dead or euthanized prior to the scheduled necropsy was necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and the testes and epididymides were preserved in neutral, phosphatebuffered 10% formalin.

Histologic examination of the tissues indicated in Table 2 were conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups (low and mid-dose) was limited to liver, kidneys, and relevant gross lesions.
Postmortem examinations (offspring):
All pups surviving to PND 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead were examined to the extent possible and discarded.
Statistics:
Various endpoints were analyzed using statistical models, including body weights, feed consumption, organ weights, etc.
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100
Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Offspring viability indices:
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

On the first day of dosing with 1000 mg/kg/day of DPnB, three females exhibited a transient, subtle, incoordinated gait which resolved within 1-2 hours after dosing and was not seen again for the remainder of the study. The only other treatment-related clinical observation was transient, post-dosing salivation (clear perioral soiling) noted sporadically in several high-dose males and females. This salivation was considered to be a local response to the taste of the test material, rather than evidence of toxicity.

Treatment-related increases in the incidence of hepatocellular hypertrophy occurred in males of all dose groups, and in females given 300 or 1000 mg/kg/day DPnB. The hypertrophy corresponded with increased liver weights in the 1000 mg/kg/day males and females, and 300 mg/kg/day males (absolute and relative weights affected). These changes were considered to be an adaptive response associated with increased hepatic metabolism of DPnB. Treatment-related increases in absolute and relative kidney weights also were found in males and females given 1000 mg/kg/day. In addition,
hyaline droplet formation in the proximal renal tubules was observed in males given 300 or 1000 mg/kg/day. Females given 1000 mg/kg/day had treatment-related higher mean absolute and relative kidney weights, but a histopathologic correlate to the higher kidney weights was lacking. There were no treatment-related effects on any reproductive parameters.

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no reproductive effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

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

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

Effect levels (F1)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
There were no treatment-related effects on any reproductive parameters. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 100 mg/kg/day, based on very slight to slight hepatocellular hypertrophy with no corresponding increases in liver weights in low-dose males. The no-observed-effect level (NOEL) for reproductive effects was 1000 mg/kg/day, the highest dose tested. According to EU classification and labeling criteria, DPnB is not classified as a reproductive toxicant and is not required to be labeled.
Executive summary:

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