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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1987- June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 414
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Dowanol-DPnB (n-butoxypropoxypropanol or
dipropylene glycol normal-butyl ether).
CAS # 29911-28-2.
Appearance: Clear, colorless liquid.
Batch No.: XZ451100.
Source: Dow Chemical Europe, Horgen, Switzerland.
Expiration Date: None specified.
Purity: >95%.
Specific Gravity: 0.91 kg/liter.
Solubility in
water: 5%.
Stability: Stable up to 200°C.
Storage: Ambient temperature in dark.
Administered as: Dilution in 1% Methocel and water.

Test animals

Species:
rat
Strain:
other: Wistar derived SPF-bred albino rats (Bor; WISW, SPF TNO)
Details on test animals and environmental conditions:
Age at dosing:       Approximately 12 weeks (females) and 13 eeks (males) of age.
Source:                Winkelmann Versuchstierzucht GmbH & Co. KG, Borchen, West-Germany.
Acclimation period:  Approximately 1 week.
Average weight (start of study):       Males: not specified Females: 186 - 209 grams.
Assignment to  groups:      Computerized, random number-based  procedure.
Diet:                "Basal Diet" (analysis provided in report)
Access to food:      Available ad libitum.
Access to water:     Available ad libitum.
Method of  Identification:      "V" notches on ears.
Housing:          Prior to mating:  males - individually, females - 5 per group in stainless steel cages with wire-mesh bottoms. 
After mating: housing-type for females not specified.
Environmental Conditions:
Temperature:         22 ± 2°C. Recording frequency not reported.
Humidity:            At least 40%. Range & recording  frequency not reported.
Air changes:         8-10 air changes per hour.
Photoperiod:         12 hr light/12 hr dark.

Administration / exposure

Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
propylene glycol
Details on exposure:
Dipropylene glycol n-butyl ether (DPnB) was applied daily to the skin of pregnant rats on gestation days 6 through 15 (detection of sperm in vaginal smears was designated day 0).  DPnB was applied to the clipped skin of two groups of Wistar rats (>20/sex/dose level) at various dilutions in propylene glycol (PG) equivalent to doses of 0 (PG-only; 1.5 ml/kg-day), 0.3 or 1.0 ml DPnB/kg-day.  These doses equate to 0, 273, or 910 mg DPnB/kg-day.  Treatment solutions were applied to the clipped dorsal trunk of each rat over an area of about 20 cm2.  Dilutions of DPnB in PG resulted in applied volumes of 1.5 ml (PG-only), 1.8 ml (1.5 ml PG & 0.3 ml DPnB), or 2.5 ml (1.5 ml PG & 1.0 ml DPnB) test solution per kg body weight.  Rats wore neck collars to prevent grooming and ingestion of test material.  Solutions were applied unoccluded since the low vapor pressure of DPnB and PG precluded evaporative loss. 
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of DPnB was determined in the test solutions prepared on July 20-1987. Samples were taken from each solution and stored at -20 celcius until further analysis. The analysis was carried out at TNO Institute CIVO-Analysis.
Details on mating procedure:
Females were placed overnight with males, e.g. two females with one male. Vaginal smears were taken to determine whether mating occured. If no sperm cells were detected, the female of question was placed with another male again. The day of detection of sperm cells was considered day 0 of pregnancy. This procedure was continued for 4 days to have sufficient number of possibly pregnant animals in each of the groups. Animals found to have mated were assigned to each group in rotation.
Duration of treatment / exposure:
Days 6-15 of gestation (day plug found was Day 0)
Frequency of treatment:
daily
Duration of test:
Treatment from Day 0 to Day 15 of pregnancy. Test until day 21 of pregnancy.
No. of animals per sex per dose:
22 in control, 21 in 273 mg/kg, and 25 in 910 mg/kg dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: until day 21 of pregnancy

Examinations

Maternal examinations:
Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Individual body weights were recorded on days 0, 6, 16, and 21 of pregnancy and food consumption was monitored over days 0 - 6, 6 - 16, and 16 - 21 of pregnancy.  At sacrifice, all animals were subjected to necropsy and examined for gross abnormalities. 
Ovaries and uterine content:
The number of corpora lutea was counted. 
Fetal examinations:
Fetuses were removed from the uterus, weighed, lengths recorded, and examined for gross abnormalities.  Early and late resorptions and live and dead fetuses were counted. Implantation sites in both uterine horns were counted and the empty uterus weighed.  Half the fetuses from each litter were eviscerated, skinned and stripped of most subcutaneous tissue, then fixed in 96% ethanol.  These fetuses were then stained with Alizarin Red S for examination for skeletal anomalies.  The remaining fetuses were fixed in Bouin's fluid, transferred to 70% ethanol and sectioned into slices (after Wilson) for soft tissue examination.  Percentages of pre- and post-implantation loss were calculated, as was the degree of ossification for each fetus.  Soft tissue and skeletal anomalies or abnormalities were recorded.
Statistics:
For the statistical analysis of difference in degree of ossification between test and control student t-test was applied. Statistical analysis for the body weight, food consumption, organ weights, litter data, foetus weights and lengths, and placenta weights was carried out by applying analysis of (co)-variance, with body weight on day 0 as covairable, followed by Dunnet's multiple comparison test, whereas skeletal and visceral anormalies were evaluated by the Chi-square test.
Indices:
Percentage pre-implantation loss (PRIL)
Percentage post-implantation loss (POIL)
Degree of ossificatin of foetus skeleton (DgO)
Transformed ossification values (DfOt)

Historical control data:
Incidence of spontaneous malformations, anomalies and variants in Albino rats of the Wistar strain (Cpb-WU; Wistar random). TNO report # v82.043/020283

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Slight skin reactions were found in the dams from all treatment groups and thus were not considered to be treatment related.  No maternal toxicity was found: clinical signs and organ or body weights did not differ between treatment and controls groups.  No deaths occurred in any groups over the course of the study.  Fecundity was comparable among groups.  

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
910 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No embryo- or fetotoxicity was evident since pre- and post-implantation loss, number of viable fetuses, and fetal weights and lengths were comparable between treatment and control groups.  DPnB did not cause frank developmental toxicity in skeletal or soft tissue.  Frank skeletal malformations were observed only in the control group (6 fetuses from 2 litters).  Skeletal variants were observed in all dose groups.  The high dose group did exhibit a slight increase (not statistically significant) in the incidence of supernumerary rudimentary thoracic ribs when compared to controls.  However, this finding was not considered biologically significant by the authors of the study since the incidence was within normal limits for these species.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
910 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
DPnB is not maternally toxic, embryo- or fetotoxic, or teratogenic in Wistar rats receiving dermal doses up to 1.0 ml/kg-d during organogenesis (days 6 - 15). The NOAEL for maternal toxicity, embryo- or fetal toxicity, or developmental toxicity is 1.0 ml/kg-d (910 mg/kg-d) and a LOAEL was not established.
Executive summary:

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

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

 

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

 

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

 

The experimental design is shown in the table below.

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

 

Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Individual body weights were recorded on days 0, 6, 16, and 21 of pregnancy and food consumption was monitored over days 0 - 6, 6 - 16, and 16 - 21 of pregnancy.  At sacrifice, all animals were subjected to necropsy and examined for gross abnormalities. The number of corpora lutea was counted. Fetuses were removed from the uterus, weighed, lengths recorded, and examined for gross abnormalities.  Early and late resorptions and live and dead fetuses were counted. Implantation sites in both uterine horns were counted and the empty uterus weighed.  Half the fetuses from each litter were eviscerated, skinned and stripped of most subcutaneous tissue, then fixed in 96% ethanol.  These fetuses were then stained with Alizarin Red S for examination for skeletal anomalies.  The remaining fetuses were fixed in Bouin's fluid, transferred to 70% ethanol and sectioned into slices (after ) for soft tissue examination.  Percentages of pre- and post-implantation loss were calculated, as was the degree of ossification for each fetus.  Soft tissue and skeletal anomalies or abnormalities were recorded. For the statistical analysis of difference in degree of ossification between test and control student t-test was applied. Statistical analysis for the body weight, food consumption, organ weights, litter data, foetus weights and lengths, and placenta weights was carried out by applying analysis of (co)-variance, with body weight on day 0 as covairable, follwoed by Dunnet's multiple comparison test, whereas skeletal and visceral anormalies were evaluated by the Chi-square test.

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

 

Slight skin reactions were found in the dams from all treatment groups and thus were not considered to be treatment related.  No maternal toxicity was found: clinical signs and organ or body weights did not differ between treatment and controls groups.  No deaths occurred in any groups over the course of the study.  Fecundity was comparable among groups.  No embryo- or fetotoxicity was evident since pre- and post-implantation loss, number of viable fetuses, and fetal weights and lengths were comparable between treatment and control groups.  DPnB did not cause frank developmental toxicity in skeletal or soft tissue.  Frank skeletal malformations were observed only in the control group (6 fetuses from 2 litters).  Skeletal variants were observed in all dose groups.  The high dose group did exhibit a slight increase (not statistically significant) in the incidence of supernumerary rudimentary thoracic ribs when compared to controls.  However, this finding was not considered biologically significant by the authors of the study since the incidence was within normal limits for these species.

 

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

 ml/kg-d during organogenesis (days 6 - 15). The NOAEL for maternal toxicity, embryo- or fetal toxicity, or developmental toxicity is 1.0 ml/kg-d (910 mg/kg-d) and a LOAEL was not established.

 

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

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