Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study conducted to GLP and although no International Test guideline available

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Adult ovariectomised rats (8 per group) were treated orally for 7 days (4 test groups + 1 vehicle control + 3 groups for positive control Diethylstilbestrol)
All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification.
All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.
GLP compliance:
yes
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 9-88 (Dipropylene glycol dibenzoate DPGDB)
- Analytical purity: Dipropylene glycol dibenzoate 89.4% (w/w)
- Impurities (identity and concentrations):
Dipropylene glycol monobenzoate - 4.98% (w/w)
Propylene glycol dibenzoate - 2.29% (w/w)
Propylene glycol monobenzoate - 0.28% (w/w)
Propenyloxy propyl benzoate - 2.35% (w/w)
- Lot/batch No.: 56635211

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY, USA.
- Age at study initiation: (P) x 9 wks
- Weight at study initiation: (P) Females: 170 -200 g
- Fasting period before study: Not applicable
- Housing: Individually in stainless steel hanging wire cages.
- Diet: Purina rodent chow (#5001), ad libitum
- Water: Mains (Montgomery County) tap water, ad libitum
- Acclimation period: 3 days prior to ovariectomy. Following a 6-7 days post surgery recovery period, evaluation of cell types was conducted daily
for one week prior to initiation of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Ranged 61°F to 76°F
- Humidity (%): Ranged 41% - 69%
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No details are available on the method of preparation of the dose solutions


VEHICLE
- Justification for use and choice of vehicle (if other than water): Test substance is poorly soluble in water
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): Mazola corn oil purchased from a local supermarket.
- Purity: Not stated but food grade
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Dosed once per day
Frequency of treatment:
Daily
Duration of test:
7 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
8 females per group
Control animals:
yes, concurrent vehicle
other: Positive control: ethylstilbestrol (DES) at 2.5, 5 & 10 µg/kg/day
Details on study design:
Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control diethylstilbestrol) were orally dosed for 7 days. All rats were observed daily for clinical signs and subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.
Statistics:
The occurrence of vaginal cornification was expressed as the number of rats showing vaginal cornification (for one or more days) over the number of rats treated. A z-test of proportions was used to compare the test groups to the control animals. The group means ± SE were calculated for body weights on day 0 and day 7 and a parametric one way analysis of variance (ANOVA) was performed. For a significant F value (p< 0.05), a Student-Newman-Keuls multiple range test was performed to determine differences among groups. Group means ± SE were also calculated for the uterine weights and uterine weight/final body weight ratios These data were compared using a non parametric one way ANOVA on ranks followed by Dunn's comparison to the designated control group.

Results and discussion

Effect levels

open allclose all
Dose descriptor:
other: estrogenic activity
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on induction of vaginal cornification compared to vehicle & positive controls
Dose descriptor:
other: uterine weight increase
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on increase in uterine weight to final bodyweight

Observed effects

Clinical signs of toxicity were apparent in 8 out of 8 ovariectomized adult rats treated with DPGDB at 2000 mg/kg/day.
These observations included rapid and or shallow breathing, tremors, piloerection, thinness, lethargy, discharge around the eyes, nose and mouth - often red in appearance, hypothermia and ataxia. One of the eight rats was found dead and two moribund animals were euthanized. The necropsy findings for these three rats included weight loss and abnormalities in the gastrointestinal tract. At necropsy no gross abnormalities were observed in the 5 surviving rats.
One rat dosed at 1500 mg/kg/day had a scruffy coat but survived the 7 days of dosing and no gross abnormalities were noted at necropsy. All other animals appeared normal during the study and at necropsy.

Any other information on results incl. tables

With respect to estrogenic activity, DES (the positive control) stimulated a dose dependent induction of vaginal cornification. The 2.5µg DES/kg/day dose was a threshold dose for this endpoint, in that the onset for the cornification response was of longer duration and more variable than in the 5.0 and 10.0 µg/kg/day groups. In addition, unlike the mid and high dose DES groups, not all rats in the low dose DES group gave a vaginal cornification response. Further, in the low dose DES group the number of rats cornified per number treated was significantly increased (p<0.05) only on days 4, 6 and 7 of the study, whereas for the mid and high dose DES groups this endpoint was significantly increased (p<0.05) from study days 3 through 7 and days 2 through 7, respectively.

In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 or 2000 mg/kg/day x 7 days. Hence by this endpoint DPGDB did not sjhow estrogenic activity.

DES tended to suppress body weight gain in a dose-dependent manner over the 7 day dosing period. Suppression of body weight gain or loss is a well known biological effect of potent estrogens. Only the high dose of DPGDB tended to decrease bodyweight gain but this effect was not significantly different for animals surviving 7 days of dosing.

DES induced a dose-dependent increase in uterine weight and uterine weight to final body weight ratio. Both parameters were increased significantly (p<0.05) at all dose levels of DES as compared to the vehicle control group. In contrast DPGDB did not stimulate an uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/ kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.

Applicant's summary and conclusion

Conclusions:
Dipropylene glycol dibenzoate did not possess estrogenic activity up to and including the maximally tolerated dose.
Executive summary:

A study was performed to investigate whether the test substance, dipropylene glycol dibenzoate, had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay. The study was conducted to GLP but no formal international test guidelines were applicable.

Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control Diethylstilbestrol (DES)) were orally dosed for 7 days. All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.

DES, the positive control, resulted in a dose dependent induction of vaginal cornification. In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/kg/day x 7 days. Hence by this endpoint the test compound showed no estrogenic activity.

DES also induced a dose dependent increase in uterine weight and uterine weight to final body weight ratio. Conversely, DPGDB did not stimulate a uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/ kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.