Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
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

Constituent 1
Reference substance name:
Benzoflex 2-45
IUPAC Name:
Benzoflex 2-45
Constituent 2
Reference substance name:
Diethylene glycol dibenzoate
IUPAC Name:
Diethylene glycol dibenzoate
Constituent 3
Chemical structure
Reference substance name:
Oxydiethylene dibenzoate
EC Number:
204-407-6
EC Name:
Oxydiethylene dibenzoate
Cas Number:
120-55-8
Molecular formula:
C18H18O5
IUPAC Name:
2-[2-(benzoyloxy)ethoxy]ethyl benzoate
Details on test material:
- Name of test material (as cited in study report): Benzoflex 2-45 (Diethylene glycol dibenzoate DEGDB)
- Analytical purity: Diethylene glycol dibenzoate - 89.9% (w/w)
- Impurities (identity and concentrations):
Diethylene glycol monobenzoate - 5.85% (w/w)
Dipropylene glycol dibenzoate - 1.50% (w/w)
Ethylene glycol dibenzoate - 0.19% (w/w)
Triethylene glycol dibenzoate - 0.15% (w/w)
Unidentified component 1 - 0.10% (w/w)
Unidentified component 2 - 0.10% (w/w)
Unidentified component 3 - 0.54% (w/w)
- Lot/batch No.: 56635211

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system 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: estrogenic activity
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 5 out of 8 ovariectomized adult rats treated with DEGDB at 2000 mg/kg/day.
These observations included aggressiveness, rapid shallow breathing, red or brown discharge on the muzzle, piloerection, tremors, kyphosis, lethargy, scruffy coat, convulsions, hypothermia, vocalization and discharge around the eyes. Of the five affected animals, two rats were found dead, two moribund
animals were euthanized and one rat survived to day 7. At necropsy the four animals found dead or euthanized prior to day 7 had considerable weight loss and exhibited abnormalities in the gastrointestinal tract. In most cases, the stomach was distended with a solid sticky material resembling food and the duodenum contained little or no food (primarily gas and some liquid). The feces within the lower gastrointestinal tract tended to be either hardened tacky/sticky. All four rats appeared to be in or a dehydrated condition (decreased peritoneal fluid hardened feces in colon). The 4 surviving rats treated with 2000 mg/kg/day had no gross abnormalities at necropsy on day 7. The rats treated with 500, 1000 and 1500 mg/kg/day appeared normal during the study and at necropsy on day 7.

Any other information on results incl. tables

A study was performed to investigate whether the test substance, diethylene glycol dibenzoate (DEGDB), had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay (Bioqual Inc 1997, Vel 001-97). The study was conducted according to GLP standards, but no formal international test guidelines were applicable at the time it was performed.

Eight ovariectomised rats per group (4 test groups + 1 vehicle control (corn oil) + 3 groups of the 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 7 days of exposure to DEGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/kg/day. Hence, based on this endpoint, DEGDB showed no estrogenic activity.

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

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

Applicant's summary and conclusion

Conclusions:
Diethylene 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, diethylene glycol dibenzoate (DEGDB), had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay (Bioqual Inc 1997, Vel 001-97). The study was conducted according to GLP standards, but no formal international test guidelines were applicable at the time it was performed.

Eight ovariectomised rats per group (4 test groups + 1 vehicle control (corn oil) + 3 groups of the 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 7 days of exposure to DEGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/kg/day. Hence, based on this endpoint, DEGDB showed no estrogenic activity.

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

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