Dibenzylbenzene, ar-methyl derivative

Endocrine disrupter mammalian screening – in vivo (level 3)

Administrative data

Endpoint:

endocrine disrupter mammalian screening – in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2020 - 29 May 2020

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 440 (Uterotrophic Bioassay in Rodents: A Short-term Screening Test for Oestrogenic Properties)

Version / remarks:

16 Oct 2007

Deviations:

no

Principles of method if other than guideline:

OECD 440 guidance document - Uterotrophic Bioassay procedure to test for antioestrogenicity (http://www.oecd.org/chemicalsafety/testing/37773994.pdf) accessed on 23-Mar-2020)

GLP compliance:

yes (incl. QA statement)

Test type:

Test for Estrogenic and Anti-Estrogenic Properties

Test material

Test material information

Constituent 1

Reference substance name:

Dibenzylbenzene, ar-methyl derivative

EC Number:

258-649-2

EC Name:

Dibenzylbenzene, ar-methyl derivative

Cas Number:

53585-53-8

Molecular formula:

C21H20

IUPAC Name:

dibenzylbenzene, ar-methyl derivative

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

RJ Han: SD (IOPS Han)

Sex:

female

State:

immature female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Date of birth: 07 May 2020
- Age at study initiation: PND 20
- Weight at study initiation: 45.8 g to 62.4 g
- If immature animals, whether or not supplied with dam or foster dam and date of weaning: nine litters with female pups were delivered with their mothers/foster mothers. The litters were culled and re-arranged at the supplier's facility to six female pups per litter. Date of weaning: 25 May 2020 (PND 19)
- Fasting period before study: n.a.
- Housing: pups and their mother were housed together in polycarbonate cages (Tecniplast 2000P, 2065 cm^2) with stainless steel lids and containing autoclaved sawdust (Le comptoir dessciures, Meyzieu, France). After weaning (PND 19), the pups were housed in the same type of cage in threes (same group). Each cage contained a rat hut and a nylabone for environmental enrichment
- Diet: SSNIFF rat/mouse pelleted maintenance diet, batch No. 63860736 (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD, WATER AND SAWDUST QUALITY
The batches of diet and sawdust are regularly analyzed by the Suppliers for composition and contaminant levels.
Bacterial and chemical analyses of sawdust, diet and water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (sawdust: pesticides and heavy metals; diet and water: pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 26 May 2020 To: 29 May 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) Carboxymethylcellulose 400-800cPs + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared as emulsion in the vehicle, according to Study No. 47136 AHS (Bezard, 2019) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study. The dosing formulations were prepared daily, on the days of treatment and were kept at room temperature until dosing.

VEHICLE
- Justification for use and choice of vehicle: Selection of the vehicle was based on previous experimental work and/or preliminary study(ies).
- Concentration in vehicle: 50, 160, 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Vehicle component 1: Drinking water treated by reverse osmosis, using ELIX 5 (Millipore SA)
- Vehicle component 2: Sodium Carboxymethylcellulose 400-800cPs (CMC), supplier: Sigma-Aldrich, batch no: SLBZ2611
- Vehicle component 3: Tween 80 (Polysorbate 80), supplier: Sigma-Aldrich, batch no: BCBZ4107

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed using the Gas Chromatography with FID detection (GC-FID) technique and the analytical method developed and validated at Charles River Laboratories Evreux (Study No. 47135 VAA (Bezard, 2019)) prior to dose formulation analysis. Test item concentrations in dose formulations were determined twice throughout the study, on days 1 and 3 by taking a sample from control and test item dose formulations and analyzed using the validated method. Deviation calculated between measured concentration and theoretical concentration should be within ± 15%.

Duration of treatment / exposure:

All animals were treated for 2 or 3 consecutive days.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 other: µg/kg bw/day

Remarks:

group 2 - reference item (17-a-Ethynylestradiol)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

group 3 - low dose

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

group 4 - mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 5 - high dose

Doses / concentrations 5

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

+ 3 µg/kg bw/day 17-a-Ethynylestradiol (reference item dose level)
group 5 - low dose + reference item

Doses / concentrations 6

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

+ 3 µg/kg bw/day 17-a-Ethynylestradiol (reference item dose level)
group 6 - mid dose + reference item

Doses / concentrations 7

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

+ 3 µg/kg bw/day 17-a-Ethynylestradiol (reference item dose level)
group 6 - high dose + reference item

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, based on the results of the following studies:
1) A preliminary dose-range finding study in pregnant rats was conducted with dose levels of 100, 500 and 1000 mg/kg bw/day. The test item was administered to eight female rats per dose level, once daily by gavage, throughout gestation and lactation (until Day 4 p.p.). Significantly decreased weight gains were observed during the first week of treatment (Days 0 to 7 p.c.) in pregnant rats at all dose levels. From Days 1 to 7 and from 1 to 20 p.c., food consumption was significantly decreased at 1000 mg/kg bw/day. The pre-birth loss index in the high-dose group was increased. In the F1 generation at 1000 mg/kg bw/day, the mean litter size was significantly reduced at birth and on Day 4 p.c. and the number of males was significantly decreased. The pup loss index at birth and the number of litters with dead pups was found to be increased at 1000 mg/kg/day.
2) A reproduction/developmental toxicity study was performed at Charles River Laboratories Evreux, OECD Test Guideline 421 (Reproduction/Developmental Toxicity Screening Test by Oral Route (Gavage) in Rats) where three groups of 10 male and 10 female rats received the test item
dibenzylbenzene, an ar-methyl derivative, at dose levels of 0, 80, 250 or 800 mg/kg bw/day from before mating, through mating and, for females, through gestation until Day 13 post-partum. There was no treatment-related mortality, clinical signs or effects on body weight or body weight change. An adverse decrease in food intake was observed in females treated at 800 mg/kg bw/day during the study. A decreased fertility index and a decreased mean number of corpora lutea/implantation sites were also observed in females at 800 mg/kg bw/day.

- Rationale for animal assignment: before the beginning of the treatment period (i.e. PND 19), all female pups showing no clinical signs were weighed. The required number of female pups (48) within the closest weight range (avoiding extremes in weight range) were allocated to the groups, according to a computerized stratification procedure, so that the average and standard deviation of body weight in each group were similar.

- Study with immature females after weaning and prior to puberty: yes, animals were weaned and allocated on PND19 and subsequently treated on PND 20-22

- 17a-ethinyl estradiol treatment (dose and volume) (if assay for antiestrogenicity): dose level: 3 µg/kg bw/day, volume administered: 5 mL/kg bw, concentration: 0.6 µg/mL

- Time of dosing: once a day at the same time of day (± 15 minutes). In order to avoid bias in the evaluation, the following order of treatment was respected: group 1, 3, 4, 5, 2, 6, 7 and 8. For groups 6, 7 and 8, on the days of treatment, the test item dose formulation was administered first by oral gavage, immediately followed by administration of the reference item formulation by oral gavage.

- Section schedule rationale: In order to avoid bias in the evaluation, the following order of treatment was respected: group 1, 3, 4, 5, 2, 6, 7 and 8.

- Time of necropsy: On completion of the treatment period, i.e. on Day 4, approximately 24 hours ± 1 hour after the last treatment, all surviving animals were euthanized

Positive control:

Name reference item: 17-a-Ethynylestradiol
Batch No.: WXBC9894V
Description: White powder
Storage conditions: At room temperature, protected from light, under argon atmosphere
Specific requirements(handling conditions): Under inactinic light
Purity: 100%
The reference item was ground to a fine powder, using a mortar and pestle and then mixed with the required quantity of vehicle. The reference substance solutions were made extemporaneously (daily preparation) and stored at room temperature, protected from light prior to use (transparent beaker covered with an aluminum foil). Particular care was taken during the preparation of reference substance formulations to avoid contamination. Each formulation was visually checked for homogeneity and this was documented before delivery to the animal room. No chemical analysis was performed of the reference item formulations.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during the acclimation period and three times a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of routine examinations. During treatment each animal was observed once a day, at approximately the same
time of day for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before group allocation (Day -1) and then on Days 1, 2 and 3 before administration and on Day 4 (day of euthanasia).

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured per cage daily.

SPECIFIC ENDPOINTS
- Wet uterine weight: Yes, for all animals
- Time schedule: Day 4 (day of necropsy)
- Blotted uterine weight: Yes, for all animals
- Time schedule: Day 4 (day of necropsy)

Optional investigations:
- Histopathological examination of uterus: Yes, of all animals
- Histopathological examination of vagina: Yes , of all animals
- Morphometric measurement of endometrial epithelium: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the treatment period, i.e. on Day 4, approximately 24 hours ± 1 hour after the last treatment, all surviving animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination in the same sequence as the test item administration (in order to avoid bias in the evaluation, there was no consecutive euthanasia from animals of the
same group). The animals were not fasted before euthanasia. On Day 3, moribund animals were euthanized in the same way.
A gross macroscopic post-mortem examination of the thoracic and abdominal cavities, focusing on the reproductive tract was performed on all animals, including those that died during the study or were prematurely euthanized. All macroscopic observations were recorded individually. For all animals, including those that died during the study or were prematurely euthanized, the uterus and vagina were preserved in 10% buffered formalin. Macroscopic lesions were preserved in 10% buffered formalin.

HISTOPATHOLOGY: Yes
All tissues required for microscopic examination were trimmed based on the RITA guidelines, embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin.
A microscopic examination was performed on the uterus (horns and cervix) and vagina of all animals (groups 1 to 8) euthanized at the end of the treatment period and for animals found dead or prematurely euthanized.

Statistics:

Provantis and PATHDATA softwares was used to perform the statistical analysis of body weight and of organ weight data (level of significance: 0.05 or 0.01) according to the flowchart attached below in section "Attached background material".

Results and discussion

Endocrine disrupting potential:

ambiguous

Maximum tolerated dose level exceeded:

yes

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the surviving groups, thin appearance and erected fur were observed in females treated with the test item alone at 800 mg/kg bw/day (group 4) on Day 4 and in one female treated with the test item at 250 mg/kg/day + the reference item (group 6) on Day 3. As these observations were also seen in prematurely euthanized group 5, 7 and 8 animals, they were considered to be test item-related.

Other clinical signs, such as hypersalivation, reflux at dosing or soiled genital region, are commonly encountered in laboratory rats and were therefore considered to be unrelated to the test- or reference-item treatments.

Mortality:

mortality observed, treatment-related

Description (incidence):

Five out of six females treated with the test item only at 1000 mg/kg bw/day (group 5) died or were prematurely euthanized on Day 3 or 4. From Day 3 decreased activity, ventral recumbency, half-closed eyes, erected fur, thin appearance and soiled urogenital region were observed in most females, associated with decreased food consumption from Day 2, and body weight loss (-19.3% on Day 3 vs. Day 1). No macroscopic findings were observed and the cause of death could not be determined (microscopic examination was performed on the uterus and vagina only). In view of the poor clinical condition on Day 3 and the occurrence of premature deaths at the high-dose level only, these deaths were considered to be related to the test item.

In the uterus of these decedents at 1000 mg/kg bw/day, similar, but less pronounced microscopic
changes than with the reference item (hypertrophy/hyperplasia of the endometrium and myometrium and apoptosis/necrosis of the endometrium) were observed.

In the vagina, minimal squamous hyperplasia was noted at microscopic examination in decedents at 1000 mg/kg bw/day. In addition, increased severity of mucification of the vaginal mucosa was noted in these decedents when compared with vehicle controls. Of note, keratinization of the vaginal mucosa was not observed in any decedents. No deaths occurred at 250 and 800 mg/kg bw/day.

Test item + reference item (groups 6 to 8)
All females treated with the reference item and the test item at 800 or 1000 mg/kg bw/day (groups 7 and 8) died or were euthanized prematurely on Day 3 or 4. From Day 3, half-closed eyes and decreased activity were observed in surviving animals, associated with mean body weight losses (approx. -15% in both groups on Day 3 vs. Day 1). In view of the poor clinical condition on Day 3, the occurrence at the intermediate- and high-dose levels only, and the absence of deaths in the group treated with the reference item alone, these deaths were considered to be related to the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item alone (groups 3 to 5): At 800 or 1000 mg/kg bw/day, mild to severe body weight losses were observed from Days 1 to 3, leading to markedly lower mean body weights on Day 3 (down to -19.3% in group 5, vs. Day 1 values). Given the magnitude and dose-relationship, these effects were considered to be test item-related. At 250 mg/kg bw/day, no test-item related effects were observed on body weight or body weight change during the study.

Test item + reference item (groups 6 to 8): At 800 or 1000 mg/kg bw/day, severe body weight losses were observed with no dose-relationship on Days 1 to 3, leading to markedly lower mean body weights on Day 3 (approximately -15% in both groups, vs. Day 1 values). Given their magnitude, these effects were considered to be test item-related. At 250 mg/kg bw/day, no test-item related effects were observed on body weight or body weight change during the study.

Reference item (group 2): No reference item-related effects were observed on body weight or body weight change during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item alone (groups 3 to 5): At 1000 mg/kg bw/day, low mean food consumption was recorded between Days 1 and 4, associated with body weight losses in the same test item-treated group over the study. Taking into account the magnitude of the changes, these effects were considered to be test item-related. At 250 or 800 mg/kg bw/day, there were no significant effects on mean food consumption.

Test item + reference item (groups 6 to 8): At 800 or 1000 mg/kg bw/day, lower mean food consumption was recorded between Days 1 and 3, associated with body weight losses in the same test item treated groups. Despite the absence of statistical analysis, these effects were considered to be test item-related given their magnitude.

Reference item (group 2): The reference item had no effect on mean food consumption.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Reference item (group 2): When compared with the vehicle control group (group 1), mean absolute and relative uterus weights (full and empty) were markedly increased in females treated with the reference item, 17-a-Ethynylestradiol. These increases correlated with hypertrophy/hyperplasia of the endometrium and myometrium and were consistent with the estrogenic effect of the reference item. The difference between the weights of the full/wet uterus compared to the empty/blotted uterus was considered to be related to the luminal fluid content and correlated with macroscopic dilatation with a translucent content at necropsy.

Test item alone (groups 3 to 5): When compared with the vehicle control group (group 1), there were no statistically significant differences on the mean uterus weights (either full or empty) in females treated with the test item alone at 250 mg/kg bw/day. There were no relevant changes in the uterus weights (either full or empty) in groups treated with the test item alone at 800 mg/kg bw/day. The lower absolute weight and higher relative weight of the uterus in the surviving female at 1000 mg/kg bw/day were considered to be related to the lower terminal body weight (-47%) compared to controls (no statistical evaluation could be performed due to the low number of surviving animals).

Test item + reference item (group 6): When compared with the reference item group (group 2), the mean absolute and relative weights of the uterus (full and empty) were slightly decreased in females treated with the test item at 250 mg/kg bw/day + reference item (however no statistical evaluation could be performed due to the low number of groups available). Of note, when compared with the vehicle control group (group 1), the weights of the uterus were slightly increased in the group treated with the test item at 250 mg/kg bw/day + reference item, which was most probably related to the reference item (estrogenic effect).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reference item (group 2): Reference item-related macroscopic changes were observed in the uterus. The uterine horns were dilated with a translucent content in all females treated with the reference item alone. This is consistent with the estrogenic effect of the reference item.

Test item alone (groups 3 to 5): No macroscopic findings were observed in females treated with the test item at 250 mg/kg bw/day alone. Macroscopic findings potentially related to the test item were observed in the kidneys at = 800 mg/kg/day and in the small intestine at 1000 mg/kg bw/day. At 800 mg/kg bw/day, the kidneys were enlarged in 2/6 females, and had an irregular color in another female. At 1000 mg/kg bw/day, in the surviving female, the kidneys had an irregular color and the renal pelvis was dilated. A yellow content was also noted in the jejunum and ileum of this female.

Test item + reference item (group 6): No macroscopic findings were observed in females treated with the test item at 250 mg/kg bw/day + reference item.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Reference item (group 2):
Microscopic findings consistent with the estrogenic effect of 17-a-Ethynylestradiol were observed in the uterus and vagina in all group 2 females, as follows:
Uterus:
• slight diffuse hypertrophy/hyperplasia of the endometrium (mucosa and glands),
• minimal or slight apoptosis/necrosis of the endometrium (mucosa and glands),
• slight or moderate diffuse hypertrophy/hyperplasia of the myometrium (smooth muscle layer).
Vagina:
• moderate diffuse squamous hyperplasia of the mucosa,
• minimal or slight diffuse keratinization of the mucosa,
• mucification of the mucosa was not observed (while it was present in all vehicle controls)

Test item alone (groups 3 to 5):
Uterus:
The same changes as those described with the reference item alone (hypertrophy/hyperplasia of the endometrium and myometrium and apoptosis/necrosis of the endometrium) were observed with a dose-related incidence and severity in females treated with the test item at 250 and 800 mg/kg bw/day and in the surviving female at 1000 mg/kg bw/day, albeit with lesser severity when compared to females treated with the reference item.
Vagina:
Minimal or slight squamous hyperplasia (of lesser severity when compared to females treated with the reference item alone), was present with a dose-related incidence in females treated with the test item at 250 and 800 mg/kg bw/day and in the surviving female at 1000 mg/kg bw/day. Minimal squamous hyperplasia was also noted in the decedents at 1000 mg/kg bw/day. Of note, keratinization of the mucosa was not observed at any dose levels.

Test item + reference item (groups 6 to 8):
Uterus:
The same changes as those described with the reference item alone (hypertrophy/hyperplasia of the endometrium and myometrium, apoptosis/necrosis of the endometrium at 250 mg/kg/day) were observed in females treated with the test item at 250 mg/kg bw/day + the reference item. However, the hypertrophy/hyperplasia of the endometrium and myometrium in this group was less pronounced than that observed with the reference item alone.
Vagina:
The same changes as those described with the reference item alone (squamous hyperplasia and keratinization of the mucosa) were observed in females treated with the test item at = 250 mg/kg bw/day + the reference item. However, the keratinization was less pronounced than that observed with the reference item alone.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Remarks:

Anti-estrogenic effects

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Remarks on result:

not determinable

Remarks:

NOAEL/LOAEL was not determinable due to the high mortality at the intermediate- and high-doses of the combination of the test item and the reference item.

2

Key result

Dose descriptor:

LOAEL

Remarks:

Estrogenic effects

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology

Remarks on result:

other:

Remarks:

At dose levels = 250 mg/kg bw/day, histopathology revealed microscopic changes suggestive of an estrogenic effect in the female reproductive organs of immature rats, although uterine weights were not increased.

3

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight

Remarks on result:

other: Test item related mortality, poor clinical condition, severe body weight loss seen in females at 800 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day, low mean food consumption was seen.

Any other information on results incl. tables

The test item concentrations in the administered dose formulations analyzed on Days 1 and 3 remained within an acceptable range of variations (+0.8 % to +10.4%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the vehicle control dose formulation.

 

Summary of mortality observed: 

Treatment	Control (vehicle for the test item)	Reference item (17-α-EE)	Test item			Test item + reference item
Group	1	2	3	4	5	6	7	8
Test item dose level (mg/kg/day)	-	-	250	800	1000	250	800	1000
Ref item dose level (µg/kg/day)	-	3	-	-	-	3	3	3
Number of animals	6	6	6	6	6	6	6	6
Total of deaths	0	0	0	0	5	0	6	6
Day(s) of death	-	-	-	-	Days 3 and 4	-	Days 3 and 4	Days 3 and 4

 

Summary of Mean Body Weights and Mean Body Weight Changes:

Group	Control	Reference item	Test item			Test item + Reference item
	1	2	3	4	5	6	7	8
Test item dose level (mg/kg/day)	0	/	250	800	1000	250	800	1000
Reference item dose level (µg/kg/day)	/	3	/	/	/	3	3	3
Number of animals	6	6	6	6	6	6	6	6
Mean body weight change (g)
Day 1 to 2	+3.6	+3.2	+1.1	-2.3*	-4.6***	+2.3	-2.3**	-2.3*
Day 2 to 3	+6.4	+5.0	0.0	-1.3*	-6.2*** (n = 5)	+2.2	-5.2*** (n = 5)	-4.3* (n = 2)
Day 3 to 4	+5.6	+5.3	+5.2	-0.2	-7.0 (n = 1)	+2.9	-	-
Days 1 to 4	+15.6	+13.4	+6.3*	-3.8***	-16.5* (n = 1)	+7.4*	-	-
Mean body weight (g)
Day 1	56.3	55.8	55.7	57.0	55.9	56.2	54.6	56.6
Day 2	59.8	59.0	56.8	54.7	51.3**	58.5	52.3*	54.3
Day 3	66.3	63.9	56.8**	53.4***	45.1*** (n = 5)	60.7	46.4*** (n = 5)	48.4*** (n = 2)
Day 4	71.9	69.2	61.9*	53.3***	37.5*** (n = 1)	63.6	-	-

 

Summary of mean food consumption:

Group	Control	Reference item	Test item			Test item + Reference item
	1	2	3	4	5	6	7	8
Test item dose level (mg/kg/day)	0	/	250	800	1000	250	800	1000
Reference item dose level (µg/kg/day)	/	3	/	/	/	3	3	3
Number of animals	6	6	6	6	6	6	6	6
Day 1 to 2	5.3	4.3	3.6	2.6	1.0	4.4	1.4	1.9
Day 2 to 3	7.7	6.5	3.4	3.3	0.0	5.2	3.5	3.7
Day 3 to 4	10.4	8.5	7.2	5.2	0.8	6.4	-	-

 

Summary of

Relevant Changes in Final Body Weight and Mean Uterus Weights at the End of the Treatment Period (% Changes Compared to Vehicle Controls):

Group	2	3	4	5
Treatment Test item Dosage	Reference item 0 mg/kg/day	Test item 250 mg/kg/day	Test item 800 mg/kg/day	Test item 1000 mg/kg/day
Number of animals	6	6	6	6
Final body weight	-3	-14*	-26**	-47°
Uterus full/wet
.absolute weight	+173**	-10	-9	-26°
.relative to body weight	+181**	+6	+21	+40°
Uterus empty/blotted
.absolute weight	+126**	-10	-10	-27°
.relative to body weight	+132**	+5	+21	+39°

 

Summary of Final Body Weight and Mean Uterus Weights (% Changes when Compared to the Reference Item Controls) in Groups 1, 2 and 6 at the End of the Treatment Period:

Group	1	2	6
Treatment Test item Dosage		Reference item 0 mg/kg/day	Test item + Reference item 250 mg/kg/day
No. animals	6	6	6
Final body weight (g)	71.5	69.3	64.1 (-7 %) °
Uterus full/wet
.absolute weight (g)	0.06	0.16	0.10 (-37%) °
.relative to body weight	0.08	0.23	0.16 (-32%) °
Uterus empty/blotted
.absolute weight (g)	0.06	0.12	0.09 (-25%) °
.relative to body weight	0.08	0.18	0.15 (-17%) °

 

Summary of Group Incidences of Relevant Macroscopic Observations at the End of the Treatment Period

Group	1	2	3	4	5	6
Treatment Test item Dosage	Vehicle 0 mg/kg/day	Reference item 0 mg/kg/day	Test item 250 mg/kg/day	Test item 800 mg/kg/day	Test item 1000 mg/kg/day	Test item + Reference item 250 mg/kg/day
No. animals	6	6	6	6	1	6
Uterus
Dilated	-	6	-	-	-	-
Kidneys
Enlarged	-	-	-	2	-	-
Irregular color	-	-	-	1	1	-
Dilated pelvis	-	-	-	-	1	-
Jejunum/ileum
Yellow content	-	-	-	-	1	-

 

Summary of Group Incidences of Relevant Microscopic Observations (Including Deaths):

Group	1	2	3	4	5	6	7	8
Treatment Test item Dosage (mg/kg/day)	V 0	RI 0	TI 250	TI 800	TI 1000	TI + RI 250	TI + RI 800	TI + RI 1000
Uterus
Hypertrophy/hyperplasia; endometrium
Minimal (grade 1)	-	-	1	2	5	2	-	3
Slight (grade 2)	-	6	-	-	1	4	6	3
Apoptosis/necrosis; endometrium
Minimal (grade 1)	-	3	1	4	1	2	-	-
Slight (grade 2)	-	3	-	-	-	4	-	-
Hypertrophy/hyperplasia; myometrium
Minimal (grade 1)	-	-	4	4	4	-	4	3
Slight (grade 2)	-	1	-	-	1	3	2	3
Moderate (grade 3)	-	5	-	-	-	3	-	-
Dilatation; lumen
Minimal (grade 1)	-	-	-	-	-	-	2	4
Slight (grade 2)	-	-	-	-	-	-	2	1
Moderate (grade 3)	-	-	-	-	-	-	1	-
Vagina
Mucification; mucosa
Minimal (grade 1)	6	-	6	6	4	-	1	2
Slight (grade 2)	-	-	-	-	2	-	-	-
Hyperplasia; squamous
Minimal (grade 1)	-	-	3	2	6	-	-	-
Slight (grade 2)	-	-	-	3	-	-	3	4
Moderate (grade 3)	-	6	-	-	-	6	3	2
Keratinization; mucosa
Minimal (grade 1)	-	1	-	-	-	5	1	2
Slight (grade 2)	-	5	-	-	-	-	5	2

 

Applicant's summary and conclusion

Conclusions:

In an oral uterotrophic bioassay performed according to OECD TG 440 and in accordance with GLP principles dibenzylbenzene, ar-methyl derivative was associated with microscopic changes suggestive of an estrogenic effect in the female reproductive organs of immature rats at dose levels = 250 mg/kg bw/day, despite the absence of increase of the uterine weights. Given the high mortality at the intermediate and high doses of the combination of the test item and the reference item, definite conclusion on the anti-estrogenic potential of the test item could not be drawn.

Executive summary:

The objective of the study was to evaluate the estrogenic and anti-estrogenic activities of the test item (dibenzylbenzene, ar-methyl derivative) following daily oral administration (gavage) to juvenile, sexually immature female rats for 3 consecutive days. The results were compared to those of a reference estrogen (17-α-Ethynylestradiol). The investigations performed at the end of the treatment period focused on uterus weight changes and microscopic examination of the uterus and vagina.

A total of 48 pre-pubertal female Sprague-Dawley rats [Post-Natal Day (PND) 20 at the start of treatment] were divided into eight groups, receiving the vehicle alone, the reference item (17-α-Ethynylestradiol) at 3 μg/kg/day, the test item at 250, 800 or 1000 mg/kg/day, or a combinaison of 250, 800 or 1000 mg/kg/day of test item 3 μg/kg/day of reference item, for 2 to 3 days.
Investigations during the study included survival, clinical signs, body weight and food consumption.
On completion of the treatment period, i.e. on Day 4, the animals were weighed and euthanized.
The uterus, with and without fluid was weighed immediately after euthanasia. A gross macroscopic post-mortem examination of the thoracic and abdominal cavities, with particular attention to reproductive organs, was performed. The uterus (horns and cervix) and vagina were preserved and examined microscopically.

The test item concentrations in the administered formulations analyzed at the beginning and end of the treatment period (Days 1 and 3) remained within the acceptable range of variations (± 15% of the nominal concentrations).

In the group treated with the test item alone at 1000 mg/kg/day (group 5) and in the groups treated with a combination of the reference item and the test item at 800 or 1000 mg/kg/day (groups 7 and 8), almost all females died or were prematurely euthanized on Day 3 or 4. Signs of poor clinical condition (such as erected fur, thin appearance, decreased activity, half-closed eyes or ventral recumbency) associated with severe mean body weight loss and decreased mean food consumption were observed prior to death/premature euthanasia. The causes of death could not be determined as no macroscopic changes were observed. However, in view of the occurrence of premature deaths at the intermediate- and high-dose levels, these mortalities were considered to be most probably related to the test item.
In the group treated with the test item alone at 800 mg/kg/day (group 4), thin appearance and erected fur were observed in some females on Day 4. These clinical signs were also observed in one female treated with a combination of the reference item and the test item at 250 mg/kg/day on Day 4. Severe body weight losses were recorded during the study in groups treated with the test item at 800 or 1000 mg/kg/day, with or without the reference item (mean body weight on Day 3 down to -19% vs. Day 1, in group 5 treated with the test item alone at 1000 mg/kg/day).
These effects were associated with lower mean food consumption when compared to controls, especially in groups treated with the test item at 1000 mg/kg/day, with or without the reference item.

Estrogenic assay
Administration of the reference item alone, 17-α-Ethynylestradiol, for 3 days to immature female rats induced marked, statistically significant increases in the weights of the full and empty uterus, correlating with macroscopic dilatation of the uterine horns and microscopic hypertrophy/hyperplasia of the endometrium and myometrium i.e. uterotrophic response, along with squamous hyperplasia and keratinization in the vagina. All these changes were consistent with estrogenic effects of the reference item. The sensitivity of the test system was therefore confirmed by the expected responses to the positive control treatment with 17-α-Ethynylestradiol.
Administration of the test item alone at 250 and 800 mg/kg/day and in the surviving female at 1000 mg/kg/day induced a uterotrophic response, namely hypertrophy/hyperplasia of the endometrium and myometrium at all dose levels tested in a dose-related manner, but changes were less pronounced than those observed with the reference item. There were no relevant changes in the uterus weights and no dilatation of the uterine horns. In the vagina, squamous hyperplasia was observed at ≥ 250 mg/kg/day, which is suggestive of an estrogenic effect. However, vaginal keratinization, which is the most specific indicator of estrogenicity, was not induced by the test item alone, at any dose levels.

Anti-estrogenic assay
Given the high mortality at the intermediate- and high-doses of the combination of the test item and the reference item, definite conclusion on the anti-estrogenic potential of the test item could not be drawn.

Under the experimental condition of this study at dose levels ≥ 250 mg/kg/day, the test item (dibenzylbenzene, ar-methyl derivative) was associated with microscopic changes suggestive of an estrogenic effect in the female reproductive organs of immature rats, although uterine weights were not increased, most likely because of effects on the terminal body weights. Given the high mortality at the intermediate- and high-doses of the combination of the test item and the reference item, definite conclusion on the anti-estrogenic potential of the test item could not be drawn.