Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Same with the substance notified in the section 1 of this dossier

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
On December 29, 2016, 110 CRL Sprague-Dawley CD® IGS rats (55/sex) arrived from Charles
River Laboratories with an assigned birth date of November 18, 2016. The rats were designated
by the supplier to be approximately 5.5 weeks of age upon arrival.
Sex:
male/female
Details on test animals and environmental conditions:
Obey with the OECD guideline 408

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Obey with the OECD guideline 408
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose levels of 0 (vehicle control), 62.5, 125, and 250 mg/kg/day of Dibenzoyl methane were
selected by the Sponsor in consultation with the Study Director, based on the results of a previous
range-finding/toxicity study (PSL 43559, 2016). The high dose was the maximum tolerable dose
with this formulation and test system, and was not expected to cause marked toxicity. The low
and intermediate dose levels were selected to derive a dose-response for any effects observed. A
NOAEL was expected to be achieved for this study.
Duration of treatment / exposure:
An appropriate amount of the vehicle control or test substance was administered daily
(7 days/week) via oral intubation to each rat for at least 90 days
Frequency of treatment:
An appropriate amount of the vehicle control or test substance was administered daily
(7 days/week) via oral intubation to each rat for at least 90 days
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
One-hundred adult Crl: Sprague-Dawley CD® IGS rats (50 males and 50 females) were distributed into six groups (10/sex/group for the main test groups and 5/sex/group for the recovery groups)
Control animals:
yes
Details on study design:
During our DRF study, we are proposing 0 (vehicle control), 250, 500, and 1000 mg/kg/day of Dibenzoyl Methane to be performed. However, we were informed by the lab that Based on the 14-Day range-finding study, they do not believe pregnant rats can tolerate the test substance at 1000mg/kg/day. So, they asked our thought on the proposed dose level selections (i.e., 500, 250 and 100 mg/kg/day) and we did agree about it.

Examinations

Observations and examinations performed and frequency:
All animals were observed at least twice daily for viability. Cage-side observations of all animals
were performed daily during the study (Deviations 1 and 2). Abnormal findings were recorded
Sacrifice and pathology:
At terminal sacrifice, all animals were euthanized by exsanguination from the abdominal
aorta under isoflurane anesthesia. All animals in the study were subjected to a full necropsy,
which included examination of the external surface of the body, all orifices, and the thoracic,
abdominal and cranial cavities and their contents. Any gross lesions were recorded.
The following tissues (of all animals sacrificed by design) were weighed wet as soon as
possible after dissection to avoid drying:
adrenals (combined) kidneys (combined) spleen
brain liver thymus
epididymides (combined) testes (combined) uterus
heart ovaries with oviducts (combined)
The following organs and tissues from all animals were preserved in 10% neutral buffered
formalin for possible future histopathological examination:
accessory genital organs ileum with Peyer’s patches rectum
(prostate and seminal vesicles) jejunum salivary glands (sublingual
adrenals kidneys submandibular, and
all gross lesions larynx parotid)
aorta liver skeletal muscle
bone (femur) lungs skin
bone marrow (from femur lymph node mandibular spinal cord - 3 levels:
and sternum) lymph node mesenteric cervical, mid-thoracic,
brain – 3 sections including mammary gland and lumbar
medulla/pons, cerebellar, nasal turbinates spleen
and cerebral cortex nose sternum
cecum ovaries stomach
cervix oviducts thymus
colon pancreas thyroid
duodenum parathyroid trachea
esophagus peripheral nerve (sciatic) urinary bladder
Harderian gland pharynx uterus
heart pituitary gland vagina
The following organs and tissues from all animals were preserved in modified Davidson’s
fixative and then stored in ethanol for possible future histopathological examination:
eyes optic nerve
epididymides testes
6.J.2 Histopathology
Histological examination was performed on the preserved organs and tissues of the main test
animals from both the control and high dose groups (Groups 1 and 4, respectively). In
addition, gross lesions of potential toxicological significance noted in any main test groups at
the time of terminal sacrifice were also examined. These examinations were extended to the
livers and kidneys of the recovery groups as well as kidneys of the low and intermediate groups at the request of Pathologist, in consultation with the Study Director and Sponsor, to
further investigate changes observed in the high dose group. The fixed tissues were trimmed,
processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope
slides, stained with hematoxylin and eosin and examined by light microscopy. Slide
preparation and histological assessment, by a board-certified veterinary pathologist, was
performed at Histo-Scientific Research Laboratories (HSRL).
Other examinations:
Ophthalmologic Evaluations
During the acclimation period, the eyes of all rats being considered for study were examined by
focal illumination, indirect ophthalmoscopy and, when indicated, slit-lamp microscopy.
Mydriatic eye drops were administered prior to ophthalmoscopy and the eyes were examined in subdued light. Subdued light was maintained in the animal room for approximately 2-3 hours
following the exam. These procedures were repeated on all main test animals prior to
termination.
Statistics:
Product Safety Labs performed statistical analysis of all data collected during the in-life phase of
the study as well as organ weight data. Dupont Haskell Laboratory provided analysis of clinical
pathology results to Product Safety Labs. The use of the word “significant” or “significantly”
indicates a statistically significant difference between the control and the experimental groups.
Significance was judged at a probability value of p<0.05. Male and female rats were evaluated
separately.
A. Statistical Methods (In-Life and Organ Weight Data)
Mean and standard deviations were calculated for all quantitative data. If warranted by sufficient
group sizes, data within groups were evaluated for homogeneity of variances and normality by
Bartlett’s test (Bartlett, 1937). Where Bartlett’s test indicated homogeneous variances, treated
and control groups were compared using a one-way analysis of variance (ANOVA). When oneway
analysis of variance was significant, a comparison of the treated groups to control by
Dunnett’s test (Dunnett, 1964, 1980) for multiple comparisons was performed. Where variances
were considered significantly different by Bartlett’s test, groups were compared using a nonparametric
method (Kruskal-Wallis non-parametric analysis of variance; Kruskal and Wallis,
1952). When non-parametric analysis of variance was significant, comparison of treated groups
to control was performed using Dunn’s test (Dunn, 1964). Statistical analysis was performed on
all quantitative data for in-life and organ weight parameters using Provantis® version 9, Tables
and Statistics, Instem LSS, Staffordshire UK.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs attributable to the test substance administration.
Mortality:
no mortality observed
Description (incidence):
Males
Incidental clinical signs included: a slightly to moderately soiled coat
(black/brown/ oily) on the back/chest/head/neck/abdomen/ano-genital area and/or
full body in 2/10 Group 1 animals, 3/10 Group 2 animals, 4/10 Group 3 animals,
and 5/10 Group 4 animals; red/orange/yellow ano-genital staining or red/brown
facial staining in 3/10 Group 1 animals, 5/10 Group 2 animals, 4/10 Group 3
animals, and 8/10 Group 4 animals; slight to moderate alopecia on the head/neck
and/or left/right forepaw/forelimb in 1/10 Group 2, 3 and 4 animals; superficial
eschar on the back/neck/left ear of 2/10 Group 1 animals, 1/10 Group 2, 3 and 4
animals; slight moist rales in 1/10 Group 2 and 4 animals; slight to moderate dry
rales in 1/10 Group 2 animals; irregular respiration in 1/10 Group 4 animals;
slight hypersalivation in 1/10 Group 3 animals; red unilateral ocular discharge in
1/10 Group 1 and 4 animals; red nasal discharge in 1/10 Group 2 animals; slight
to moderate diarrhea in 2/10 Group 2 and 4 animals; soft feces in 1/10 Group 1, 2
and 3 animals and 7/10 Group 4 animals; dark feces in 1/10 Group 2 animals; a
lesion on the back, left ear and neck (1 cm x 2 cm, white with a shiny center and
red edges) of 1/10 Group 3 animals; visible swelling on the chest and/or right
axillary area/forelimb of 1/10 Group 2 and 4 animals; malocclusion of the
upper/lower incisors in 1/10 Group 1 and 3 animals; broken tooth (right/left
upper/lower incisors) in 3/10 Group 1 and 1/10 Group 2 animals; a broken
toenail (left forepaw/hindpaw) in 1/10 Group 1 and 2 animals; an injured tongue
in 1/10 Group 2 animals; and left/right eye shut in 1/10 Group 3 animals and 1/10
Group 4 animals.
Corresponding detailed clinical observations included: noticeable lacrimation in
1/10 Group 1 animals; eyes halfway shut in 1/10 Group 3 and 1/10 Group 4
animals; eyes completely shut in 1/10 Group 3 animals; exophthalmos in 1/10 Group 3 and 1/10 Group 4 animals; enophthalmos in 1/10 Group 3 animals;
unkempt coat in 3/10 Group 3 and 4 animals; staining/wetness on the fur/skin of
3/10 Group 1 animals, 4/10 Group 2 animals (1/10 black/oily), 4/10 Group 3
animals, and 8/10 Group 4 animals; alopecia in 1/10 Group 2, 3 and 4 animals;
rales (moist/dry) in 1/10 Group 2 animals; rapid respiration pattern in
1/10 Group 4 animals; soft feces in 4/10 Group 4 animals; diarrhea in 1/10 Group
2 and 1/10 Group 3 animals; a broken toenail in 1/10 Group 1 animals; unibilateral
broken upper incisors in 2/10 Group 1 animals; a visible swelling/mass
in 1/10 Group 2 and 1/10 Group 4 animals; and slow or absent pupillary reflex in
1/10 Group 3 animals.
Females
Incidental clinical signs included: a slightly soiled coat (oily) on the head/neck of
1/10 Group 3 animals; yellow ano-genital staining in 1/10 Group 1, 2 and 3
animals and 6/10 Group 4 animals; slight alopecia on the left/right
forelimb/hindlimb and/or ano-genital area in 1/10 Group 2 animals; slight moist
rales in 1/10 Group 3 animals; soft feces in 1/10 Group 3 and 4 animals;
malocclusion (upper/lower incisors) in 1/10 Group 4 animals; and a broken tooth
(upper/lower incisors) in 1/10 Group 4 animals.
Corresponding detailed clinical observations included: unkempt coat in 1/10
Group 3 animals; staining/wetness on the fur/skin of 1/10 Group 2 animals, 1/10
Group 3 animals, and 5/10 Group 4 animals; alopecia in 1/10 Group 2 animals;
decreased muscle tone in 1/10 Group 4 animals; rales (moist/dry) in 1/10 Group
3 animals; broken upper/lower incisors in 1/10 Group 4 animals; and a
malocclusion of the upper/lower incisors in 1/10 Group 4 animals.
2 Recovery
Males
Incidental clinical signs included: a slightly to moderately soiled coat (oily) on
the neck/abdomen/chest/head/ano-genital area of 1/5 Group 5 animals and 4/5
Group 6 animals; red/orange/ yellow ano-genital staining and/or brown facial
staining in 2/5 Group 5 and 5/5 Group 6 animals; slight moist rales in 1/5 Group
5 animals; irregular respiration in 1/5 Group 6 animals; slight diarrhea in 1/5
Group 6 animals; soft feces in 4/5 Group 6 animals; slight vocalization in 1/5
Group 5 animals; malocclusion (upper incisors) in 1/5 Group 6 animals; a broken
tooth (right/left upper incisors) in 2/5 Group 5 and 1/5 Group 6 animals; and a
broken toenail (left/right forepaw) in 2/5 Group 6 animals.
Corresponding detailed clinical observations include: vocalization in 1/5 Group 5
animals; unkempt coat in 1/5 Group 5 and 3/5 Group 6 animals; staining/wetness
on the fur/skin (orange/yellow ano-genital staining) of 2/5 Group 5 animals and
5/5 Group 6 animals; alopecia in 1/5 Group 6 animals; rales (moist/dry) in 1/5
Group 5 animals; rapid respiration in 1/5 Group 6 animals; broken upper incisors
in 1/5 Group 5 and 6 animals; malocclusion in 1/5 Group 6 animals; and a broken
toenail in 1/5 Group 6 animals.
Females
Incidental clinical signs included: a slightly soiled coat (oily) on the neck of 1/5
Group 6 animals; yellow ano-genital staining in 4/5 Group 6 animals; slight to
extreme alopecia on the left/right forepaw/forelimb in 1/5 Group 5 and 6 animals;
clear unilateral ocular discharge in 1/5 Group 6 animals; and slight swelling on
the left/right forepaw/hindpaw in 1/5 Group 6 animals.
Corresponding detailed clinical observations included: unkempt coat in 1/5
Group 6 animals; staining/wetness on the fur/skin (yellow ano-genital staining) in
3/5 Group 6 animals; and alopecia in 1/5 Group 5 and 6 animals.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
1. Main Test
Mean weekly body weights for male and female rats in Groups 2-4 were comparable to their respective control Group 1 animals throughout the study. Mean daily body weight gain for male and female rats in Groups 2-4 was comparable to their respective control Group 1 animals throughout the study
2 Recovery
Males
Mean weekly body weights for male rats in Groups 6 were comparable to the recovery control Group 5 animals throughout the study. Statistically significant decreases (p<0.05) in mean daily body weight gain occurred on Days 57-64, 85-92, and for the overall main study period (Days 1-92) for Group 6 males as compared to recovery control Group 5 males. An incidental statistically significant increase (p<0.05) in mean daily body weight gain occurred for Group 6 males on Days 106-113 of the recovery period.
Without a correlating effect in body weight change these variations in body weight gain are considered to be non-toxicologically relevant.
Females
Mean weekly body weights for female rats in Groups 6 were comparable to the recovery control Group 5 animals throughout the study. Mean daily body weight gain for Group 6 females was comparable to the recovery control Group 5 females, with the exception of an incidental statistically significant increase (p<0.05) on Days 78-85 of the main study period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Main Test
Males
Mean daily food consumption for male rats in Groups 2-4 was comparable to
control Group 1 throughout the study.

Females
Mean daily food consumption and mean food efficiency for female rats in
Groups 2-4 was comparable to control Group 1 throughout the study.

Recovery
Males
Statistically significant increases (p<0.01-0.05) in mean daily food consumption
occurred on Days 29-36, 43-71, 78-92 and for the overall study (Days 1-120) for
male rats in Group 6 as compared to recovery control Group 5. These increases
were not associated with significant increases in body weight and are considered
to be non-toxicologically relevant.

Females
Mean daily food consumption for female rats in Group 6 was comparable to the
recovery control Group 5 throughout the study, with the exception of incidental
statistically significant increases (p<0.05) on Days 57-71 and 78-92 of the main
study period.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Main Test
Males
Mean food efficiency for male rats in Groups 2-4 was generally comparable to
control Group 1 throughout the study. Statistically significant decreases (p<0.01-
0.05) occurred in Group 4 males on Days 57-71 and for the overall main study
period (Days 1-92). Without correlating changes in mean body weight or food
consumption, these variations in food efficiency are considered to be nontoxicologically
relevant.
Females
Mean daily food consumption and mean food efficiency for female rats in
Groups 2-4 was comparable to control Group 1 throughout the study.
Recovery
Males:
Statistically significant decreases (p<0.01-0.05) in mean food efficiency occurred
on Days 22-43, 57-71, 85-92, 106-113, for the overall main study period (Days
1-92) and overall study (Days 1-120) for Group 6 males as compared to recovery
control Group 5 males. These were likely due to non-toxicological relevant
changes in body weight gain that without correlating changes in mean body
weight or food consumption are interpreted to have no toxicological relevance.
Females:
Mean food efficiency for female rats in Group 6 was comparable to the recovery
control Group 5 throughout the study, with the exception of an incidental
statistically significant increase (p<0.05) on Days 78-85 of the main study period.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no ophthalmological findings in either male or female rats attributed to the
administration of Dibenzoyl methane. Both eyes of all animals on study were examined by focal
illumination, slit lamp biomicroscopy, and indirect ophthalmoscopy prior to study initiation and
near termination of the study (Day 87). On Day 87, a Group 4 male (Animal 7047) had a left eye
that was reduced in size due to severe inflammation and resultant scarring (phthisis bulbi). The
pupil was occluded with fibrous tissue and the posterior segment could not be visualized. The
most likely explanation for this finding, given a normal fellow eye, is trauma. These findings
were not attributed to exposure to the test substance; therefore, the test substance is not
considered an ocular toxicant. All other animals included in the study were normal upon
ophthalmic exam
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology
Main Test
There were no test substance-related changes in hematology parameters on
Day 79.
Statistically significant changes in hematology parameters were as follows:
 Hemoglobin (HGB) values decreased (p<0.05) in Group 4 males and in
Group 3 females.
 Mean corpuscular volume (MCV) decreased (p<0.05) in Group 4 females.
 Mean corpuscular hemoglobin (MCH) decreased (p<0.05) in Group 3 and 4
females.
 Mean corpuscular hemoglobin concentration (MCHC) decreased (p<0.05) in
Group 3 and 4 males and in Group 4 females.
These small decreases in magnitude that are within PSL historical data base
range are not of toxicological relevance as they did not correlate with changes in
other hematological parameters, such as red blood cell counts or hematocrit, for
Group 3 and 4 males and females.
Recovery
There were no test substance-related changes in hematology parameters on
Day 114.
Red cell distribution width (RDW) was significantly decreased (p<0.05) in
Group 6 males. This decrease is not of toxicological relevance as other
hematological parameters are normal for Group 6 males.
Coagulation
Main Test
There were no test substance-related changes in coagulation parameters on
Days 93 and 94.
One difference in coagulation parameters that was statistically significant is listed
below. It was observed in a non-dose dependent manner, is within PSL historical
data base range, and is interpreted to be within expected biological variation and
is not toxicologically relevant:
 Prothrombin time (PT) decreased (p<0.05) in Group 3 females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Chemistry
Main Test
Test substance related changes in serum chemistry parameters on Day 79
consisted of:
 Total bilirubin (BILI) increased (p<0.05) in Group 4 males and females.
 Total serum protein (TP) increased (p<0.05) in Group 3 and 4 males
 Albumin (ALB) values increased (p<0.05) in Group 2-4 males.
 Total cholesterol (CHOL) increased (p<0.05) in Group 4 males and females.
 Triglyceride (TRIG) decreased (p<0.05) in Group 3 and 4 females.
 Chloride (CL) values decreased (p<0.05) in Group 4 males.
All the changes observed are within PSL historical range and have no adverse
histopathological correlate, were not observed at the end of the recovery period
and are therefore interpreted to be non-adverse.
Blood creatinine (CREA) values decreased significantly (p<0.05) in Group 3 and
4 females. Decreases in creatinine do not have clinical or pathological
significance and are therefore interpreted to have no toxicological relevance.
Other differences in serum chemistry parameters that were statistically
significant are listed below. They were observed in a non-dose dependent
manner, are within PSL historical data base range, and are interpreted to be
within expected biological variation and are not toxicologically relevant:
 Globulin (GLOB) values increased (p<0.05) in Group 3 females.
 Alkaline phosphatase (ALKP) values decreased (p<0.05) in Group 2 and 3
females.
 Serum aspartate aminotransferase (AST) values decreased (p<0.05) in
Group 2 females.
 Total serum protein (TP) increased (p<0.05) in in Group 3 females.
Recovery
There were no test substance-related changes in serum chemistry parameters on
Day 114.
Decreases in bilirubin and cholesterol observed in Group 6 males do not have
clinical or pathological significance and are therefore interpreted to have no
toxicological relevance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis
Main Test
On day 79, urine volume was increased in Group 4 males and correlated with the
histopathology observation of dilated renal tubules. Urobilinogen (URO) values
increased significantly (p<0.05) in Group 3 and 4 males.
Recovery
There were no test substance-related changes in urinalysis parameters on Day 114.
There were no test substance-related changes in hematology or coagulation parameters. Test
substance-related changes in serum chemistry parameters consisted of increased bilirubin and
cholesterol in Group 4 males and females; increased serum protein in Group 3 and 4 males;
increased albumin in Group 2-4 males; decreased triglycerides in Group 3 and 4 females and
decreased chloride in Group 4 males. All the changes observed are within PSL historical range,
do not have an adverse histopathological correlate, were not observed at the end of the recovery
period and are therefore interpreted to be non-adverse. Urine volume was increased in Group 4
males and correlated with the histopathology observation of dilated renal tubules and
urobilinogen values increase in Group 3 and 4 males; these changes were completely reversed
after the 28-day recovery period.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Main Test (Day 93/94)
Males
Statistically significant organ weight changes were limited to increases (p<0.001-
0.05) in absolute and relative to body and brain kidney and liver weights
observed in Group 2-4 males. These findings correlated with microscopic
observations of hepatocellular hypertrophy in Group 4 animals only and renal
tubular dilation observed at dose levels. Microscopic renal changes in the Group
2 animals are considered negligible and non-adverse and thus the renal weight
changes observed at this dose level are interpreted also be non-adverse.
Females
Organ weight changes, with or without statistical significance (p<0.001-0.05),
were limited to increases in absolute and relative to body and brain kidney and
liver weights were observed in Group 2-4 females. These findings correlated
with microscopic observations of hepatocellular hypertrophy and renal tubular
dilation observed in Group 4 animals and are interpreted to be non-adverse in
Group 3 and 2 animals.
Recovery (Day 122)
Males
At the end of the recovery period, the liver weights were comparable to
concurrent control values and a very slight recovery was observed for absolute
and relative renal weights.
A statistically significant increase (p<0.05) in the relative heart-to-body weight
ratios was observed in the Group 6 males. Without correlating increases in
absolute heart changes, this increase was considered to be of no toxicological
relevance.
Females
A recovery in the magnitude of liver weights differences was observed for
Group 6 females. The magnitude of increases in absolute and relative renal
weights was similar to what was observed at the end of the dosing period.
A statistically significant increase (p<0.05) in the absolute heart weight ratios
were observed in Group 6 females. Without correlating increases in relative to body or brain weight ratios, this increase was considered to be of no toxicological
relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no mortalities associated with the administration of Dibenzoyl methane. There no
test substance-related macroscopic observations. Increased absolute and relative liver and kidney
weights were observed for Groups 2-4 males and females, with a recovery in the magnitude of
change observed in the male and female liver weights and a slight recovery observed in male
kidney weights. Compensatory centrilobular hepatocyte hypertrophy, compatible with the
absorption, metabolism and excretion, was observed in all main Group 4 animals and correlated
with increased liver weights. This change was not observed at the end on the 28-day recovery
period and was interpreted to be non-adverse. Compensatory dilation of renal tubules, compatible
with increased glomerular filtration and excretion, was also observed in Group 4 animals and
correlated with increases in kidney weights. It was still present microscopically with similar
severities in Group 6 recovery rats and correlated with kidney weight increases. Tubular dilation
was noted for Group 3 males (mild) and Group 2 males (minimal). Minimal renal changes in
Group 2 males were not associated with cellular degeneration, or other abnormal clinical blood or
urinalysis alterations noted, that could be ascribed to test substance toxicity on the kidneys, the
significance of these renal findings was interpreted to be negligible and non-adverse.
Neuropathological findings:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes were found in the liver and kidneys of all Group 4
rats. In the liver, these consisted of hepatocellular hypertrophy, which was found
in 10 out of 10 Group 4 males as well as 10 out of 10 Group 4 females. It was
not found in any of the control rats of either sex. The hypertrophy was generally
mild, and due to increased hepatocyte cytoplasmic contents contributing to
increased liver cell size, particularly surrounding the central veins, when
compared to same-sex controls. The appearance of these livers was consistent
with that expected with hepatocellular metabolism of an orally-administered test
substance. Also noted in many hepatocytes was an increase in fine, round,
cytoplasmic vacuoles, presumably lipid (although fat-sensitive staining was not
conducted). This was remarkable in 7 out of 10 Group 4 males and 6 out of 10
Group 4 females, and more common in hepatocytes associated with the central
veins. Lipid-like vacuoles were also recorded in 2 out of 10 Group 1 males, but
the pattern was essentially periportal when seen in control animals (and so noted
in the Pristima® data via histologic notation). Although the appearance of the
lipid-like vacuoles was more frequent in Group 4 rats when compared to
controls, it was interpreted to be related to cytoplasmic deposition of the corn oil
vehicle and not specifically related to test substance toxicity.
In the kidneys, there was a mild (males) or minimal (females) dilation of renal
tubular lumens noted in 10 out of 10 Group 4 males and 10 out of 10 Group 4
females. This was not noted in controls of either sex. Dilated tubular lumens
contained varying amounts of eosinophilic-staining material, presumably related
to increased excretion of test substance and/or associated metabolites.
Kidney sections were also processed and examined from all Group 2 and 3 main
test (Day 93/94 termination) rats. Tubular dilation was noted microscopically in
10 out of 10 Group 2 males to a minimal degree, and 10 out of 10 Group 3 males
to a mild degree. No tubular dilation was noted microscopically in any of the
Group 2 or 3 females.
There were no other findings of toxicologic significance in these two tissues,
such as cellular degeneration or necrosis or lesion-related-inflammatory
component(s). The hypertrophy in the livers and tubular dilation (increased
excretory content of tubules) in the kidneys of both sexes of Group 4 rats
correlated with the statistically significant increase in organ weights noted in
these groups in the organ weight data determined at necropsy. Similarly, the
microscopic results seen in Group 2 and 3 male kidneys were consistent with the
observed organ weight differences between Group 2/3 male kidneys and
corresponding controls (i.e., organ weight increases and tubular dilation appeared
to be dose-related).
The morphologic liver changes were consistent with absorption/metabolism of
the administered test substance, and with full recovery in both sexes, did not
appear to be adverse at any dose level.The kidney findings (simple tubular dilation) appeared to be related to
absorption/excretion of the Dibenzoyl methane or a metabolite. The no-observedadverse-
effect level for the kidney change in females was the intermediate dose
(125 mg/kg/day). With no cellular degeneration, or other abnormal clinical
blood or urinalysis alteration noted, that could be ascribed to test substance
toxicity on the kidneys, the significance of these renal findings was interpreted to
be negligible. Thus, the no-observed-adverse-effect level for the renal effect in
males was determined to be the low dose (62.5 mg/kg/day).
A minor microscopic finding in the lungs of many treated and control rats
consisted of mixed cell infiltration. This was generally minimal (and focal) to
mild (and multifocal) and consisted of isolated mononuclear cell infiltrates
around small blood vessels, with monocyte, macrophage and neutrophil
collections extending into interstitial spaces and associated alveoli. As these
were seen in a majority of the controls as well as most treated Group 4 rats, this
observation was clearly unrelated to Dibenzoyl methane administration. These
findings were consistent with very minute amounts of corn oil being regurgitated,
and are commonly seen in long-term, repeat-oral studies utilizing corn oil as a
vehicle. These lung lesions were insufficient to affect animal health or adversely
affect study objectives.
Other microscopic findings recorded were common age-associated,
developmental or inflammatory changes typically seen in laboratory rats of this
strain and were incidental to administration of the test substance or vehicle.
Product Safety
Recovery Sacrifice (Day 122)
Based on the observed findings in the terminal sacrifice rats, the liver and
kidneys were processed for microscopic examination from Group 5
(0 mg/kg/day) and 6 (250 mg/kg/day) recovery rats. The centrilobular hepatocyte
hypertrophy noted in rats terminated on Day 93/94 was not discernible in the
recovery rats given the same dosage (250 mg/kg/day) when allowed a 28-day
recovery period. However, the renal tubular dilation noted at the end of the
treatment period in main test rats was still present microscopically – and at
similar severities – in all (5 out of 5 males, mild severity; 5 out of 5 females,
minimal severity) Group 6 recovery rats. The observed tubular dilation in Group
6 rats also correlated with statistically significant organ weight increases, when
Group 6 values were compared to those from Group 5 control rats.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study and based on the toxicological endpoints evaluated, the
no-adverse-effect level (NOAEL) for administration of Dibenzoyl methane was determined to be
125 mg/kg/day for female and 62.5 mg/kg/day for male Sprague Dawley rats
Executive summary:

One-hundred adult Crl: Sprague-Dawley CD® IGS rats (50 males and 50 females) were

distributed into six groups (10/sex/group for the main test groups and 5/sex/group for the

recovery groups). For the main test groups, dose levels of 62.5, 125 and 250 mg/kg/day (6.25,

12.5 and 25 mg/mL, respectively) of Dibenzoyl methane, as well as a vehicle control (corn oil),

were selected and administered for at least 90 days. For the recovery groups, vehicle control and

250 mg/kg/day of Dibenzoyl methane were administered for the same length as the main study

animals and were given a recovery period of 28 days following the end of dosing.

An appropriate amount of the vehicle control or test substance was administered daily

(7 days/week) via oral intubation to each rat for at least 90 days. The test substance was

administered as 6.25, 12.5 or 25 mg/mL w/v mixtures in corn oil. Samples of the neat test

substance were collected at the beginning (Day 1), in the middle (Day 43), and at the end of the

in-life phase of the study (Day 93) and analyzed to evaluate stability. Samples were also

collected from the dose formulation mixtures to verify homogeneity (Day 1) and dose

concentration (Days 43 and 93).

The test substance was determined to be stable under the conditions of storage at PSL over the

course of this study. Based on the overall neat test substance stability, dose preparation

homogeneity, and concentration verification analysis, animals were considered to have received

target concentrations of Dibenzoyl methane.

Prior to study initiation and again on Day 87, the eyes of all rats were examined by focal

illumination and indirect ophthalmoscopy. The animals were observed for viability, signs of

gross toxicity, and behavior changes at least once daily during the study, and weekly for a battery

of detailed clinical observations. Both eyes of all animals on study were examined by focal

illumination, slit lamp biomicroscopy, and indirect ophthalmoscopy prior to study initiation and

near termination of the study (Day 87). Body weights were recorded twice during acclimation,

including prior to test initiation (Day 1), weekly thereafter, and prior to sacrifice. Food

consumption measurements were taken to coincide with body weight measurements. Urine and

blood samples were collected on Day 79 from all main test animals and on Day 114 from the

recovery animals for urinalysis, hematology, and clinical chemistry determinations. Coagulation

assessments were performed for all main test animals on Day 93 or 94 (males and females,

respectively), prior to necropsy. Gross necropsies and histological evaluation of selected organs

and tissues were performed on all study animals.

There were no ophthalmological findings in either male or female rats attributed to the

administration of Dibenzoyl methane. There were no mortalities during the course of the study.

There were no clinical signs attributable to the test substance administration.

There were no test substance-related changes in mean weekly body weights, daily body weight

gain, daily food consumption, and food efficiency in male or female rats.

There were no test substance-related changes in hematology or coagulation parameters. Test

substance-related changes in serum chemistry parameters consisted of increased bilirubin and

cholesterol in Group 4 males and females; increased serum protein in Group 3 and 4 males;

increased albumin in Group 2-4 males; decreased triglycerides in Group 3 and 4 females and

decreased chloride in Group 4 males. All the changes observed are within PSL historical range,

do not have an adverse histopathological correlate, were not observed at the end of the recovery

period, and are therefore interpreted to be non-adverse. Urine volume was increased in Group 4

males and correlated with the histopathology observation of dilated renal tubules and

urobilinogen values increase in Group 3 and 4 males; these changes were completely reversed

after the 28-day recovery period.

There were no mortalities associated with the administration of Dibenzoyl methane. There no

test substance-related macroscopic observations. Increased absolute and relative liver and kidney

weights were observed for Groups 2-4 males and females, with a recovery in the magnitude of

change observed in the male and female liver weights and a slight recovery observed in male only

kidney weights. Compensatory centrilobular hepatocyte hypertrophy, compatible with the

absorption, metabolism and excretion, was observed in all main Group 4 animals and correlated

with increased liver weights. This change was not observed at the end on the 28-day recovery

period and was interpreted to be non-adverse. Compensatory dilation of renal tubules, compatible

with increased glomerular filtration and excretion, was also observed in Group 4 animals and

correlated with increases in kidney weights. It was still present microscopically with similar

severities in Group 6 recovery rats and correlated with kidney weight increases. Tubular dilation

was noted for Group 3 males (mild) and Group 2 males (minimal). Minimal renal changes in

Group 2 males were not associated with cellular degeneration, or other abnormal clinical blood or

urinalysis alterations noted, that could be ascribed to test substance toxicity on the kidneys, the

significance of these renal findings was interpreted to be negligible and non-adverse.

Under the conditions of the study and based on the toxicological endpoints evaluated, the

no-adverse-effect level (NOAEL) for administration of Dibenzoyl methane was determined to be

125 mg/kg/day for female and 62.5 mg/kg/day for male Sprague Dawley rats.