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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study. Published in peer reviewed literature.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
based on the draft of enhanced guideline 407
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
CAS no: 103-23-1
Lot no: LDJ4348
Purity: 99.8%
Supplier: Wako Pure Chemical Industries (Tokyo, Japan)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crj:CD (SD) rats were purchased from Charles River
Japan, Inc (Shiga, Japan). Animals were weighed,
weight-ranked, and randomly assigned to each of the
treatment groups and control group before administration,
and then housed individually in stainless steel,
wire-mesh cages throughout the study. Rats were provided
with water automatically and with a commercial
diet (MF, Oriental Yeast Co., Tokyo, Japan). The animal
room was maintained at a temperature of 23±2C
and a relative humidity of 55±5%, and was artificially
illuminated with fluorescent light on a 12-h light/dark
cycle (0700–1900 h). All animals were cared for
according to the principles outlined in the guide for
animal experimentation prepared by the Japanese
Association for Laboratory Animal Science.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Vehicle: corn oil, lot no. WAL7450, obtained from Wako Pure Chemical Industries (Tokyo, Japan).
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
At least 28 days
Frequency of treatment:
Daily treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 200, 1000 mg/kg/day
Basis:
other: oral gavage
No. of animals per sex per dose:
10 male and 10 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rats were orally gavaged with 0, 40, 200, and 1,000 mg/
kg/day DEHA for at least 28 days from 8 weeks of age.
The doses were selected on the basis of our preliminary
test. A vehicle control group was gavaged with corn oil
alone. The volume of the corn oil solution containing
DEHA for gavage was 10 ml/kg. The concentration and
stability of the DEHA were confirmed. Each group
consisted of ten males and ten females. Animals were
killed by exsanguination under ether anesthesia, and
blood samples were obtained from the abdominal aorta
and examined for hematological, clinical biochemistry
and hormonal parameters.
Positive control:
No positive control.

Examinations

Observations and examinations performed and frequency:
General observations:
Clinical signs were recorded daily. Once before the first
dose and once a week thereafter, detailed clinical
observations of all animals were made outside the home
cage. The signs for which the animals were examined
included changes in skin, fur, eyes, and mucous membranes,
the frequency of urine and feces, and autonomic
activity (e.g., lacrimation, piloerection, pupil size,
respiratory pattern). Changes in gait, posture, response
to handling, the occurrence of clonic or tonic movements,
stereotypes (e.g., excessive grooming, circling), or
bizarre behavior (e.g., self-mutilation, walking backwards),
were also recorded. In the 4th week, a functional
observation battery (FOB) that tested sensory reactivity
to stimuli of different types (e.g., auditory, visual, and
proprioceptive), assessed of grip strength, and assessed
motor activity, was also conducted.

Body weight and food consumption:
Individual body weight was recorded twice weekly and
immediately before necropsy. Food consumption was
measured weekly.

Hematology:
The following were examined in the hematology examinations:
red blood cell count, white blood cell count,
hemoglobin concentration, hematocrit value, mean
corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, prothrombin time, activated
partial thromboplastin time, and differential leukocyte
count.

Clinical biochemistry:
Serum levels of the following were measured in the
clinical biochemistry examination: glutamic-oxaloacetic
transaminase, glutamic-pyruvic transaminase, alkaline
phosphatase, cholinesterase, c-glutamyl transpeptidase,
total cholesterol, triglyceride, glucose, total protein,
albumin, blood urea nitrogen, creatinine, total bilirubin,
calcium, inorganic phosphorus, sodium, potassium and
chlorine, and the albumin–globulin ratio was calculated.

Organ weights:
The following organs were weighed after necropsy: testes,
epididymides, ventral prostate, dorsolateral prostate,
seminal vesicles, ovaries, uterus, adrenals, liver,
spleen, kidneys, heart, brain, and thymus, as fresh organs,
and the thyroid and pituitary gland after organ
fixation.

Histopathology:
The following organs were fixed in 10% neutral buffered
formalin and examined: prostate, including ventral
prostate and dorsolateral prostate, seminal vesicles,
ovaries, uterus, vagina, mammary gland, brain, thyroid,
adrenals, liver, spleen, kidneys, stomach, intestine,
pancreas, thymus, parathyroids, and pituitary glands.
The epididymes and testes were fixed in Bouin’s solution
before examining them.

Sacrifice and pathology:
Animals were
killed by exsanguination under ether anesthesia, and
blood samples were obtained from the abdominal aorta
and examined for hematological, clinical biochemistry
and hormonal parameters.
Other examinations:
Other examinations were: hormone analysis, spermatology and estrous cycling.
Statistics:
Body weight, food consumption, hematological data,clinical biochemical data, organ weight, spermatological
data (sperm counts) and FOB data were analyzed by the Bartlett test for homogeneity of variance.
When the variance was homogeneous at a significance level of 5%, one-way analysis of variance was performed.
Following a significant difference in this analysis, the difference between the control group and each of the treatment groups was analyzed by the Dunnett’s test. When the
variances were not homogeneous, the Kruskal-Wallis test was used. Following a significant difference in this test, the difference between the control group and each of the treatment groups was analyzed by the nonparametric
Dunnett’s test. FOB countable data and spermatological data (sperm morphological data) were
analyzed using the Kruskal-Wallis test. When there was a significant difference in this analysis, the difference between the control group and each of the treatment
groups was analyzed by the nonparametric Dunnett’s
test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: At 1000 mg/kg bw/day increased relative liver (m/f) and kidney weight (m) was observed. In addition, histopathology revealed increased eosinophilic and hyaline droplets in the kidney (m/f). Increased follocle atresia in the ovary (f) were found.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Increased kidney weight without histopathological changes were observed in the male in the 200 mg/kg group, at 1000 mg/kg the increase in organ body weight was seen in both males and females, accompanied by histopathological changes in the kidney. In addition, increased liver weight was also detected in the male and female rats in the 1,000 mg/kg group, and this change appeared to be a toxic effect of DEHA.

As a marginal (but statitistically significant) relative increase kidney weight at 200 mg/kg was seen in males only, and was not accompanied by histopatholgical changes in the kidney, this was not considered as adverse. Therefore the no-observed-effect level for DEHA at 200 mg/kg/day.

Applicant's summary and conclusion