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Administrative data

Description of key information

one Guideline study on subacute repeated dose toxicity available.

one pre-OECD Guideline study on subchronic repeated dose toxicity available.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
unknwon
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
only summary available in english
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
2,2'-azobis (2,4-dimethylvaleronitrile) (available from Wako Pure Chemical Industries, Lot No. ALJ4132, purity 100.0%) is a white crystalline at room temperature and stored at cool place after availability.
Stability of the test substance, the infrared absorption spectrum was measured before and after dosing period, the test substance was confirmed to be stable during the administration period
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
according to Guideline
Sex:
male/female
Details on test animals and environmental conditions:
65 female and 55 male Crl: CD (SD) rat (SPF), breed at Charles River Japan (Hino Care Center)
being 8 weeks old were purchased and acclimatised for 8 days including quarantine for 6 days.. By
weight stratified random sampling method to the administration after 2 days ago, they were grouped
so that the average body weight of each group are substantially equal. Week-old administration at the
start of the animals in both sexes 9 weeks of age, body weight range is male 323.8 ~ 359.2g, female
was 205.9 ~ 236.6 g.
Animals, throughout the entire rearing period, including during the quarantine, acclimatization period,
temperature 21 ~ 25 ° C, relative humidity of 40 to 70%, and ventilation rate for 10 to 15 times / hour,
light-dark cycle 12-hour intervals (7 lighting, 19:00 darkness)
The animal received autoclaved diet (chow, MF, Oriental Yeast) and tap water with added chlorine ad
libitum.
Route of administration:
oral: gavage
Details on route of administration:
Preparation of dosing solutions are added olive oil (Fudjimi pharmaceutical plants) the test substance
was suspended dosing solution of high dose, low dose and medium dose was diluted prepared from
higher doses. The uniformity and the 14-day stability of the test substance preparation liquid was c
onfirmed by high performance liquid chromatography. Administration solution, and store it in a cool,
dark place until the administration, and used within 14 days after preparation. It confirmed the test
substance concentration in the administration solution at the start of administration
Vehicle:
olive oil
Details on oral exposure:
Preparation of dosing solutions are added olive oil (Fudjimi pharmaceutical plants) the test substance
was suspended dosing solution of high dose, low dose and medium dose was diluted prepared from
higher doses. The uniformity and the 14-day stability of the test substance preparation liquid was c
onfirmed by high performance liquid chromatography. Administration solution, and store it in a cool,
dark place until the administration, and used within 14 days after preparation. It confirmed the test
substance concentration in the administration solution at the start of administration
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The uniformity and the 14-day stability of the test substance preparation liquid was confirmed by high
performance liquid chromatography.
Duration of treatment / exposure:
Males 42 days, females from 14 days before mating to day 4 of lactation
Frequency of treatment:
daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
based on pre-test
Administered dose, 14-day repeated dose toxicity studies and repeated oral dose toxicity, reproduc
tive and developmental toxicity annexation attempt
The results of the test were determined on the basis of. In other words, the test substance at a dose
of 0,10,50,250 and 1,000 mg / kg / day, 1 group of male and female each of the three animals of Crl
: CD (SD) 14-day repeated oral administration as a result of the rat, 1,000 mg of male and female /
kg locomotor activity decreased in group, respiratory rate reduction, eyelid closure, food poor, feces
amount decrease, prone position with the same group of female, after was breathing deep large
Nadogami, each male and female until the administration 7 days 2 examples died. In the survivors of
250 mg / kg or more groups of male and female, liver weight high, swelling and centrilobular hepat
ocellular hypertrophy was observed. In addition, the extension of the APTT was observed at 10 mg /
kg or more groups of females. Although these results were carried out repeated oral dose toxicity,
reproductive and developmental toxicity Combined study at a dose of 0,2,10 and 50 mg / kg / day, a
few changes in the weight highs and stomach of the liver at 50 mg / kg group since was only seen in
cases, in this study, a 250 mg / kg / day to organic changes in the liver are expected to be seen as a
high dose, the following in common ratio 5 50 and 10 mg / kg / day the three doses has been set.
Positive control:
none
Observations and examinations performed and frequency:
Parental animals: Observations and examinations
1) the general state
Male, were observed in twice and dissection day every day until the day before dissection from th
e start date of administration. Female, twice daily until recovery group administered the last day of
the period, the animals to be subjected to mating was observed in the labor situation from the start
date of administration, twice daily until delivery four days, including the nursing state and delivery 5
days (anatomy date) . During the recovery period, it was observed once a day both to dissection Date
male and femal
2) detailed the general state observation
For male and female all cases, once before the start of the administration, after the start of the ad
ministration were observed in the weekly-blind after administration, we were scoring. Blinded rearr
anges all of the animals by a random number, using a test label, was a state in which the administr
ation group can not be determined. However, in the case of the inspection target animal is one exam
ple at the time of inspection of labor four days did not blinded.
Reaction when extracting from the cage was observed near or hands to hold the animal, the ease
and utterance out of the animal when the animal is given an external stimulus, etc. hold. Detailed o
bservation for the hands, muscle tone, decreased body temperature and the presence or absence of
a nap, of the hair condition (dirt, SoTsuyoshi), of the skin and mucous membrane color (pallor, redne
ss, cyanosis), abnormalities of the eye (lacrimation, eye protruding, pupil diameter), to observe the
presence or absence of salivation and secretions. Observation of the action in the arena, attitude at
1 minute animal in the arena, activity, breathing, eyelid closure, walking state, tremors, spasms, con
vulsions, were observed for the presence of stereotyped behavior and abnormal behavior. In addition
, it was recorded Haikuso number of times (the number of feces) and frequency of urination of one m
inute (the number of pool of urine).
3) function testing
Male, was examined after administration of the administration last week for five which were selected
in order of small animal number for each dose. Female, before fasting for delivery four days for five c
lose of delivery date of each dose of the calving animals, recovery group were examined after admin
istration of the administration last week. Inspection of during the recovery period, in order to change
to doubt the effect of the test substance administered in the inspection of during the administration p
eriod was not observed, it was not carried out.
Reactivity test, observing the animal's reactions when given the appropriate external stimulus to a
sensory organ to be inspected, subjected to scoring. Inspection was carried out in continued blind to
detailed observation. Visual (approach and touch), the closer the ball-point pen of the sheath to the
face before 3 cm, was recorded the reaction of when held as it is 4 seconds. Hearing, it was recor
ded the reaction of when you play a finger in the overhead. Pain was recorded reaction when sandwic
hing the third ridge portion of the tail with clothespin. Pupillary, after blocking the light it was recorde
d and the reaction of the pupil when exposed to light. Aerial SeiMuko reflections were recorded rea
ction when dropped animals abdominal from a height of approximately 30 cm in a state of facing upwa
rd.
Grip strength measurements, grip strength meter (FGC-2, Meitisu) was measured in the blind using.
Forelimbs, hind limb both measured twice, and the average value was used as the grip strength va
lue of an individual's forelimb and hind limb.
Locomotor activity measurement, rats for momentum measurement device (ACTIMO-10, Shintek
uno) was used to measure the animal's momentum. The number of crossed an infrared beam as a
measurement value was measured (6 times at 10 minute intervals) for 1 hour.
4) body weight Male was measured 1,3,7,14,21,28,35,42 and 43 day administration (carry-out at the time, fasted
state).
Female, 1, 3, 7 and 14 days administration, pregnancy 0,7,14,17 and 20 days, delivery 0 (delivery
date), was measured in the 4 and 5 days (carry-out at the time, fasted state). Female of the satellite
was measured in male and the same day. During the recovery period, it was measured in both sexes
1,3,7,14 and 15 days (carry-out at the time, fasted state).
5) food consumption
Male administration 1,3,7,14,28,35 and 42 days, female, administered 1, 3, 7 and 14 days, p
regnancy 0,7,14,17 and 20 days, delivery 0 (calving day) and was measured 4 days. Female of the
satellite was measured in male and the same day. During the recovery period, it was measured in
both the 1, 3, 7 and 14 days male and female. For both sexes each period, in search of food cons
umption from the measurement date to the next measurement date, to calculate the average daily
food consumption.
) hematology
After overnight fasting at the free drinking water from the afternoon to the anatomy of the adminis
tration the last day of the period or the recovery period the last day (16 to 20 hours), 5 males in the
ascending order of the animal number out of each dose of the surviving animals, females each f
or male and female all cases of five and recovery group close to the dose with calving date, it was
bled EDTA-2K as an anti-coagulant, fully automated comprehensive hematology analyzer (CEL
L-DYN3500, Abbott Laboratories) by the number of red blood cells (RBC) , white blood cell count
(WBC), hemoglobin concentration (Hb), hematocrit value (Ht), mean corpuscular volume (MCV), m
ean corpuscular hemoglobin (MCH), to measure the mean corpuscular hemoglobin concentration
(MCHC) and the number of platelets (platelet) . In addition, comprehensive hematology testing equi
pment (ADVIA 120, Siemens) by, to measure the reticulocyte count ratio (Reticulo) and white blood
cell percentage (Differentiation of leukocyte). Furthermore, blood was collected 3.2% aqueous soluti
on of sodium citrate as an anticoagulant, using the obtained isolated from blood plasma, the blood
coagulation automatic measuring apparatus (KC-10A, Amerungu), prothrombin time (PT) and ac
tivated It was measured partial thromboplastin time (APTT).
7) Blood biochemical tests
For animals was performed hematology, with serum, the automatic biochemical analyzer (7180
Automatic Analyzer, Hitachi, Ltd.), aspartate aminotransferase (AST), alanine aminotransferase
(ALT), total protein (T- Protein), albumin (albumin), total bilirubin (T-Bil), alkaline phosphatase (ALP),
cholinesterase (ChE), γ- guru Tamil trans peptidase (γ-GTP), total cholesterol (T-Cho), triglyceride
(TG ), glucose (glucose), urea nitrogen (BUN), was measured creatinine (Creatinine), calcium (Ca)
and inorganic phosphorus (IP). Furthermore the electrolyte analyzing apparatus (PVA-EXII, A &
T) was thus measured sodium (Na), potassium (K) and chlorine (Cl). A / G ratio (A / G ratio) was
calculated from the total protein and albumin.
Sacrifice and pathology:
8) autopsy
For all cases, the day after the administration the last day of the period (excluding the recovery group)
and recover the last day of the period, body surface, openings, subcutaneous, cranial cavity, chest
cavity, abdominal cavity, the naked eye observation of the pelvic cavity and its contents went It was.
Ovary is the number of pregnancy corpus luteum, uterus was recorded the number of implantation
marks after the incision.
9) organ weight and tissue collection
For all cases, (including the duodenum - the rectum, the Peyer's patches), trachea and lungs, stom
ach, intestines, liver, heart, kidney, bladder, testis, epididymis, prostate, seminal vesicles, ovary,
uterus, vagina, brain (cerebrum , including the cerebellum and pons), spinal cord, sciatic nerve,
bone marrow (femur), lymph nodes (axillary and mesenteric lymph nodes), spleen, thymus, pituitary
gland, thyroid (including parathyroid) and the adrenal glands taken and, liver, heart, kidney, testis, epi
didymis, brain, spleen, the weight of thymus and adrenal gland were measured. Moreover, to calculat
e the relative weight of the organs based on the weight at unloading. That was well taken gross les
ions. Trachea, lung and bladder, was taken after the injection of a 10% neutral buffered formalin s
olution. Stomach and intestines, after injecting 10% neutral buffered formalin, and immersed in the
same solution, and then the contents removed by washing with water. Harvested organs and tissues
were fixed in 10% neutral buffered formalin. However, the testis and epididymis were fixed in Bouin's
solution
10) histopathological examination
Each dose, five in the order male small control group and 250 mg / kg group of animal numbers, fema
le for five close to that of one calving date of birth animal, trachea, lung, stomach, intestines, liver, he
art, kidney , bladder, testis, epididymis, prostate, seminal vesicles, ovary, uterus, vagina, brain, spinal
cord, sciatic nerve, bone marrow, axillary lymph nodes, mesenteric lymph nodes, spleen, thymus, pit
uitary gland, thyroid and adrenal glands It was examined. Tests, to prepare a paraffin-embedded thin
sections, was subjected to hematoxylin-eosin staining was performed on the optical microscopic. O
ther 250 mg / kg organs and tissues change doubt the impact of the administration of the test subst
ance was observed in the group, was also tested for all of the other dose groups and recovery group
. In addition, it was examined gross lesions of the histopathological examination subject animal. F
emale of this addition to infertility were examined ovaries, the uterus and vagina in order to investigate
the cause of the infertility. Female all Haraji died were examined stomach before in order to examin
e the condition of the mother animal, glandular stomach, liver, heart, kidney, spleen, thymus and a
drenal glands.
In HE staining samples of 250 mg / kg group of male, eosinophilic bodies increase in kidney was
observed, in order to confirm the association between alpha 2u- globulin, for one each example
of the control group and 250 mg / kg group, by anti-alpha2u- globulin antibody was performed
immunohistochemistry. In addition, small leaves around hepatocellular vacuolization was seen in
one patient of liver of female in the control group, for the accumulation of fat in the liver cells was
suspected, were oil red O staining of the liver.
Other examinations:
none
Statistics:
Of the parent animal body weight (except when carried out), food consumption, grip strength and
motor activity, hematology item, blood biochemical test items, organ weight, the average of the
period the number of days, number of days required mating, pregnancy period, pregnancy corpus
luteum the number and implantation marks the number, birth number of births, stillbirths number of
children, birth live number of children, birth children ratios, 0-day survival children __ number, 0-day
survival child sex ratio, 4 Nissei number of children, 4-day survival child sex ratio and body weight, pe
rforms a test by Bartlett method, was one-way analysis of variance when the equal variance was observed in 5% significance level. If the significant differences in variance analysis, between the control
group and each dosage group were assayed by Dunnett method. If the equal variance is not obser
ved, to test the significance of all groups in the rank sum test method of the Kruskal-Wallis, the case
was significant difference, non-parametric and the control group in a multiple comparison assay of
Dunnett the significant difference between went.
Haikuso number of times and frequency of urination conducted a test of Kruskal-Wallis.
Abnormalities cycle incidence of parental animals, mating rate, insemination rate, conception rate and
fertility rate, birth child sex ratio of offspring, 0-day survival child ratios and 4-day survival child
sex ratio is, in Fisher's exact test and significant difference tests were performed between the control
group.
Of the parent animal implantation rate, pre-implantation embryo loss rate, post-implantation embryo
loss rate and birth rate, fertility rate of birth, 0-day survival, the outer table abnormal rate and the 4-
day survival rate, rank sum of the Kruskal-Wallis It was tested for significance of all groups in assays.
Body weight of the offspring were treated to every male and female of the litter as a sample unit.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the administration period, in males, in the group of 50 mg / kg or more, immediately after ad
ministration, transient salivation was found in all 12 subjects at each dose. Besides this, upper incisor
teeth defects were found in one of the 10 mg / kg group on administration 35 to 41 days, but the rela
tionship with the dose was not clear change. In females, transient salivation immediately after adm
inistration was observed in 3 of 12 cases during pregnancy in the 50 mg / kg group, all in pregnanc
y and pregnancy in 250 mg / kg group, 7 For example, in all 5 cases of 250 mg / kg group of satelli
tes. In addition, one patient in the control group showed scarification on 3 to 5 days of nursing. No
abnormality was found in the 10 mg / kg group.
No abnormality was observed in both males and females during the recovery period
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the administration period, both during the recovery period, significant difference in the male
and female of the test substance administered group was not observed.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the administration period, in the males, the 10 mg / kg group showed a low value on the 28 th
to 42 th day of administration, but the relationship with the dose was not clear change. There was no
significant difference in the 50 and 250 mg / kg groups.
In females, high levels were observed on the 4th day of nursing in the 10 and 250 mg / kg group, but
there was no significant difference in the 50 mg / kg group and the dose relationship was unclear and
it was judged to be an accidental change.
During the recovery period, there was no significant difference in the test substance-administered
group in both males and females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Description (incidence and severity)
At the end of the administration period, in the male, but the low value of the hemoglobin concentration
and hematocrit value in the group of more than 50 mg / kg were found, none of which are differed on
the value of the 50 and 250 mg / kg group, dose and it related is not clear, since the abnormality in r
ed blood cell count is not seen, it is determined that the accidental changes.
Significant differences in 10 mg / kg group was observed. In females, significant difference in all of the
test substance administered group was not observed.
During the recovery period, in the male, MCHC at 250 mg / kg group, height of the platelet count
and lymphocyte ratio, but low value of neutrophil ratio was observed, that the same change is not
observed at the time of the end of the administration period , from the fact that no abnormality w
as observed in the number of red blood cells and white blood cell count, it is determined that the
accidental change. In females, significant difference in the 250mg / kg group were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, 250 mg / kg high of γ-GTP in the group, the low
value of triglycerides was observed. Low value of total bilirubin that is dependent on the dose in this
addition to 50 mg / kg or more groups, calcium higher in the 250 mg / kg group, but the low value of
the chlorine was observed, that a change to any relevant is not seen from, it is determined that the
accidental changes. Significant differences in 10 mg / kg group was observed. In females, although
low levels of potassium were observed at 10 mg / kg group, for association with dose is not clear, it
is determined that the accidental changes. Significant differences in 50 and 250 mg / kg group was
observed.
During the recovery period, in the male, but the low value of AST at 250 mg / kg group was seen,
from the fact that the relevant change is not observed, it is determined that the accidental change. In
addition, although high calcium in the same group was observed, as compared with at the end of the
administration period, the degree had been reduced. In females, but the low value of albumin in the
250 mg / kg group was seen, from the fact that the relevant change is not observed, it is determined
that the accidental change.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
ministration period. In females, the high value of locomotor activity was observed in 0 to 10 minutes in
the 250 mg / kg group, but there was no abnormality in the total value of 0 to 60 minutes due to trans
ient change, general condition observation, arena Since there was no change related to behavior wit
hin the judge, it was determined to be an accidental change. No abnormality was found in the 10 and
50 mg / kg groups.
The examination during the recovery period was not conducted because there was no doubt about
the influence of administration of the test substance in the examination during the administration
period in both males and females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, height of the absolute and relative weight of the
liver was observed in the 250 mg / kg group.
Significant differences in 10 and 50 mg / kg group was observed. In females, the peak value of the
absolute and relative weight of the liver that is dependent on the dose was observed in the group of
more than 50 mg / kg. Absolute and relative weight of the high price of the thymus in this addition to
250 mg / kg group, but the low value of the absolute and relative weight of the adrenal glands were
observed, because the changes that any relevant is not seen, toxicologically significant changes It
was determined not to be. Significant differences in 10 mg / kg group was observed.
During the recovery period, in the male, but high in the absolute and relative weight of the liver was
observed in the 250 mg / kg group, as compared with at the end of the administration period, the d
egree was reduced. The absolute weight of the low value of the adrenal gland in this addition to 250
mg / kg group was observed, since the change of the same at the end of the administration period
is not observed, it is determined that the accidental change. In females, but high relative weight of
the liver was observed in the 250 mg / kg group, as compared with at the end of the administration
period, the degree was reduced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, enlargement of the liver is one case in 12 cases of
50 mg / kg group, was seen in seven patients all patients of 250 mg / kg group. Nodule and the white
part of the epididymis was observed in one case each in this addition to 250 mg / kg group. No abnormalities were observed in the 10 mg / kg group. In females, enlargement of the liver was observed in
12 patients all patients in the 250 mg / kg group. Mucosa recessed portion of the front stomach in this
other control group, mucosa black part and the size of the thymus is one example of the glandular
stomach, mucous membranes black part is one example of the glandular stomach at 10 mg / kg gr
oup, the glands in the 50 mg / kg group mucosa the black part of the stomach was observed in two pa
tients.
During the recovery period, no abnormalities were observed in male and female with 250 mg / kg
group. In addition, the white part is an example of the epididymis in male control group, dark red of th
e thymus was observed in another example.
No abnormalities were observed in each example of the female in the control group and 10 mg / kg
group of infertility.
One thing in the 10 mg / kg group of females in which the wholeborn died on the first day of nursing
showed miniaturization of the thymus.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, liver of diffuse liver cell hypertrophy at 250 mg /
kg group 5 cases all cases, is eosinophilic bodies increase in the kidney was observed in one patient.
50 mg / kg 1 of 5 patients are solitary cyst of the medulla of the kidney in the group as this addition
to naturally occurring lesions, 1 case localized myocarditis of heart at 250 mg / kg group, bilateral
in another example away and out inhibition and basolateral stagnation of the sperm cells of mature
sperm cells of the testes, sperm granuloma of one side of the epididymis was observed in two patien
ts. In addition, although lymphocyte infiltration of the prostate was observed in 4 cases of three
patients and 250 mg / kg group in 5 patients in the control group, the extent of 250 mg / kg group is
weaker than the control group, revealed to be associated with dose because not, it is determined
that the accidental change. No abnormalities were observed in the 10 mg / kg group. Eosinophilic b
odies increase in the kidney that was observed in 250 mg / kg group, as a result of the immunohisto
chemistry with an anti-alpha 2u- globulin antibodies for one each example of the control group and
250 mg / kg group, the anti-alpha that 2u- globulin antibody-positive substances have accumulated
it has been confirmed. In females, 250 mg / kg small centrilobular liver cell hypertrophy of the liver
in the group were seen in 5 cases all cases. Besides this erosion of previous stomach in the control
group as a natural occurrence lesions, stomach bottom gland mucosal necrosis and lobular perip
heral liver cell fat of the liver is one example of the glandular stomach, small leaves surrounding liver
cells good of the liver to another example basified, basophilic tubules of the kidney, the renal tubular
epithelial degeneration and necrosis, atrophy in 2 cases of thymus, cell infiltration is one example of
the cortex of both sides of the adrenal glands, stomach glandular stomach at 50 mg / kg group one e
xample is the bottom gland mucosal necrosis, cell infiltration of the cortex of the mineral deposits and
both sides of the adrenal glands of Peyer's patches of the jejunum was observed in one patient in
the 250 mg / kg group. For small leaves around hepatocellular vacuolization of the liver was observed
in one patient in the control group, as a result of the oil red O staining, placed in liver cells
Le Red O-positive material was observed in a large amount, that the fat is accumulated was
confirmed in the hepatocytes
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Conclusions:
The No-Observed-Effect-Level (NOEL) for the repeated dose toxicity was 10 mg/kg/day for males and females based on salivation in males and females at 50 mg/kg and above, enlargement of the liver in males at 50 mg/kg and above, and increased absolute and relative weights of the liver in females at 50 mg/kg and above.
It was concluded that the No-Observed-Effect-Level (NOAEL) for the repeated dose toxicity of 2,2'-azobis(2,4-dimethylvaleronitrile) was 250 mg/kg/day for males and females.
Executive summary:

Effects of the test substance were observed in the liver and kidneys.

As effects on the liver, absolute and relative weights were increased in males at 250

mg/kg and females at 50 mg/kg and above. Enlargement of the liver was observed in males at

50 mg/kg and above and females at 250 mg/kg on necropsy. In the histopathological

examination, diffuse hypertrophy of the hepatocytes in males and hypertrophy of the

centrilobular hepatocytes in females were observed at 250 mg/kg. On blood chemistry,

increased γGTP and decreased triglycerides were observed in males at 250 mg/kg. At the end

of the recovery period, these changes were not observed, except for a slight increase in liver

weight.

As ef島cts on the kidney, increased eosinophilic bodies was observed in males at 250

mg/kg. This change was con五rmed to be alpha 2u・globulin nephropathy on

immunohistochemical examination. Increased eosinophilic bodies and basophilic tubules were

observed in the kidney at the end of the recovery period.

Salivation was observed just after dosing in males and females at 50 mg/kg and above.

Concerning other parameters, there were no changes attributable to the test

substance on detailed clinical observation, reflex, grip strength, locomotor activity, body

weight, food consumption or hematology.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 15 to September 14, 1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: Procedures for the testing of international food additives to establish their safety for use. Second report of the joint FAO/WHO expert committee on food additives. Published jointly by FAO and WHO and issued also as WHO. Technical report series, No. 144.
Principles of method if other than guideline:
The present studies were mostly carried out according
to the methods of toxicity tests for food additives reported
by The Joint FAO/WHO Expert Committee (1957)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
according to current Guidelines
Sex:
male/female
Details on test animals and environmental conditions:
Wistar-strain rats of either sex (4 weeks old) were
purchased from Nihon Rat Co. Ltd. and they were fed free
access to the commercial pelleted dry diet CE2 (Nihon
Clea) for a week. After examination for physical wellbeing
a total of 200 · male and female rats (100 males and 100 females),
weighing 90 to 120 g, were selected from the large population
and employed in the experiments.
The animals were housed individually in steinless steel
cages and kept in an air-conditbned room maintained a
temperature at 23 + 2°C and a relative humidity at 55±5%
during the experimental period.
The drug diet was given freely and tap water was available at
libitum ..
Route of administration:
oral: feed
Details on route of administration:
Since V-65 is unstabl·e to heat, V-65-containing feed was prepared
freshly before use by mixing up V-65 with commercial food.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Since V-65 is unstabl·e to heat, V-65-containing feed was prepared
freshly before use by mixing up V-65 with commercial food.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 d / 13 weeks
Frequency of treatment:
daily
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
1 % in diet, The doses were calculated from the values obtained by estimating average body weights of a rat as 200g and by estimating feed consumption per a day as 20 g
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
0.3 % in diet,The doses were calculated from the values obtained by estimating average body weights of a rat as 200g and by estimating feed consumption per a day as 20 g
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
0.1 % in diet,The doses were calculated from the values obtained by estimating average body weights of a rat as 200g and by estimating feed consumption per a day as 20 g
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): not stated
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: no satellite groubs
- Section schedule rationale (if not random): not defined
Positive control:
none
Observations and examinations performed and frequency:
General behavioral changes
Body weights
Feed consumption and drug intake
Efficiency of feed utilization
Hematological investigations
Serum analyses
Sacrifice and pathology:
Organ weights
Anatomicopathological studies
Histopathological studies
Other examinations:
Some rats of each group were sacrificed once a month
after the onset of the trial and observed as indicated under "Sacrifice and pathology"
Statistics:
none
Clinical signs:
no effects observed
Description (incidence and severity):
All the rats ®:x.:~·e:pit the dead ones showed normal signs (motor
activities, muscle tone etc.) and no significant differences
were observed between the control and V-65-treated group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male and 2 females in 1.0% group died during lst
week of the experiment (Table 2). The dead animals consumed
nothing of the diet and caused a marked decrease in body weight.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth curves
Growth curves of the V-65-treated group throughout the trial
were shown in Figures 1 and 2. Body weight gains showed a
tendency to depression only in the male rats of 1.0% group
till 8th week. As shown in Table 15, the body weight gains
in the males of 0.3% group were depressed at the 9th and the
lOth week. These manifestations were reversed to the control
levels after the llth week. Each female group showed the
similar patterns till the 9th week. The body weight gains were
depressed in all the groups other than 0.1% group, which was
recovered to the control levels after the llth week (Table 16).
Body weight gains, feed consumption and efficiency of feed
utilization are shown in Tables 15 and 16, and in Figures 3
and 4. A decrease in body weight gains was observed in the
male rats of each group and in the females of 1.0 and 0.3%
group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Body weight gains, feed consumption and efficiency of feed
utilization are shown in Tables 15 and 16, and in Figures 3
and 4. There were no marked differences in feed consumption
between the control and V-65-treated group.
The average intake of V-65 is represented in Table 3.
The rats were maintained on the powdered diet containing 1.0%,
0.3% or 0.1% of V-65 for 3 months.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains, feed consumption and efficiency of feed
utilization are shown in Tables 15 and 16, and in Figures 3
and 4. The efficiency
of feed utilization in each group increased as the dosage level
of V-65 decreased. The values in the females of 0.1% group
were, however, higher than those of the control group.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The results of hematological findings are shown in
Tables _4,5 and 6. Red blQod cell counts were higher in the
male rats of 0.1% group at the lst month and in the females of
1.0% group at the 2nd month of the trial. White blood cell
counts were higher in the females of 1.0% group at 3rd month
of the trial. Hemoglobin levels were decreased in the males
of 1.0% and 0.3% group and in the females of 1.0% group at the
2nd month of the trial. In general, however, appreciable differences
in the hematological findings between the control
and V-65-treated group were found only sporadically.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Tables 4,5 and 6 show the results of serum analyses.
An increase in total cholesterol was observed only in the male
rats of 1.0% group at the lst month after the initial administration.
GOT activities were lowered in the male and female
rats of 1.0% group at the Jrd month of trial.
A decrease in GPT activities was observed in the males of
0.1% group at the lstmonth and in the females of 1.0% group
at the Jrd month of the experiment. Total protein decreased
in the females of O.J% group at the lst month and increased
in the males of 1.0% group at the 2nd month of the trial.
In general, no marked differences in the biochemical findings
between the control and V-65-treated group were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weights are presented in Tables 10, 11 and 12.
In the females of 1.0% group, the weight of the liver decreased
at the lst month and in the males, incr~ased at the Jrd month
of the trial. In the males of 0.1% group, loss in the weight
of the heart and an increase in the weights of the testes were
observed at the lst month of the trial. Marked differences
in the o.rgan weights of all groups were not observed at the
2nd month after the initial administration. In general, there
were no marked differences in the organ weights hetween the
control and V-65-treated group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant gross pathological changes were noted in
the tissues and organs examined histologically.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological studies in V-65-treated groups at
the lst month of trial
No significant pathological changes were recognized in
nerve and glia cells of the cerebrum. Subpial hyperemia was
observed only in the male rats o·f 1. 0% and o .1% group.
There were no significant pathological changes in cortex and
medulla of the cerebellum and glandular cells of the pituitary
in the groups treated with V-65. In the liver, slight or
distinct cloudy swelling and an increase in interlobular bile
ducts were observed in all rats treated with V-65. This
increase of the bile ducts seems to be slightly doserelated.
Restricted and localized necroses of hepatocytes were found
in the rats treated with the 1%-V-65 diet. Cloudy swelling
in epithelia of kidney tubules was observed only in the females
of 0.3% group. In the spleen, atrophy of splenic nodules,
degeneration of lymphocytes and pathological changes in red
pulps and blood vessels were not observed. There were no
special pathological changes in seminal epithelia and stroma
of the testes and in Graafian follicular epithelia, connective
tissues and blood vessels of the ovaries.
Histopathological studies in V-65-treated groups at
the Jrd month of trial
Any significant pathological changes in the cerebrum,
cerebellum and pituitary were not observed. There were no
pathological changes in cortex and meddula 0£ the thymus,
£ollicular epithelia and stroma 0£ the thyroid, the heart,
lungs, adrenals, stomach, and small and large intestines.
Cloudy swelling of epithelia of kidney tubules was found in
some rats of O.J% group. In the liver, slight or distinct
cloudy swelling of hepatic cells was observed in all the rats
treated with V-65. The cloudy swelling was also observed in
some rats of the control group. An increase in interlobular
biliary ducts was recognized only in the males of 1.0% group
and the females of o.1% group. Any significant pathological
changes in seminal epithelia and stroma of the testes and in
Graafian follicular epithelia, blood vessels and connective
tissues of the ovaries were not observed.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
In the histopathological studies, a slight or distinct
increase in interlobular bile ducts of the liver was recognized
in all the V-65-treated groups at the lst month of the trial.
The slight increase in the bile ducts was observed in the
males of 1.0% group and in the females of 0.1% group at the
3rd month of the trial. Necroses confined within narrow
limits were found in hepatic cells in 0.1% group at the lst
month of the trial. Small cell infiltration in the live~
was observed only in the females of 0.1% group at the 3rd
month. However, this infiltration was also observed in the
liver of the females in the control group at the lst month of
the experiment. In the kidney, cloudy swelling of epithelia
of kidney tubules was observed in the females of 0.3% group
at the lst month and in the rats of both sexes of 0.3% group
at the 3rd month of the trial.

No significant histopathological changes in all
organs other than the liver and kidney were observed both
in the control and drug-treated group.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 701 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 764 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Conclusions:
From these results, it can be concluded that V-65 might
exert no appreciable toxic effects on the rats when V-65
in the doses of 700.8 mg/Kg/day or less for males and 764.0
mg/Kg/day or less for females was continuously administered
orally for 3 months
Executive summary:

The present studies were undertaken to elurc.idate the

subacute toxicity of three compounds, V-65, DISN and PVC.

V-65 is short for 2,2' -azobis-(2,4-dimethyl valeronitrile),

a new polymerization initiator, DISN for dimethyl diisobutyl

succinonitrile, the decomposition product obtained by heating

V-65, and PVC for polyvinyl chloride produced by using V-65

as a polymerization initiator.

The subacute toxicity tests were carried out for 3

consecutive months using rats of both sexes. The drugs were

continuously administered to the rats orally. The results

obtained suggest that V-65, DISN and PVC might exert no

appreciable toxic effects on the rats which received oral

administration of them for 3 consecutive months in the daily

dose, ·the administration period and the method of administration

used in the present studies.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
700 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
two studies available
System:
other: no toxicological affected system identified

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

as no adverse effects are observed, no mode of action can be established

Additional information

Justification for classification or non-classification

the available information is conclusive but not sufficient for classification