2,2'-azobis[2-methylpropionamidine] dihydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.05.-27.09.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

minor deviations in the experimental procedure which were rated uncritical (see Results and discussion)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

EC Number:

221-070-0

EC Name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

Cas Number:

2997-92-4

Molecular formula:

C8H18N6.2ClH

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylpropanimidamide) dihydrochloride

Constituent 2

Reference substance name:

2,2'-azobis(2-amidinopropane)-dihydrochloride

IUPAC Name:

2,2'-azobis(2-amidinopropane)-dihydrochloride

Test material form:

other: granules

Details on test material:

- Name of test material (as cited in study report): 2,2'-azobis(2-amidinopropane)-dihydrochloride
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.1 %
- Lot/batch No.: I59596
- Expiration date of the lot/batch: 31. Dec. 2012
- Storage condition of test material: ambient temperature, dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Groups of five animals of the same sex in open macroloncages type 2000P
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended in tab-water. For the high dose group (75 mg/kg BW), 750 mg of the test item were suspended in 40 ml of sterile filtered water, yielding a concentration of 18.75 mg/m:. The preparation was intended for an spplication volume of 4 ml per kg body weight. therefore, 4 mL of this high-dose suspension contained 75 mg of the test item. For the medium-dose group (25 mg/kg bw) an aliquot of the high-dose suspension was diluted 1 in 3; it contained 6.25 mg/mL. For the low-dose group (8.3 mg/kg bw) an aliquot of the medium-dose suspension was diluted 1 in 3. The low-dose suspension contained 2.08 mg/mL

VEHICLE
- Amount of vehicle (if gavage): 4 mL per kg body weight

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
75 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
25 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
8.3 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: based on dose-range finding study
- Rationale for animal assignment (if not random): random

Positive control:

non

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly,
- IRWIN test

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately before sacrificing
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately before sacrificing
- Animals fasted: No
- How many animals: all

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: last exposure week
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity (beam walking test) / grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

Descriptive statistics
The data for body weight, organ weight, grip strength and haematological/biochemical
examination were documented for each individual animal. Food- and water consumption
were documented in gender groups. For each experimental group, means and standard
deviations were calculated. The test item groups were analysed in comparison to the vehicle
group by calculation of statistical significance using a two-tailed unpaired student´s t-test
(p ≤ 0,05). Statistic significances were represented when appropriate in graphical form or in
the text of the study report.
Additionally, for the haematological/biochemical data, the median, the 0,25 quantil and the
difference of median and mean were calculated in %. These calculations give basic
information about the sample distribution of the respective data sets in relation to a Gaussian
(normal) distribution. In case of differences higher or lower than 10% the Study Director
decided whether statistical analysis was still appropriate.
The main decision-tree, documented in the appendix, displays the general approach of the
deductive statistical analysis.
Most statistical hypotheses of this study are best characterised as “many to one”
comparisons, for example: a vehicle control vs. three treatment groups. Therefore, the
adequate analytic method was a One-Way ANOVA (ANAlysis Of VAriance), followed by a
post hoc t-test.
For interval-scaled data, it was intended to apply a One-Way ANOVA supplemented by
Dunnett’s post-hoc t-test. Ordinal-scaled data should be analysed using the Kruskal-Wallis
test, supplemented by Dunn’s t-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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see Details

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see Details

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see Details

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Analyses of haematology and serum biochemistry returened normal values in the vast majority of the monitored parameters. The minor effects observed in individual red blood cells related parameters, the thrombocytes as well as the total number of leucocytes were assessed to be of no toxicological relevance.
A test item related effect on the potassium concentration in the blood of male animals of the high dose group was found to be associated with increased kidney weight and decreased size of the adrenal gland.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A decrease of Potassium concentration in the blood along with increased kidney weight and decreased size of the adrenal gland.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A NOAEL of 25 mg/kg bw/ day for the 28-day repeated dose toxicity has been established.

Executive summary:

Aim of the study:

The aim of this study was to assess data on the subacute toxicity of the chemical substance 2,2' – azobis (2-amidinopropane)-dihydrochloride suspended in water.

Experimental model:

The test item 2,2' – azobis (2-amidinopropane)-dihydrochloride was administered daily by oral gavage at dose levels of 75, 25 and 8 mg/kg bodyweight per day to 5 male and 5 female Wistar rats per dose group over a period of 28 days. Additionaly, 10 male and 10 female rats received the same volume of sterile water as vehicle control. All solutions were administered at an application volume of 4 mL/kg bodyweight.

Assessed parameters:

During the in-life phase, viability, general and detailed clinical signs, food and water consumption, body weight, were recorded.

At the end of the in-life phase, grip strength and reactivity to sensory stimuli (beam walking test) were detected and blood samples from all animals were collected to provide data on haematology and serum biochemistry. All animals were sacrificed immediately after bleeding and examined by gross necropsy. Weights of selected organs were recorded and tissues and organs were preserved according to the study plan. All samples were processed for histopathological examination. The examination was conducted on samples from the high dose groups and the vehicle groups.

Assessment of the Results:

Detailed clinical signs, food and water consumption, motoric activity, and reactivity to sensory stimuli showed no distinct abnormalities. The grip strength test also revealed no differences between the different experimental groups.

The mean body weight increase of all experimental groups was within the normal range for rats of this strain and age.

Analyses of haematology and serum biochemistry returned normal values in the vast majority of the monitored parameters. The minor effects observed in individual red blood cells related parameters, the thrombocytes as well as the total number of leucocytes were assessed to be of no toxicological relevance. A test item related effect on the potassium concentration in the blood of male animals of the high dose group was found to be associated with increased kidney weight and decreased size of the adrenal gland.

Morphological and histological examination did not reveal morphological changes related to the test item. Macroscopic findings in all animals as well as histopathological findings in animals of the high dose group did not differ significantly or the differences were regarded as coincidental. All findings are considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of this strain and age.

Conclusion:

A daily oral administration of the test item, 2,2'-azobis(2-amidinopropane)-dihydrochloride, to Wistar rats at a dose level of 75, 25 and 8 mg/kg body weight over a time period of 28 days resulted in minor systemic effects in the male high dose group. Concerning the findings in kidney weight, adrenal-gland size and potassium levels in the male high dose group, the NOAEL in this study was 25 mg/kg body weight over a 28 day application period.