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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2016 - October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Updated and adopted 26 September 2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Erythrocyte micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[[3-(dimethylamino)propyl]amino]propiononitrile
EC Number:
274-159-1
EC Name:
3-[[3-(dimethylamino)propyl]amino]propiononitrile
Cas Number:
69852-45-5
Molecular formula:
C8H17N3
IUPAC Name:
3-{[3-(dimethylamino)propyl]amino}propanenitrile
impurity 1
Chemical structure
Reference substance name:
3,3'-[[3-(dimethylamino)propyl]imino]bispropiononitrile
EC Number:
275-331-9
EC Name:
3,3'-[[3-(dimethylamino)propyl]imino]bispropiononitrile
Cas Number:
71326-27-7
Molecular formula:
C11H20N4
IUPAC Name:
3,3'-{[3-(dimethylamino)propyl]imino}dipropanenitrile
Test material form:
liquid
Remarks:
colorless
Specific details on test material used for the study:
ID: 3-[[3-(dimethylamino)propyl]amino]propiononitrile
Purity : >96%
Molecular Weight: 155.24068 g/mol
Chemical Name: 3-[[3-(dimethylamino)propyl]amino]propiononitrile
Batch/Lot No.: AAE1131000
Expiration Date: 27-July-2018 (provided by Sponsor)
Description: Clear colorless liquid
Storage Conditions: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: 165.3 - 191.1g
- Assigned to test groups randomly: [no/yes, under following basis: ] Yes (Excel method)
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 +/- 3 °F
- Humidity (%): 50 +/- 2%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12hr dark/ 12 hr light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Deoionised water
Details on exposure:
Dose formulations were administered at a volume of 10 mL/kg by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles).
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Since no significant differences in toxicity or mortality were observed in the dose range-finding test, only male rats were used for the micronucleus assay.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Femoral bone marrow was collected at approximately 24 or 48 hours after the final dose, as indicated above. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least five positive control slides) was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and will be archived at report finalization. Stained slides will be discarded prior to report finalization. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Evaluation criteria:
A test substance was considered to have induced a positive response if:

a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.

A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).

If the response was neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Possible additional work may include scoring additional cells (where appropriate) or performing an additional experiment that could employ the use of modified experimental conditions. Such additional work was only carried out following consultation with, and at the request of, the Sponsor.

In some cases, even after further investigations, the data set precluded making a conclusion of positive or negative, at which time the response was concluded to be equivocal. In such cases, the Study Director used sound scientific judgment and reported and described all considerations.
Statistics:
Statistical analysis was performed on the micronucleus frequency (MnPCE%) and PCE% using the animal as the unit. The mean and standard deviation of MnPCE% and PCE% were presented for each treatment group.

The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significant level of p  0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post hoc analysis to compare each dose group to the concurrent vehicle control.

A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p 0.01).

A pair-wise comparison (Student’s T-test) was used to compare the positive control group to the concurrent vehicle control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Definitive Assay - Clinical Signs



Treatment 

Observation

Number of Animals With Observed Signs/Number of Surviving Animals

Number of Animals Found Dead/Total Number of Animals Dosed

Males

Day 1

Day 2

Day 3

Males

Pre-Dose

Post-Dose

1 Hr

2 Hr

Deionized water

Normal

10/10

10/10

10/10

10/10

5/5

0/10

3-[[3(dimethylamino)propyl] amino]propiononitrile

250 mg/kg

 

 

Normal

 

 

5/5

 

 

5/5

 

 

5/5

 

 

5/5

 

 

N/A

 

 

0/5

500 mg/kg

Normal

5/5

5/5

5/5

5/5

N/A

0/5

1000 mg/kg

Normal

10/10

10/10

10/10

10/10

5/5

0/10

Definitive Assay - Group Mean Body Weights

 

 

Group Mean Body Weights (g ± SD)

 

% Change¹

 

Treatment

 Sex

Day 1

Day of Euthanasia

 

Day of Euthanasia

 

Mortality²

Deionized water

24 Hour

M

200.2

210.7

5.2%

0/5

±2.8

±2.4

48 Hour

M

203.1

213.3

5.0%

0/5

±6.4

±7.1

3-[[3(dimethylamino)propyl]
amino]propiononitrile

250 mg/kg/day

M

201.5

206.8

2.6%

0/5

±3.4

±2.5

500 mg/kg/day

M

203.2

202.0

-0.6%

0/5

±6.0

±3.2

1000 mg/kg/day

 

 

 

 

 

 

 

 

24 Hour

M

204.8

202.9

-0.9%

0/5

±4.8

±5.5

48 Hour

M

203.8

196.1

-3.8%

0/5

±7.6

±20.9

 

 

 

 

 

 

 

 

 

SD = Standard deviation

¹% Change =[(Post-treatment weight - Pretreatment weight) x 100]/Pretreatment weight

²Reported as number of animals found dead after dose administration/total number tested.

Summary of Bone Marrow Micronucleus Assay

 

Time

%PCE

Change from Control

% MnPCE

Number of

Treatment

Gender

(Hrs)

Animals

(Mean +/- SD)

(%)

(Mean +/- SD)

MnPCE/PCE Scored

Deionized water

 

 

 

 

 

 

M

24

5

54.1

±

1.7

---

0.06

±

0.03

12

/20000

 

 

 

 

 

 

3-[[3-(dimethylamino)propyl]
amino]propiononitrile

 

 

 

 

 

 

250 mg/kg/day

M

24

5

53.2

±

1.0

-2

0.06

±

0.02

11

/20000

 

 

 

 

 

 

500 mg/kg/day

M

24

5

52.5

±

0.7

-3

0.07

±

0.02

13

/20000

 

 

 

 

 

 

1000 mg/kg/day

M

24

5

52.3

±

0.7

-3

0.11

±

0.03*

21

/20000

 

 

 

 

 

 

Deionized water

 

 

 

 

 

 

M

48

5

54.8

±

1.9

---

0.05

±

0.02

9

/20000

 

 

 

 

 

 

3-[[3-(dimethylamino)propyl]
amino]propiononitrile

 

 

 

 

 

 

1000 mg/kg/day

M

48

5

50.8

±

1.3

-7

0.07

±

0.03

14

/20000

 

 

 

 

 

 

CP

 

 

 

 

 

 

40 mg/kg/day

M

24

5

42.4

±

1.8**

-22

2.30

±

0.18**

459

/20000

 

 

 

 

 

 

PCE – Polychromatic Erythrocytes; MnPCE – Micronucleated Polychromatic Erythrocytes

*p < 0.05 or **p < 0.01, One-Way ANOVA with Post-Hoc Dunnett's Test or T-Test

24 Hrs MnPCE Male GLM P-value = 0.015, R-sqr = 47.27%

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, 3-[[3-(dimethylamino)propyl]amino]propiononitrile was concluded to be negative for the induction of micronucleated polychromatic erythrocytes.
Executive summary:

The test substance,3-[[3-(dimethylamino)propyl]amino]propiononitrile, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow. Deionized water was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg by single oral gavage.

In the dose range-finding assay (DRF), the maximum dose tested was 2000 mg/kg. The dose levels tested were 500, 1000, and 2000 mg/kg in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 1000 mg/kg, which is the maximum tolerated dose (MTD).

The definitive assay dose levels tested were 250, 500, and 1000 mg/kg.

A statistically significant increase in the incidence of MnPCEs was observed in animals 24 hours after treatment with 1000 mg/kg relative to the vehicle control. However, the increase was within the 95% historical vehicle control range. The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range. 

Under the conditions of this study, the administration of3-[[3-(dimethylamino)propyl] amino]propiononitrileat doses up to and including a dose of 1000 mg/kg was concluded to be negative in the Micronucleus assay.