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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of Aradur 1019 was assessed by an Ames test using salmonella tryphinum carried out before the publication of modern methods such as OECD guidelines and a chromosome aberration assay according to OECD test guideline 473.

Both studies conclude that there were no genetoxic effects caused by the test substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch no. Zd 429-10A III
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomes and co-factors
Test concentrations with justification for top dose:
5, 10, 50, 100, 500 ,1000 and 5000 µg/0.1 ml
Vehicle / solvent:
Twice distilled water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: Minimum agar, plus salts and glucose.

The plates were incubated for about 48 hours at 37°C in darkness.
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Material

Concentration of testing sample

(µg / plate)

Presence of S9 Mix

Back Mutation                 Number of colunies/plate (mean)

Base Conversion Type

Frame Shift Type

TA100

TS1535

WP2UBrA

TA98

TA1537

TA1538

Vehicle Control

 

-

91

15

20

19

15

14

TK12271

 

5

-

87

18

20

28

14

18

10

-

89

20

26

29

14

14

50

-

95

23

15

27

9

12

100

-

100

15

19

25

14

20

500

-

86

15

21

20

13

14

1000

-

83

15

20

25

15

14

5000

-

88

15

19

25

12

14

Vehicle Control

 

+

78

11

16

46

8

33

TK12271

 

5

+

71

12

24

44

7

30

10

+

76

10

21

42

5

29

50

+

83

12

13

44

5

27

100

+

78

12

17

46

12

26

500

+

81

15

18

51

6

30

1000

+

81

12

13

44

6

28

5000

+

69

7

19

45

9

30

Conclusions:
In the experiments performed without and with microsomal activation, comparison of the number of histidine- or tryptophan-prototrophic mutants in the controls and after treatment with TK 12271 revealed no marked differences.
Executive summary:

TK 12271 was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimirium and on a tryptophan-auxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back- mutation to histidine- or tryptophan- prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of TK 12271 revealed no marked deviations.

No evidence of the induction of point mutations by TK 12271 or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains ofS. typhimuriumandE. coliused in these experiments.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November 2016 to 17 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: chromosome aberration
Specific details on test material used for the study:
Identification: 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent
Purity 98.5%
Molecular Formula C15H14N2O
Molecular Weight 238.28 g/mol
Commercial Name Aradur 3123 ES
EC No. 428-170-5
CAS No. 185554-99-8
Physical State Solid (powder)
Color White-beige
Batch/ Lot Number AED0041000
Expiration Date 18 August 2016
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose level, the lowest precipitating dose level, and the highest soluble dose level was measured. Dose levels for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) or visible precipitate at the conclusion of the treatment period.
Vehicle / solvent:
Vehicle solvent used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
mitomycin C
Details on test system and experimental conditions:
Test System
Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Preparation of Target Cells
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Identification of Test System
Prior to treatment, each flask was identified by the BioReliance study number, dose level, test phase, treatment condition, activation system and/or replicate design.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3330, Exp. Date: 09 Sep 2016) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and added to the test system at 20% (v/v). The final concentrations of the components in the test system are indicated below:

Component Final Concentration in Cultures
NADP (sodium salt) 1 mM
Glucose-6-phosphate 1 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
S9 homogenate 20 μL/mL

Solubility Determination
Solubility tests were conducted, using sterile water, DMSO, ethanol, acetone, DMF, acetonitrile, and tetrahydrofuran (THF), to determine the vehicle, selected in order of preference that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Experimental Design
The in vitro mammalian chromosome aberration assay was conducted using standard procedures (Galloway et al, 1994; Preston et al, 1981; Swierenga et al, 1991) by exposing Chinese hamster ovary (CHO) cells to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.

Preliminary Toxicity Test for Selection of Dose Levels
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose level, the lowest precipitating dose level, and the highest soluble dose level was measured. Dose levels for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) or visible precipitate at the conclusion of the treatment period.

Chromosome Aberration Assay
Seven to twelve dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was based on visible precipitate at the conclusion of the treatment period. Two additional dose levels were included in the microscopic evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assay)
The pH at the highest test substance concentration was measured prior to dosing using a pH meter. Treatment was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 500 μL of test substance dosing solution or vehicle alone. Untreated controls were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions.
For the definitive assay only, the non-activated 20 hour treatment group cultures with visible precipitate were to be washed with CMF-PBS prior to Colcemid® treatment to avoid precipitate interference with cell counts (see Deviations). Two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Preliminary Results:

In the preliminary toxicity assay, CHO cells were exposed to nine dose levels of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent, ranging from 0.195 to 1950 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test substance formed workable suspensions in DMSO from 5.85 to 195 mg/mL and formed clear solutions in DMSO from 0.0195 to 1.95 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment

Condition

Treatment

Time

Visible precipitate

At the beginning of Treatment period

At the conclusion of Treatment period

Non-activated

4 hr

≥ 19.5 μg/mL

≥ 195 μg/mL

20 hr

≥ 19.5 μg/mL

≥ 585 μg/mL

S9-activated

4 hr

≥ 19.5 μg/mL

≥ 195 μg/mL

The osmolality in treatment medium was measured as follows:

Dose tested

Dose levels

(μg/mL)

Osmolality

(mmol/kg)

Vehicle

0

431

Highest soluble

195

435

Lowest Precipitating

585

441

Highest

1950

392

The osmolality of the test substance doses in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 7.5.

The results of the evaluation of cell growth inhibition are presented in Tables 1, 2 and 3. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 585 μg/mL in the non-activated 4-hour exposure group, at 1950 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 58.5 μg/mL in the non-activated 20-hour exposure group.

Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment

Condition

Treatment

Time

Recovery

Time

Dose levels

(μg/mL)

Non-activated

4 hr

16 hr

10, 25, 50, 100, 150, 200, 250

20 hr

0 hr

2.5, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250

S9-activated

4 hr

16 hr

10, 25, 50, 100, 150, 200, 250

Chromosome Aberration Assay

In the chromosome aberration assay, the test substance formed workable suspensions in DMSO from 10 to 25 mg/mL and formed clear solutions in DMSO from 0.25 to 7.5 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment

Condition

Treatment

Time

Visible precipitate

At the beginning of Treatment period

At the conclusion of Treatment period

Non-activated

4 hr

≥ 100 μg/mL

≥ 100 μg/mL

20 hr

≥ 100 μg/mL

≥ 100 μg/mL

S9-activated

4 hr

≥ 100 μg/mL

≥ 100 μg/mL

The pH of the highest dose level of test substance in treatment medium was 7.0.

Toxicity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 16% at 100 μg/mL, the highest test dose level evaluated for chromosome aberrations (Table 4). The activity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent in the induction of chromosome aberrations is presented by treatment flask in Table 5 and summarized by group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 100 μg/mL, was 2% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 25, 50, and 100 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Toxicity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 7% at 100 μg/mL, the highest test dose level evaluated for chromosome aberrations (Table 6). The activity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent in the induction of chromosome aberrations is presented by treatment flask in Table 7 and summarized by group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 100 μg/mL, was 13% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 25, 50, and 100 μg/mL. The percentage of cells with structural aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). A statistically significant increase (2.7%) in numerical aberrations was observed at 50 μg/mL (p ≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase in numerical chromosome aberrations was within the historical control range of 0.00% to 9.50% and within the 95% control limit of historical data (0.00% - 6.37%). Therefore, the statistically significant increase was considered to be biologically irrelevant. The percentage of structurally aberrant cells in the CP (positive control) treatment group (13.3%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Toxicity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was not observed at 100 μg/mL, the highest test dose level evaluated for chromosome aberrations (Table 8). The activity of 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent in the induction of chromosome aberrations is presented by treatment flask in Table 9 and summarized by group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 100 μg/mL, was 62% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 25, 50, and 100 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (14.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Conclusions:
The positive and vehicle controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

The test substance, 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.195 to 1950 μg/mL. The top dose tested was based on limited solubility of the test substance in DMSO. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 585 μg/mL in the non-activated 4-hour exposure group, at 1950 μg/mL in the S9-activated 4-hour exposure group, and at doses ≥ 58.5 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 195 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at doses ≥ 585 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 10 to 250 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 2.5 to 250 μg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose in the S9-activated 4-hour and the non-activated 20-hour exposure groups. Cytotoxicity was observed at 250 μg/mL in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 100 μg/mL in all three treatment conditions. The dose levels selected for microscopic analysis were 25, 50, and 100 μg/mL for all three treatment conditions.

No significant or dose-dependent increases in structural aberrations were observed in any of the test substance treated groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase (2.7%) in numerical aberrations was observed at 50 μg/mL (p ≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase in numerical chromosome aberrations was within the historical control range of 0.00% to 9.50% and within the 95% control limit of historical data (0.00% - 6.37%). Therefore, the statistically significant increase was considered to be biologically irrelevant.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

Under the conditions of the assay described in this report, 1-[(2-methyl-1H-imidazol-1-yl)methyl]-2-naphthalenol Monoconstituent was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The genetic toxicity of Aradur 1019 was assessed by a Mammalian Erythrocyte Micronucleus assay according to OECD test guideline 474.

The test concluded that Aradur 1019 was negative in terms of genetic toxicity.

Link to relevant study records
Reference
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
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
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
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

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

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%

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification