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Genetic toxicity: in vivo

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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
GLP compliance:
no
Remarks:
published summary of a GLP study
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: 99.86%
CAS number: 110-18-9
Supplied by MACH 1 Inc., King of Prussia, PA
Target gene:
Reversion to histidine / tryptophan independence
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:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
5000 ug/mL, the maximum concentration as cited in the guideline.
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
Bacterial salmonella strains were received from Dr. Ames (University of California) and Escherichia coli strain was obtained from Pharmacia and Upjohn Co., Kalamazoo, Michigan.
S-9 mix or water was added to tubes containing 2.0 mL of top agar supplemented with histidine-biotin or tryptophan. An aliquot of the bacteria was added (0.1 mL) followed by the test article or vehicle control. The tube was mixed for 2-3 seconds and the contents were evenly distributed over a Vogel-Bonner bottom agar plate. The plates were allowed to set and the plates were inverted and incubated at 37 degrees C for 72 hours. The revertant colonies were counted using automated scoring.
Evaluation criteria:
Positive response was considered to be an increase in revertant number of 2-fold (strains TA98 and TA100) or 3-fold (strains TA1535, TA1537 and WP2 uvrA) the concurrent control.
Statistics:
None.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Results in the absence of S-9 mix

Treatment (mcg/plate)

Mean revertants per plate

TA98

TA100

TA1535

TA1537

WP2 uvrA

0 (vehicle)

23

111

20

9

16

500

19

102

10

7

16

750

23

90

14

6

16

1000

22

101

17

5

18

3000

18

103

10

5

18

5000

20

117

10

8

22

Positive control

765

525

759

608

520

 

Results in the presence of S-9 mix

Treatment (mcg/plate)

Mean revertants per plate

TA98

TA100

TA1535

TA1537

WP2 uvrA

0 (vehicle)

37

65

20

11

23

500

29

69

20

6

21

750

3

83

22

11

18

1000

40

84

23

19

26

3000

34

69

22

13

22

5000

37

106

18

8

18

Positive control

1019

583

334

354

229

Conclusions:
TMEDA did not induce increases in the number of revertants for any tester strains in the presence or in the absence of metabolic activation when tested in strains TA98, TA100, TA1535, TA1537 and WP2 uvrA.
Executive summary:

An Ames test was conducted with TMEDA in accordance with OECD test guideline 471. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA were tested in the absence and in the presence of aroclor 1254 induced rat liver S9 fraction. Plate incorporation methodology was employed. TMEDA showed no increases in revertant number which were 2 -fold the concurrent control (strains TA98 and TA100) of 3 -fold the concurrent control (strains TA1535, TA1537 and WP2 uvrA) when tested in two independent assays. TMEDA has shown no evidence of mutagenicity when tested up to 5000 ug/plate, the maximum concentration specified in the guideline.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
no
Remarks:
published summary of a GLP study
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity: 99.86%
CAS number: 110-18-9
Supplied by MACH 1 Inc., King of Prussia, PA
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Clone CHO-W-B1
Cytokinesis block (if used):
Colcemid (0.1 ug/mL) was present during the final two hours of incubation.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone-induced rat hepatic S9 fraction
Test concentrations with justification for top dose:
Seven concentrations ranging from 10 to 5000 mcg/mL in the Range-Finder Experiment
Concentrations of 50, 100, 500, 1000, 2500 and 5000 mcg/mL in the Main Experiment
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
The cells were suspended in cell culture medium (McCoy's 5A). A 5 mL sample was seeded into a T-25 tissue culture flask. The flasks were incubated at 37°C for 20-24 hours prior to treatment. The cells were exposed to the test article for 3 hours. The medium was removed and the cells were rinsed with phosphate buffer solution, replenished with 5 mL of fresh medium and incubated for an additional 15 hours. Colcemid (0.1 mcg/mL) was present during the final two hours. The monolayer of cells was dissociated with 0.05% trypsin and resuspended in medium. An aliquot of cell suspension was counted using an electronic cell counter. The number of cells per flask was calculated for each concentration and the relative cell growth (RCG) was calculated as a percentage of the number of cells in the test flask divided by the number of cells in the solvent flask. The remaining cell suspension was collected by centrifugation (800 rpm), ruptured in 0.075M KCl and fixed with methanol:glacial acetic acid (3:1) and dropped onto microscope slides. The slides were air-dried, stained with 5% Giemsa and mounted in Cytoseal using cover glasses. The slides were scored for Mitotix Index (MI). For each concentration, 1000 cells were scored and the numbers of dividing cells were recorded. The MI and Relative Mitotic Index (RMI) for each concentration was calculated as follows:

MI=number of dividing cells from 1000 cells divided by 10.
RMI=test concentration MI divided by solvent control MI expressed as a percentage.

Cytotoxicity was evaluated on the basis of the reduction in RCG and/or RMI. Chromosome aberrations were scored from 3 concentrations of test material, the vehicle and positive control. Two hundred 200 metaphases were scored from each concentrations and the controls.
Evaluation criteria:
The test material was considered to have caused a positive response in this assay if the test article showed a positive concentration response trend and a statistically significant increase over the solvent controls in the percentage of cells with chromosome aberrations, at one or more concentrations.
Statistics:
Chi-squared test was used for statistical analysis
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A positive response was seen only at the highest concentration tested, which exceeds the highest recommended concentration in the current guideline of 10mM, equivalent to 1140 mcg/mL.
Remarks on result:
other:
Remarks:
Clastogenic

Chromosomal aberration in the absence of S-9 mix

Treatment

Relative cell growth

Percentage of aberrations (mean ± standard deviation)

0 (water)

100 ± 0

0.5 ± 0.7

1000 ug/mL

160 ± 10

1.0 ± 0.0

2500 ug/mL

117 ± 8

1.5 ± 0.7

5000 ug/mL

90 ± 8

13.5 ± 5.0a

Positive control

67 ± 10

40.5 ± 0.7a

a            Statistically significant (p<0.05)

 

Chromosomal aberrations in the presence of S-9 mix

Treatment

Relative cell growth

Percentage of aberrations (mean ± standard deviation)

0 (water)

100 ± 19

1.5 ± 0.7

1000 ug/mL

75 ± 0

0.5 ± 0.7

2500 ug/mL

88 ± 1

2.0 ± 2.8

5000 ug/mL

67 ± 3

12.0 ± 0.0a

Positive control

63 ± 1

35.5 ± 3.5a

a            Statistically significant (p<0.05)

Conclusions:
TMEDA was negative in this assay when tested up to 2500 mcg/mL, a concentration which exceeds the highest concentration of 10mM based on the OECD test guideline 473. TMEDA was positive only at 5000 mcg/mL, a concentration which far exceeded the 10mM concentration in the OECD TG.
Executive summary:

TMEDA was assessed for clastogenicity in an in vitro chromosomal aberration study in CHO cells based on OECD test guideline 473. Concentrations of 1000, 2500 and 5000 ug/mL were examined for chromosome aberrations in the absence and in the presence of S-9 following a 3 hour treatment and 15 hour recovery. The increases in the number of aberrations at 5000 ug/mL in the absence and presence of S-9 were statistically significant compared to the concurrent vehicle controls when data were analysed at the 5% level using the Chi-squared test. However, test concentrations of 1000 and 2500 ug/mL showed no statistically significant increases in the number of aberrations. The highest concentration recommended by the OECD guideline 5000 ug/mL, 5 mcL/mL or 10mM, whichever is the lowest. A concentration of 10 mM is equivalent to 1140 ug/mL. TMEDA is considered to be not clastogenic when tested up to 2500 ug/mL, a concentration which exceeds the maximum concentration recommended for this assay.

Data source

Reference
Reference Type:
publication
Title:
Genotoxicity assessment of two hypergolic energetic propellant compounds
Author:
Reddy G, Song J, Mecchi MS & Johnson MS
Year:
2010
Bibliographic source:
Mutation Research 700: 26-31

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Remarks:
published summary of a GLP study
Type of assay:
other: In vivo mouse micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N,N',N'-tetramethylethylenediamine
EC Number:
203-744-6
EC Name:
N,N,N',N'-tetramethylethylenediamine
Cas Number:
110-18-9
Molecular formula:
C6H16N2
IUPAC Name:
[2-(dimethylamino)ethyl]dimethylamine
Test material form:
liquid
Details on test material:
clear, colourless to pale yellow
Specific details on test material used for the study:
Purity: 99.86%
CAS number: 110-18-9
Supplied by MACH 1 Inc., King of Prussia, PA

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
No further details reported.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Harlan Sprague Dawley, Inc
Diet: Purina Certified Rodent Diet
Water: Tap water; ad libitum
Room temperature: 17 to 23°C
Humidity: 17-54%
Photoperiod: 12 hour light/dark cycle
Acclimatisation period: At least 7 days prior to testing

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Water
Details on exposure:
The estimated maximum tolerated dose was 250 mg/kg bw based on an initial dose range-finding test which was conducted with 5, 10, 50, 100, 200 and 300 mg/kg bw with three mice/sex/group. The maximum dose volume was 10 mL/kg bw.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide 80 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow smears prepared and allowed to air dry.
Polychromatic erythrocytes (PCE) and Normochromatic erythrocytes (NCE) examined.
Details of tissue and slide preparation:
Bone marrow smears allowed to air dry. The slides were fixed in methanol, stained in Wright-Giemsa stain, rinsed in distilled water, allowed to air dry completely and mounted in Cytoseal using cover glasses.
Evaluation criteria:
The test material was considered to have caused a positive response in the assay if the material showed a positive dose-response trend and a statistically significant increase in the number of micronucleated polychromatic erythrocytes. at one or more dose levels, relative to that of the concurrent vehicle control. In the event that a statistically significant increases was observed with an unusually low number of micronucleated polychromatic erythrocytes (less than 0.05%) in the concurrent vehicle control, the data from that dose was compared to the historical control data. In the event that there was no positive dose-response trend, at least two consecutive test doses must have produced a statistically significant increase in the number of micronucleated polychromatic erythrocytes to be considered positive. The test article was considered to have caused a negative response if none of the test doses showed a statistically significant increase in the number of micronucleated polychromatic erythrocytes when compared to the vehicle control.
Statistics:
The slides were scored blind. The number of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) among 2000 erythrocytes (PCE+NCE) per animal was determined. The number of micronucleated polychromatic erythrocytes (MPCE) was then determined for 2000 PCE per animal. Data were analysed separately for male and female animals. The frequency of MPCE in each dose group was compared to that in the respective vehicle control group using a one-tailed Student's t-test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no statistically significant increases in the number of MnPCEs in the treated groups at any dose level for either 24 or 49-hour harvest time as compared to the concurrent vehicle control group or the historical vehicle control group. There was no reduction in the percentage of PCEs at any dose level for either harvest time of more than 20% which would be an indicator of toxicity. The positive controls showed a statistically significant increase at the 5% level in the number of MnPCEs when comapared to the concurrent vehicle controls using the Students t-test.

Any other information on results incl. tables

Micronucleus data for 24-hour harvest

Sex

Dose (mg/kg bw)

Number of MN PCEs/2000 PCEs

(mean ± standard deviation)

NCE/PCE per animal

(mean ± standard deviation)

Male

0 (water)

0.6 ± 0.5

0.6 ± 0.1

62.5

0.4 ± 0.5

0.8 ± 0.3

125

0.8 ± 0.8

0.6 ± 0.2

250

0.4 ± 0.5

0.9 ± 0.2

Positive control

28.8 ± 4.8a

0.3 ± 0.1

Female

0 (water)

0.0 ± 0.0

0.7 ± 0.2

62.5

0.6 ± 0.5

0.9 ± 0.3

125

0.0 ± 0.0

0.7 ± 0.2

250

0.6 ± 0.5

0.6 ± 0.1

Positive control

23.2 ± 6.8a

0.8 ± 0.1

a            Statistically significant increases when data were compared to the vehicle control using the Students t-test at the 5% level.

Micronucleus data for 48-hour harvest

Sex

Dose (mg/kg bw)

Number of MN PCEs/2000 PCEs

(mean ± standard deviation)

NCE/PCE per animal

(mean ± standard deviation)

Male

0 (water)

0.8 ± 0.4

1.5 ± 0.4

62.5

2.2 ± 1.3

0.9 ± 0.2

125

0.0 ± 0.0

1.4 ± 0.1

250

0.4 ± 0.9

1.2± 0.3

Female

0 (water)

0.0 ± 0.0

1.3 ± 0.1

62.5

0.0 ± 0.0

1.4 ± 0.2

125

0.2 ± 0.4

1.3 ± 0.2

250

0.8 ± 0.4

1.3 ± 0.4

 

Applicant's summary and conclusion

Conclusions:
The results indicate that TMEDA is negative in this assay.
Executive summary:

An in vivo micronucleus study was conducted with CD-1 mice using TMEDA dose levels of 0, 62.5, 125 or 250 mg/kg bw. Bone marrow sampling was conducted at 24 and 48 hours post administration. Five mice of each sex were treated for each concentration and sampling time. An additional 5 mice of each sex were treated with the positive control, cyclophosphamide, with a sampling time of 24 hours. There were no statistically significant increases in the number of micronucleated polychromatic erythrocytes for any of the TMEDA treated animals when compared to the concurrent vehicle control treatments or the historical vehicle control ranges. The positive control treated animals showed a statistically significant increase in the number of micronucleated polychromatic erythrocytes. The overall conclusion was that TMEDA had shown no evidence of cytogenicity under the conditions of this study.