Ethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987 or before

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Salmonella typhimurium/ microsome mutagenicity assay.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediamine

EC Number:

203-468-6

EC Name:

Ethylenediamine

Cas Number:

107-15-3

Molecular formula:

C2H8N2

IUPAC Name:

ethane-1,2-diamine

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Without S9: 0, 0.01, 0.03, 0.1, 0.3 and 1 mg/plate; confirmatory study TA100 and TA1535: 0, 1, 3 and 5 mg/plate
With S9: 0, 0.1, 0.3, 1, 3 and 5 mg/plate; confirmatory study TA100 and TA1535: 0, 1, 2, 3, 4, 5 and 6 mg/plate

Vehicle / solvent:

Distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminoanthacene

Details on test system and experimental conditions:

A 100 ml sample of EDA was received for testing at BRRC on November 21, 1986. The sample was assigned the unique BRRC Sample Number of 49-428 and was stored at room temperature. Gas chromatographic analysis was performed by the sponsor indicated that the sample was 99.58% EDA.
The sample was completely soluble in water; thus, water was used as the solvent for dilution of the test agent. Dilutions were prepared fresh on the morning of each test day and the accuracy of dilution was verified by gravimetric analysis. The undiluted test agent and the test agent diluted in water were indicated by the sponsor to be stable and no stability testing was performed in the the test system.

Control Substances
1. Solvent: Water; BRRC #49-27; CAS #7732—18—5
2. Positive Controls: ‘
a) 4-nitro-o-phenylenediamine; BRRC #44—71; CAS #99—56-9
b) 9-aminoacridine; BRRC #44-233; CAS #90—45—9
c) sodium azide; BRRC #44—72; CAS #26625—22—8
d) 2-aminoanthracene; BRRC #44—67 and 50-168; GAS #613—13-8

Metabolic Activation
S9 liver homogenate, prepared from Aroclor 1254 induced, Sprague-Dawley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 ml of $9 mix containing 50 pl of S9 was added per plate.

The assay was performed according to BRRC Standard Operating Procedures 7.4.1A through 7.4.7A, 7.4.12A, and 7.4.13.

Sample Preparation:
For definitive testing, an initial stock solution of the test substance was prepared by mixing EDA in water to achieve a concentration of 50 mg/ml. All subsequent dilutions were made in the same solvent. Dilutions of the test substance were made fresh each day of testing.
All dilutions for the mutagenicity tests were analysed gravimetrically to determine actual concentrations.

Dose Selection:
A preliminary toxicity test was performed using strain TA100 to determine the level of toxicity of the test substance. Ten doses were tested for toxicity with a plate assay performed in the manner used for mutagenicity determinations. Toxicity was assessed at 48 hours after treatment by observing growth inhibition of the background lawn and/or a reduction in the number of spontaneous mutants.

Testing:
The test chemical was tested in triplicate at five doses chosen to span a range which included moderately toxic to relatively nontoxic concentrations. Testing was performed both with and without metabolic activation. Concurrent solvent and positive controls were tested in each experiment.

Quality Control
Data from each test was checked for accuracy and integrity by a second investigator. Compliance of testing procedures with Good Laboratory Practices was audited by the independent Quality Assurance Unit at the Bushy Run Research Center.

Evaluation criteria:

The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.

Statistics:

No statistical analysis

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Ambiguous results in strain TA100 with metabolic activation. Negative in all other strains.

Any other information on results incl. tables

Dose	Table 1 Ethylenediamine without S9 activation
mg/plate	TA98	TA100	TA1535	TA1537	TA1538
0	26+3.5	113+19.7	19+2.5	6 +3.1	8+2.5
0.01	23+9.6	112+7.0	21+5.7	6 +1.5	7+1.7
0.03	25 +3.1	119+13.9	17+10.5	5+1.5	10+5.0
0.1	28+2.9	136+26.3	27+1.5	6+1.5	9+4.7
0.3	32 +10.5	152+5.9	35+10.4	7 +2.5	11+4.5
1	Toxic	Toxic/s	Toxic	Toxic	Toxic
Pos Control	1140+63.5	2554+115	2369+117	269+53.3	1470+102.4
	4 -NPD	NaN3	Na N3	9 -AA	4 -NPD
Dose	Table 2 Ethylenediamine with S9 activation
mg/plate	TA98	TA100	TA1535	TA1537	TA1538
0	34+7.4	114+4.6	6+1.2	5+0.6	28+1.7
0.1	36+5.8	119+21.4	11+5.2	4+0.6	25+8.1
0.3	30+2.3	128+9.6	8+3.1	6+3.6	27+6.4
1	27+6.4	121+9.2	8+2.0	7+4.0	17+4.0
3	29+4.2T	214+0.7S	15+7.5	9+4.0	20+8.5
5	16+9.1T	212+32.7	17+9.3	7+1.0	8+1.4S
Pos Control	1226 +249.5	818 +172.5	32 +8.7	49 +7.6	106 +15.5
	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
Dose	Table 3 Ethylenediamine (repeat) without S9 activation
mg/plate	TA100	TA1535
0	92+10.8	13+3.2
1	135+10.0	11+4.4
3	165+10.1	16+3.2
5	161+22.9	12+3.5S
Pos Control	940+152.0	42+7.4
	2 -AA	2 -AA
Dose	Table 4 Ethylenediamine (repeat) with S9 activation
mg/plate	TA100	TA1535
0	116+14.0	16+4.4
1	139+5.3	9+2.5
2	148+15.3	12+2.5
3	157+28.5	16+1.2
4	193+17.9	16+6.6
5	201+25.0	16+3.6
6	149 S	14+2.1
Pos Control	1564+108.8	114+26.2
	2 -AA	2 -AA
T-Toxic
S-Sparse growth of background lawn

4-NPD: 4-NITRO-0 -PHENYLENEDIAMINE; 9-AA: 9-AMINOACRIDINE;

NaN3: SODIUM AZIDE; 2-AA: 2-AMINOANTHRACENE

+ indicates standard deviation of mean value

Applicant's summary and conclusion

Conclusions:

Because of a less than 2-fold increase obtained with TA100 tested with S9, EDA was considered not to be mutagenic in this in vitro bacterial assay. No increases were seen using strains TA 98, TA 1535, 1537 and 1538 with and without metabolic activation.

Executive summary:

Ethylenediamine (EDA) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test concentrations for the Ames test were chosen from data obtained in a preliminary study with strain TA 100 performed both with and without a rat liver S9 activation system.

In tests without S9, a dose level of 1.0 mg/plate allowed only sparse growth of the background lawn and reduced the numbers of revertant colonies to less than half the control level. In contrast, tests with S9 activation allowed confluent growth of the background lawn at dose levels ranging from 0.01 to 3.0 mg/plate. A slightly higher dose of 5.0 mg/plate allowed only sparse growth of the background lawn. Based on these results, five doses ranging from 0.01 to 1.0 mg/plate were tested in the definitive test without S9 and a higher range of 0.1 to 5 mg/plate was tested in the presence of the S9 metabolic activation system. These concentrations were tested with five different strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) using triplicate cultures at each dose level for each strain.

In the definitive mutagenicity test performed without S9, no evidence of positive or dose related mutagenic activity was observed with any of the five strains. In tests performed in the presence of an Aroclor 1254 induced rat-liver S9 metabolic activation system, only strain TA100 showed a slight increase in numbers of revertant colonies (the magnitude of the response at the highest concentration levels ranged from 1.7 to 1.9 times the concurrent controls) but this was less than a 2-fold increase. In the first test with S9, TA1535 showed a slight increase; however, this response was within the historical control range of variation for this strain and was not reproduced in two additional experiments. Overall, because of a less than 2-fold increase obtained with TA100 tested with S9, and no increases were seen using strains TA 98, TA 1535, 1537 and 1538 with and without metabolic activation, EDA was considered not to be mutagenic in this in vitro bacterial assay.