N,N,N',N'-tetramethylethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests are available for the submission substance ATMEDAHP (N,N,N′,N′-tetramethylethylenediamine). Both studies report negative results; however one published study is slightly deficient when compared to the current guideline. A negative mouse lymphoma assay is also available. An in vitro chromosome aberration assay reports a positive response at concentrations exceeding the limit concentration recommended by the current OECD guideline.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Data published in 1987

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Strain TA102, WP2 uvrA or WP2 uvrA pKM101 not tested

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1989

Deviations:

yes

Remarks:

Salmonella strain TA102 or E.coli strains WP2 uvrA or WP2 uvrA pKM101 not tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

CAS number: 110-18-9
Supplier: Sigma
Purity: 99%

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9 (Sprague Dawley and Syrian Hamster)

Test concentrations with justification for top dose:

10000 ug/plate. The highest concentration was tested above the recommended maximum test concentration of 5000 ug/plate; toxicity was apparent in some strains.

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (strain TA98 in the absence of S-9) and 2-aminoanthracene (all strains in the presence of S-9)

Details on test system and experimental conditions:

Bacterial strains were received from Dr. Ames (University of California). Bacterial cultures were grown overnight at 37 degrees C, with shaking, in Oxoid broth. Phenotypes were analysed. The test material, bacteria and S-9 mix or buffer were incubated at 37 degrees C, with shaking, for 20 minutes. The top agar was added and the resulting contents mixed and poured on to Vogel Bonner test plates. The revertant colonies were counted following 2 days incubation at 37 degrees C. Counting was automatic, or hand-counted if the presence of precipitate.

Evaluation criteria:

Toxicity was considered to be evidence of a decrease in revertant colonies or a diminution of the backgound bacterial lawn. A positive mutagenic response was considered to be a dose related increase in the number of revertant colonies over the concurrent control. Scientific judgement was applied in interpreting increases in revertant numbers at a single concentration or a non-concentration related increase. Reprodubility between two experimental occassions were also considered.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the presence of hamster S-9

Vehicle controls validity:

not specified

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:

cytotoxicity

Remarks:

in the absence of S-9 and in the presence of hamster S-9

Vehicle controls validity:

not specified

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:

not specified

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:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Revertant numbers in the absence of metabolic activation

	Strain
Concentration (mcg/plate)	TA98	TA100	TA1535	TA1537
0 (water)	16	98	15	6
100	11	98	17	4
333	19	78	16	7
1000	11	89	17	7
3333	13	78	15	6
10000	13	78 S	12	5
Positive control	637	360	340	298

 

Revertant numbers in the presence of 10% metabolic activation (hamster liver)

	Strain
Concentration (mcg/plate)	TA98	TA100	TA1535	TA1537
0 (water)	25	117	7	8
100	31	114	7	5
333	37	115	10	3
1000	28	107	7	7
3333	27	127	15	6
10000	0 S	81 S	17	5
Positive control	579	1207	460	341

 

Revertant numbers in the presence of 10% metabolic activation (rat liver)

	Strain
Concentration (mcg/plate)	TA98	TA100	TA1535	TA1537
0 (water)	24	115	8	5
100	28	107	12	10
333	22	115	9	4
1000	25	109	7	5
3333	17	106	12	4
10000	22	107	6	5
Positive control	131	449	200	129

Conclusions:

It was concluded that the test material was not mutagenic under the conditions of testing.

Executive summary:

An Ames testr was conducted using pre-incubation methodology. N,N,N'N'-Tetramethylethylenediamine was tested at concentrations of 100, 333, 1000, 3333 and 10000 mcg/plate in the absence of metabolic activation, in the presence of 10% Aroclor-1254 induced rat liver S-9 and in the presence of 10% Aroclor-1254 induced hamster liver S-9. Testing was conducted in strains TA98, TA100, TA1535 and TA1537. There was some evidence of toxicity at the highest concentration tested in some of the tester strains. There was no evidence of increases in the number of revertants for any of the strains in either the absence or in the presence of metabolic activation (rat or hamster).

Reference 2

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.

Reference 3

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 ± 5.0^a
Positive control	67 ± 10	40.5 ± 0.7^a

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 ± 0.0^a
Positive control	63 ± 1	35.5 ± 3.5^a

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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 August - 10 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

ATMEDAHP (N,N,N′,N′-Tetramethylethylenediamine)
CAS 110-18-9
Batch 17F-1069978
Purity >99.4%
Expiry June 2018

Target gene:

Thymidine kinase (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Clone -3.7.2C
Doubling time 10-12h
Cloning efficiency >50%
Near diploid (40+/-2) karyotype

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Male Wistar rat liver S9 fraction (phenobarbital / beta-naphthoflavone induced)

Test concentrations with justification for top dose:

The highest concentration tested was 10 mM (the limit concentration), in the absence of limiting solubility or toxicity.

Vehicle / solvent:

RPMI culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

A preliminary study was used to assess the toxicity of the test material. No precipitation of the test material was observed.

Based on the results of this assay, test material concentrations of up to 10 mM (the limit concentration for this study) were used in the main assay, both in the absence and presence of metabolic activation. For the main assay, approximately 1x10e7 cells were suspended in culture medium and exposed to concentrations of the test material for 4 hours in the absence or presence of exogenous metabolic activation (male Wistar rat liver S9 fraction; phenobarbital / beta-naphthoflavone induced). Following the exposure period cells were washed, resuspended in medium and cultured for a further 2 days (expression period). After the expression period, the cloning efficiency was assessed by seeding cells from each culture in 96-well plates and culturing for 6 days. Cultures were also seeded in selective medium containing TFT, and were scored following growth for approximately 12 days. At the end of the selection period, the mutant frequency was calculated for each culture.

Rationale for test conditions:

The concentrations used in the study were selected in line with the test guideline: four concentrations up to and including the limit of 10 mM were selected for each assay.

Evaluation criteria:

The calculated mutation frequencies for each culture were compared to the Global Evaluation Factor (GEF), which is defined as 126 for the microwell method.
The assay was considered to be acceptable if it met the following criteria:

At least three of the four 96-well plates were scorable

Cloning efficiency of the negative/solvent controls was in the range 65-120%

Spontaneous mutation frequency for the negative/solvent controls was in the range 50-170/10e6 cells

Cell number of the negative/solvent controls should increase 8-32 fold during the 2-day growth period

Positive controls (MMS and B(a)P) should produce an induced mutation frequency of at least 300/10e6 cells, with at least 40% of the positive colonies being small , or with a small colony mutant frequency of at least 150/10e6 cells. RTG should be >10%.

An assay is considered to be positive if the following criteria are met:

The induced mutant frequency meets or exceeds the GEF (126/10e6 cells)

A concentration-response relationship is apparent

An increased occurrence of small colonies (≥40% of total colonies) is considered to be indicative of clastogenic effects.

Biological relevance is considered first for interpretation of the results, statistical methods may also be used.

The assay is considered to be negative if the induced mutant frequency is less than the GEF and the trend is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data, using mutation frequencies for the negative/solvent controls as reference.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Summary of results

Exposure	-S9					+S9
	RCE (%)	RTG (%)	MF (/10e6 cells)	IMF (/10e6 cells)	+/-	RCE (%)	RTG (%)	MF (/10e6 cells)	IMF (/10e6 cells)	+/-
Untreated	100	100	63.8	---	-	100	100	74.6	---	-
0.5 mM	101.5	83.8	60.0	-3.8	-	98.3	106.0	70.6	-4.0	-
1 mM	106.9	66.3	58.4	-5.4	-	96.8	111.6	80.3	5.7	-
2 mM	105.1	39.8	54.2	-9.6	-	101.4	114.0	73.1	-15	-
4 mM	99.9	24.1	64.0	0.2	-	109.8	84.8	47.5	-27.1	-
6 mM	89.3	17.4	70.0	6.2	-	95.4	66.2	71.2	-3.4	-
8 mM	99.9	18.8	71.2	7.4	-	101.4	55.6	92.7	18.1	-
10 mM	101.5	18.6	47.7	-16.1	-	95.4	45.1	102.0	27.4	-
EMS	75.7	56.6	702.7	638.9	+	---	---	---	---	---
MMS	61.9	37.0	594.3	530.5	+	---	---	---	---	---
B(a)P	---	---	---	---	---	76.6	67.5	806.8	732.2	+

RCE: relative cloning efficiency

RTG: relative total growth

MF: mutation frequency

IMF: induced mutation frequency

+/-: assessment compared to GEF

Conclusions:

No evidence of mutation was seen for TMEDA under the conditions of this assay in the absence and presence of metabolic activation, at concentrations of up to 10 mM (limit concentration).

Executive summary:

The mutagenicity of ATMEDAHP (N,N,N′,N′-tetramethylethylenediamine) was investigated in a mouse lymphoma assay (L5178Y cells, tk locus) in the absence and presence of metabolic activation (male Wistar rat liver S9 fraction; phenobarbital / beta-naphthoflavone induced). Concentrations of the test material used were based on the results of a preliminary assay which did not reveal any limiting solubility. Cultures of approximately 10e7 cells were exposed to concentration of the test material (dissolved in culture medium) of up to 10 mM (limit concentration) for 4 hours, prior to an expression period of 2 days and a selection period of 12 days prior to calculation of the induced mutation frequency. There was no biologically or statistically significant increase in the induced mutation frequency at any concentration of the test material, either in the absence or presence of metabolic activation.  Appropriate responses to the negative control (culture medium) and positive controls (EMS, MMS, B(a)P) confirmed the sensitivity of the assay. The results of this study are therefore concluded to be negative for ATMEDAHP (N,N,N′,N′-tetramethylethylenediamine).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo mouse micronucleus studies are available for the submission substance ATMEDAHP (N,N,N′,N′-tetramethylethylenediamine): both studies report negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 July to 08 September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Micronucleus

Specific details on test material used for the study:

ATMEDA
Batch/lot number: 378/5F-5904
Purity: 99.8%
Appearance: Clear colourless liquid
Storage conditions: Room temperature; protected from light
Expiry date: July 1996

Species:

mouse

Strain:

ICR

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., Frederick, MD
Age of mice at initiation of study: 6-8 weeks
Body weights at randomisation: 29.0-32.4 g (males, pilot study); 25.4-27.2 g (females, pilot study); 29.7-32.6 g (males, toxicity study); 22.3-25.3 g (females, toxicity study); 27.9-33.9 (males, micronucleus assay); 25.5-31.8 g (females, micronucleus assay)
Acclimatisation period: At least 5 days.
Temperature: 68-80 degrees F (approx. 20-26.7 degrees C)
Humidity: 30-70% (with the exception of a period of at least 4 hours where the limit of humidity was exceeded; the Study Director considered this to have no adverse impact on the study).
Photoperiod: 12 hour light/dark cycle
Housing: Plastic caging with filter tops; up to 5 animals of same sex/cage; hardwood chips for bedding
Food: Purina Certified Rodent Chow 5002; ad libitum
Water: Tap water; ad libitum

Route of administration:

intraperitoneal

Vehicle:

Distilled water

Details on exposure:

Prior to the main Micronucleus assay, a pilot test and toxicity assay were conducted. In the pilot test, male mice were dosed by intraperitoneal injection with 1, 10, 100 or 1000 mg/kg bw (two per group) and male and female mice were dosed with 5000 mg/kg bw (five animals per sex). In the toxicity study, male and female mice were dosed by intraperitoneal injection with 200, 400, 600 or 800 mg/kg bw (five animals per sex per group.

The mice were assigned to 13 experimental groups of five males and five females.
Three groups were tested with the vehicle control, low dose (67.5 mg/kg bw), mid dose (135 mg/kg bw) and high dose (270 mg/kg bw). The first group had bone marrow collected 24 hours following dosing, the second group 48 hours after dosing and the third group 72 hours after dosing. One group of positive control animals was dosed with cyclophosphamide at 60 mg/kg bw and the bone marrow was collected after 24 hours
An additional group was used as replacement animals and dosed with the highest test dose (270 mg/kg bw).

The test item was administered by intraperitoneal injection at a constant volume of 20 mL/kg body weight. The animals were weighed immediately prior to dosing and the dose volume was based on body weight. Mice were observed after administration for signs of clinical effects.

Duration of treatment / exposure:

24, 48 and 72 hours following exposure

Frequency of treatment:

Once

Post exposure period:

Animals sacrificed at end of 24, 48 or 72 hour exposure period.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Dose / conc.:

67.5 mg/kg bw/day (actual dose received)

Dose / conc.:

135 mg/kg bw/day (actual dose received)

Dose / conc.:

270 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide 60 mg/kg bw

Tissues and cell types examined:

Bone marrow was examined.
Polychromatic erythrocytes and normochromatic erythrocytes were examined.

Details of tissue and slide preparation:

At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by carbon dioxide asphyxiation. The femurs were exposed, cut above the knee and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL of foetal bovine serum. The bone marrow cells were pelleted by centrifugation at 100 g for 5 minutes and the supernatant removed leaving a small amount of serum and the cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a lean glass slide. Two to four slides were prepared for each mouse, The slides were fixed in methanol and stained with May-Gruenwald Giemsa and permanently mounted.

Evaluation criteria:

Micronuclei were defined as round darkly stained nuclear fragments having a sharp contour with diameters usually 1/20 to 1/5 of the erythrocyte. The incidence of micronucleated polychromatic erythrocytes were determined for each treatment group.
The proportion of polychromatic erythrocytes to total erythrocytes are reported for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Statistics:

Statistical significance was determined using Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality, clinical effects and reductions in ration of PCE to total erythrocytes.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

One male and one female receiving 270 mg/kg bw were found dead on Day 2 following dose administration and were replaced at the time of bone marrow collection with animals from the replacement group which had also been dosed with 270 mg/kg bw. Mortality was observed in 1/20 males and 1/20 females. Clinical signs following dose administration included lethargy in male and female mice at 135 and 270 mg/kg bw. All other mice treated with test and control articles appeared normal during the study. Reductions up to 26% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article treated groups relative to the respective vehicle controls. A moderate reduction (38%) was observed in the female dose group 48 hours after treatment with 270 mg/kg bw, suggesting that there was bioavailability of the test article to the bone marrow target tissue. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test article treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (p>0.05, Kastenbaum-Bowman Tables). Cyclophosphamide induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman Tables).

All ten Sprague-Dawley rats receiving a single, one hour whole-body exposure to 4649 ppm of Balub 600 as a vapour, survived the exposure and 14-day post-exposure observation period. Signs of treatment included respiratory, secretory and ocular irritation during the exposure. While abatement of the former two responses was seen during the subsequent observation period, the ocular lesions were apparently irreversible. A transient, adverse effect upon body weigh was also produced by treatment. Gross post-mortem observations confirmed the ocular lesions, but were otherwise unremarkable.

Results of bone marrow collected 24 hours following dose administration

Treatment	Sex	No. of mice	PCE/Total erythrocytes	No. of micronucleated PCEs per 1000 PCEs (mean ± SD)	No. of micronucleated PCEs per PCEs scored
Water (20 ml/kg)	M	5	0.58	1.0±0.71	5/5000
ATMEDA (67 mg/kg bw)	M	5	0.58	1.2±1.1	6/5000
ATMEDA (135 mg/kg bw)	M	5	0.50	0.4±0.55	2/5000
ATMEDA (270 mg/kg bw)	M	5	0.51	0.6±0.89	3/5000
Cyclophosphamide (60 mg/kg)	M	5	0.52	75±22.57	375/5000
Water (20 ml/kg)	F	5	0.53	0.4±0.89	2/5000
ATMEDA (67 mg/kg bw)	F	5	0.53	0.2±0.45	1/5000
ATMEDA (135 mg/kg bw)	F	5	0.52	0.4±0.55	2/5000
ATMEDA (270 mg/kg bw)	F	5	0.53	0.5±0.55	2/5000
Cyclophosphamide (60 mg/kg)	F	5	0.52	33.4±12.88	167/5000

  

Results of bone marrow collected 48 hours following dose administration

Treatment	Sex	No. of mice	PCE/Total erythrocytes	No. of micronucleated PCEs per 1000 PCEs (mean ± SD)	No. of micronucleated PCEs per PCEs scored
Water (20 ml/kg)	M	5	0.65	2.2±2.28	11/5000
ATMEDA (67 mg/kg bw)	M	5	0.66	1.2±1.10	6/5000
ATMEDA (135 mg/kg bw)	M	5	0.64	1.2±1.30	6/5000
ATMEDA (270 mg/kg bw)	M	5	0.48	1.4±1.34	7/5000
Water (20 ml/kg)	F	5	0.61	0.6±0.89	3/5000
ATMEDA (67 mg/kg bw)	F	5	0.60	0.8±1.10	4/5000
ATMEDA (135 mg/kg bw)	F	5	0.54	0.6±0.55	3/5000
ATMEDA (270 mg/kg bw)	F	5	0.38	0.8±1.30	4/5000

 

Results of bone marrow collected 72 hours following dose administration

Treatment	Sex	No. of mice	PCE/Total erythrocytes	No. of micronucleated PCEs per 1000 PCEs (mean ± SD)	No. of micronucleated PCEs per PCEs scored
Water (20 ml/kg)	M	5	0.61	1.8±1.64	9/5000
ATMEDA (67 mg/kg bw)	M	5	0.66	1.4±0.89	7/5000
ATMEDA (135 mg/kg bw)	M	5	0.58	1.2±0.84	6/5000
ATMEDA (270 mg/kg bw)	M	5	0.58	1.2±0.45	6/5000
Water (20 ml/kg)	F	5	0.57	1.4±0.55	7/5000
ATMEDA (67 mg/kg bw)	F	5	0.56	0.6±0.89	3/5000
ATMEDA (135 mg/kg bw)	F	5	0.54	1.0±01.00	5/5000
ATMEDA (270 mg/kg bw)	F	5	0.62	0.6±0.55	3/5000

 

Conclusions:

All criteria for a valid test were met. Under the conditions of the assay, ATMEDA did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using both male and females ICR mice.

Executive summary:

An in vivo micronucleus study was conducted with ICR mice at concentrations of ATMEDA of 0, 67.5, 135 or 270 mg/kg bw. Sampling was conducted at 24, 48 and 72 hours post dose 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 increases in the number of micronucleated polychromatic erythrocytes for any of the ATMEDA treated animals when compared to the concurrent vehicle control treatments. The positive control treated animals showed a statistically significant increase in the number of micronucleated polychromatic erythrocytes. The overall conclusion was that ATMEDA had shown no evidence of cytogenicity under the conditions of this study.

Reference 2

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

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 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

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:

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

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

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

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.

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.

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 ± 4.8^a	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 ± 6.8^a	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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The positive response reported for clastogenicity in vitro was observed only at concentrations exceeding the limit concentration specified in teh current OECD guideline and is not considered to be of toxicological significance. Two in vivo mouse micronucleus assays report negative responses.

Justification for classification or non-classification

Based on the weight of evidence from in vitro and in vivo studies, the submission substance ATMEDAHP (N,N,N′,N′-tetramethylethylenediamine) is not classified for germ cell mutagenicity according to CLP criteria. A positive response reported for clastogenicity in vitro was observed only at concentrations exceeding the limit concentration specified in teh current OECD guideline and is not considered to be of toxicological significance. Two in vivo mouse micronucleus assays report negative responses.