Hexamethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1980-08-11 to 1980-10-29 and from 1980-10-31 to 1980-12-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The highest concentration tested showed no sign of cytotoxicity: higher concentrations could have been tested. This study does not correspond to any international guideline and was not conducted under GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of these studies was to employ the BALB/3T3 Clone A31 mouse cell line in the presence and in the absence of an exogenous metabolic activation system in order to investigate the in vitro morphological transforming potential of Hexamethylene Diamine.
The 3T3 cells were selected for their good cloning efficiency, responsiveness to post confluence inhibition of cell replication, sensitivity to chemical mutagens/carcinogens, and morphology in response to chemical carcinogens.
These in vitro mammalian cell transformation tests were performed according to modified methods of Schechtman and Kouri. Target cells were treated in suspension for 2 hours at 36 ± 2°C with different concentrations of test article as well as positive and negative controls in the presence of exogenously supplied mammalian metabolizing activity, after which they are plated for analysis of the cytotoxic effects of treatment and the induction of phenotypic transformation.
The transforming potential of the test article was determined by its ability to induce a significant increase in the number of morphologically transformed foci when compared to the negative control.

GLP compliance:

no

Remarks:

Study performed before GLP establishment

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylenediamine

EC Number:

204-679-6

EC Name:

Hexamethylenediamine

Cas Number:

124-09-4

Molecular formula:

C6H16N2

IUPAC Name:

hexane-1,6-diamine

Details on test material:

- Name of test material (as cited in study report): Hexamethylene Diamine, T1623
- Molecular weight (if other than submission substance): 116.21
- Substance type: no data
- Physical state: Supplied as a colorless or light yellow vitrous solid. At the time of testing, the test article appeared as a colorless solid.
- Analytical purity: 99.5%
- Impurities (identity and concentrations): Known : Bis-hexamethyentriamin (0.2%), diaminocyclohexan (0.04%), water (0.10%), ammonia (0.15%), imine/ACN (traces), azacycloheptan, ¿-aminocapronitril.
- Lot/batch No.: 79/519
- Expiration date of the lot/batch: no data
- Stability under test conditions: indicated as "yellowing with air/heat, formation of carbonate if exposed to air".
- Storage condition of test material: in the dark at room temperature
- Other: Melting point of 40.9°C
Boiling point of 105°C/40mbar
Flash point of 87.5°C

Method

Target gene:

Genes which can increase significantly the number of morphologically transformed loci when compared to the negative control.

Species / strain

Species / strain / cell type:

mammalian cell line, other: BALB/3T3 Clone A31 mouse embryo cells

Details on mammalian cell type (if applicable):

- Type and identity of media: complete medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

See bellow table 7.6.1/1

Vehicle / solvent:

No vehicle

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 12.5 µg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine, 0.5 µg/mL

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension and on plate

DURATION
- Preincubation period: no data
- Exposure duration: without metabolic activation: 20-24 h; with metabolic activation: 2 h
- Expression time (cells in growth medium): approximately 7-10 days after initiation of the experiment for the cytotoxicity assay
approximatively 4-6 weeks after initiation of the experiment for the phenotypic transformation assay

NUMBER OF REPLICATIONS: triplicate per condition for the cytotoxicity assay; 12-15 dishes per condition for the phenotypic transformation assay

NUMBER OF CELLS EVALUATED:
no data

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
no data

OTHER: no data

Evaluation criteria:

The relative plating efficiency of the negative control must be ¿ 15%. The relative survival of cells exposed to the test substance must be ¿ 40% for at least one dose. The number of Type III foci in the negative control must not exceed 1 focus per total replicate dishes. The positive control must induce a significant (P¿0.05) number of Type III foci relative to the negative control.
The cytotoxic effects of the test article and positive control were compared to that of the negative control. Cell survival of the positive control and test article treatment groups expressed as relative colony-forming efficiency (RCE), were reported relative (normalized) to 100% survival in the negative control.
The number of cells at risk (#CAR) for each treatment group was calculated from the RPE and the number of cells seeded in the transformation assay dishes for all replicates per condition.

Statistics:

The transformation potential of each of the treatment groups was compared to the negative control by a special application of the Poisson distribution.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: BALB/3T3 Clone A31 mouse embryo cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

other: No vehicle tested in this study

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No additional data

Remarks on result:

other: strain/cell type: BALB/3T3 Clone A31 mouse embryo cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Cytotoxic effects of T 1623 to BALB/3T3 Cl. A31 cells exposed in suspension in the presence of exogenous metabolic activation.

Treatment	Concentration	RPE^b(%)	RCE^c(%)
Negative control PBS	--	21.3	100.0
Positive Control BaP	12.5 µg/mL	14.0	65.6
Compound T1623	100 µg/mL 30 µg/mL 10 µg/mL	22.4 20.5 21.3	105.0 96.3 100.0

^a2-3 ×10⁶cells were incubated at 36 ± 2°C in the presence or absence of test article and S9 + cofactors in suspension for 2 hours. Following treatment, cells were washed, seeded at 250 cells/dish, and incubated in complete growth medium for 7-10 days. Cells were fixed, stained with giemsa, and scored for colony formation.

^bRPE = relative plating efficiency, i.e. the number of colonies forming relative to the number of cells seeded per condition.

^cRCE = relative colony-forming efficiency, i.e. the number of surviving colonies per condition relative (normalized) to 100% survival in the negative control.

 

Table 7.6.1/3: Cytotoxic effects of T 1623 to BALB/3T3 Cl. A31 cells exposed in suspension in the absence of exogenous metabolic activation.

Treatment	Concentration	RPE^b(%)	RCE^c(%)
Negative control PBS	--	51.7	100.0
Positive Control MNNG	0.5 µg/mL	7.9	15.2
Compound T1623	100 µg/mL 30 µg/mL 10 µg/mL	34.0 42.0 46.7	65.7 81.2 90.2

Table 7.6.1/4: Transformation potential of T 1623 to BALB/3T3 Cl. A31 cells exposed in suspension in the presence of exogenous metabolic activation.

Treatment	Concentration	# CAR^b (×10^3)	# Type II Foci	# Type III  Foci	TF^c(×10^4)
Negative control PBS	--	23.43	0	1	0.43
Positive Control BaP	12.5 µg/mL	19.60	1	6	3.06
Compound T1623	100 µg/mL 30 µg/mL 10 µg/mL	17.92 30.75 31.95	0 0 0	1 1 2	0.56 0.33 0.63

^a2-3 ×10⁶cells were incubated at 36 ± 2°C in the presence or absence of test article and S9 + cofactors in suspension for 2 hours. Following treatment, cells were washed, seeded at 250 cells/dish, and incubated in complete growth medium for 7-10 days. Cells were fixed, stained with giemsa, and scored for colony formation.

^b#CAR = number of cells at risk for each treatment group.

^cTF = transformation frequency for the number of Type III foci per number of cells at risk.

Table 7.6.1/5: Transformation potential of T 1623 to BALB/3T3 Cl. A31 cells exposed in suspension in the absence of exogenous metabolic activation.

Treatment	Concentration	# CAR^b (×10^3)	# Type II Foci	# Type III  Foci	TF^c(×10^4)
Negative control PBS	--	77.5	1	0	<0.13
Positive Control MNNG	0.5 µg/mL	11.06	13	18	16.27
Compound T1623	100 µg/mL 30 µg/mL 10 µg/mL	47.60 63.00 60.71	1 0 1	1 2 6	0.21 0.32 0.99

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative for transforming activity

Under the conditions employed in the assay, the data suggest that notable transforming activity was not observed for the test article with and without metabolic activation; statistically significant (P ¿ 0.05) induction of focus transformation by the test article was not detectable.

Executive summary:

The purpose of this study was to investigate the in vitro morphological transforming potential of Hexamethylene Diamine (HMD) by using the BALB/3T3 Clone A31 mouse cell line in the presence of an exogenous metabolic activation system. This in vitro mammalian cell transformation test was performed according to modified methods of Schechtman and Kouri.

Target cells were treated in suspension for 2 hours with exogenously supplied mammalian metabolizing activity (S9) and for 20-24 hours without metabolism activation. Test article was tested at 10, 30 and 100 µg/mL (based upon a preliminary dose range-finding test) as well as positive and vehicle controls. The transforming potential of the test substance was determined by its ability to induce a significant increase in the number of morphologically transformed foci when compared to the negative control, 4-6 weeks after exposure to test substance.

No cytotoxicity was observed (up to 100 µg/mL) in the absence of metabolic activation whereas cytotoxicity, as indicated by a decrease up to 34% at 100 µg/mL of the colony-forming efficiency (RCE) of 3T3 cells, was observed without metabolic activation. Positive controls BaP and MNNG reduced the colony-forming efficiency of 3T3 cells and increased the number of type III foci

Negative and positive controls fulfilled the requirement for determination of a valid test. Under the conditions of the assay, any transforming activity was not observed for HMD in the absence and the presence of activation system. This study is considered as acceptable for in vitro mutagenicity endpoint.