Cyclohex-1,2-ylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

5 October 1984 - 22 February 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, the study is comparable to OECD 476. The purity is not indicated and analytical certificate is not available.

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 Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

mammalian cell gene mutation 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
- Physical state: clear liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Lot/batch No.: no data
- Stability under test conditions: no data
- Storage condition of test material: in the plastic container received from the sponsor, maintained in frozen aliquots in a Revco Ultra-low Freezer.

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
serum free medium F12 (for wash)
F12FCM5 (for plate)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

At dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation.
At doses levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.
See below Table 7.6.1/1

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water supplied by Pharmakon Research International.
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate (EMS) and Dimethylnitrosamine (DMN)

Remarks:

Positive controls: EMS was used at 200 µg/mL without S9 activation and DMN was used at 100 µg/mL with S9 activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in suspension for preliminary cytotoxicity and mutagenicity screen
- in agar (plate incorporation) for the main test


DURATION
- Preincubation period: No
- Exposure duration: 5 h
- Selection time (if incubation with a selection agent): 7 days


SELECTION AGENT (mutation assays): hypoxanthine


NUMBER OF REPLICATIONS:
Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

- The test substance was tested to the level of at least 10% survival or to a level deemed acceptable by the sponsor.
- At least three of the five test substance concentrations should produce toxicity ranging from 10 to 100% survival in the cytotoxicity assay.
- ideally, at least one concentration should induce toxicity of less than 50% survival in the assay.
- The plating efficiency of the solvent control was at least 50%.
- The spontaneous mutation frequency of the solvent control was within 2 standard deviations of the historical mean frequency for Pharmakon Research International, Inc.

Statistics:

Dose-response analyses were performed on transformed mutation frequency data by the one-way analysis of variance method outlined by Snee and Irr (1981). Dose-response was considered significant if p < 0.01.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

No additional data

Remarks on result:

other: strain/cell type: CHO-K1-BH4

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary cytotoxicity Screen

		Relative Cell Survival (%)
		S.9 concentrations
Test article	µg/mL	0%	1%	2%	5%	10%
Untreated	-	100^a	77.6	103.6	99.5	102.0
HMD	3	105.0	144.1	130.2	96.1	100.3
HMD	9	109.9	116.2	128.8	123.6	107.9
HMD	30	117.6	112.1	99.7	101.6	96.1
HMD	91	98.0	108.3	117.6	109.1	103.5
HMD	304	53.2	115.3	109.8	97.6	89.0
HMD	912	0	0	0	0	0
HMD	3039	0	0	0	0	0
HMD	9116	0	0	0	0	0

^aAbsolute cloning efficiency = 63.5 %

Table 7.6.1/3: Preliminary mutagenicity Screen

Compound	µg/mL	S.9 (±)	Relative^aInitial Survival (%)	Total No. of Mutants (5 plates)	Cloning Efficiency (%)	Mutant Frequency (Mutants/10^6clonables cells)	Mean Mutation Frequency
	1% S.9 Concentration
Untreated	- -	- -	99.6 100.4	13 8	108.3 97.5	12.0 8.2	10.1
	- -	+ 1% + 1%	102.9 98.9	4 9	96.0 86.8	4.2 10.4	7.3
HMD	100 100	+ 1% + 1%	85.2 86.8	6 2	63.8 96.0	7.2 2.1	4.6
	400 400	+ 1% + 1%	2.4 3.1	4 11	91.5 83.0	4.4 13.3	8.9
	600 600	+ 1% + 1%	- -	- -	- -	- -	-
	2% S.9 Concentration
Untreated	- -	- -	99.6 100.4	13 8	108.3 9705	12.0 8.2	10.1
	- -	+ 2% + 2%	88.5 102.9	12 5	92.8 95.2	12.9 5.3	9.1
HMD	100 100	+ 2% + 2%	91.2 94.9	6 10	87.7 96.8	6.8 10.3	8.6
	400 400	+ 2% + 2%	55.4 53.2	4 3	96.3 85.8	4.2 3.5	3.9
	600 600	+ 2% + 2%	35.7 25.8	5 2	111.5 87.7	4.5 2.3	3.4
	5% S.9 Concentration
Untreated	- -	- -	99.6 100.4	13 8	108.3 97.5	12.0 8.2	10.1
	- -	+ 5% + 5%	97.7 91.0	9 5	99.5 92.2	9.0 5.4	7.2
HMD	100 100	+ 5% + 5%	85.5 86.0	5 10	86.7 105.7	5.8 9.5	7.7
	400 400	+ 5% + 5%	66.1 52.6	13 4	99.5 98.0	13.1 4.1	8.6
	600 600	+ 5% + 5%	23.8 27.6	9 3	98.5 80.5	9.1 3.7	6.5
	10% S.9 Concentration
Untreated	- -		99.6 100.4	13 8	108.3 97.5	12.0 8.2	10.1
	- -	+ 10% + 10%	95.4 83.9	12 11	105.5 100.7	11.4 10.9	11.1
HMD	100 100	+ 10% + 10%	86.2 77.9	10 10	90.2 85.7	11.1 11.7	11.4
	400 400	+ 10% + 10%	68.9 62.6	7 8	81.8 92.5	8.6 8.6	8.6
	600 600	+ 10% + 10%	37.4 38.3	7 16	89.3 87.2	7.8 18.3	13.0
DMN	100 100	+ 10% + 10%	28.9 28.1	146 126	61.5 64.2	237.4 196.3	216.9
	200 200	- -	72.4 53.2	161 158	83.2 71.2	193.5 221.9	207.6
	0% S.9 Concentration
Untreated	- -	- -	98.4 101.6	5 8	92.5 97.0	5.4 8.2	6.8
HMD	50 50	- -	103.8 96.2	2 3	76.2 85.3	2.6 3.5	3.1
	100 100	- -	60.7 64.8	8 2	75.3 89.5	10.6 2.2	6.4
	200 200	- -	51.5 39.5	4 3	76.8 95.3	5.2 3.1	4.1
	250 250	- -	28.4 22.7	11 22	87.0 78.5	12.6 28.0	20.3
EMS	200 200	- -	54.1 52.3	156 172	70.2 69.8	222.2 246.4	234.3

^aAbsolute cloning efficiency: -    Mean for 1-2-5-10% = 79.9

                                          -    Mean for 0% = 68.4

Table 7.6.1/4: Mutagenicity Data

Compound	µg/mL	S.9 (±)	Relative^aInitial Survival (%)	Total No. of Mutants (5 plates)	Cloning Efficiency (%)	Mutant Frequency (Mutants/10^6clonables cells)	Mean Mutation Frequency
	1% S.9 Concentration
HMD	25 25 25	- - -	109.4 105.6 99.5	4 5 7	90.2 94.3 86.0	4.4 5.3 8.1	6.0
	50 50 50	- - -	83.4 98.6 74.3	1 0 16	86.3 93.7 84.7	1.2 0^b 18.9	6.7
	100 100 100	- - -	66.0 62.4 47.4	4 3 2	84.7 86.0 79.2	4.7 3.5 2.5	3.6
	175 175 175	- - -	58.3 54.9 38.7	1 4 0	88.7 93.3 86.7	1.1 4.3 0^b	1.8
	250 250 250	- - -	35.5 15.5 39.6	6 4 3	93.5 83.8 74.8	6.4 4.8 4.0	5.0
HMD	50 50 50	+ 5% + 5% + 5%	81.1 82.1 98.9	9 3 14	84.8 87.7 105.8	10.6 3.4 13.2	9.0
	100 100 100	+ 5% + 5% + 5%	85.3 83.1 78.4	8 6 2	91.5 92.7 87.0	8.7 6.5 2.3	5.9
	300 300 300	+ 5% + 5% + 5%	76.7 69.3 73.5	3 5 7	100.5 108.3 94.5	3.0 4.6 7.4	5.0
	450 450 450	+ 5% + 5% + 5%	55.4 47.1 40.3	2 3 5	91.8 76.7 88.7	2.0 3.8 6.0	4.0
	600 600 600	+ 5% + 5% + 5%	36.1 40.6 30.5	1 15 12	91.8 76.7 86.7	1.1 19.6 13.5	11.4

^aAbsolute cloning efficiency: Mean = 93.5

^bNot scorable mutants.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay

Under the test conditions, Hexamethylene diamine was found not to induce mutations in this in vitro CHO/HGPRT mammalian cell forward gene mutation assay.

Executive summary:

The ability of Hexamethylene Diamine to induce mutations at the hypoxanthine-guanine phosphoribosyl transférase (hgprt) locus in Chinese hamster Owary (CHO) cells (clone K1 designated CHO-K1-BH4) was tested following directives of the OECD guideline 476 and in compliance with GLP.

A Preliminary Cytotoxicity Screen was performed to evaluate HMD for cytotoxicity in CHO at 8 dose levels with and without metabolic activation. Hexamethylene Diamine was then evaluated in a Preliminary Mutagenicity Screen at several dose levels, with and without metabolic activation. Based on the results of the Preliminary Mutagenicity, the test article mutagenicity was tested with and without a 5% concentration of metabolic activation preparation.

Hexamethylene Diamine was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation and at dose levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.

The positive control articles, EMS and DMN, indicate that the integrity of the test system was valid.

No statistically significant increases of mutants in HMD treated cultures were observed when compared to the negative controls. Results for Hexametylene Diamine were negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at the dose levels and S9 concentrations tested.

Under the test conditions, HMD didn't induce mutagenicity in mammalian cells in the absence and the presence of activation system.