Hexamethylenediamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: United States FDA Good Laboratory Practices regulations (21 CFR 58), the study is comparable to OECD 474. The purity is not indicated and analytical certificate is not available.

Justification for type of information:

- please see the read-across justification document attached in section 13

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:

other: Toxicity report Series of the National Toxicology Program

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

At the end of the 13-week inhalation study, smears were prepared from peripheral blood samples obtained by cardiac puncture of all exposed and control mice.
The slides were stained with Hoechst 33258/pyronin Y (Mac Gregor et al., 1983). Ten thousand normochromatic erythrocyres and 2000 polychromatic erythrocytes from each animal were scored for micronuclei.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylenediammonium dichloride

EC Number:

227-977-8

EC Name:

Hexamethylenediammonium dichloride

Cas Number:

6055-52-3

IUPAC Name:

hexane-1,6-diamine dihydrochloride

Constituent 2

Reference substance name:

1,6 Hexanediamine Dihydrochloride

IUPAC Name:

1,6 Hexanediamine Dihydrochloride

Details on test material:

- Name of test material (as cited in study report): 1,6-hexanediamine (HDA)
- Molecular formula (if other than submission substance): C6H16 2HCl
- Molecular weight (if other than submission substance): 185.2
- Smiles notation (if other than submission substance): no data
- InChl (if other than submission substance): no data
- Structural formula attached as image file (if other than submission substance): no data
- Substance type: no data
- Physical state: no data
- Analytical purity: 70.9 % (purchased as a 70% aqueous solution)
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 70% aqueous solution
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: PT-031985
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in amber or foil-wrapped bottles
- Other: periodic chemical reanalyses at 4-month intervals indicated no breakdown of the chemical during storage

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 to 7 weeks of age
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Hazelton H-2000 stainless steel and glass exposure chambers (Hazelton Systems, Inc., Aberdeen, MD) of 2 m^3 volume
- Diet: Pelleted NIH-07 feed (Zeigler Brothers, Inc., Gardners, PA) ad libitum during the nonexposure periods
- Water: water ad libitum during the nonexposure period
- Acclimation period: 11-14 days


ENVIRONMENTAL CONDITIONS
- Temperature: at all times 72° ± 3°F; except during exposure 72° to 78°F
- Humidity: at all times 50% ± 15%; except during exposure 70% to 80%
- Air changes: at all times 12-15 per hour; except during exposure 15 per hour (500 L/min)
- Photoperiod: 12 hours of subdued fluorescent light per day


IN-LIFE DATES: From: June To: September 1987

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

For the inhalation studies, 1,6-hexanediamine was converted to 1,6-hexanediamine dihydrochloride (HDDC) by acidification with concentrated hydrochloric acid under a stream of nitrogen. The final pH was adjusted within the range of 4.5 to 5.5 before storage and again before use in the inhalation chambers.

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: no data
- Method of holding animals in test chamber: housed continuously in exposure chambers with chamber doors closed except during animal husbandry procedures.
- Source and rate of air: no data
- Method of conditioning air: no data

- System of generating particulates/aerosols: the 70% aqueous HDDC solution was placed in a 9-liter glass reservoir and pressurized with N2 gas. HDDC was delivered to 5 Sonimist Ultrasonic Spray Nozzles (Model HS600-2, Heat Systems-Ultrasonics, Inc., Farmingdale, NY) by a positive displacement metering pump. Up this point, stainless steel lines carried the test substance. The nebulizer reservoir was kept in a separate exposure chamber (H-1000, Hazelton Systems, Inc., Aberdeen, MD). This chamber served as a mixing plenum where large droplets and nonnebulized liquid were impacted or sedimented out of the test atmosphere before the aerosol was delivered to the inhalation chambers. The HDDC aerosol was mixed with compressed breathing air that had been filtered through an ENMET (ENMET Air Filtration Panel, Model AFP-82, Enmet Co., Ann Arbor, MI) and supplied at 50 psi to generate an aerosol at a concentration equal to the highest exposure concentration. The resulting aerosol was transported to the inhalation chambers through a manifold constructed of 3-o,ch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount HEPA/charcoal-filtered room air to obtain the desired test concentratino, then delivered to the inhalation chamber.

- Temperature, humidity, pressure in air chamber: 21 to 27°C, 70% to 80% relative humidity
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data


TEST ATMOSPHERE
- Brief description of analytical method used: no data
- Samples taken from breathing zone: Gravimetric sampling was conducted with 25 mm glass fiber filter paper (Gelman Sciences, Inc., Ann arbor, MI). Gravimetric analysis was performed on a Perkin Elmer AS-2Zmicrobalance (Perkin Elmer, Norwalk, CT) by weighing filters to the nearest 0.01mg before and after sampling and again after storing the filters in a desiccator overnight.


VEHICLE (if applicable)
- Justification for use and choice of vehicle: no data
- Composition of vehicle: no data
- Type and concentration of dispersant aid (if powder): no data
- Concentration of test material in vehicle: no data
- Lot/batch no. of vehicle (if required): no data
- Purity of vehicle: no data

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours plus T90 (30 minutes) per day, 5 days per week for 13 weeks

Post exposure period:

No

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.6, 5, 16, 50, 160 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10 animals/sex/species/exposure group

Control animals:

yes

Positive control(s):

none

Examinations

Tissues and cell types examined:

Peripheral blood

Details of tissue and slide preparation:

At the end of the 13-week inhalation study, smears were prepared from peripheral blood samples obtained by cardiac puncture of all exposed and control mice. The slides were stained with Hoechst 33258/pyronin Y (MacGregor et al., 1983). Ten thousand normochromatic erythrocytes and 2000 polychromatic erythrocytes from each animal were scored for micronuclei.

Evaluation criteria:

Frequency of Micronuclei in the normochromatic erythrocytes and the polychromatic erythrocytes.

Statistics:

Log transformation of the normochromatic erythrocyte (NCE) data, and testing for normality by the Shapiro-Wilk test and for heterogeneity of variance by Cochran's test were performed before statistical analyses. The frequency of micronucleated cells among NCEs was analyzed by analysis of variance using the SAS GLM procedure. The NCE data for each dose group were compared with the concurrent solvent control using Student's t-test. The frequency of micronucleated cells among polychromatic erythrocytes (PCEs) was analyzed by the Cochran-Armitage trend test, and individual dose groups were compared to the concurrent solvent control by Kastenbaum- Bowman's (1970) binomial test. The percentage of PCEs among total erythrocytes was analyzed by an analysis of variance on ranks (classed by sex) and individual dose groups were compared with the concurrent solvent control using a t-test on ranks.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

No significant increases were seen in the frequencies of micronucleated normochromatic erythrocytes (NCEs) or polychromatic erythrocytes (PCEs) in male or female mice. The percentage of PCEs among the total erythrocyte population was increased at the highest exposure levels for male and female mice.

Any other information on results incl. tables

Table 1: Frequency of micronuclei in mouse peripheral blood erythrocytes following inhalation of 1,6 -Hexanediamine Dihydrochloride

	Micronucleated Cells/1000 Cells^1
	Treatment (mg/m^3)	PCEs	NCEs	PCEs (%)
Males	0	1.74 ± 0.42	1.93 ± 0.15	2.07 ± 0.21
	16	1.61 ± 0.25	1.82 ± 0.15	2.10 ± 0.18
	50	2.70 ± 0.47	2.19 ± 0.15	4.31 ± 1.09**
	160	2.26 ± 0.52	1.79 ± 0.12	2.59 ± 0.31*
		P=0.180^2	P=0.972	P=0.016
Females	0	1.90 ± 0.42	1.38 ± 0.13	1.83 ± 0.11
	16	1.17 ± 0.43	1.35 ± 0.19	2.07 ± 0.12
	50	0.93 ± 0.27	1.14 ± 0.07	1.79 ± 0.06
	160	2.31 ± 0.44	1.21 ± 0.11	2.76 ± 0.17**
		P=0.052	P=0.218	P<0.001

¹Values presented as mean ± standard error of the treatment group. PCE=polychromatic erythrocytes, NCE=normochromatic erythrocytes

²Cochran-Armitage trend test for PCEs, analysis of variance using the SAS GLM procedure for NCEs, and analysis of variance on ranks for %PCE.

* P¿0.05;t-tests on ranks for %PCE.

** P¿0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, 1,6-hexanediamine dihydrochloride was negative in this micronucleus assay.

Executive summary:

A 13-week study of the toxicity of the dihydrochloride salt of hexane 1,6-diamine (HDDC) was conducted in B6C3F₁ mice (both sexes) exposed by whole body inhalation in compliance with US-FDA GLP. Animals were exposed to HDDC at concentrations of 0, 1.6, 5, 16, 50 and 160 mg HDDC/m³ for 6 hours/day, 5 days/week.

At the end of the 13-week inhalation study, smears were prepared from peripheral blood samples obtained by cardiac puncture of all exposed and control mice. The slides were stained with Hoechst 33258/pyronin Y. Ten thousand normochromatic erythrocytes (NCEs) and 2000 polychromatic erythrocytes (PCEs) from each animal were scored for micronuclei.

A statistically significant increase in the percent of PCEs in the total erythrocytes population was observed for males inhaling HDDC for 13 -weeks at 50 mg/m3 and higher (not dose-related) and females exposed at 160 mg/m3, without a statiscally significant increase in the frequency of micronucleated normochromatic and polychromatic erythrocytes. Under the test conditions, HMD didn't induce micronucleated erythrocytes in rats exposed by whole-body inhalation for 90 days. This study is considered as acceptable and used as a key study for in vivo cytogenicity (Mammalian Erythrocyte Micronucleus Test).