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Toxicological information

Genetic toxicity: in vivo

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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 based on grouping of substances (category approach)
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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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 Cells1

 

Treatment (mg/m3)

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=0.1802

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 (in vivo cytogenicity study: Mammalian Erythrocyte Micronucleus Test).