Carbamic acid ester

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:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study similar to the guideline under GLP.

Data source

Reference

Reference Type:

other: study report in the public domain

Title:

Unnamed

Year:

1993

Report date:

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

Remarks:

stated to be performed under GLP, no study director or QA statement included

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

The test material is an anlogue of the expected metabolite of the substance

Identity: Hexane diamine (identified by infrared spectroscopy)
Lot: PT-031985
Purity: 70.9% (purchased as a 70% aqueous solutionfrom E. I. DuPont de Nemours and Company, Inc. (Wilmington, DE))
Stability: stable for 4 months
Storage: at room temperature in amber or foil-wrapped bottles

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: 6-7 weeks
- Average weight at study initiation: males 20.6-24.8 g; females 17.9-19.8 g
- Housing: individually
- Diet: Pelleted NIH-07 feed (Zeigler Brothers, Inc., Gardners, PA) ad libitum during non-exposure
- Water: ad libitum during non-exposure
- Acclimation period: 11-14 days

ENVIRONMENTAL CONDITIONS (non-exposure)
- Temperature (°C): ca 22 °C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

none

Details on exposure:

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.
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 HS6002, Heat Systems-Ultrasonics, Inc., Farmingdale, NY) by a positive displacement metering pump. Up to 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-inch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount of HEPA/charcoal-filtered room air to obtain the desired test concentration, then delivered to the inhalation chamber. After exiting the chambers, the test atmospheres were delivered to a common duct and cleansed of the test substance by a Mystaire HS-7CM scrubber (Heat Systems Ultrasonics).


Method of holding animals in test chamber: individually in compartments of multi compartment wire mesh cages, during exposure in stainless steel andglass exposure chambers of 2 m3 volume, with 15 air changes per hour (500 L/min). During inhalation exposures, chambers were maintained at 22°C to 25°C and 70% to 80% relative humidity.

TEST ATMOSPHERE
- Brief description of analytical method used: forward light scatter with RAM-S real-time aerosol monitors (GCA Corporation,Technology Division, Bedford, MA) and gravimetric analyses of filter samples collected from each exposure chamber
- Samples taken from breathing zone: no, 6 RAM-S readings and 3 gravimetric samples were taken from each exposure chamber on each day of exposure.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 16, 30 and 150 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10/sex/concentration

Control animals:

yes

Positive control(s):

none
The blood is derived from animals that were included in a 13 week toxicity study.

Examinations

Tissues and cell types examined:

peripheral blood samples obtained by cardiac puncture and slides are scored for immature erythrocytes (polychromatic or PCE) that are less than 48 hr old, and mature erythrocytes 2-35 days old (normochromatic or NCE)

Details of tissue and slide preparation:

slides were stained with Hoechst 33258/pyronin Y (MacGregor et al., 1983)

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.
Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the slide-based micronucleus data, the micronucleus frequency in any dose group is considered significantly elevated over the control group if the P value is equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same data.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

percentage PCE of erythrocytes significantly increased at 50 (males only) and 160 mg/m3

Vehicle controls validity:

not examined

Additional information on results:

No significant increase in the number of PCEs or NCEs was reported at any of the concentrations examined.

Any other information on results incl. tables

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

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

² Cochran-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
In an in vivo micronucleus test the substance did not induce a significant increase of micronucleated erythrocytes. Therefore the substance is considered to be non-clastogenic.

Executive summary:

Mice (10/sex/treatment) were exposed to 0, 16, 50 and 150 mg/m3 during 13 weeks. At termination bloodsmears were evaluated for the presence of micronucleated polychromatic and normochromatic erythrocytes. No significant increase of micronucleated erythrocytes was observed. Therefore the substance is considered to be non-clastogenic.