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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 17 MAY 1990 to 1 DEC 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, comparable to current standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1993

Materials and methods

Principles of method if other than guideline:
standard subacute method
GLP compliance:
yes
Remarks:
according to EPA GLP Regulations (40 CFR 792)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohex-1,2-ylenediamine
EC Number:
211-776-7
EC Name:
Cyclohex-1,2-ylenediamine
Cas Number:
694-83-7
Molecular formula:
C6H14N2
IUPAC Name:
cyclohexane-1,2-diamine
Details on test material:
- Name of test material (as cited in study report): 1,2-Diaminocyclohexane, DCH
- Physical state: liquid
- Analytical purity: 93.3%
- Composition of test material, percentage of components:
93.3% DCH
5.87% 2-Methylpentamethylenediamine
0.22% Hexamethylenediamine
0.08% Water
- Stability under test conditions: test material was expected to be stable under the conditions of the study

Test animals

Species:
rat
Strain:
other: Crl:CD*BR
Sex:
male
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approximately ranging from 207 to 243 grams
- Housing: individually housed in stainless steel, wire mesh cages
- Diet: ground Purinae Certified Rodent Chow #5002, ad libiitum
- Water: tap water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C) targeted: 23 +/-2
- Humidity (%): targeted: 50 +/-10

IN-LIFE DATES: From: 8 JUN 1990 To: 6 JUL 1990

Administration / exposure

Route of administration:
other: inhalation of an aerosol/vapour mixture
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Aerosol size (MMDA): ranging from 2.6 to 5.8 micrometres
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 29 litre exposure chambers (made of glass)
- Method of holding animals in test chamber: restrainers (perforated stainless steel or polycarbonate cylinders with conical nose pieces, which were inserted into face plates to yield nose-only exposure conditions)
- System of generating particulates/aerosols: Atmospheres containing aerosol and vapour test material were generated by pumping the test material with a Harvard compact infusion pump to a glass J-tube containing 6 nm glass beads. A Goodburn Model T2 electronic torch heated the air entering the J-tube to about 60 to 70°C. The torch temperature was controlled by an Omega Model CS-6001-J temperature controller. The control chamber also had a torch at similar temperatures controlled by an Omega temperature controller. The aerosol/vapour mixture entered the chamber and was dispersed by a glass baffle to promote uniform distribution of the test material throughout the exposure chamber.
- Method of particle size determination: determined weekly for each exposure concentration with a Sierra Series 210 Cascade Impactor
- Treatment of exhaust air: Chamber atmospheres were exhausted through an emissions-abatement train consisting of a water scrubber (240 cg/ms chambers only), a cold trap, and an HSA cartridge filter prior to discharge into a fume hood.

TEST ATMOSPHERE
- Brief description of analytical method used:
The exposure chambers were monitored approximately hourly for the test material during the animal exposures. A minimum of 4 samples were
taken per exposure. For test material analysis, a measured volume of the chamber atmosphere was drawn through a glass-fiber filter in tandem with the methanol impinger. The filter trapped the test material aerosol particles, the methanol-filled impinger trapped the vapour.
The atmospheric concentration of test material aerosol was calculated from the pre- and post-sampling filter weights which were determined with a Cahn Model 26 Automatic Electrobalance. For the GC analysis, methanol trapped samples were analysed using a Hewlett-Packard Model 5890A GC equipped with a flame-ionization detector. Samples were chromatographed isothermally at 160°C on a 30 meter X 0.53 mm I.D. fused-silica column lined with polyphenylmethylsiloxane. Improved test material detection was obtained in the last 4 exposures using a Hewlett-Packard Model 5890A GC equipped with a nitrogenn-phosphorous detector. Samples were chromatographed isothermally at 160°C on a 10 meter X 0.53 mm I.D. fused-silica column lined with crosslinked 50% phenylmethylsilicone. Test material chamber concentrations were determined by comparing the GC response of the chamber samples with those of standard samples which were prepared by quantitatively diluting the test material in methanol.
Airborne particle size (reported as mass median aerodynamic diameter and percent of mass less than 10 micrometers aerodynamic diameter) was
determined weekly for each exposure concentration with a Sierra Series 210 Cascade Impactor. During each exposure, chamber temperatures were
measured continually with thermocouples during each exposure. Temperatures were averaged and recorded hourly by means of Grant 1200 Series Squirrel Meter/Logger Relative humidity was measured twice per exposure with a Belfort Instrument Co. Model 566 Electric Psychrometer, and chamber oxygen concentration was measured twice per exposure with a Biosystems Model 3100R Oxygen Monitor.
- Samples taken from breathing zone: no

VEHICLE (if applicable)
- Justification for use and choice of vehicle: due to substance characteristics and resulting aerosol/vapour mixture generation via heat-evaporation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see "Details on inhalation exposure"
Duration of treatment / exposure:
two weeks
Frequency of treatment:
6 hours per day, 5 days a week, for two weeks (i.e. 10 exposures in total) followed by a 14-day recovery period
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 10, 49 and 240 mg per cubic metre
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 10, 50 and 250 mg per cubic metre
Basis:
nominal conc.
No. of animals per sex per dose:
10 males per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure concentrations in the present study were selected based on results of rangefinding studies
- Post-exposure recovery period in satellite groups: 14 days (5 animals of each treatment group as well as control)

Examinations

Statistics:
Mean body weights and body weight gains for exposed rats were compared to control rats during the exposure and recovery periods.
Data were statistically analysed by one-way analysis of variance. Exposure group values were compared to controls by the least significant difference test when the ratio of variance (F) indicated a significant among-to-within group variation. Significant differences were declared at the 0.05 probabiility level. The statistical analyses used to evaluate the clinical pathology and the pathology data are described below:
A one-way analysis of variance (ANOVA) and Bartlett's test were calculated for each sampling time. When the F-test from ANOVA was
significant, the Dunnett test was used to compare means from the control group and each of the groups exposed to the test material. When the results of
the Bartlett test were significant (p < 0.005), the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the
control group and each of the groups exposed to the test material. Significance was judged at the 5% probability level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
high dose group: 1 out of 10 rats showed black nasal discharge, 2 out of 10 rats showed alopecia
Mortality:
mortality observed, treatment-related
Description (incidence):
high dose group: 1 out of 10 rats showed black nasal discharge, 2 out of 10 rats showed alopecia
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
dose related lesions confined to the upper respiratory tract; fully reversible (inflammation and necrosis in the nasal mucosa and the larynx/pharynx region, of only minimal to mild severity for low and mid dose group, but moderate in the high dose group)
Details on results:
CLINICAL SIGNS AND MORTALITY
all groups: clinical signs as commonly found in restrained animals (clear nasal and ocular discharge, discoloured and wet fur)

HISTOPATHOLOGY: NON-NEOPLASTIC
all dose groups: dose dependent nasal lesions consisted of inflammation and necrosis which occurred in the nasal mucosa immediately after the two week exposure

- low dose group:
# nasal lesions, which were considered to be of minimal severity.
# minimally inflammation and necrosis in the larynx/pharynx region in 1 out of 5 rats.
- mid dose group:
# nasal lesions, which were considered to be of mild severity;
# minimally inflammation and necrosis in the larynx/pharynx region in 1 out of 5 rats.
- high dose group:
# nasal lesions, which were considered to be of moderate severity.
# minimal to moderate inflammation and necrosis in the larynx/pharynx region in 2 out of 5 rats.
#minimal to mild inflammation of the tracheal muscle in 2 out of 5 rats

After the two week recovery period none of the above mentioned nasal lesions were observed in any test material exposed rats.

Effect levels

Dose descriptor:
LOAEC
Effect level:
10 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: dose dependent effects seen on the upper respiratory tract (e.g. nasal lesions) which were from minimal to moderate severity and were fully reversible within the 14-day recovery period

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chamber atmosphere analysis summary

 Conc. [mg/cubic metre] Mean (SD) (a)  [%] Aerosol / [%] Vapour (b)  Particle size - MMD [micrometre] (c) Particle size - [%] < 10 micrometres (d)
          Control chamber
 10 (2)  33/67 (e) 4.8   79
 49 (8.7) 3/97  3.6  82 
240 (66)   36/64  5.1  80
       
a. Total test material chamber atmospheric concentration (combined aerosol and vapour). Mean and standard deviation of 10 exposures based on a minimum of 4 samples per exposure. b. Mean % of test material in the chamber atmosphere which was in the aerosol or vapour form. c. Mass median aerodynamic diameter, one measurement per week. (mean of two samples) d. Percent by weight of particles with aerodynamic diameter less than 10 micrometres. From cascade impactor determinations (mean of two samples). e. Percent aerosol for the 10 mg/m3 chamber increased dramatically during the second veek of exposures. See chamber atmosphere discussion below for further interpretation of this finding.

Chamber atmosphere discussion

As expected from the low vapour pressure of the test material, the proportion of test material which was in the aerosol form was highest (36%) at the highest concentration tested (240 mg/m3 ). Low aerosol/vapour ratios were measured in the first week of exposure in the 10 and 49 mg/m3 chambers; the 10 mg/m3 chamber averaged 4.4% aerosol, and the 49 mg/m3 chamber averaged 3.2%. However, the average percent aerosol in the 10 mg/m3 chamber increased from 4.4% in the first week to 61% in the second week. The reason for the increase in the aerosol percentage for the 10 mg/m3 chamber in the second week of exposure is not known. There were no changes reported in the atmosphere generation system, however unobserved pressure or temperature changes in the system could have affected the vapour formation rate. A possible related change was a significant increase in the relative humidity (RH) in this chamber of about 20% in the second week. This was about twice the increase observed in the other chambers during this period. Changes in RE have been observed to change condensation aerosol formation rates. To determine whether the increase in aerosol/vapor ratio in the 10 mg/m3 group affected the biological outcome of the study would require further investigation. However, the effects observed in the rats at 10 mg/m3 were only minimal and completely reversible.

Applicant's summary and conclusion

Conclusions:
In this subacute inhalation toxicity study male rats were exposed for 6 hours per day and overall 10times within two weeks to a aerosol/vapour mixture of the test material. Concentrations used were 0, 10, 49 and 240 mg/m³ (analytical). Due to the fact that effects on the respiratory tract were seen in all concentration groups tested and these effects were clearly dose related a no observed adverse effect level could not be determined. The LOAEC of this study was established to be 10 mg/m³.
Executive summary:

10 male rats were exposed via inhalation for 6 hours per day, 5 times a week for two weeks to an aerosol/vapour mixture containing test material in concentrations of 0, 10, 49 and 240 mg/m3 (analytical concentration). There were no adverse effects observed in any of the test groups with respect to the haematologic, clinical chemical, urinalysis, and body weight parameters measured. As expected from its chemical basicity, inhaled test material produced respiratory tract irritation in exposed rats. However, the lesions (inflammation/necrosis) observed occurred only in the upper respiratory tract (mainly the nose, larynx, and pharynx), were moderate at the highest concentration tested, and were decreased in severity at the lower test concentrations. Effects observed in the 49 mg/m3 group were mainly mild and those in the 10 mg/m3 group were minimal. The observed effects were completely reversible after a two-week recovery period even at the 240 mg/m3 concentration. Although a no-adverse-effect concentration was not determined in this study, the respiratory tract effects observed at the lowest tested concentration (10 mg/m3 ) were considered minimal and reversible.