2-methylpentane-1,5-diamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 17 NOV 1989 to 14 JUN 1991

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:

1991

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

Test material information

Constituent 1

Reference substance name:

2-methylpentane-1,5-diamine

EC Number:

239-556-6

EC Name:

2-methylpentane-1,5-diamine

Cas Number:

15520-10-2

Molecular formula:

C6H16N2

IUPAC Name:

2-methylpentane-1,5-diamine

Details on test material:

- Name of test material (as cited in study report): DYTEK A Amine; 1,5-Pentanediamine, 2-methyl-; MPMD
- Physical state: liquid
- Analytical purity: 99%
- Composition of test material, percentage of components:
99% MPMD
0.7% Methylcyclopentanediamine (4 isomers)
0.07% Ethyltetramethylenediamine
0.02% Water
0.02% Other
- 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. 7 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): 23 +/-2
- Humidity (%): targeted: 50 +/-10; but due to cold weather conditions combined with poor humidity control in the laboratory during most of the study, the animal room and chamber humidity readings were below 40%.

IN-LIFE DATES: From: 27 NOV 1989 To: 22 DEC 1989

Administration / exposure

Route of administration:

other: inhalation of a aerosol/vapour mixture

Type of inhalation exposure:

nose only

Vehicle:

other: air and nitrogen

Remarks on MMAD:

MMAD / GSD: Aerosol size (MMDA): ranging from 4 to 13 micrometres

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 150 litre exposure chambers (mado stainless steel and glass)
- Method of holding animals in test chamber: restrainers (perforated stainless steel cylinders with conical nose pieces, which were inserted into face plates to yield nose-only exposure conditions)
- System of generating particulates/aerosols: Aerosol and vapour atmospheres of the test material were generated by pumping the test material to a heated (140-186°C) 500 ml Instatherm flask. Nitrogen introduced at the flask swept the aerosol/vapour mixture into the chambers. Dilution air was added to the stream just prior to the entry port of the chamber.
- Temperature, humidity, pressure in air chamber: 23 to 25 °C; usually less than 40% humidity, as nothing else is specified in the study report standard pressure is assumed
- 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, 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 teas material during the animal exposures. A minimum of 6 samples were
taken per exposure. For test material analysis, a measured volume of the chamber atmosphere was drawn through one of two sampling systems. The first system (A) used a glass midget impinger containing methanol to trap combined test material aerosol/vapor for subsequent analysis by gas chromatography (GC).
The second system (B) used a glass-fiber filter in tandem with the methanol impinger. The filter trapped the test material aerosol particles, the impinger trapped the vapor. During the first 2 exposures, one sample per exposure was used to analyse the test item aerosol and vapor separately (sampling system B), the remainder of the samples in the first 2 exposures were used to analyse for combined test material aerosol/vapor by GC
(sampling system A). For the remaining 8 exposures, aerosol and vapor concentrations were determined separately using sampling system B (see
note below).
The atmospheric concentration of test material aerosol was calculated from the pre- and post-sampling filter weights which were determined with a Cahna Model 26 Automatic Electrobalancc. 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 125'C an a 30 meter X 0.53 mm O.D. fused-silica column lined with polydinethylsiloxane. 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 once or twice with mercury thermometers, relative humidity was measured once with a Reuter-Stokes Model RSS-230 Electric Psychrometer, tnd chamber oxygen concentration was measured once with a Biosystems Model 3100 Oxygen Monitor.

Note: After the first two exposures it was observed that the chamber test material aerosol/vapour ratio was not constant during the exposures
due to the test material evaporation methods used. To better follow the varying aerosol/vapour ratlos, hourly samples using system B were initiated during the 3rd exposure.

- 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 ihnalation 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 9.2, 59 and 250 mg per cubic metre
Basis:
analytical conc.

Doses / concentrations 2

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 range finding studies which showed dose-related weight losses in rats exposed to the test material for 3 days at 250 mg/m³ .
- 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 probability 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: two out of 10 animals died; clinical signs during exposure period ranged from lung noise, irregular respiration, hunched posture, red nasal and ocular discharge to lethargy

Mortality:

mortality observed, treatment-related

Description (incidence):

high dose group: two out of 10 animals died; clinical signs during exposure period ranged from lung noise, irregular respiration, hunched posture, red nasal and ocular discharge to lethargy

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose group: body weights were depressed compared to controll during the exposure and the first week of the recovery period

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:

effects observed, treatment-related

Description (incidence and severity):

high dose group: increase in relative numbers of red blood cells, in haemoglobin concentration and in haematocrit percentage (together with findings from urinalysis these are signs of dehydration/haemoconcentration)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

high dose group: decrease in lymphocytes (thought to be stress induced)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

high dose group: decrease in urine volume and increase in urine osmolality (together with findings from haematology these are signs of dehydration/haemoconcentration)

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

high dose group: mean absolute and lung-to-body weight ratios increased immediately after the exposure and post recovery period (other differences in mean and relative organ weights are thought to be secondary to the depressed body weights in these rats)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dose related lesions confined to the respiratory tract; partially reversible

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

inflammation, necrosis, regeneration and squamous metaplasia in respiratory and olfactory epithelim (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
Red nasal and ocular discharges were observed in all groups, a clinical sign commonly seen among rats under restraint. No adverse clinical signs of toxicity were observed in the 10 or 50 mg/m³ exposure groups. In the 250 mg/m³ exposure group, clinical signs observed during the exposure phase of the study were lung noise, irregular respiration, hunched posture, red nasal and ocular discharges, and lethargy. One rat showed slight lung noise an the 6th and 7th days of the recovery period.
One rat in the 250 mg/m³ exposure group was found dead an the morning of the 8th exposure, and another an the 2nd day of recovery. Cause of the deaths was not determined, however the deaths were attributed to test chemical exposure.

BODY WEIGHT
low dose group: similar to control group
mid dose group: similar to control group
high dose group: the body weights of the rats group were depressed during the exposure phase of the study and during the first week of the recovery period, but were no longer statistically different from the controls during the second week of the recovery period.

GROSS PATHOLOGY
low dose group: -
mid dose group:-
high dose group: multifocal areas of discolouration present in lungs immediately after exposure and after the 14 day recovery period

HISTOPATHOLOGY: NON-NEOPLASTIC
all dose groups: dose dependent nasal lesions consisted of inflammation, necrosis, regeneration, and squamous metaplasia which occurred in the respiratory and olfactory epithelium

- low dose group:
# nasal lesions, which were considered to be of minimal to mild severity. The nasal lesions were the only effects seen in rats of this dose group.

- mid dose group:
# nasal lesions, which were considered to be of minimal to mild severity;
# minimal inflammation in the larynx/pharynx region; minimal hyperplasia, hypertrophy, and necrosis of the respiratory epithelium in the tracheal area; The tracheal and laryngeal lesions were reversible after the 14-day recovery period.
# Pulmonary changes consisted of minimal hypertrophy and hyperplasia of bronchial and bronchiolar epithelium. These changes were partially resolved after the 14-day recovery period.
# Subacute to chronic peribronchiolar inflammation, was observed in one rat after the 14-day recovery period.
# Masson's trichrome stain for collagen was used in the 14-day recovery rats to identify a slight increase in fibrous connective tissue at sites of peribronchiolar inflammation. This test was positive for the one rat.

- high dose group:
# nasal lesions, which were considered to be of mild to moderate severity. The nasal lesions were partially resolved after the 14-day recovery period;
# mild inflammation in the larynx/pharynx region; mild hyperplasia, hypertrophy, and necrosis of the respiratory epithelium in the tracheal area; The tracheal and laryngeal lesions were reversible after the 14-day recovery period.
# Pulmonary changes consisted of hypertrophy and hyperplasia of bronchial and bronchiolar epithelium which were considered to be minimal to mild. These changes were partially resolved after the 14-day recovery period.
# Subacute to chronic peribronchiolar inflammation, which was mainly mild in severity, was observed at both sacrifice times
# Masson's trichrome stain for collagen was used in the 14-day recovery rats to identify a slight increase in fibrous connective tissue at sites of peribronchiolar inflammation. This test was positive in all four rats.

Effect levels

Dose descriptor:

LOAEC

Effect level:

9.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: effects seen on the respiratory tract (e.g. nasal lesions), which were only partially reversible after 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
9.21 (2.19)	73/27	4.1; 9.2	65; 51
58.8 (75)	89/11	13; 12	46; 47
242 (191)	93/7	4; 6	74; 59
a. Total test material chamber atmospheric concentration (combined aerosol and vapour). Mean and standard deviation of 10 exposures based on a minimum of 6 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. (measurment of week 1; week 2)d. Percent by weight of particles with aerodynamic diameter less than 10 micrometres. From cascade impactor determinations.

Particle size analysis discussion

Rats were exposed to a wide range of test material aerosol particle sizes and a more variable particulate test atmosphere than normally encountered in inhalation studies. Particle size determinations with a cascade impactor showed mass median diameters (MMDs) among chambers that ranged from 4 to 13 micrometres, with the highest values occurring in the 50 mg/m3 chamber. This variability in particle size was considered to be due to the chemical/physical properties of the test material coupled with a relative lack of control over the aerosol formation in the generation system and test chambers. It is likely that a carbonate compound is

formed by the test material vapour in air which may agglomerate forming larger particles than would normally be expected with condensed aerosols. In addition, these aerosol particles may grow by forming hydrates in air.

It is not known why the measured aerosol particle size was higher in the 50 mg/m3 chamber than in the other chambers.

Cascade impactor determinations produced results vhich were not quantitative, were poorly reproducible, and the resulting particle sizes were not log-normally distributed. It was denonstrated with the test material in a subsequent experiment under similar test conditions, that significant sample losses (approx. 74% at a concentration of approx. 80 to 100 mg/m3 ) occurred in the inlet of the cascade impactor preseparator while the chamber atmospheres were sampled. It is not known how accurate the test material particle size determinations were given these substantial losses, however, in the subsequent experiment, cascade impactor measurements

were obtained both with, and without the presence of the preseparator, and the resulting MMDs were 1.7 and 3.4 micrometre, respectively. This is not considered a large difference in toxicology studies. In any case, in the 2-week Inhalation study, animals were exposed to a wide range of particle sizes including small respirable particles (<3.5 um) and these were considered sufficient to conduct an adequate inhalation toxicity evaluation of the test material. In addition, these Inhalation experiments may be expected to simulate aerosol behavior which would likely occur in a plant exposure situation, although the exposure concentrations in the plant would be lower. Despite some uncertainty in the particle size measurements, the hourly aerosol and vapour concentration measurements during the study are considered to be an accurate representation of the atmospheric test material concentrations to which the test animals were exposed.

Applicant's summary and conclusion

Conclusions:

In this subacute inhalation toxicity study male rats were exposed for 6 hours per day and overall 10 times within two weeks to a aerosol/vapour mixture of the test material. Concentrations used were 0, 9.2, 59 and 250 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 9.2 mg/m³.

Executive summary:

The effects observed in rats exposed to MPMD ranged from severe (including death) in the 250 mg/m³ group to minimal in the 9.2 mg/m³ group. Two rats in the 250 mg/m³m³ group died, one on the 8th day of the exposure period and one on the 2nd day of the recovery period. The body weights of the rats in the 250 mg/m³m³ group were significantly lower than those of the control rats during the exposure period and in the first week of the recovery period. Lung weights were increased in this group immediately after the 2-week exposure period and after the 2-week recovery period.

Clinical laboratory results for rats in the 250 mg/m³m³ suggested the presence of dehydration/haemoconcentration indicated by increases in relative numbers of red blood cells, in haemoglobin concentration, and in haematocrit percentage, and by decreases in urine volume and increases in urine osmolality. In addition, there was a decrease in lymphocytes which was considered to be generalized reaction to stress.

Following the 14-day recovery period there was no evidence of dehydration/ haemoconcentration or lymphopenia.

Gross examination at necropsy showed multifocal areas of discoloration present in the lungs of the rats exposed to 250 mg/m³ immediately after the 2-week exposure and 2 weeks later.

Microscopically, exposure-related lesions were confined to the respiratory tract and were dose related. In the 59 and 250 mg/m³ groups, lesions were observed in the nose, trachea, larynx/pharynx, and lung. These lesions were mainly minimal to mild in the 50 mg/m³ group and mild to moderate in the 250 mg/m³ group. Nasal lesions consisted of inflammation, necrosis, regeneration, and squamous metaplasia of the olfactory and respiratory epithelium; laryngeal/pharyngeal lesions consisted of subacute inflammation; tracheal lesions observed were hyperplasia, hypertrophy, and necrosis of the respiratory epithelium; pulmonary lesions consisted of hypertrophy and hyperplasia of bronchial and bronchiolar epithelium. After the 2 -week recovery period, the above nasal and pulmonary lesions were diminished in severity and the tracheal and laryngeal/pharyngeal lesions were no longer present. At this time, there was a slight increase in fibrous connective tissue (collagen formation) associated with pulmonary inflammatory lesions in the 250 mg/m³ group and in one rat in the 59 mg/m³ group.

In rats exposed at 9.2 mg/m³ only the nose was affected. Minimal to mild inflammation of the nasal epithelium was observed in this group immediately after the 2-week exposure period. After a 2-week recovery period the nasal lesions in this group were diminished in severity and were only seen in 2 of 5 rats.

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 9.2 mg/m³.