Carbamic acid ester

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

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 conform the guideline under GLP. Limited reported, not all findings are included in the report.

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:

no guideline followed

Principles of method if other than guideline:

The study is a 90-day inhalation study with inclusion of a reproductive toxicity trial. The parameters assessed are very similar to those assessed in the protocol for the OECD 422 repeated dose reproduction test and described below.

GLP compliance:

yes

Remarks:

no QA statement and/or study director statement included in the report, but GLP is claimed

Limit test:

no

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 analogue 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 solution from 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:

rat

Strain:

Fischer 344

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 142-150 g; females 112-114 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

Type of inhalation exposure:

whole body

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 microns, with a geometric standard deviation of 1.52 to 1.53.

Details on inhalation 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Twice monthly during the 13-week studies, glass fiber filter samples from each chamber were analyzed by gas chromatography with flame ionization detection for total hexanediamine, using the technique supplied by Midwest Research Institute. Measured concentrations of HDDC in the exposure chambers were within 6% of the target concentrations in all samples.
Spatial homogeneity of the aerosol within the exposure chambers was determined using the calibrated RAM-S monitors. Chamber concentrations were measured at 12 points within each chamber and then were compared to a fixed reference point. Time spans required to reach stable concentrations after start up and to reach background concentrations at the end of exposure were determined by taking measurements of aerosol concentrations every 60 seconds. The time span required after start up to reach 90% of the target concentration was identified as the T90; the time span required after the end of the exposure period to reach 10% of the target concentration was identified as the T10.
Triplicate particle size measurements were obtained for each exposure chamber once in the first week and monthly thereafter, using an APS 3300 aerodynamic particle sizer (TSI, Inc., Minneapolis, MN). In addition, a CFM Ambient Impactor (Flow Sensor, McLean, VA) cascade impactor was used to determine the particle size distribution in the highest exposure level chamber once during the 13-week studies. The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 microns, with a geometric standard deviation of 1.52 to 1.53. All control chamber respirable mass concentration values were less than 0.005 mg/m3.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h (+ T90 (=30 min))/day, 5 d/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.6, 5, 16, 50, and 160 mg HDDC/m3 (6 hours/day, 5 days/week)
Basis:
analytical conc.

No. of animals per sex per dose:

main study: 10/sex/concentration
mating study: 20 males and 40 females/concentration

Control animals:

yes

Details on study design:

- Dose selection rationale: based on weight gain depression and inflammation and ulceration of the nasal cavity and larynx seen in both sexes of rats at the higher concentrations in the 2-week range finding study
- Rationale for selecting satellite groups: used for reproduction parameters

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

Examinations in main study animals

CAGE SIDE OBSERVATIONS: Yes no details

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT (GAIN): Yes
- Time schedule for examinations: at start, weekly thereafter and at necropsy

FOOD CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes on all animals
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: CO2:O2 (70:30) gas mixture
- Parameters checked: erythrocyte (RBC), leukocyte (WBC), and platelet (PLAT) counts, hemoglobin (HGB) concentration, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and methemoglobin (METH), Differential leukocyte count, reticulocyte count.
Morphology of erythrocytes, leukocytes and platelets during the leukocyte differential count

CLINICAL CHEMISTRY: Yes on all animals
- Time schedule for collection of blood: at termination
- Animals fasted: No data
- Parameters checked: urea nitrogen (UN), creatinine, alanine aminotransferase (ALT), alkaline phosphatase (AP), sorbitol dehydrogenase (SDH), and glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sperm morphology in control and 3 highest concentrations
- Time schedule: at termination
- Parameters checked: density, motility and non-motility on 4 slides per animals (extruded from the right epididymal tail)

OTHER: vaginal cytology in control and 3 highest concentrations
- Time schedule daily from day 7 before termination until termination
- Parameters checked: cytology: leukocytes, nucleated epithelial cells, and large squamous epithelial cells in the lavage fluid to identify the stages of the estrous cycle

Sacrifice and pathology:

GROSS PATHOLOGY: Yes on all animals
ORGAN WEIGHT: Yes on all animals:
thymus, heart, right kidney, lungs, brain, liver, and right testis
HISTOPATHOLOGY: Yes on all animals of the control and high dose group:
adrenal gland, bone and bone marrow, brain, bronchial lymph node, cecum, clitoral/preputial glands, colon, duodenum, epididymis, esophagus, gallbladder (mice), heart, ileum, jejunum, kidney, larynx, lung and mainstem bronchi, liver, mammary gland, mandibular lymph node, mediastinal lymph node, mesenteric lymph node, nasal cavity and nasal turbinates, ovary, pancreas, prostate gland, pituitary gland, parathyroid gland, rectum, salivary gland, skin, spleen, stomach, seminal vesicle, testis, thyroid gland, thymus, trachea, urinary bladder, uterus, and all gross lesions.
On all animals in intermediate groups:
nasal cavity (3 standard sites) and larynx

Other examinations:

see under toxicity to reproduction

Statistics:

Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of
continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response (Dunnett, Dunn). If the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or Williams' test.

Vaginal cytology by multivariate analysis of variance (Morrison, 1976)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

nasal discharge in all animals

Mortality:

no mortality observed

Description (incidence):

nasal discharge in all animals

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in males at 160 mg/m3

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):

in females at 16, 50 and 160 mg/m3

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

in males at 16, 50 and 160 mg/m3, in females at 5, 16, 50 and 160 mg/m3

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

at all dose levels

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 16, 50 and 160 mg/m3 in males and females

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY: no treatment related effects

CLINICAL SIGNS: nasal discharge in males at 5 and 16 mg/m3 and in females at 0, 1.6, 5, 16 and 50 mg/m3

BODY WEIGHT AND WEIGHT GAIN: slightly decreased in males at 160 mg/m3 (7%)

HAEMATOLOGY:
dose related significant decrease in leukocytes in females at 50 and 160 mg/m3
dose related significant decrease in segmented neutrophils in females at 16, 50 and 160 mg/m3

CLINICAL CHEMISTRY:
Significantly increased Alkaline Phosphatase in males at 50 and 160 mg/m3

ORGAN WEIGHTS:
significantly decreased lung weights (abs + rel) in all treated animals (no relationship with dose)
significantly decreased thymus weight in females at 16, 50 and 150 mg/m3 (no relationship with dose)

GROSS PATHOLOGY: no treatment related effects

HISTOPATHOLOGY:
inflammation of the larynx in males at 50 and 160 mg/m3 and in females at 160 mg/m3
degeneration of respiratory epithelium of the nose in males and females at 16, 50, and 160 mg/m3
inflammation of the nasal pasages/squamous metaplasia in males and/or females at 16, 50, and 160 mg/m3
degeneration of the olfactory epithelium in males and females at 160 mg/m3

SPERM: No treatment related effects

OESTRUS CYCLE: no treatment related effects.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects on the upper respiratory tract and the lungs

2

Dose descriptor:

NOAEC

Effect level:

160 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of systemic effects. All effects seen are considered secondary to local inflammation

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Local effects in the nasal cavity were found in males and females at 16 mg/m3 and above. The NOAEC is 5 mg/m3.
No systemic effects were noted at any of the concentrations tested.

Executive summary:

Rats (10/sex/concentration) were exposed to the substance at 0, 1.6, 5, 16, 50, and 160 mg/m3 during 13 weeks (6h/day, 5 d/week). No mortalities were reported and the nasal discharge seen in all concentrations (including controls) is considered not related to treatment with the substance. At termination body weight in males at 160 mg/m3 was slightly decreased). Effects on haematology (decreased number of leukocytes and segmented neutrophyls) and clinical chemistry (increased ALP) were seen at 16 mg/m3 and above. Lung weights were decreased at all concentrations in both males and females with no relationship to dose. No effects on sperm motility and the oestrus cycle were found. No gross findings, but histopatholgy showed inflammation of the nasal region including degeneration of the respiratory epihelium at 16 mg/m3 and above. Degeneration of the olfactory epithelium was reported at 160 mg/m3.

The NOAEC for local effects is 5 mg/m3. No treatment related systemic effects were reported at any of the concentrations tested.