Carbamic acid ester

Endpoint summary

Description of key information

Propylene glycol: oral feed rat NOAEL 1700 mg/kg bw 
Propylene glycol: inhalation rat NOAEC 1000 mg/m3 (systemic effects), 160 mg/m3 (local effects nasal cavity)
1,6-hexanediamine:  inhalation rat and mouse NOAEC 160 mg/m3 (systemic effects), 5 mg/m3 (local effects to the upper respiratory tract)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited report, non-GLP, the information in the publication is limited to what is included in the summary. The results are indicative for low toxicity

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

study performed before OECD/EC guideline became available. limited report, limited number of animals included, no investigation of blood chemistry, limited urinary parameters checked

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: not indicated
- Weight at study initiation: males 120-150 g; females 120-140 g
- Housing: no data
- Diet: Spillers' Laboratory Small Animal Diet ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± I°C
- Humidity (%): 50-60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION: no data

VEHICLE: no data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 years (additional group at 0 and 50000 ppm 15 weeks)

Frequency of treatment:

continuously in diet

Remarks:

Doses / Concentrations:
0, 6250, 12,500, 25,000 and 50,000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
200, 400, 900 and 1700 mg/kg bw in males and 300, 500, 1000 and 2100 mg/kg bw in females
Basis:
actual ingested

No. of animals per sex per dose:

30/sex/dose

Control animals:

yes

Details on study design:

Earlier studies found some haematological effects (including haemolysis) and therefore 15 rats/sex were treated at 50000 ppm (1700-2100 mg/kg bw) for 15 weeks. These animals (including additional controls) were examined for renal concentration (gravity and volume) and activity of aspartate aminotransferase (AST) in urine samples collected over 6 hours. In addition haematological examination and assessment of blood-chemistry parameters (urea, AST and ALAT) was assessed. At autopsy brain, heart, liver, spleen, kidneys, adrenals, gonads and pituitary were weighed.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: 2 weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: 2 weekly
- Compound intake measured over a 24 period before weighing

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, collected from tail veins
- Time schedule for collection of blood: in week 13, 21, 52 and 80 on 8/sex from control, 25000 and 50000 ppm, in week 54 on 8/sex from control, 6250 and 12500 ppm, in week 104 on all survivors
- Anaesthetic used for blood collection: No
- Animals fasted: No
- Parameters checked:
in week 13 and 21: haemoglobin content, packed cell volume and counts of erythrocytes, total leucocytes and differential leucocytes.
in week 52, 54 and 80 haemoglobin content, packed cell volume and counts of erythrocytes, reticulocytes, total leucocytes and differential leucocytes.
in week 104: haemoglobin content and differential leucocytes count

CLINICAL CHEMISTRY: No (performed on additional group of 15 animals/sex)

URINALYSIS: Yes
- Time schedule for collection of urine: in week 13, 30 and 52 on selected rats from 0, 25,000 and 50,000 ppm.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data; urine collection during water deprivation period of 6 hours; waterload of 25 mL/kg bw for two hours followed by urine collection over 4 hour period starting 16 hours thereafter
- Parameters checked:
deprivation: volume, gravity, cell count
waterload: volume, gravity

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full
HISTOPATHOLOGY: Yes , brain, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine, caecum, salivary gland, trachea, aorta,
thymus, lymph nodes, pituitary, urinary bladder, colon, rectum, pancreas, uterus, muscle and gross lesions
ORGAN WEIGHTS: Yes, brain, heart, liver, spleen, kidneys, adrenals, gonads, stomach, small intestine and caecum

Statistics:

Yes, but no details

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Description (incidence and severity):

limited parameters investigated in additional group

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY: males 18, 14, 16, 19 and 19 at 0, 6250, 12500. 25000 and 50000 ppm; females 15, 18, 11, 16 and 15 at 0, 6250, 12500. 25000 and 50000 ppm

CLINICAL SIGNS : no treatment related effects

BODY WEIGHT(gain): no treatment related effects

FOOD CONSUMPTION; no treatment related effects

HAEMATOLOGY: no treatment related effects

CLINICAL CHEMISTRY: no treatment related effects (in the additional control and 50000 ppm animals treated during 15 weeks)

URINALYSIS: no treatment related effects

ORGAN WEIGHTS (absolute and relative to body weight): no treatment related effects

GROSS PATHOLOGY: no treatment related findings
At all doses and controls
Kidney: yellow discoloration
Liver: fatty changes,

HISTOPATHOLOGY: NON-NEOPLASTIC: no treatment related effects
At all doses and controls
Kidney: nephropathy (no relationship with treatment)
Liver: portal lymphocyte infiltration, nodular hyperplasia, hepatocyte vacuolation , bile duct proliferation, cirrhosis and fibrosis (no relationship with treatment)
Lung: chronic infection (peribronchial and perivascular lymphocyte cuffing, thickening and collapse of the alveoli), pneumonia (polymorphonuclear leucocyte infiltration of the alveoli) (no relationship with treatment, but cause of most deaths during the study)

HISTOPATHOLOGY: NEOPLASTIC incidental findings within normal ranges for rats of this strain and age
females: mammary fibroadenomas, pituitary adenomas and subcutaneous fibrosarcomas
males: subcutaneous fibrosarcomas

Dose descriptor:

NOAEL

Effect level:

1 700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Dose descriptor:

NOAEL

Effect level:

2 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

Conclusions:

The NOAEL is 1700 mg/kg bw

Executive summary:

In a dietary study, the substance was fed to rats (30/sex/dose) for 104 weeks at 0, 6250, 12500, 25000 and 50000 ppm. Mortality was high and related to pulmonary disease, but showed no relationship with treatment. No treatment related effects were found on body weight(gain), food consumption, haematology, biochemistry (in additional group treated for 15 weeks at 50000 ppm) and urinalysis. Effects on kidney, liver and lungs were considered not related to treament. Some neoplasms were found but considered within normal ranges for rats of this age. Therefore it is concluded that the NOAEL is set at 50000 pp (1700 mg/kg bw for males and 2100 mg/kg bw for females). The substance is of low toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 700 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Test on the expected metabolite propylene glycol. Limited report, non-GLP, the information in the publication is limited to what is included in the summary. The results are indicative for low toxicity

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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.

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

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

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

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

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)

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.

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

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

160 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

NTP study on1,6-hexanediamine

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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.

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

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

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

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

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)

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.

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

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

NTP study on1,6-hexanediamine

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The available information on the metabolites of the substance shows that both are expected to be of low toxicity with 1,6-hexanediamine (analogue of the metabolite) showing slightly higher toxicity. Therefore in a weight of evidence approach the studies on 1,6 -hexanediamine will be taken as starting point for the derivation of the NOAEL.

Justification for classification or non-classification

Based on the information on the metabolites, the substance does not need to be classified according to CLP (Regulation EC No 1272/2008).