Adipic acid, compound with hexane-1,6-diamine (1:1)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

males: 1987-06-03 to 1986-09-03; females: 1987-06-02 to 1987-09-02 (dosing period)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1993

Reference 2

Reference Type:

publication

Title:

Inhalation Toxicity of 1,6-Hexanediamine Dihydrochloride in F344/N Rats and B6C3F1 Mice.

Author:

Hébert, CD et al.

Year:

1993

Bibliographic source:

Fund Appl Toxicol 20: 348-359.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylenediammonium dichloride

EC Number:

227-977-8

EC Name:

Hexamethylenediammonium dichloride

Cas Number:

6055-52-3

IUPAC Name:

hexane-1,6-diamine dihydrochloride

Details on test material:

Component of the test substance

- Name of test material (as cited in study report): 1,6-hexanediamine dihydrochloride, purchased as 70% aqueous solution
- Physical state: liquid
- Analytical purity: 70.9% by gas chromatography
- Lot/batch No.: PT-031985
- Stability under test conditions: confirmed by analysis
- Storage condition of test material: 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
male and female Fischer 344/N rats
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 142 - 150 g (males); 112 - 114 g (females)
- Fasting period before study: no
- Housing: in individual compartments of multi-compartment stainless steel wire mesh cages; during exposure: in Hazelton H-2000 stainless steel and glass exposure chambers (Hazelton Systems, Inc., Aberdeen, MD) of 2 m³ volume
- Diet (ad libitum): pelleted NIH-07 feed
- Water (ad libitum): tap water
- Acclimation period: 11 - 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 - 24 °C (original value: 72 +/- 3°F); during exposure: ca. 22 - 26 °C (72 - 78°F)
- Humidity (%): 35 - 65 % ; during exposure: 70 - 80 %
- Air changes (per hr): 12 - 15; during exposure: 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

clean air

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 . All control chamber respirable mass concentration values were less than 0.005 mg/m³.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

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 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 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. 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 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.

TEST ATMOSPHERE
Concentrations of HDDC in the exposure chamber, exposure room, and exhaust were monitored by measuring the forward light scatter with RAM-S real-time aerosol monitors and by gravimetric analyses of filter samples collected from each exposure chamber. Six RAM-S readings and 3 gravimetric samples were taken from each exposure chamber on each day of exposure. Gravimetric sampling was conducted with 25 mm glass fiber filter paper. Gravimetric analysis was performed by weighing filters to the nearest 0.01 mg before and after sampling and again after storing the filters in a desiccator overnight. 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.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours plus T90 (30 minutes) per day; 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.6 other: mg HDDC/m³ (analytical)

Doses / concentrations 2

Dose / conc.:

5 other: mg HDDC/m³ (analytical)

Doses / concentrations 3

Dose / conc.:

16 other: mg HDDC/m³ (analytical)

Doses / concentrations 4

Dose / conc.:

50 other: mg HDDC/m³ (analytical)

Doses / concentrations 5

Dose / conc.:

160 other: mg HDDC/m³ (analytical)

Doses / concentrations 6

Dose / conc.:

1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Doses / concentrations 7

Dose / conc.:

3.1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Doses / concentrations 8

Dose / conc.:

10 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Doses / concentrations 9

Dose / conc.:

31 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Doses / concentrations 10

Dose / conc.:

100 other: mg HMD/m³

Remarks:

corresponding HMD concentration

No. of animals per sex per dose:

10 males and 10 females per base group;
20 male and 40 females per satellite group (mating trial)

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The test concentrations were chosen based on the reported inhalation LCLo of 750 mg/m³ in mice and because of the lack of information on inhalation toxicity of HDDC in rats and on the results of a 2-week inhalation study (weight gain depression and the inflammation and ulceration of the nasal cavity and larynx seen in both sexes; see other entry in this section).
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: mating trials

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded at study start, weekly, and at the end of the study.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: Yes (CO2:O2 90:30)
- Animals fasted: No data
- How many animals: all 13-week inhalation base-study rats
In addition, blood samples were taken from 10 mating-trial rats/sex/exposure (group after 3 and 13 exposures (Days 4 and 18).
- Parameters examined: 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)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No data
- How many animals: all 13-week inhalation base-study rats
In addition, blood samples were taken from 10 mating-trial rats/sex/exposure (group after 3 and 13 exposures (Days 4 and 18).
- Parameters examined: urea nitrogen (UN), creatinine, alanine aminotransferase (ALT), alkaline phosphatase (AP), sorbitol dehydrogenase (SDH), glucose

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Sperm Morphology/Vaginal Cytology and Mating Trials
Sperm morphology and vaginal cytology evaluations (SMVCE) and mating trials were performed at the end of the 13-week studies. Sperm morphology and vaginal cytology were evaluated in base-study rats from the control, 16, 50, and 160 mg HDDC/m³ exposure groups. Mating trials were performed on supplemental rats exposed to 0, 16, 50, or 160 mg HDDC/m³. See section 7.8.1.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy performed; tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with H&E for microscopic examination. The following tissues were examined microscopically from all high-dose and control animals: adrenal gland, bone and bone marrow, brain, bronchial lymph node, cecum, clitoral/preputial glands, colon, duodenum, epididymis, esophagus, 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.

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity related to HDDC exposure were seen in the study. Nasal discharge occurred in male rats in the 5 and 16 mg/m³ exposure groups and in female rats in all exposure groups (including the control group) except those in the 160 mg/m³ group. Similarly, rales occurred in all female groups but-not in exposed males. However, because these signs appeared late in the study and because the incidence was not dose related, the signs were not considered to be the result of specific HDDC toxicity.

Mortality:

no mortality observed

Description (incidence):

All rats exposed to HDDC by inhalation for 13 weeks survived to the end of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The final mean body weights of most groups of rats exposed to HDDC were slightly lower than the mean body weights of the controls (Table 7, see attached file); these differences, however, were not statistically significant.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At Day 4, the only change noted in the hematology parameters of rats exposed by inhalation to HDDC was a slight decrease in the mean platelet count in female rats in the lowest exposure group. At Day 18, hematocrit values were increased in female rats in the 2 highest exposure groups and segmented neutrophil counts were decreased minimally in male rats in the highest exposure group. By Day 94, there was a significant decrease in leukocyte and lymphocyte counts in females in the highest exposure groups, and in segmented neutrophil counts in females in the 3 highest exposure groups (16, 50, and 160 mg/m³). Female rats in the 2 lowest exposure groups had increased hematocrit values. A slight decrease in erythrocyte count was noted in male rats in the 16 mg/m³ exposure group, and a minor increase in mean cell hemoglobin values occurred in female rats in the 160 mg/m³ exposure group and male rats in the 50 mg/m³ exposure group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry changes on Day 4 included a small increase in alanine aminotransferase activity in male rats in the lowest exposure group (1.6 mg/m) and a slight increase in the urea nitrogen level in female rats in the 5 mg/m³ exposure group. By Day 18, concentrations of urea nitrogen increased in male rats in the 2 highest exposure groups (50 and 160 mg/m³) and female rats in the 4 highest exposure groups (5, 16, 50, and 160 mg/m³). Sorbitol dehydrogenase (SDH) activity was slightly elevated in female rats in the highest exposure group. At Day 94, alkaline phosphatase activity was slightly increased in male rats in several exposure groups (1.6, 50, and 160 mg/m³), and SDH activity was elevated in males in the 50 mg/m3 exposure group. No other significant clinical chemistry changes occurred in male or female rats at Day 94.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only consistent changes in organ weights seen in rats were decreases in absolute and relative lung weights compared to those of the controls. However, all control male and female rats had inflammatory lesions in the lungs and had lung weights that were greater than those of historical controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross lesions attributed to HDDC exposure.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) of male and female rats in the 2 highest exposure groups (Table 8, attached file). The morphology, incidence, and severity of microscopic lesions were similar for males and females, and there was a dose-related increase in the incidence and severity of these lesions. For details, see attached file.

Description (incidence and severity):

Sperm Morphology/Vaginal Cytology and Mating Trials
Administration of HDDC to rats by inhalation caused no changes in any of the sperm morphology or vaginal cytology parameters evaluated.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

160 mg/m³ air (nominal)

Based on:

other: HDDC

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

16 mg/m³ air (nominal)

Based on:

other: HDDC

Sex:

male/female

Basis for effect level:

other: irritating effects

3

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

100 mg/m³ air (nominal)

Based on:

other: corresponding HMD concentration

Sex:

male/female

Basis for effect level:

other: systemic toxicity

4

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

10 mg/m³ air (nominal)

Based on:

other: corresponding HMD concentration

Sex:

male/female

Basis for effect level:

other: irritating effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a 13-week inhalation study, 10 rats of each sex were exposed to 0, 1.6, 5, 16, 50, or 160 mg HDDC/m3 for 6 hours per day, 5 days per week for 13 weeks. In addition special groups of 20 male and 40 female rats (mating trial animals) at each exposure level were included to assess the effect of HDDC on reproduction. All rats in the base-study groups survived to the end of the studies, and there were no exposure-related changes in body weight. No exposure-related changes in absolute or relative organ weights and no exposure-related clinical signs or gross lesions were seen. In female rats, a dose-related decrease in white blood cell count was observed. Chemical-related microscopic lesions in male and female rats were limited to the upper respiratory tract (larynx and nasal passages) in the 2 highest exposure groups. These lesions included minimal to mild focal erosion/ulceration, inflammation, and hyperplasia of the laryngeal epithelium as well as degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or in the length of the estrous cycle of rats.

The NOAEC was 160 and 16 mg/m³ for systemic toxicity and local irritating effects, respectively.