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

Endpoint summary

Description of key information

After repeated administration of AH-salt to rats by gavage for 28 days, liver (hepatocytic necrosis), kidney (renal tubular degenerative changes) and stomach (gastric mucosal necrosis) were the target organs after exposure to 5000 mg/kg bw/day. Deaths were observed after 5000 mg/kg bw/day .
The NOAEL was 1000 mg/kg bw/day.
One component of AH salt was investigated in a separate study. Adipic acid (2 year study) gave no indication of specific target organs in dietary studies in rats. NOAEL of approx. 500 - 1000 mg/kg bw/day was obtained.
No data are available for the inhalation of the AH-salt. Additionally, there is no inhalation toxicity information available on adipic acid, one component of the AH-salt. Therefore, the other component of the AH-salt, 1,6 -hexanediamine and its salt 1,6 -hexanediamine dihydrochlorid were used for assessment. Exposure to aqueous 1,6 -hexanediamine aerosols for 13 weeks at analytical concentrations of 0, 12.8, or 51 mg/m³ induced respiratory and conjunctival irritation in Sprague-Dawley rats at the 51 mg/m³ 1,6 -hexanediamine test level.
For one compound of AH salt, The NOAEC is considered to be 10 mg/m³ for local toxicity and 31 mg/m³ for systemic toxicity.
Inhalation of 1,6-hexanediamine dihydrochloride caused increased absolute and relative liver weights in male mice but no effects in female mice and Fischer rats of either sex besides local effects possibly due to the nonphysiological pH. The local NOAEC was 16 mg/m³ of HDDC corresponding to 10 mg/m3 of HMD in both species, the systemic NOAECs were 16 mg/m³ of HDDC corresponding to 10 mg/m3 of HMD for mice and 160 mg/m³ for rats (corresponding to 100 mg/m³ HMD).
In contrast to the AH salt (CAS 3323-53-3) which possesses a pH value of 7.8 and exhibits no irritant activity, 1,6-hexanediamine has a pH value of 12.4 and is corrosive.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981-04-28 to 1981-05-28 (treatment period)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and female Charles River Crl: CD (TM)(SD)BR rats
- Source: Charles River Breeding Laboratories
- Age at study initiation: males: 270 - 323 g; females: 179 - 216 g (test day 1)
- Weight at study initiation: ca. 7 weeks (test day 1)
- Housing: individual cages (suspended, open mesh stainless steel)
- Diet (ad libitum): Ralston-Purina Certified Rodent Chow #5002
- Water (ad libitum): tap water from public supply
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 - 23 °C (original value: 70 - 74 °F)
No further data

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
As appropriate to assure accurate dosing, the test material was administered neat and/or diluted with water. Water was be administered to control animals. All doses were administered on a constant volume/kg body weight basis.

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

daily

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily for mortality and as appropriate for clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations was performed every week.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight for each animal was determined every week and at sacrifice.

FOOD CONSUMPTION: Yes
Food consumption for each animal was determined every week.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study end
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined: White blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study end
- Animals fasted: No data
- How many animals: all survivors
- Parameters examined: Total protein, blood urea nitrogen, glucose, glutamic pyruvic transaminase, alkaline phosphatase, creatinine, cholesterol, calcium, and thyroxin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All animals surviving the study and animals that died or were sacrificed in extremis were submitted for necropsy. Animals surviving to study termination were fasted overnight prior to necropsy. Necropsy procedures included a thorough examination of the nasal, cranial, thoracic, abdominal and scrotal cavities. The following tissues were retained for histopathologic examination (organs in capital letters were weighed): Aorta, adrenals, femur with marrow, brain, esophagus, eyes, TESTES, ovaries, heart, KIDNEYS, LIVER, lung with bronchi, duodenum, jejunum, ileum, colon, urinary bladder, grossly evident lesions, mammary gland, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spleen, stomach, thymus, trachea, thyroid, parathyroid, uterus, and mesenteric lymph nodes.

Statistics:

- Dunnett's test (two-tailed) and by inspection for body weight and food consumption data.
- Bartlett's test to assess the variability.
- Hematology and serum chemistry: Dunnett's test and by inspection.
- Terminal body weights and absolute organ weights: analysis of variance and Dunnett's test.
- Organ weights/terminal body weight ratios: Mann-Whitney test using the Bonferoni Inequality Procedure.
- Incidence of microscopic abnormalities: Fisher Exact test with the Bonferoni Inequality Procedure.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No significant clinical observations were noted at the control, 200 or 1000 mg/kg dose levels. Significant changes at the 5000 mg/kg level included loose stools, rough coats, hypoactivity and red nasal and ocular discharges. Two high dose females also had urine stained fur. One of the females had difficulty in breathing, paleness and was sacrificed in extremis.

Mortality:

mortality observed, treatment-related

Description (incidence):

The highest dose level caused the death or sacrifice in extremis of 10/10 males within 5 days and 6/10 females within 14 days of exposure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The surviving females of the high-dose group had lower mean body weights than the control group at day 8, but their body weights were similar to the controls at study end. 1000 and 200 mg/kg bw produced no body weight changes for either sex.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Initial food consumption data among females at the high dose level reflected a decrease when compared to their controls, but were similar to their controls during the remaining three weeks of testing. No food consumption effect occurred in either sex at 1000 and 200 mg/kg bw.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant increases in red blood cells and hematocrits in males at the mid and low dose levels were observed (+12% for the mid-dose, +9.6% for the low dose group as compared to the control group). However, the values remained within normal limits of this strain and therefore is not considered as biologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum chemistry was not altered.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights among test animals of both sexes that survived to the final necropsy, did not differ significantly from the control group. There was no difference in absolute and relative testes weights between treated groups and controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, no changes were observed for the low level males. Animals of the high-dose group had gaseous distention of the stomach (assumed to be due to gasping during the terminal period of life). One mid dose female had renal congestion/redness at the corticomedullary junction. In the high dose group, enlarged adrenals, renal congestion/redness at the corticomedullar were each observed in one female. Two females that died prior to the end of the study had gastric dilatation, and three high dose females which survived to the end of the study had flattening of the gastric mucosal rugae. In the highest dose group 2 males and 3 females had lung congestion, 3 males had cortical congestion/hemorrhages of adrenals.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological changes at levels of 5000 mg/kg bw included renal tubular degenerative changes in 5 of 10 male rats and 3 of 10 female rats. The same dose level produced focal gastric mucosal necrosis in 3 of 10 male rats. Both focal changes were of a non-inflammatory necrotic nature. These changes were not detected in animals receiving 1000 mg/kg bw. Hepatocytic necroses were found in each 2 males and females of the high dose groups and in 1 control female. No changes were found in the pituitaries, testes and ovaries. There were no significant microscopic changes in the mid dose males and females.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

not specified

Conclusions:

Based on the findings observed, the NOAEL was 1000 mg/kg bw/d under the conditions of this study.

Executive summary:

Abstract

A four week study was conducted using 40 male and 40 female Sprague-Dawley rats administered Nylon Salt 6/6 by gavage (groups of 10 rats/sex). Dosing solutions were prepared daily to give 0, 200, 1000, and 5000 mg/kg/day. Daily checks were made for mortality and obvious signs of toxicity. Weekly body weight and food consumption measurements were taken. All animals were necropsied. Survivors at study termination were bled for hematology and blood chemistry, and their livers, kidneys, arid testes were weighed.

The highest dose level (5000 mg/kg/day) produced the death or sacrifice in extremis of 10/10 males and 6/10 females within 14 days of exposure. Gross pathologic examination of these rats revealed gaseous distention of the stomach. Microscopic findings included renal tubular degenerative changes for both sexes and gastric mucosal changes for males only. At necropsy three of the four surviving females had flat gastric mucosa, but microscopically no significant digestive tract changes were found in these animals. Overall, males were more sensitive to the toxicity of Nylon Salt 6/6.

There was a slight but not statistically significant increase in serum alkaline phosphatase in mid dose level females (1000 mg/kg/day) and statistically significant increases in erythroid parameters in males at the mid and low dose levels (1000 and 200 mg/kg/day respectively). However, the values remained within normal limits making these changes of questionable toxicological significance.

The NOAEL was 1000 mg/kg bw/d under the conditions of this study.

Endpoint conclusion

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Organ:

kidney

liver

stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

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

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

Dose / conc.:

1.6 other: mg HDDC/m³ (analytical)

Dose / conc.:

5 other: mg HDDC/m³ (analytical)

Dose / conc.:

16 other: mg HDDC/m³ (analytical)

Dose / conc.:

50 other: mg HDDC/m³ (analytical)

Dose / conc.:

160 other: mg HDDC/m³ (analytical)

Dose / conc.:

1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

3.1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

10 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

31 other: mg HMD/m³

Remarks:

corresponding HMD concentration

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

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.

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.

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

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

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

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

males: 1987-06-10 to 1986-09-10; females: 1987-06-09 to 1987-09-09 (dosing period)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and female B6C3F1 mice
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 20.6 - 24.8 g (males); 17.9 - 19.9 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

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

Dose / conc.:

1.6 other: mg HDDC/m³ (analytical)

Dose / conc.:

5 other: mg HDDC/m³ (analytical)

Dose / conc.:

16 other: mg HDDC/m³ (analytical)

Dose / conc.:

50 other: mg HDDC/m³ (analytical)

Dose / conc.:

160 other: mg HDDC/m³ (analytical)

Dose / conc.:

3.1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

10 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

31 other: mg HMD/m³

Remarks:

corresponding HMD concentration

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 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

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: No

CLINICAL CHEMISTRY: No

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 mice from the control, 16, 50, and 160 mg HDDC/m³ exposure groups. Mating trials were performed on supplemental mice 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, gall bladder, 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No exposure-related clinical signs were observed in male or female mice.

Mortality:

no mortality observed

Description (incidence):

All mice survived to the end of the study (Table 11, see attached file).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no exposure-related changes in body weight (Table 11, see attached file).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in organ weights and organ-weight-to-body-weight ratios in mice treated with HDDC for 13 weeks are shown in Table 12 (see attached file). A statistically significant increase occurred in the absolute and relative lung weights of female mice in the highest exposure group (160 mg/m³). Absolute and relative liver weights were significantly increased in male mice in the 2 highest exposure groups (50 and 160 mg/m³); liver-weight-to-body-weight ratios were also increased in male mice in the 5 and 16 mg/m³ exposure groups. Other changes in organ weights were not considered to be specifically related to HDDC toxicity.

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):

Exposure-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) of male and female mice in the 2 highest exposure groups (50 and 160 mg/m³). The morphology, incidence, and severity of microscopic lesions were similar for males and females; there was a dose-related increase in the incidence and severity of these lesions (Table 13; attached file).

Description (incidence and severity):

Sperm Morphology/Vaginal Cytology and Mating Trials
Administration of HDDC to mice by inhalation caused no changes in the sperm morphology parameters evaluated, with the exception of an increase in sperm motility in the 16 and 160 mg/m³ exposure groups. However, this change was not dose related, and the values for sperm motility were all well within the range for historical controls for NTP studies. Consequently, the increase in sperm motility was not interpreted as an adverse effect.

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

16 mg/m³ air (nominal)

Based on:

other: HDDC

Sex:

male/female

Basis for effect level:

other: systemic toxicity

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

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

10 mg/m³ air (nominal)

Based on:

other: corresponding HMD concentration

Sex:

male/female

Basis for effect level:

other: systemic toxicity

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

Critical effects observed:

not specified

Executive summary:

In a 13-week inhalation study, 10 mice 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 mice (mating trial animals) at each exposure level were included to assess the effect of HDDC on reproduction. All mice in the base-study groups survived to the end of the studies, and there were no exposure-related changes in body weight. In the mating trials, 3 female mice exposed to 16 mg/m³ and 1 female and 1 male mouse exposed to 50 mg/m³ died before scheduled termination. These deaths, however, were not considered to be chemical related. In male mice in the base study, liver weights were increased relative to controls in the 2 highest exposure groups. No exposure-related changes in absolute or relative organ weights and no exposure-related clinical signs or gross lesions were seen. Chemical-related microscopic lesions in male and female mice 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 mice.

The NOAEC was 160 mg HDDC/m³ for systemic toxicity and 16 mgHDDC/m³ irritating effects, respectively or 100 HMD mg/m³ or 10 mg HMD/m³.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Sprague-Dawley-derived rats were exposed to the test compound aerosols for 6 hr/day, 5 days/week for 13 weeks at different concentrations. Body weight was determined weekly. Animals were observed at the end of each exposure period. Signs of toxicity were recorded. Indirect ophthalmoscopic examination as well as clinical laboratory and hematological determinations were conducted.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no further data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: water

Mass median aerodynamic diameter (MMAD):

1.1 µm

Geometric standard deviation (GSD):

3.9

Remarks on MMAD:

>97% were respirable (>10 µm)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass Rochester-type chambers
- System of generating particulates/aerosols:
Dry air was passed through the atomizer to generate the aerosol. The aerosolized test material was then directed into 1-m³ stainless steel and glass Rochester-type chambers and diluted with the main chamber air. Each chamber had an effective exposure volume of 760 liters and was operated dynamically at flow rates between 232 and 290 liters/ min.
- Method of particle size determination:
Particle size distribution was determined each chamber three times during Study Week 1 and once weekly thereafter using a Casella cascade impactor. Once per week the distribution of the test material was measured in each chamber. Two air samples were drawn through the glass fiber filter simultaneously, one from above and one from below the test animals. The amount of HMD collected was determined using the titrimetric analysis method. These values were compared to determine any loss of test material in the chamber.

No further data.

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber concentrations of HMD were analyzed at least three times/day. Each sample was drawn from the breathing zone of the rats at a rate of 10 liters/min for 7 min, 5 liters/min for 5 min, and 5 liters/min. for 2 min for the low, mid, and high exposure levels, respectively. The test material was collected through a Gelman glass fiber filter holder containing one AE glass fiber filter paper (1 µm pore size). The amount of HMD collected was determined by titration with 0.05 N HCI to a phenolphthalein endpoint after desorption in 0.01 N NaOH at 45°C for 5 min.
- Samples taken from breathing zone: yes

VEHICLE: water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

tritrimetry; see above, item "Generation of test atmosphere"

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week; 6 hours per day

Dose / conc.:

12.8 mg/m³ air (analytical)

Remarks:

corresponding to 0.0128 mg/L

Dose / conc.:

51 mg/m³ air (analytical)

Remarks:

corresponding to 0.051 mg/L

Dose / conc.:

215 mg/m³ air (analytical)

Remarks:

corresponding to 0.215 mg/L

Dose / conc.:

69 mg/m³ air (nominal)

Dose / conc.:

196 mg/m³ air (nominal)

Dose / conc.:

834 mg/m³ air (nominal)

No. of animals per sex per dose:

15 males and 15 females per group

Control animals:

other: exposure to "0 mg/m³"; not further specified

Details on study design:

Rats were exposed to target concentrations of 0, 12, 50, or 200 mg/m³ 6 hr/day, 5 days/week for 13 weeks.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at the end of each exposure period and signs of toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighted prior to study and weekly during the test period.

FOOD CONSUMPTION and FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Rats underwent indirect ophthalmoscopic examination pretest and after 13 weeks of exposure.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pretest and after 5 weeks of treatment.
- Anaesthetic used for blood collection: No data
- Animals fasted: no data
- How many animals: five animals/sex/group
- Parameters examined:
-- hemoglobin(HGB), hematocrit (HCT), erythrocyte count (RBC),and total leukocyte (WBC) count
-- calcium, potassium, glucose (GLU), blood urea nitrogen (BUN), total protein, albumin, globulin (by difference), total cholesterol, direct and total bilirubin, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase, and serum lactic
dehydrogenase.
The same parameters were measured in all rats surviving until termination.
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At termination of the study, fasted rats were exsanguinated under ethyl ether anesthesia. All animals which died or were killed underwent a gross necropsy. Organ weights relative to brain and body weights were calculated for the following organs from survivors at study termination (Week 13): adrenal glands, brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, and thyroid/parathyroid glands. Representative samples of the following tissues were taken from each animal: abdominal aorta, adrenals, bone and marrow, brain (three sections), esophagus, eyes with contiguous Harderian gland and optic nerve, ovaries, testes with epididymides, heart, intestine (five sections), kidneys, liver (two sections), lungs with mainstem bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, nasal passages (two sections) including paranasal sinuses, pituitary, prostate, salivary glands, sciatic nerve with muscle, seminal vesicles, skin, spinal cord (two sections), spleen, stomach (two sections), thymus, trachea, thyroid/parathyroids, urinary bladder, uterus, vagina, and all grossly observable lesions. Tissue sections were prepared by conventional histopathologic techniques, stained with hematoxylin and eosin, and examined by light microscopy.

Statistics:

Body weights, hematology values, clinical laboratory parameters, and organ weights were statistically evaluated by a test for analysis of variance, and, where appropriate, by the necessary tests for multiple comparison. A minimum level of significance of p < 0.05 was used throughout.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of respiratory and conjunctival irritation were observed in rats at both the 51 and 215 mg/m³ exposure levels.

Mortality:

mortality observed, treatment-related

Description (incidence):

Because of exposure-related deaths in the 215 mg/m³ group, this group was terminated during the seventh week of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was significantly reduced in both sexes exposed to 215 mg/m³.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the 5-week study interval, slight hemopoietic stimulation of peripheral blood parameters was observed in rats of both sexes exposed to 215 mg/m³.

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects were reported with regard to ovaries and testes organ weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic lesions were seen only in rats exposed to 215 mg/m³ and were confined to the trachea, nasal passages, and lungs. No effects were reported with regard to to the microscopic evaluation of testes, epididymides, mammary gland, prostate, seminal vesicles, ovaries, uterus and vagina.

Details on results:

For details, see attached files.

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

12.8 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: respiratory and conjunctival irritation

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

0.013 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: respiratory and conjunctival irritation

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

51 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: mortality, reduced body weight gain

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.051 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: mortality, reduced body weight gain

Mean nominal atmospheric concentrations ±SD of HMD achieved in the three treated groups in this study were 69 ± 13.5, 196 ± 7.1, and 834 ± 270 mg HMD/m³.

Rats were exposed to cumulative mean analytical chamber concentrations ±SD of 0, 12.8 ± 2.5, and 51 ± 9 mg/m³ HMD for 13 weeks. An additional group of rats exposed to 215 ± 62 mg/m³ HMD was terminated during the seventh week.

Executive summary:

Four groups of 15 male and 15 female Sprague-Dawley-derived (CD) rats each were exposed to aqueous hexamethylenediamine (HMD) aerosols for 6 hr/day, 5 days/week for 13 weeks at mean analytical concentrations of 0, 12.8, or 51 mg/m³. Because of exposure-related deaths in a group of male and female rats similarly exposed to 215 mg/m³ HMD, this group was terminated during the seventh week of the study. Signs of respiratory and conjunctival irritation were observed in rats at both the 51 and 215 mg/m³ HMD test levels. Body weight gain was significantly reduced in both sexes exposed to 215 mg/m³ HMD. At the 5-week study interval, slight hemopoietic stimulation of peripheral blood parameters was observed in rats of both sexes exposed to 215 mg/m³ HMD. Treatment-related microscopic lesions were seen only in rats exposed to 215 mg/m³ and were confined to the trachea, nasal passages, and lungs.

The no effect level in this study is considered to be 51 mg/m³ HMD.

Endpoint conclusion

Dose descriptor:

NOAEC

100 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Sprague-Dawley-derived rats were exposed to the test compound aerosols for 6 hr/day, 5 days/week for 13 weeks at different concentrations. Body weight was determined weekly. Animals were observed at the end of each exposure period. Signs of toxicity were recorded. Indirect ophthalmoscopic examination as well as clinical laboratory and hematological determinations were conducted.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no further data

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: water

Mass median aerodynamic diameter (MMAD):

1.1 µm

Geometric standard deviation (GSD):

3.9

Remarks on MMAD:

>97% were respirable (>10 µm)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass Rochester-type chambers
- System of generating particulates/aerosols:
Dry air was passed through the atomizer to generate the aerosol. The aerosolized test material was then directed into 1-m³ stainless steel and glass Rochester-type chambers and diluted with the main chamber air. Each chamber had an effective exposure volume of 760 liters and was operated dynamically at flow rates between 232 and 290 liters/ min.
- Method of particle size determination:
Particle size distribution was determined each chamber three times during Study Week 1 and once weekly thereafter using a Casella cascade impactor. Once per week the distribution of the test material was measured in each chamber. Two air samples were drawn through the glass fiber filter simultaneously, one from above and one from below the test animals. The amount of HMD collected was determined using the titrimetric analysis method. These values were compared to determine any loss of test material in the chamber.

No further data.

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber concentrations of HMD were analyzed at least three times/day. Each sample was drawn from the breathing zone of the rats at a rate of 10 liters/min for 7 min, 5 liters/min for 5 min, and 5 liters/min. for 2 min for the low, mid, and high exposure levels, respectively. The test material was collected through a Gelman glass fiber filter holder containing one AE glass fiber filter paper (1 µm pore size). The amount of HMD collected was determined by titration with 0.05 N HCI to a phenolphthalein endpoint after desorption in 0.01 N NaOH at 45°C for 5 min.
- Samples taken from breathing zone: yes

VEHICLE: water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

tritrimetry; see above, item "Generation of test atmosphere"

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days per week; 6 hours per day

Dose / conc.:

12.8 mg/m³ air (analytical)

Remarks:

corresponding to 0.0128 mg/L

Dose / conc.:

51 mg/m³ air (analytical)

Remarks:

corresponding to 0.051 mg/L

Dose / conc.:

215 mg/m³ air (analytical)

Remarks:

corresponding to 0.215 mg/L

Dose / conc.:

69 mg/m³ air (nominal)

Dose / conc.:

196 mg/m³ air (nominal)

Dose / conc.:

834 mg/m³ air (nominal)

No. of animals per sex per dose:

15 males and 15 females per group

Control animals:

other: exposure to "0 mg/m³"; not further specified

Details on study design:

Rats were exposed to target concentrations of 0, 12, 50, or 200 mg/m³ 6 hr/day, 5 days/week for 13 weeks.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at the end of each exposure period and signs of toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighted prior to study and weekly during the test period.

FOOD CONSUMPTION and FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Rats underwent indirect ophthalmoscopic examination pretest and after 13 weeks of exposure.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pretest and after 5 weeks of treatment.
- Anaesthetic used for blood collection: No data
- Animals fasted: no data
- How many animals: five animals/sex/group
- Parameters examined:
-- hemoglobin(HGB), hematocrit (HCT), erythrocyte count (RBC),and total leukocyte (WBC) count
-- calcium, potassium, glucose (GLU), blood urea nitrogen (BUN), total protein, albumin, globulin (by difference), total cholesterol, direct and total bilirubin, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase, and serum lactic
dehydrogenase.
The same parameters were measured in all rats surviving until termination.
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At termination of the study, fasted rats were exsanguinated under ethyl ether anesthesia. All animals which died or were killed underwent a gross necropsy. Organ weights relative to brain and body weights were calculated for the following organs from survivors at study termination (Week 13): adrenal glands, brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, and thyroid/parathyroid glands. Representative samples of the following tissues were taken from each animal: abdominal aorta, adrenals, bone and marrow, brain (three sections), esophagus, eyes with contiguous Harderian gland and optic nerve, ovaries, testes with epididymides, heart, intestine (five sections), kidneys, liver (two sections), lungs with mainstem bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, nasal passages (two sections) including paranasal sinuses, pituitary, prostate, salivary glands, sciatic nerve with muscle, seminal vesicles, skin, spinal cord (two sections), spleen, stomach (two sections), thymus, trachea, thyroid/parathyroids, urinary bladder, uterus, vagina, and all grossly observable lesions. Tissue sections were prepared by conventional histopathologic techniques, stained with hematoxylin and eosin, and examined by light microscopy.

Statistics:

Body weights, hematology values, clinical laboratory parameters, and organ weights were statistically evaluated by a test for analysis of variance, and, where appropriate, by the necessary tests for multiple comparison. A minimum level of significance of p < 0.05 was used throughout.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of respiratory and conjunctival irritation were observed in rats at both the 51 and 215 mg/m³ exposure levels.

Mortality:

mortality observed, treatment-related

Description (incidence):

Because of exposure-related deaths in the 215 mg/m³ group, this group was terminated during the seventh week of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was significantly reduced in both sexes exposed to 215 mg/m³.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the 5-week study interval, slight hemopoietic stimulation of peripheral blood parameters was observed in rats of both sexes exposed to 215 mg/m³.

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects were reported with regard to ovaries and testes organ weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic lesions were seen only in rats exposed to 215 mg/m³ and were confined to the trachea, nasal passages, and lungs. No effects were reported with regard to to the microscopic evaluation of testes, epididymides, mammary gland, prostate, seminal vesicles, ovaries, uterus and vagina.

Details on results:

For details, see attached files.

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

12.8 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: respiratory and conjunctival irritation

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

0.013 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: respiratory and conjunctival irritation

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

51 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: mortality, reduced body weight gain

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.051 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: mortality, reduced body weight gain

Mean nominal atmospheric concentrations ±SD of HMD achieved in the three treated groups in this study were 69 ± 13.5, 196 ± 7.1, and 834 ± 270 mg HMD/m³.

Rats were exposed to cumulative mean analytical chamber concentrations ±SD of 0, 12.8 ± 2.5, and 51 ± 9 mg/m³ HMD for 13 weeks. An additional group of rats exposed to 215 ± 62 mg/m³ HMD was terminated during the seventh week.

Executive summary:

Four groups of 15 male and 15 female Sprague-Dawley-derived (CD) rats each were exposed to aqueous hexamethylenediamine (HMD) aerosols for 6 hr/day, 5 days/week for 13 weeks at mean analytical concentrations of 0, 12.8, or 51 mg/m³. Because of exposure-related deaths in a group of male and female rats similarly exposed to 215 mg/m³ HMD, this group was terminated during the seventh week of the study. Signs of respiratory and conjunctival irritation were observed in rats at both the 51 and 215 mg/m³ HMD test levels. Body weight gain was significantly reduced in both sexes exposed to 215 mg/m³ HMD. At the 5-week study interval, slight hemopoietic stimulation of peripheral blood parameters was observed in rats of both sexes exposed to 215 mg/m³ HMD. Treatment-related microscopic lesions were seen only in rats exposed to 215 mg/m³ and were confined to the trachea, nasal passages, and lungs.

The no effect level in this study is considered to be 51 mg/m³ HMD.

Reference 2

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

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

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

Dose / conc.:

1.6 other: mg HDDC/m³ (analytical)

Dose / conc.:

5 other: mg HDDC/m³ (analytical)

Dose / conc.:

16 other: mg HDDC/m³ (analytical)

Dose / conc.:

50 other: mg HDDC/m³ (analytical)

Dose / conc.:

160 other: mg HDDC/m³ (analytical)

Dose / conc.:

1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

3.1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

10 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

31 other: mg HMD/m³

Remarks:

corresponding HMD concentration

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

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.

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.

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

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

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

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

Critical effects observed:

not specified

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.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

males: 1987-06-10 to 1986-09-10; females: 1987-06-09 to 1987-09-09 (dosing period)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and female B6C3F1 mice
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 20.6 - 24.8 g (males); 17.9 - 19.9 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

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

Dose / conc.:

1.6 other: mg HDDC/m³ (analytical)

Dose / conc.:

5 other: mg HDDC/m³ (analytical)

Dose / conc.:

16 other: mg HDDC/m³ (analytical)

Dose / conc.:

50 other: mg HDDC/m³ (analytical)

Dose / conc.:

160 other: mg HDDC/m³ (analytical)

Dose / conc.:

3.1 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

10 other: mg HMD/m³

Remarks:

corresponding HMD concentration

Dose / conc.:

31 other: mg HMD/m³

Remarks:

corresponding HMD concentration

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 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

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: No

CLINICAL CHEMISTRY: No

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 mice from the control, 16, 50, and 160 mg HDDC/m³ exposure groups. Mating trials were performed on supplemental mice 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, gall bladder, 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.

Clinical signs:

no effects observed

Description (incidence and severity):

No exposure-related clinical signs were observed in male or female mice.

Mortality:

no mortality observed

Description (incidence):

All mice survived to the end of the study (Table 11, see attached file).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no exposure-related changes in body weight (Table 11, see attached file).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in organ weights and organ-weight-to-body-weight ratios in mice treated with HDDC for 13 weeks are shown in Table 12 (see attached file). A statistically significant increase occurred in the absolute and relative lung weights of female mice in the highest exposure group (160 mg/m³). Absolute and relative liver weights were significantly increased in male mice in the 2 highest exposure groups (50 and 160 mg/m³); liver-weight-to-body-weight ratios were also increased in male mice in the 5 and 16 mg/m³ exposure groups. Other changes in organ weights were not considered to be specifically related to HDDC toxicity.

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):

Exposure-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) of male and female mice in the 2 highest exposure groups (50 and 160 mg/m³). The morphology, incidence, and severity of microscopic lesions were similar for males and females; there was a dose-related increase in the incidence and severity of these lesions (Table 13; attached file).

Description (incidence and severity):

Sperm Morphology/Vaginal Cytology and Mating Trials
Administration of HDDC to mice by inhalation caused no changes in the sperm morphology parameters evaluated, with the exception of an increase in sperm motility in the 16 and 160 mg/m³ exposure groups. However, this change was not dose related, and the values for sperm motility were all well within the range for historical controls for NTP studies. Consequently, the increase in sperm motility was not interpreted as an adverse effect.

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

16 mg/m³ air (nominal)

Based on:

other: HDDC

Sex:

male/female

Basis for effect level:

other: systemic toxicity

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

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

10 mg/m³ air (nominal)

Based on:

other: corresponding HMD concentration

Sex:

male/female

Basis for effect level:

other: systemic toxicity

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

Critical effects observed:

not specified

Executive summary:

In a 13-week inhalation study, 10 mice 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 mice (mating trial animals) at each exposure level were included to assess the effect of HDDC on reproduction. All mice in the base-study groups survived to the end of the studies, and there were no exposure-related changes in body weight. In the mating trials, 3 female mice exposed to 16 mg/m³ and 1 female and 1 male mouse exposed to 50 mg/m³ died before scheduled termination. These deaths, however, were not considered to be chemical related. In male mice in the base study, liver weights were increased relative to controls in the 2 highest exposure groups. No exposure-related changes in absolute or relative organ weights and no exposure-related clinical signs or gross lesions were seen. Chemical-related microscopic lesions in male and female mice 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 mice.

The NOAEC was 160 mg HDDC/m³ for systemic toxicity and 16 mgHDDC/m³ irritating effects, respectively or 100 HMD mg/m³ or 10 mg HMD/m³.

Endpoint conclusion

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subchronic

Species:

rat

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

Oral

AH salt, as an aqueous preparation (48-50 %,w/w), was tested in a 28-day gavage study in Sprague-Dawley rats at dose levels of 0, 200, 1000, and 5000 mg/kg bw/day. 10 animals per sex/dose were used. The highest dose level caused the death or sacrifice in extremis of 10/10 males within 5 days and 6/10 females within 14 days of exposure. The surviving females of the high-dose group had lower mean body weights than the control group at day 8, but not at day 28. 1000 and 200 mg/kg bw/day produced no effects on body weight or food consumption in either sex, nor were any significant clinical observations noted. Organ weights among test animals of both sexes that survived to final necropsy, did not differ significantly from the control group. There was no difference in absolute and relative testes weights between treated groups and controls. Histopathological changes at the toxic level of 5000 mg/kg bw/day included renal tubular degenerative changes in both sexes (5/10 males and 3/10 females) and gastric mucosal necrosis in 3/10 males. Hepatocytic necroses were found in 2 males and 2 females of the high dose groups, and in 1 control female. No changes were found in all other organs including the pituitaries, testes and ovaries. There were no significant microscopic changes among mid dose males and females. Statistically significant increases in red blood cells and hematocrit in males at the mid and low dose levels were observed. However, the value remained within the normal limits and was therefore not considered as biologically significant. The serum chemistry was not altered. The NOAEL was 1000 mg/kg bw/day (Monsanto, 1982).

 

Additional information on the repeated dose toxicity of AH salt can be deduced from studies with adipic acid. This study gave no indication of specific target organs for systemic toxicity.

 

In a chronic study rats were fed either the basal laboratory diet, or the diet to which adipic acid was added (males: 0.1, 1, 3, and 5 % equivalent to ca. 50 -100; 500 -1000; 1500 -3000; and 2500 -5000 mg/kg bw/day, respectively; 20 animals/dose and 20 controls; females: 1% equivalent to ca. 500 -1000 mg/kg bw/day, 19 animals/dose group, 10 controls). After 2 years, surviving rats were weighed, killed, and examined grossly. Ten organ weights were recorded for approximately half of each group of males, and 4 organ weights were recorded for females. Microscopic examination of 15 tissues was done on a representative number of animals from each group. Thus, the study does not fully comply with the guidelines for a chronic study. In male rats, there were no body weight differences throughout the 2-year period in the 0.1 or 1% exposure group. During the rapid growth period, the weight gains of the 3.0 and 5.0% adipic acid groups were significantly less than the control groups. At the end of the study the body weight of males was reduced by 10% and more in the two highest exposure groups. There was slight, but consistent, reduction in feed consumption at 5% exposure. Throughout the study, the following clinical signs were observed among all groups, including controls: wheezing, blood-tinged crust about the noses and eyes, and body sores. The incidence of these findings did not appear to be significantly different among the groups although a lower incidence of signs indicative of respiratory infection and body sores occurred in the 5% dose group. The incidence of lung pathology and tumor growth appeared to be equally distributed among all groups, including the controls. When the surviving males were sacrificed at the end of the study, there were no significant differences in organ weights or microscopic examination. In females, there were no significant differences in body weight gains or food consumption. Clinical signs noted in control and test groups included blood-tinged crust about the eyes and nose, unthriftiness, and body sores. There were no significant differences in organ weights, gross, or microscopic pathology (Horn et al, 1957). The NOAEL for adipic acid was 1% (ca. 500-1000 mg/kg bw/day).

 

Inhalation of 1,6-hexanediamine or its dihydrochloride produced lesions in the upper respiratory tracts of Sprague Dawley rats (12.8 - 215 mg/m³) or of Fischer 344 rats and B6C3F1 mice (1.6 - 160 mg/m³ of HDDC corresponding to 1 – 100 mg/m3 of HMD) that could be attributed to the nonphysiological pH of these compounds and therefore such effect was not expected for AH-salt. Significantly increased absolute and relative liver weights were seen from 50 mg/m³ of HDDC (corresponding to 31 mg/m3 of HMD) onwards, only in male mice. The no observed adverse effect concentrations (NOAECs) for local irritation in these studies were in rats 16 and 10 mg/m³ for 1,6-hexanediamine dihydrochloride and 1,6-hexanediamine, respectively. In mice, the local NOAEC was 16 mg/m³ for 1,6-hexanediamine dihydrochloride and 10 mg/m3 for HMD. The NOAECs for systemic toxicity of 1,6 hexamethylene dihydrochloride were 160 mg/m³ for rats and 16 mg/m³ for mice, respectively. For 1,6-hexanediamine the systemic NOAEC for rats was 100 mg/m³ (NTP, 1993; Johannsen et al., 1987).

 

Justification for classification or non-classification

There is no need to classify AH-Salt for repeated toxicity according to the Directive 67/548/EC or EU GHS criteria (Regulation (EC) N° 1907/2006).