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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2 and 3 January 1986 (first concentration exposure) to 3-4 September 1986 (necropsy)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study comparable to OECD 413 conducted in compliance with GLP from US-FDA, no major deviation from the protocol.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
other: Toxicity report Series of the National Toxicology Program
Title:
Unnamed
Year:
1993
Reference Type:
publication
Title:
Inhalation Toxicity of 1,6-Hexanediamine Dihydrochloride in F344/N Rats and B6C3F1 Mice
Author:
Hébert C.D., Elwell M.R., Travlos G.S., Zeiger E., French J.E. and Bucher J.R.
Year:
1993
Bibliographic source:
Fundamental and Applied Toxicology, 20:348-359

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
some examinations were not performed (ophtalmoscopic examination, urinalysis, food and water consumption)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,6-hexanediamine Dihydrochloride (HDDC)*
- Molecular formula (if other than submission substance): C6H16 N2 2HCl
- Molecular weight (if other than submission substance): 189.2
- Physical state: Liquid
- Analytical purity: 101% (HDA)
- Purity test date: no data
- Lot/batch No.: PT-011882 (HDA)
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature i amber or foil-wrapped bottles
- Other:
* 1,6 hexanediamine (HDA) was purchased from E.I.DuPont (purity and batch number, see above), and was used as HDDC (see "Any other information on materials and methods" for more details)
* periodic chemical reanalyses at 4-month intervals indicated no breakdown of the chemical during storage

Test animals

Species:
rat
Strain:
other: Fischer 344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratories, Inc (Gilroy, CA)
- Age at study initiation: 6 to 7 weeks of age
- Weight at study initiation: no data
- Fasting period before study: 11 to 14 days
- Housing: individual compartments of multi-compartment stainless steel wire mesh cages.
- Diet (e.g. ad libitum): pelleted NIH-07 feed ( Zeigler Brothers, Inc, Gardners, PA) ad libitum during nonexposure periods
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 11 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 µm, with a geometric standard deviation of 1.52 to 1.53. All control chamber respirable mass concentration vaues were less than 0.005 mg/m3.
Details on inhalation exposure:
1,6-hexanediamine (HDA or HMD) was converted to HDA dihydrochloride (HDDC) by acidification with concentrated hydrochloricacid 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.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: no data
- Method of holding animals in test chamber: housed continuously in exposure chambers with chamber doors closed except during animal husbandry procedures.
- Source and rate of air:
- Method of conditioning air: no data

- System of generating particulates/aerosols: the 70% aqueous HDDC solution was placed in a 9-liter glass reservoir and pressurized with N2 gas. HDDC was delivered to 5 Sonimist Ultrasonic Spray Nozzles (Model HS600-2, Heat Systems-Ultrasonics, Inc., Farmingdale, NY) by a positive displacement metering pump. Up this point, stainless steel lines carried the test substance. The nebulizer reservoir was kept in a separate exposure chamber (H-1000, Hazelton Systems, Inc., Aberdeen, MD). This chamber served as a mixing plenum where large droplets and nonnebulized liquid were impacted or sedimented out of the test atmosphere before the aerosol was delivered to the inhalation chambers. The HDDC aerosol was mixed with compressed breathing air that had been filtered through an ENMET (ENMET Air Filtration Panel, Model AFP-82, Enmet Co., Ann Arbor, MI) and supplied at 50 psi to generate an aerosol at a concentration equal to the highest exposure concentration. The resulting aerosol was transported to the inhalation chambers through a manifold constructed of 3-o,ch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount HEPA/charcoal-filtered room air to obtain the desired test concentration, then delivered to the inhalation chamber.

- Temperature, humidity, pressure in air chamber: 21 to 27°C, 70% to 80% relative humidity
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: use an APS 3300 aerodynamic paticle sizer
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: concentrations of HDDC in the exposure chamber were monitored by measuring the forward light scatter with RAM-S real-time aerosol monitors.Spatial homogeneity of the aerosol within the exposure chambers was measured using calibrated RAM-S monitors.
- Samples taken from breathing zone: Gravimetric sampling was conducted with 25 mm glass fiber filter paper (Gelman Sciences, Inc., Ann arbor, MI). Gravimetric analysis was performed using an aerodynamic particle sizer as a Perkin Elmer AS-2Zmicrobalance (Perkin Elmer, Norwalk, CT) by weighing filters to the nearest 0.01mg before and after sampling and again after storing the filters in a desiccator overnight.

VEHICLE: the HDDC aerosol was mixed with compressed breathing air filtered through an EMMET (EMMET Air filtration Panel, Model AFP-82, Emmet Co., Ann Arbor, MI)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 (6 readings; GCA Corporation, Technology Division, Bedford, MA) and by gravimetric analyses (3 samples) of filter samples collected from each exposure. Twice montly during the 13-week study, glass fiber filter samples from each chamber were analysed by gas chromatography with flame ionization detection for total hexanediamine, using the technique supplied by Midwest Research Institue. Measured concentrations of HDDC in the exposure chambers were within 6% of the target concentrations in all samples.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours plus T90 (30 minutes) per day, 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1.6, 5, 16, 50 and 160 mg HDDC/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 1.0, 3.1, 10, 31 and 100 mg/m3 HMD mg/m3
Basis:
other: calculated from HDDC analytical concentrations
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Base on effects seen in dose range finding study. Because of the weight gain depression and the inflammation and ulceration of the nasal cavity and larynx seen in both sexes of rats at the higher concentrations used in the 13 weeks studies were 0, 1.6, 5, 16, 50, and 160 mg HDDC/m3 corresponding to 0, 1, 3.1, 10, 31 and 100 mg HMD/m3 (using HDDC molecular mass = 189.2 g/mol and HMD molecular mass = 116.2 g/mol)
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes (observation of deaths)
DETAILED CLINICAL OBSERVATIONS: yes, clinical signs for animals in the base study groups were recorded weekly
BODY WEIGHT: Yes, body weights were recorded at study start, weekly, and at the end of the study.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
Blood samples were collected from all 13-week inhalation base-study rats at the end of the study. Erythrocyte, leukocyte and platelet morphologies were evaluated during the leukocyte differential count. Methemoglobin concentrations were measured.
CLINICAL CHEMISTRY: Yes, from all animals at the end of the study.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, a complete necropsy was performed on all treated and control animals.
Other: The thymus, heart, right kidney, lungs, brain, liver, and right testis of each animal were weighed.
HISTOPATHOLOGY: Yes, all tissues from control and high-exposure groups were examined microscopically.
Other examinations:
Sperm morphology and vaginal cytology evaluations were performed on base study rats from the control and the 3 highest exposure groups (16, 50 and 160 mg/m3).
Epididymal sperm motility was evaluated at necropsy. The number of motile and non-motile sperm were counted, sperm density was determined and sperm morphology was examined. Vaginal saline lavage was performed on females for 7 consecutive days prior to scheduled termination. The relative preponderance of leukocytes, nucleated epithelial cells and large squamous epithelial cells in the lavage fluid were used to identify the stages of the estrous cycle.
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 were analysed using the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955). Clinical chemistry and hematology data were analysed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheer's test was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (William, Shirley) was more appropriate for pairwise comparisons. 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 William's test. The outlier test of Dixon and Massey (1951) was employed to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or, at most, half of the next smallest value.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
An exposure-related decrease in lymphocytes, segmented neutrophils counts and leukocytes was observed. These effects were not relevant as overall, the hematologic changes in rats were minor, sporadic and not accompanied by related clinical pathology findi
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Significant decrease of the absolute and relative lung weights of HDC-exposed rats by comparison with the lung weights of the study controls (due to inflammation) but within the normal range (historical data).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 7.5.3/1 (below)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No clinical signs of toxicity related to HDDC exposure were seen in the 13-week rat 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 occured 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.
- No mortality occurred during the study

BODY WEIGHT AND WEIGHT GAIN
The final mean body weights of most groups of rats exposed to HDDC were slightly lower than the mean body weights of the controls. These differences, however, were not statistically significant.

HAEMATOLOGY
- 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 occured in female rats in the 160 mg/m3 exposure group and male rats in the 50 mg/m3 exposure group.

CLINICAL CHEMISTRY
- Clinical chemistry changes on Day 4 included a small increase in alanine aminotransferase activity in male rats in the lowest exposure group (1.6mg/m3) and a slight increase in the urea nitrogen level in male 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).
- At day 94, alkaline phosphatase activity was slightly increased in male rats in several exposure groups (1.6, 50 and 160 mg/m3) and Sorbitol dehydrogenase activity was elevated in males in the 50 mg/m3 exposure group.

ORGAN WEIGHTS
The only consistent changes in organ weights seen in rats were decreased 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 lungs weights that were greater than those of historical controls.

GROSS PATHOLOGY
There were no gross lesions attributed to HDDC exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
Chemical-related microscopic lesions were limited to the upper respiratory tract of male and female rats in the 2 highest exposure groups. 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.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

HISTORICAL CONTROL DATA (if applicable)
Data concerning the lesions and weights of the lungs (see NTP, 1990)

OTHER FINDINGS
Sperm morphology and vaginal cytology examinations did not reveal any compound-related abnormalities.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
(HDDC)
Effect level:
16 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology: nose/nasal passages as respiratory epithelium degeneration
Dose descriptor:
NOAEC
Remarks:
(HMD)
Effect level:
10 other: mg/m3 air (calculated from the HDDC NOAEC)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology: nose/nasal passages as respiratory epithelium degeneration

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.3/1: Incidence and Severity of Histopathologic lesions in F344/N rats

 

HDDC (mg/m3)

0

1.6

5.0

16

50

160

Male

 

Larynx

Inflammation

Erosion/Ulcer

Hyperplasia

 

1 (2.0)

0

0

 

0

0

0

 

0

0

0

 

0

0

0

 

2 (1.0)

0

0

 

7 (1.4)

2 (2.0)

1 (1.0)

Nose/Nasal Passages

Respiratory Epithelium

Degeneration

Erosion/Ulcer

Inflammation

Squamous metaplasia

 

Olfactory epithelium

Degeneration

Erosion/Ulcer

Inflammation

 

 

0

0

2 (1.0)

0

 

 

0

0

0

 

 

0

0

0

0

 

 

0

0

0

 

 

0

0

0

0

 

 

0

0

0

 

 

0

0

2 (1.0)

0

 

 

0

0

0

 

 

3 (1.0)

0

2 (1.0)

0

 

 

1 (1.0)

0

0

 

 

10 (2.0)

2 (1.0)

5 (1.4)

4 (1.2)

 

 

10 (2.1)

0

1 (1.0)

Female

 

Larynx

Inflammation

Erosion/Ulcer

Hyperplasia

 

2 (2.0)

0

0

 

0

0

0

 

0

0

0

 

0

0

0

 

0

0

0

 

5(2.6)

4 (2.0)

1 (3.0)

Nose/Nasal Passages

Respiratory Epithelium

Degeneration

Erosion/Ulcer

Inflammation

Squamous metaplasia

 

Olfactory epithelium

Degeneration

Erosion/Ulcer

Inflammation

 

 

0

0

4 (1.5)

0

 

 

1 (1.0)

0

1 (1.0)

 

 

0

0

0

0

 

 

0

0

0

 

 

0

0

0

0

 

 

0

0

0

 

 

1 (1.0)

0

6 (1.7)

0

 

 

0

0

0

 

 

4 (1.2)

1 (1.0)

8 (1.5)

1 (1.0)

 

 

0

0

0

 

 

8 (1.8)

4 (1.5)

8 (1.6)

4 (1.0)

 

 

9 (2.2)

0

0

1n=10 for all groups. The average severity score ( ) was based on the number of animals with lesions from each group; 1=minimal, 2=mild, 3=moderate, 4=marked.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEC (HDDC) = 16 mg/m3/day for local respiratory damage (nasal respiratory epithelium degeneration), corresponding to a NOAEC (HMD) = 10 mg/m3/day
Executive summary:

A 13-week study of the toxicity of the dihydrochloride salt of HMD (HDDC) was conducted in male and female Fischer 344/N rats using whole body inhalation exposure (NTP, 1993). The method followed was comparable to OECD 413 and was conducted according to the US-EPA GLP which were similar to the EU-GLP.

HDDC has the same organic backbone as HDA and its use would allow detection of any specific toxicity associated with that backbone while avoiding the causticity and the stability problems that would be encountered with the use of HDA. Therefore, the use of HDDC aerosol is relevant to study the toxicity of the HDA aerosol in a read-across strategy.

Animals were exposed to HDDC aerosol at concentrations of 0, 1.6, 5, 16, 50 and 160 mg HDDC/m3 for 6 hours plus T90 (30 minutes) per day, 5 days/week for 13 weeks corresponding to 0, 1.0, 3.1, 10, 31 and 100 mg HMD /m3. The aerosol mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 µm, with a geometric standard deviation of 1.52 to 1.53. All control chamber respirable mass concentration vaues were less than 0.005 mg/m3. Mortality, clinical signs, body weights, hematology, clinical chemistry, sperm morphology, vaginal cytology, organ weight, microscopic and macroscopic examinations were recorded.

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

No treatment related changes in body weight.

The only consistent changes in organ weights seen in rats were significant decreases in absolutes and relative lung weights of HDDC-exposed rats by comparison with the lung weights of the study controls. However, the apparent reduction was considered to be an artefact resulting from the greater lung weights of the control due to inflammation. Hence, the lung weights of the exposed rats were well within the normal range (historical data of the NTP).

By day 94, a dose-related decrease in lymphocytes and leucocytes was observed in females at both highest doses while an increased hematocrit values were noted at both lowest dose. A statistically significative decrease in segmented neutrophil counts was also observed in females exposed from 16 mg HDDC/m3 (slight decrease) to 160 mg HDDC/m3/d(moderate decrease). A slight decrease in erythrocyte count was noted in males rats in the 16 mg HDDC/m3 exposure group while a minor increase in mean cell hemoglobin values occurred only in males exposed at 50 mg HDDC/m3. Hence, the hematological changes were minor and/or sporadic without any related histopathological finding. Moreover, the overall decrease in the leukocyte count, in the absence of evidence of bone marrow toxicity, was consistent with the inflammatory changes in the larynx and nasal passages inducing increased margination to tissues or the marginal pool, a decreased release to circulation or increased destruction. Therefore, these effects were considered as a secondary effect of the HMD related respiratory inflammation.

Some clinical chemistry changes in rats (such as urea nitrogen on day 18 in both sexes) were not considered as biologically significant since these effects were minor and/or sporadic and were not accompanied by any related histological finding and were probably related to causes such as dehydration, increased protein catabolism, diet.

There were no gross lesions attributed to HDDC exposure. No treatment related changes in organ weight. excepted in Chemical related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in both highest exposure groups (50 and 160 mg HDDC/m3) of both sexes without difference in the morphology, incidence and severity of microscopic lesions between the sexes. These lesions included minimal to mild erosion, ulceration, inflammation and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory (focal areas with thinning of the olfactory epithelial layer) and respiratory nasal epithelium. A minimal degeneration of the respiratory nasal epithelium was observed at 16 mg/m3 HDDC exposure in one animal. HDDC caused no significant changes in sperm morphology or vaginal cytology parameters.

Based on the results observed in the rat after subchronic inhalation of HDDC, the NOAEC for both males and females was 16 mg HDDC/m3 for local respiratory damage, corresponding to 10 mg HMD/mg3.

This subchronic study by inhalation is considered as acceptable. It does satisfy the guideline requirement for a 13-week repeated dose toxicity study for a OECD 413 guideline in the rat. Hence, this study can be considered as valide for classification.

Under the test conditions, no adverse effects were observed at HDDC concentrations up to 16 mg/m3 corresponding to 10 mg HDA/m3 .