Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 AUG 1985 to 8 NOV 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: comparable to guidelines
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
but only minor deviations e.g. slightly elevated humidity and temperature during exposure phase in comparison to recommendations given in the technical guidance, some examinations were not performed (urinalysis, food and water consumption)
GLP compliance:
yes
Remarks:
according to US EPA GLP Standards
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
bis(6-aminohexyl)amine; hexane-1,6-diamine
EC Number:
907-605-7
Cas Number:
68815-47-4
Molecular formula:
C6H16N2 (HMD) C12H29N3 (BHT)
IUPAC Name:
bis(6-aminohexyl)amine; hexane-1,6-diamine
Details on test material:
- Name of test material (as cited in study report): BHMT mixture; Bis(Hexamethylene)triamine
- Substance type: black liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 54 days
- Weight two days before study initiation: males: 227-337 g; females: 136-199 g
- Housing: individually housed
- Diet: Pzrina Laboratory Certified Rodent chow #5002 (Ralston Purina Company, St. Louis, Missouri, USA), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.5
- Humidity (%): 35-60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD: 2.36-6.32 micrometers (with only two of the 15 values being above 3.96)
% of particles with less than 10 micrometers: 64.8-96.9 (with only two of the values being below 82.9)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: four chambers of 1.75 cubic meter New York University-type glass and stainless steel chambers with pyramidal tops and bottoms
- Method of holding animals in test chamber: individually in wire mesh cages suspended within the chamber (weekly rotation of position in order to guarantee that all animals receive similar exposure concentrations)
- System of generating aerosols: This was achieved by delivering a metered rate of test material (diluted in water) via a Harvard Apparatus Compact infusion pump through a plastic capillary tube into a nebulizer.
- Temperature, humidity, pressure in air chamber: 22.8-28.4 °C, 29.3-82.3%, normal pressure
- Air flow rate: 340 l/min
- Method of particle size determination: gravimetrically via a non-viable, nine-stage Andersen Impactor (Andersen Samplers Inc., 4215-C Wendell Dr., Atlanta, Georgia 30336)

TEST ATMOSPHERE
- Brief description of analytical method used: Test material concentrations in air were measured three to four times daily for each treatment chamber by collection on impingers containing 2-propanol. Analytical determinations of BHMT in the 2-propanol samples were done by gas chromatography.
- Samples taken from breathing zone: no, but chamber distribution measurements were also performed at periodic intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analyses of the test material were performed before, during, and after the study. These were done by comparing peak areas of gas chromatograms obtained from the test material and analytical grade BHMT (approximately 99% pure) that was kept frozen in the dark.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
once daily at 5 days/week for 13 weeks (i.e. totals 65 exposures)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 10, 31 and 62 mg/m³
Basis:
other: analytical concentration of BHMT (which accounts for 63.25% of the test material)
Remarks:
Doses / Concentrations:
0, 15.8, 49 and 98 mg/m³
Basis:
other: calculated to the submission substance (i.e. 100% )
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
- Dose selection rationale: based on results of range finding study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (before and after exposure and at comparable times on non-exposure days)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure began and at study week 12 (exposure day 58)
- Dose groups that were examined: in the beginning all animals were examined, at day 58 only control and high-dose animals were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study, before sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight, water available)
- How many animals: all
- Parameters checked: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, differential leukocyte count, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets and reticulocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study, before sacrifice
- Animals fasted: Yes (overnight, water available)
- How many animals:
- Parameters checked: alkaline phosphatase, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, total protein, glucose, albumin, globulin, creatinine, cholesterol and serum electrolytes (i.e. sodium, potassium, phosphorus, calcium and chloride).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following list of tissues were taken from all surviving animals and microscopically examined after processing on the control and high dose groups. For the low and mid dose groups, nasal passages, trachea and lungs were also examined microscopically.
Abdominal Aorta
Adrenals
Bone (femur)
Bone marrow (femur)
Brain (longitudinal section)
Diaphragm
Esophagus
Eyes (with optic nerve)
Gonades (Ovaries, Testes with epididymides)
Heart
Intestine (Duodenum, Colon, Ileum)
Kidneys (2)
Liver (2 sections, at least 2 lobes and mainstem bronchi)
Lymph nodes (mesenteric & thymic)
Mammary gland
Nasal passages (3 sections)
Pancreas
Pituitary
Prostate
Salivary gland (submandibular)
Sciatic nerve
Skeletal muscle
Skin
Spinal cord (thoracolumbar)
Spleen
Stomach (longitudinal)
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus (corpus and cervix)
Vagina
Gross lesions
Other examinations:
The following organ weights were determined at the end of the study: adrenals (together), brain, heart, kidneys (together), liver, spleen, testes with epididymides (together)
Statistics:
Body weight (weekly) and organ weight (absolute and relative to body and brain weights at terminal sacrifice) data were analyzed using a one-way analysis of variance (ANOVA) and Dunnett's (two-tailed) test to detect significant differences between means of treatment groups compared to the respective control group.
Clinical chemistry data were analyzed using Dunnett's test only. Bartlett's test was used to assess the variability of these data.
Statistical analysis of organ-to-body weight ratios were performed using the Mann-Whitney Test with Bonferroni's Inequality Procedure. Differences in the frequency of the lesions were determined using Fisher's exact test with Bonferroni's Inequality Procedure.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no animals died; high dose animals showed respiratory wheezing, and mostly in females at all exposure levels a discolouration of the fur was noted
Mortality:
mortality observed, treatment-related
Description (incidence):
no animals died; high dose animals showed respiratory wheezing, and mostly in females at all exposure levels a discolouration of the fur was noted
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in males of the high exposure level group the mean body weights were significantly reduced beginning in study week 2 (approx. 9-17%), in females also reduced starting from week 2, but differences to control group were not always signifcant (approx. 5-9%),
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
in the high dose females: elevated red blood cell counts with associated increases in hemoglobin and hematocrit
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
in high dose males: ALT, AST and phosphorous were elevated significantly; in high dose females: lowered serum glucose levels
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
tendency toward decreased absolute and increased rel. organ weights (particularly in high dose males) were attributed to decreaed body weights rather than a direct compound effect on the organs
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
dose related increase in incidence of pulmonary emphysema
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
related to the test item
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
related to the test item
Details on results:
Body weight:
- high dose group: In males the mean body weights were significantly reduced beginning in study week 2 (approx. 9-17%). In females mean body weights were also reduced starting from week 2, but differences to control group were not always signifcant (approx. 5-9%).
- mid dose group: In males the body weights were slightly lower than those of the control group. In females the mean body weights were comparable to the control.
- low dose group: No effects seen.

Clinical chemistry:
Effects seen in high dose males were mostly attributed to only a few animals of the group (i.e. in case of ALT: 2 animals, AST and phosphorous: 3 animals each).
Moreover all the effects seen were not substantiated by pathological data.
Other than the above mentioned differences in clinical chemistry seen at the low and mid-dose level (i.e. decreases blood urea nitrogen, increased sodium), were within normal ranges and were not considered to be treatment-related as they were not detected in the high dose animals.

Gross pathology/Histopathology:
The pulmonary emphysema noted at gross necropsy was related to the histopathological changes and may have resulted in hypoxia and associated increases in red blood cell production.

Lesions associated with treatment were restricted to the respiratory tract. Three target organs were affected;
the nasal passages, trachea and lungs
all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium. The incidence and severity of the lesions was increased as a function of higher exposure levels, being most notable in the two highest dose groups and much less severe at the low dose level.

Effect levels

Dose descriptor:
LOAEC
Effect level:
15.8 mg/m³ air (analytical)
Based on:
other: submission substance
Sex:
male/female
Basis for effect level:
other: lesions of the respiratory tract (nasal passages, trachea and lungs all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results from pathological section affecting the respiratory tract

1) Gross necropsy

            males (n=12/dose group)           females (n= 12/dose group)
   control low dose  mid dose  high dose   control low dose  mid dose  high dose 
 lung emphysema 0  2  6  10  0  1  9  10 

2) microscopic findings

     males (n=12/dose group)                    females (n=12/dose group)
     control low dose  mid dose  high dose   control low dose  mid dose  high dose 
 lung  peribronchial lymphoid hyperplasia/ cuffing  1  2  2  0  -  -  -  -
   interstitial pneumonia  1  0  1  2  1  2  2  1
   accumulation of alveolar macrophages  1  0  9*  9*  0  0  7*  12*
   emphysema  0  2  7*  12*  0  0  7*  12*
   bronchiointerstitial pneumonia, chronic  0  0  4  10*  0  0  3  12*
   squamous metaplasia (bronchi or bronchioles)  0  6  11* 10*   0  3  6  11*
   bronchioal ulceration, chronic  0  0  1  9*  0  0  0  4
   haemorrhage, acute  1  1  0  1  2  0  0  1
   hypertrophy with vacuolar change, bronochial epithelium  0  9*  10*  7*  0  11* 12*   11*
   bronchiolitis obliterans  0  0  1  1  0  0  0  1
     foamy basophilic material within bronchi/bronchioles/alveoli  0  1  6  7*  0  0  5  11*
 nose/ turbinates  inflammation, mixed mucosal to submucosal 2  6   8  10*  -  -  -  -
   squamous metaplasia  5  8  12*  12*  1  5  10*  12*
   epithelial hyperplasia  0  6  12*  9*  0  3  10*  10*
   hypertrophy with vacuolar change/respiratory epithelium  0  11*  12*  12* 0   9*  12*  12*
   eosinophilic granularity, olfactory epithelium  0  12*  11*  12*  0  10*  12*  12*
   ulceration  0  0  0  9*  0  0  0  9*
 trachea squamous metaplasia   0  0  9*  11*  0  1  3  11*
   hypertrophy/vacuolar change, epithelial  0  11*  12*  5  0  6  11*  9*
   inflammation, chronic, mononuclear, submucosa  0  0  1  4  1  0  0  1
   hyperplasia, submucosal gland  0  0  1  1  0  0  0  1
   foamy basophilic material in lumen  0  0  1  0  0  0  0  1

* significantly different from control using fishers exact test with bonferroni inequality

Applicant's summary and conclusion

Conclusions:
The submission substance was subjected to test subchronic toxicity in rats (equivalent to OECD 413, GLP). For 13 weeks (5 days/week, 6 h/d) the rats (12 males and 12 females/group) were exposed to aerosol containing BHMT at analytical concentrations of 0, 10, 31, and 62 mg/m³/day. This holds for 0, 15.8, 49 and 98 mg of the submission substance per m³ air.
Based on the fact that the lesions of the respiratory tract were seen throughout all exposure concentrations tested in this study the lowest observable adverse effect concentration is 15.8 mg of submission substance per cubic metre (LOAEC).
Executive summary:

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance being given as aerosol for 13 weeks (5 d/week, 6 h/d; equivalent to OECD 413, GLP). Mean analytical values for the three expsoure levels were 10, 31 and 62 mg BHMT/m3. Recalculating this BHMT exposure levels to the submission substance concentrations leads to 0, 15.8, 49 and 98 mg submission substance per cubic metre. The nominal to analytical ratios ranged from 3.2 to 5.9 which resulted from substantial deposition of test material on chamber surfaces; however chamber distribution data indicated acceptable dispersion of the aerosol. Generally, greater than 90% of the test material particles were in the respirable size range. No animals died on study. Clinical signs of toxicity were confined to respiratory wheezing in high dose animals and discoloration of fur in females. High dose animals exhibited significantly decreased body weight in comparison to control animals. Elevated red blood cell counts with associated increases in hemoglobin and hematocrit were observed in the high dose females (maybe as compensatory response to hypoxia which was secondary to the pulmonary changes/emphysema). ALT, AST and phosphorous were elevated in high dose males; high dose females had lowered serum glucose levels. Other differences in clinical chemistry seen at the low and mid-dose level were within normal ranges and were not considered to be treatment-related. In the high-dose animals, several organs showed reduced absolute weights and increased relative weights which was attributed to overall body weight reductions. Dose related increases in the incidence of pulmonary emphysema were noted at gross necropsy. Lesions associated with treatment were restricted to the respiratory tract. Three target organs were affected; the nasal passages, trachea and lungs all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium. The incidence and severity of the lesions was increased as a function of higher exposure levels, being most notable in the two highest dose groups and much less severe at the low dose level. Hypertrophy and eosinophilic granularity of the olfactory epithelium was noted at all levels of treatment and was considered to be an age-related change exacerbated by treatment. Hypertrophy, hyperplasia, squamous metaplasia and ulceration were found in the nasal passages, trachae and lungs with secondary inflammation of the ciliary epithelium. These responses were consistent with those expected for a corrosive substance; there was no evidence that these changes represented a pre-neoplastic condition. The pulmonary emphysema noted at gross necropsy was related to the histopathological changes and may have resulted in hypoxia and associated increases in red blood cell production.

CONCLUSION: Exposure of rats to the submission substance at 98 mg/m3 for thirteen weeks resulted in pathological lesions of the respiratory tract, reduced body weight, increased RBC count and pulmonary emphysema. Lesions of the respiratory tract were observed at the 15.8 and 49 mg/m3 treatment level, although with much lesser incidence and severity at the low-dose level. Therefore a no-effect level could not be determined for this study.The LOAEC is 15.8 mg/m3.