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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 6th, 1984 to June 6th, 1985
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
No information on the purity, the source and age of the animals, the period of acclimation, on housing and environmental conditions, no analytical method used to monitor the concentration of test material and how the dose was selected
GLP compliance:
[not mandatory at the time of the study]
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Name of test material (as cited in study report): tetramethylhexamethylene diamine
- Physical state: Liquid
- Stability under test conditions: Unlimited in closed container
- Storage condition of test material: Room temperature

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
- Source: Charles River, Kingston, New York
- Age at study initiation: No indicated
- Weight at study initiation: 130-180 g
- Fasting period before study: Animals had no access to food or water during exposure.
- Housing: During non-exposure periods, rats were housed singly in suspended stainless steel cages, During exposure animals were housed individually in plastic tubes.
- Diet (e.g. ad libitum): Purtna Rodent Chow #5001 ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

- Temperature and humidity were recorded hourly during the exposure.
- Light cycle 12 hours light/12 hours darkj

IN-LIFE DATES: From: December 12th, 1984 To: December 21st, 1984

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
head only
Details on inhalation exposure:
- Exposure apparatus: DeVilbiss Nebuliser fed through teflon tubling by a plastic syringe on a Sage Pump Model 355
- Method of holding animals in test chamber: Head-only
- Source and rate of air: 10L/min
- Method of particle size determination: Andersen Cascade Impactor

- Brief description of analytical method used: Samples were taken at hourly intervals and analysed by a gravimetric technique. The airborne aerosol concentration was calculated by dividing the weight of the material collected on a Millipore microfiber glass filter paper in a millipore filter paper holder, by the volume of air sampled (liters).
Analytical verification of test atmosphere concentrations:
Gravimetric technique
Duration of exposure:
4 h
0.41 ( ± 0.15) mg/L
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:toxic effects were noted twice daily from day 1 to 14. Prior to exposure animals were observed and detailed observations were recorded. Body weights were recorded for Groups Ia and IIA on days 0, 1 and 4. Groups Ib and IIb were recorded on days 0, 1, 4, 7, 11 and 14.
- Necropsy of survivors performed: yes/no
- Other examinations performed: Neurological screening was carried out on days 0, 1, 4, 7 and 14 for Group Ib and IIB and on days 0, 1, 4 for Groups Ia and IIA. Groups Ia and IIa were satellite groups included to allow assessment of posisble reversible (by days 14) histopathological changes. Groups Ia and IIa were sacrificed on day 4 and groups Ib and IIb were sacrificed 14 days after exposure. Microscopic examination was carried out on selected tissues from the brain, cervical spinal cord, trachea, lungs, thymus., kidneys and spleen from all anmals, and a small number of other tIssues tound to have apparent macrospcopic lesions at necropsy.
The analysis of variance were used to compare the treated group versus the control group s for body weight changes or organ weight changes. The Duncan's procedure is used if F for analysis of variance is significantly high to delineate which groups differ from the controls. If Bartlett's test indicated heterogeneous variance, the F max was used for each group versus the control. If these individual F max tets were not significant or if N1=N2 the Cochran t-test or the Wilcoxon Rank Suan test were used. Contingency table-type data were analyzed by Fischer's Exact Test or RXC Chl Square Test, as appropriate.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 0.41 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 40% mortality at this exposure concentration
At 0.41 mg/L. 2/5 males and 2/5 females died.
Clinical signs:
other: The following clinical signs were reported in the treated animals yellow staining of the anal-genital region, brown staining of the anal-genital region, red or mucoid, nasal discharge. dried material around nose, eyes and/or mouth, moist rales,
laboured breathing, gasping, rapid breathing, cold to touch, rough coat, reduced activity and thinness in both sexes, with poor condition and distended abdomen observed in males only.
Body weight:
Mean body weights for treated animals of both sexes were significantly reduced compared to controls on days 1 to 7. Treated males showed a significant loss compared to control from day 1 until study termination. (day 14) while treated female showed a significant reduction compared to controls until day 7.
Gross pathology:
Treatment related gross necropsy observations included alopecia, and dried material around the nose, mouth and eyes, gaseous dilatlon of the lntestines and/or stonmach. and dark red colored lungs (discolored lungs were observed in all four spontaneous death animals).
Two males and two female rats died during the course of the study. One male showed marked lymphoid depletion of both the thymus and spleen that were deemed to be treatment related. This animal had also multifocal marked haemorrhagic gastritis. Subtle treatment related changes in the spleen and thymus were reported in Group IIa (terminated 4 days after exposure). The splenic changes were characterised by necrosis of the erythrocytic precursor cells. This was reversed by day 14 in Group IIb. Splenic extramedullary haematopoiesis was slightly increased in some rats in Group IIb. It may have been associated with a rebound phenomena secondary from a prior treatment-induced depression of erythrocytic production. Other tissue alterations were noted however these were not deemd to be treatment related.
Lymphoid depletion/necrosis of the thymus was noted in all mals and 3/5 females after 4 days post exposure but was reversed by day 14.
Other findings:
Changes in absolute thymus and spleen weight were considered statistically significant in both sexes in Group IIa when compared to control. Changes in absolute brain, liver, testes, and both kidneys weight were considered statistically significant in maless in Group IIa when compared to control. Statistically significant difference was also reported in male in absolute thymus weights in Group IIb at study termination when compared to control. Absolute thymus weights were significantly decreased in males until study termination. One set of relative kidney weights were increased.

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
40 % mortality was seen in this study at the single exposure concentration of 0.41 mg/L. Based on the results of this study, the LC50 can be estimated to fall in the range 0.5 - 1.0 mg/L. Classification of the substance in Category III for acute inhalation toxicity is therefore considered appropriate.
Executive summary:

Groups of five Fischer 344 rats/sex were exposed to a single dose of the undiluted test material for 4 hours at 0.41 mg/L TMHDA. Groups Ia (control) and IIa (treated animals) were terminated after a 4 day observation period. Groups Ib (control) and IIb (treated animals) were terminated after a 14 day observation period. The animals were observed for toxic effects twice daily. Neurological screens were performed on Days 0, 1, 4, 7 and 14 for animals in groups Ib and IIb and on days 0, 1 and 4.   Body weights were recorded on days 0, 1 and 4 for Groups Ia and IIa and on days 0, 1, 4, 7, 11 and 14 for Groups Ib and IIb. Necropsy was carried out at the end of the study on all animals. Organ weights and relative organ weights (brain, thymus, lungs with trachea, liver, spleen, each kidney and gonads) were also recorded. At necropsy, gross pathology was performed and microscopic evaluation of brain, cervical spinal cord, trachea, lungs, thymus, kidneys and spleen from all animals, and a small number of other tissues found to have apparent macroscopic lesions at necropsy. Two out of 5 males and 2 out of 5 females died during the course of the study. Local effect such as respiratory and nasal irritation was reported. Mean body weights for all treated animals were statistically significantly lower than controls between day 1 and 7 post treatment. Reduction in bodyweight gain was also noted in treated males when compared to control between day 1 and study termination. Females also showed lower bodyweight gain until day 7. At gross pathology and microscopic evaluation, reversible effects on the spleen and thymus were noted. These conclusions were based on a reduction in absolute thymus weights and necrosis of erythrocytic precursor cells (extramedullary haematopoiesis). Based on a 40% mortality at 0.41 mg/L, as a conservative approach it is considered that the LC50 is between 0.5 and 1 mg/L and therefore the substance is classified as "Toxic if inhaled" and is assigned H331.