Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
It is considered justified to utilise information on this substance in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.
Reason / purpose:
read-across source
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.
Dose descriptor:
LOAEC
Remarks:
local irritation
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equal 18.7 mg/m³
Critical effects observed:
not specified
Conclusions:
In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.
Executive summary:

It is considered justified to utilise information on dimethylamine in a read across approach since, like the registered substance, it is an aliphatic amine with saturated alkyl groups. Furthermore, dimethylamine is considered to be a potential metabolite of the registered substance.

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Principles of method if other than guideline:
Male and female F-344 rats and B6C3F1 mice were exposed by inhalation to 0, 10, 50, or 175 ppm dimethylamine (DMA) for 6 hr/day, 5 days/week for 12 months. Groups of 9-10 male and female rats and mice were necropsied after 6 and 12 months of exposure.
The purpose of this study was to investigate the toxicity associated with chronic inhalation exposure of F-344 rats and B6C3F1 mice to DMA for 2 years. This report summarises the clinical and pathologic data found for the first 12-month period.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
- Source: Air Porducts and Chemicals, Inc. (Fogelsville, Pa.)
- Purity: 99.97 %

Test animals

Species:
other: rat and mouse
Strain:
other: F-344 rats and B6C3F1 mice
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River breeding laboratories, Kingston, New York, USA
- Age at study initiation: 4-8 weeks
- Housing: individually in hanging stainless steel wire mesh cages in the exposure chambers
- Diet: NIH-07 open formula diet, Ziegler Brothers, Gardners, Pa.; analyzed for contaminants by Lancaster Labs, Lancaster, Pa., ad libitum during periods of non-exposure
- Water: tap water via an automatic watering system, ad libitum during periods of non-exposure
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 - 76 °F
- Humidity (%): 45 - 65 % ( real: 35 - 74 %)
- Air: airflow of 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: n.a.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated by metering pure DMA directly from the cylinder via flowmeters (Fischer-Porter, Warminster, Pa., or Calibrating and Measuring Equipment, Inc., Manassas, Va.) into the supply air stream.
- Method of holding animals in test chamber: rats and mice were exposed and housed in 8-m3 stainless steel and glass whole body chambers operated with a dynamic airflow of approximately 2200 liters/min (HEPA-filtered room air) and at slightly subatmospheric pressure (0.2-0.3 in. of water). The cages within a rack were rotated once per week using a computer-generated randomisation procedure designed to ensure that each animal spent an equal amount of time in all areas of the chamber.

- Method of conditioning air: HEPA filter
- System of generating particulates/aerosols: DMA directly entered the air stream
- Temperature, humidity, pressure in air chamber: slightly subatmospheric pressure (0.2-0.3 in. of water).
- Air flow rate: 2200 liters/min
- Air change rate: approx. 15
- Method of particle size determination: n.a.

TEST ATMOSPHERE
- Brief description of analytical method used: IR spectrophotometer
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test atmospheres was performed four times per hour by infrared spectrometry at a wavelength of 3.5 µm and a path length of 20.25 m (MIRAN 801, Foxboro-W&s, Norwalk, Conn.). The spectrophotometer was set to zero optical density with ultra zero air containing approximately 350 ppm Co, (Ma&son Gas, Morrow, Ga.) and adjusted to approximately 50 % relative humidity. Temperature and relative humidity were recorded hourly.

The mean time-weighted average (TWA) analytical chamber concentrations for the 12 month period, derived from daily TWA concentrations, with standard deviations and ranges were
175 ppm +/- 2.0 (167-188),
50.0 ppm +/- 1.0 (45.8-54.0), and
10.0 ppm +/- 0.3 (9.0-10.8).

The average percentage ratios of analytical/nominal concentrations (+/- standard deviations) were 81 +/- 8, 87 +/- 7, and 76 +/- 15 % for the 175-, 50-, and 10-ppm chambers, respectively, thus indicating a fairly constant loss of DMA on the chamber surfaces and animals for each chamber. The distribution of the analytical concentration of DMA in the chambers was determined to be uniform within +6 % of target concentrations.
Duration of treatment / exposure:
12 month
Frequency of treatment:
5 days/week, 6 hours/day
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
175 ppm (nominal)
No. of animals per sex per dose:
95 animals
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: dose levels were selected on the basis of preceding studies. F-344 rats exposed to 175 or 250 ppm DMA (6 hr/day, 5 days) or 500 ppm DMA (6 hr/day, 3 days) had ulcerative rhinitis, severe congestion, and squamous metaplasia in the respiratory tract.These lesions were most severe in the anterior sections of the nasal passages.

Examinations

Observations and examinations performed and frequency:
The animals were observed for clinical abnormalities twice daily and were weighed once per week for the first 13 weeks, and biweekly thereafter.
Sacrifice and pathology:
After 6 and 12 months, 9-10 animals of each species, sex, and treatment group were fasted overnight and weighed just prior to necropsy. Male mice were not included in the 12-month sacrifice because of the high rate of unscheduled mortality in this group.

Animals were anesthetised with pentobarbital by i.p. injection, and blood was drawn from the heart for haematology and serum chemistry. Each animal was examined for gross abnormalities, and 45 tissues and any gross lesions were collected and placed in 10 % buffered formalin. The nasal passages were flushed, the lungs inflated, and the lumen of the gastrointestinal tract infused with formalin. The liver, kidneys, and brain were weighed. All tissues from the control and 175 ppm exposed animals and target tissues (nasal turbinates) from the 10- and 50- ppm exposed animals were processed for light microscopic examination. Tissues containing bone were placed in 10 % buffered formalin for 48 hr, desiccated, then rinsed with tap water for at least 4 hr, and replaced in formalin. Tissues were then dehydrated in graded alcohols, cleared with xylene, and embedded in paraffin wax. Transverse blocks of the nose were cut and prepared to yield histological sections at the following levels: (1) just anterior to the incisor teeth, (2) approximately one third of the distance from the posterior aspect of the incisor teeth to the incisive papilla, (3) at the incisive papilla, (4) at the crest of the second palatial ridge, and (5) at the centre of the second molar tooth. Embedded tissues were sectioned at 5 pm and stained with haematoxylin and eosin for light microscopic examination. For photography, selected tissues were removed from paraffin re-embedded in glycol methacrylate (GMA), and 2- to 3- am-thick sections were cut and stained with Lee’s methylene blue. Selected nasal sections were stained with Alcian blue at pH 2.5 for acidic mucous glycoproteins.
Other examinations:
Hematological data were collected using a Coulter S Plus II counter. Sodium and potassium determinations were performed with a flame photometer. Chloride analyses were performed with a Coming Chloride 920 M meter (Coming Scientific Inst., Mechield, Mass.). An Abbott VP clinical analyser was used for determination of serum chemistries.
Statistics:
Data for body weights, serum chemistry, haematology, and organ weights were analyzed using an analysis of variance. Dunnett’s test was employed to detect differences between control and treatment groups (Steel and Torrie, 1980). Red blood cell morphology and histopathological findings were analyzed using the Kolmorgomov-Smimov test (Daniel, 1978). In all cases, the preselected significance level was p ≤ 0.05.

Results and discussion

Results of examinations

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant loss at 175 ppm ( 90 % of control)
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
decreased platelet counts (175 ppm), increased counts of atypical lymphocytes in females (175 ppm), decreased mean red blood cell volume (females, 175 ppm)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
decreased protein (175 ppm), increased alkaline phosphatase (females, 175 ppm)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(1) LESIONS IN RESPIRATORY EPITHELIUM
The lesions in the respiratory area were most severe on the anterior septum, just posterior to the vestibule, and on the free margins of the naso- and maxilloturbinates, with lesser involvement of the lateral wall. In the 175 ppm exposure group, there was variable destruction of the anterior portions of the naso- and maxilloturbinates, and fenestration of the nasal septum. In areas of destruction of the turbinates and septum, the surface was covered by nonkeratinizing squamous epithelium. Evidence of both acute and chronic inflammatory response included focal to diffuse mucosal and submucosal infiltration of mononuclear leukocytes and some neutrophils. Exudate was minimal or absent. Other lesions included epithelial hypertrophy and hyperplasia, focal epithelial ulceration, and focal to diffuse squamous metaplasia. Goblet cell hyperplasia of mild to moderate severity was observed on the ventral aspect of the nasal septum in rats, but not in mice. Globules of eosinophilic material were observed in the respiratory epithelium of the anterior nasal passages. The globules were present in the basal half of the affected cells, and the dense, oval to round nucleus appeared to be compressed between the globules and the basement membrane. The cell type containing the globules was not identified. Small, basophilic bodies, which were laminated and presumably mineralized, with the globules in mice.
At the 50-ppm exposure level, lesions in the respiratory epithelium were minimal in both rats and mice. Changes were confined to focal squamous metaplasia on the free margins of the turbinates in mice after 6 months of exposure, and eosinophilic globules with mild inflammation and epithelial hypertrophy and hyperplasia after 12 months. A slightly higher incidence of chronic inflammation was observed in the vestibule and the respiratory epithelium of rats in the 10-ppm exposure group.

(2) LESIONS IN THE OLFACTORY REGION
DMA exposure induced a concentration-dependent destruction of the olfactory epithelium which was most severe in the middle third of the dorsal meatus, with variable involvement of the free margins of the ectoturbinates. The most widespread change, which was found consistently in DMA exposed rats and mice, was degeneration of olfactory sensory cells with variable vacuolation of the olfactory epithelium. Another lesion often observed in areas of the olfactory epithelium was characterized by accumulation of hyaline, eosinophilic material in sustentacular cells, which were often markedly hypertrophic. The eosinophilic material was also present as large globules in the overlying airway, suggesting that it may be a secretory product of the sustentacular cells. Similar material was present in the large submucosal glands at the junction of olfactory and respiratory epithelium. These lesions were almost always accompanied by atrophy of the olfactory nerves in the lamina propria. Bowman’s glands exhibited or focal hyperplasia. In the more severely affected cases, olfactory epithelium was replaced by well-differentiated, ciliated respiratory epithelium. These metaplastic areas of ciliated respiratory epithelium were found, in several cases, to be continuous with ciliated ducts of hypertrophic or hyperplastic Bowman’s glands. There were foci of fusiform cells near the basal layer in rats, but not in mice, in areas of respiratory metaplasia in the dorsal meatus. In the underlying, oedematous connective tissue, the basement membrane appeared thickened and separated from the epithelium. Basal cell hyperplasia was frequently observed in the olfactory epithelium of rats, but was not observed in mice. This lesion was characterized by a zone of polyhedral to fusiform cells lying beneath any remaining sustentacular cells. The basal cells had indistinct cytoplasmic boundaries and dark round nuclei and formed cords, sheets, or acinar arrangements. At the 50-ppm exposure level, lesions were much less severe than at 175 ppm and were confined to loss of sensory cells and olfactory nerves, primarily in the middle third of the dorsal meatus. However, most of the animals exposed to 50 ppm exhibited olfactory epithelial lesions. After 12 months, only a few animals at the l0-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.
Details on results:
Treatment-induced lesions were confined to the nasal passages, and were very similar in nature and grade between the species and sexes. In mice, there was no apparent progression or increase in severity of the nasal lesions from 6 to 12 months, while in rats, increased exposure time was associated with more extensive involvement of the olfactory area. The lesions were present in two areas of the nose: (1) the respiratory epithelium and underlying tissues adjacent to the vestibule, and (2) the olfactory epithelium in the medial portion of the dorsal meatus with variable involvement of more posterior olfactory areas. There was a distinct concentration-response relationship for the severity and frequency of both lesions. After 12 months of treatment, clear effects were seen at 50 and 175 ppm, whereas at 10 ppm lesions were only seen in few animals.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Deaths at 175 ppm (= 327 mg/m³) in rats and mice were secondary to severe local effects, but should nevertheless be considered to represent systemic toxicity.
Dose descriptor:
LOAEC
Remarks:
local irritation
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on the respiratory epithelium in some of the rats and mice at 12 months after study initiation. Using a conversion factor (1 ppm = 1.87 mg/m³), 10 ppm equals 18.7 mg/m³

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Lesions in Nasal Passages of Rats and Mice Exposed to DMA for 12 Months

DMA (ppm)

 

Squamous epithelium (level I)a

Respiratory epithelium (levels I, II)b

Olfactory epithelium
(levels II, III, IV)c

Chronic inflammation (levels I, II, III)

0

Rats

+/-

+

Mice

10

Rats

+/-

+

+

Mice

+/-

+

50

Rats

+

++

++

Mice

+

++

+

175

Rats

+/-

+++

+++

++

Mice

+/-

+++

+++

+

Notes. — =No abnormality detected. +/- = very slight changes of doubtful significance, + = minimal change, ++ = moderate change, +++ = severe change.
aFor levels of the nose, see Methods.
bLesion largely confined to the respiratory epithelium adjacent to the vestibule.
cLesions especially severe in the dorsal meatus.
dLesions were somewhat more extensive in rats than mice.

 

Applicant's summary and conclusion

Conclusions:
In this study the LOAEC for local effects was 10 ppm dimethylamine (DMA), as evidenced by lesions of the respiratory and olfactory epithelium of rats and mice. The NOAEC for systemic toxicity was 50 ppm DMA in both species. Local irritation is the primary mode of action of all free aliphatic amines, and this finding can be transferred to N,N-dimethylbutylamine for assessment.
Executive summary:

Rats (95 Fischer 344/sex and dose) and mice were exposed to atmospheres containing dimethylamine at 0, 10, 50, 175 ppm (exposure duration 12 months, 5 days/week, 6 hours/day). The concentration was monitored using IR spectrophotometry. Groups of rats (and mice) were sacrificed at 6 and 12 months after study initiation. Examinations included clinical signs body weights, clinical chemistry, haematology, necropsy and histopathology.

After 12 months of exposure, the only effect noted was a dose-dependent irritation of the nasal respiratory and olfactory epithelium, both in rats and mice, without any gender or species related differences. At 10 ppm the effects were minimal but present in several animals. Deaths occurred at 175 ppm in high incidence in male mice (Buckley et al, 1985).

 

The results indicate that the mode of action of the free amine is local irritation/corrosion at the point of contact, followed by inflammatory processes with concomitant alterations of the blood cell counts. Systemic toxicity is a minor issue and there was no indication in this study for any kind of systemic toxicity or target organ other than the airways. This result is in line with observations made with other saturated aliphatic amines. Therefore, the result can be read across to related substances, including N,N-dimethylamine, and can be used for assessment.. In this study the LOAEC for local effects was 10 ppm DMA, the NOAEC for systemic toxicity was 50 ppm DMA.