Dimethylamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well-documented publication, which meets basis scientific principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well-documented publication, which meets basis scientific principles

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Male and female F-344 rats and B6C3Fl 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 B6C3Fl mice to DMA for 2 years. This report summarizes the clinical and pathologic data found for the first 12-month period.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River breeding laboratories, Kingston, New York, and Portage, Michigan, respectively
- Age at study initiation: 4-8 weeks
- Housing: individually in hanging stainless steel wire mesh cages in the exposure chambers
- Diet (e.g. ad libitum): 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 (e.g. ad libitum): 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 changes (per hr): airflow of 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

99,97 % pure
Analysis of the test atmospheres was performed four times per hour by infrared spectrometry at a wavelength of 3.5 pm 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 distribution of the analytical concentration of DMA in the chambers was determined to be uniform within ± 6 % of target concentrations.

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.

Duration of treatment / exposure:

6 h /day, 5 days a week, for 12 month

Frequency of treatment:

daily

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 / sex

Control animals:

yes, concurrent no treatment

Details on study design:

In studies at our laboratory (unpublished observations), 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.

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 hi rate of unscheduled mortality in this group.

Animals were anesthetized with pentobarbital by ip injection, and blood was drawn from the heart for hematology 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, decakSed in Cal-ex solution (Fisher Scientific, Raleigh, N.C.) for 2 days, 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 pa&in wax. Transverse blocks of the nose were cut and prepared to yield histologic sections at the following levels: (1) just anterior to the incisor teeth, (2) approximately onethird 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 center of the second molar tooth. Embedded tissues were sectioned at 5 pm and stained with hematoxylin and eosin for light microscopic examination. For photography, selected tissues were removed from pan&n, reembedded 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 (Coulter Electronics, Inc., Hialeah, Ha.). Sodium and potassium determinations were performed with a Flame photometer 343 (Instrument Lab, Lexington, Mass). Chloride analyses were performed with a Coming Chloride 920 M meter (Coming Scientific Inst., Mechield, Mass.). An Abbott VP clinical analyzer (Abbott Labs, Chicago, Ill.) was used for determination of serum chemistries.

Statistics:

Data for body weights, serum chemistry, hematology, 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 histopathologic findings were analyzed using the Kolmorgomov-Smimov test (Daniel, 1978). In all cases, the preselected significance levei was p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-induced lesions were confined to the nasal passages (very similar in nature 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant loss at 175 ppm (90 % of control)

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):

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)

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-induced lesions were confined to the nasal passages (very similar in nature between the species and sexes). In rats, increased exposure time (from 6 to 12 month) 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.

(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. 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 rats. 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, 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 in areas of respiratory metaplasia in the dorsal meatus. In the underlying, edematous connective tissue, the basement membrane appeared thickened and separated from the epithelium. Basal cell hyperplasia was frequently observed in the olfactory epithelium of rats. 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 10-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

Additionally a number of neoplastic and non-neoplastic lesions were found, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A number of neoplastic and non-neoplastic lesions were found, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Details on results:

A number of neoplastic and non-neoplastic lesions were found, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

10 ppm

Based on:

other:

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Treatment-induced lesions were confined to the nasal passages (very similar in nature 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.

(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, edematous 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 10-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

TABLE 1. 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	+/-	+++	+++	+
Note. — =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.

 

Conclusions:

The highest concentration: 175 ppm induced severe alterations in the nasal passages.
The middle concentration of 50 ppm did cause significantly lower alterations and the lowest dose of 10 ppm only caused slight alterations in a few animals.

Executive summary:

The mean body weight gain of rats and mice exposed to 175 ppm DMA was depressed to approximately 90 % of control after 3 weeks of exposure. The only other treatment-related changes were concentration-related lesions in the nasal passages. Two distinct locations in the nose were affected~ the respiratory epithelium in the anterior nasal passages, and the olfactory epithelium, especially that lining the anterior dorsal meatus. There was focal destruction of the anterior nasoturbinate and nasal septum, local inflammation, and focal squamous metaplasia of the respiratory epithelium in rats and mice. Mild goblet cell hyperplasia was observed only in rats. The olfactory epithelium exhibited extensive loss of sensory cells with less damage to sustentacular cells. There was also loss of olfactory nerves, hypertrophy of Bowman’s glands, and distension of the ducts of these glands by serocellular debris in regions underlying degenerating olfactory epithelium. At the 175-ppm exposure level, rats had more extensive olfactory lesions than mice, with hyperplasia of small basophilic cells adjacent to the basement membrane being present in rats but not mice. After 12 months of exposure to 10 ppm DMA, minimal loss of olfactory sensory cells and their axons in olfactory nerve bundles was observed in the nasal passages of a few rats and mice. These results indicate that the olfactory sensory cell is highly sensitive to the toxic effects of DMA, with minor lesions being produced in rodents even at the current threshold limit value of 10 ppm.

The following effect level were derived: LOAEC = 10 ppm = 18.45 mg/m³ for local effects and NOAEC (systemic) = 50 ppm = 92.26 mg/m³ for systemic effects (conversion of ppm value into mg/m³ value was performed taking into account the temperature of 76 °F of the Buckley study).

Data source

Reference

Reference Type:

publication

Title:

The Toxicity of Dimethylamine in F-344 Rats and B6C3Fl Mice following a 1 -Year Inhalation Exposure

Author:

Buckles, L.A., Morgan, J.A., Swenberg, R.A., James, T.E., Hamm, J.R., and Barrow, C.S.

Year:

1985

Bibliographic source:

Fundamental and Applied Toxicology, Vol. 5, pp. 341-352 (1985)

Materials and methods

Principles of method if other than guideline:

Male and female F-344 rats and B6C3Fl 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 atIer 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 B6C3Fl mice to DMA for 2 years. This report summarizes the clinical and pathologic data found for the first 12-month period.

GLP compliance:

no

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylamine

EC Number:

204-697-4

EC Name:

Dimethylamine

Cas Number:

124-40-3

Molecular formula:

C2H7N

IUPAC Name:

N-methylmethanamine

Details on test material:

optained from Air Porducts and Chemicals, Inc. (Fogelsville, Pa.)
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 randomization procedure designed to ensure that each animal spent an equal amount of time in all areas of the chamber.

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River breeding laboratories, Kingston, New York, and Portage, Michigan, respectively
- Age at study initiation: 4-8 weeks
- Housing: individually in hanging stainless steel wire mesh cages in the exposure chambers
- Diet (e.g. ad libitum): 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 (e.g. ad libitum): 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 changes (per hr): airflow of 2200 liters/min
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

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.

Details on analytical verification of doses or concentrations:

99,97 % pure
Analysis of the test atmospheres was performed four times per hour by infrared spectrometry at a wavelength of 3.5 pm 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 distribution of the analytical concentration of DMA in the chambers was determined to be uniform within ± 6 % of target concentrations.

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.

Duration of treatment / exposure:

6 h /day, 5 days a week, for 12 month

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

10 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

50 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

175 ppm (nominal)

No. of animals per sex per dose:

95 animals / sex

Control animals:

yes, concurrent no treatment

Details on study design:

In studies at our laboratory (unpublished observations), 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 hi rate of unscheduled mortality in this group.

Animals were anesthetized with pentobarbital by ip injection, and blood was drawn from the heart for hematology 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, decakSed in Cal-ex solution (Fisher Scientific, Raleigh, N.C.) for 2 days, 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 pa&in wax. Transverse blocks of the nose were cut and prepared to yield histologic sections at the following levels: (1) just anterior to the incisor teeth, (2) approximately onethird 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 center of the second molar tooth. Embedded tissues were sectioned at 5 pm and stained with hematoxylin and eosin for light microscopic examination. For photography, selected tissues were removed from pan&n, reembedded 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 (Coulter Electronics, Inc., Hialeah, Ha.). Sodium and potassium determinations were performed with a Flame photometer 343 (Instrument Lab, Lexington, Mass). Chloride analyses were performed with a Coming Chloride 920 M meter (Coming Scientific Inst., Mechield, Mass.). An Abbott VP clinical analyzer (Abbott Labs, Chicago, Ill.) was used for determination of serum chemistries.

Statistics:

Data for body weights, serum chemistry, hematology, 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 histopathologic 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Alopecia, 56 death

Mortality:

mortality observed, treatment-related

Description (incidence):

Alopecia, 56 death

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant loss at 175 ppm

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:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased glucose (females, 175 ppm)

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the 50-ppm exposure level, lesions in the nasal passages 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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-induced lesions were confined to the nasal passages (very similar in nature 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. 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.

(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. No Goblet cell hyperplasia was observed on the ventral aspect of the nasal septum 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 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, edematous connective tissue, the basement membrane appeared thickened and separated from the epithelium. No basal cell hyperplasia was observed in the olfactory epithelium of in mice. 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 10-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

Additionally, a number of neoplastic and nonneoplastic lesions were found, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

A number of neoplastic and nonneoplastic lesions were found, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Details on results:

A number of neoplastic and nonneoplastic lesions were found in both rats and mice, including variable degrees of hair loss, ovarian cysts, testicular discoloration and atrophy, enlargement of the preputial glands, and other minor findings. These changes were typical of animals of these strains and ages and were not considered to be related to DMA exposure.

Effect levels

1

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

2

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Treatment-induced lesions were confined to the nasal passages (very similar in nature 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.

(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, edematous 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 10-ppm exposure level were affected, and lesions were confined to focal degeneration of the olfactory epithelium in the dorsal meatus.

TABLE 1. 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	+/-	+++	+++	+
Note. — =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:

The highest concentration: 175 ppm induced severe alterations in the nasal passages.
The middle concentration of 50 ppm did cause sigfnificantly lower alterations and the lowest dose of 10 ppm only caused slight alterations in a few animals.

Executive summary:

The mean body weight gain of rats and mice exposed to 175 ppm DMA was depressed to approximately 90 % of control after 3 weeks of exposure. The only other treatment-related changes were concentration related lesions in the nasal passages. Two distinct locations in the nose were affected: the respiratory epithelium in the anterior nasal passages, and the olfactory epithelium, especially that lining the anterior dorsal meatus. There was focal destruction of the anterior nasoturbinate and nasal septum, local inflammation, and focal squamous metaplasia of the respiratory epithelium in rats and mice. The olfactory epithelium exhibited extensive loss of sensory cells with less damage to sustentacular cells. There was also loss of olfactory nerves, hypertrophy of Bowman’s glands, and distension of the ducts of these glands by serocellular debris in regions underlying degenerating olfactory epithelium. At the 175-ppm exposure level, rats had more extensive olfactory lesions than mice, with hyperplasia of small basophilic cells adjacent to the basement membrane being present in rats but not mice. After 12 months of exposure to 10 ppm DMA, minimal loss of olfactory sensory cells and their axons in olfactory nerve bundles was observed in the nasal passages of a few rats and mice.

These results indicate that the olfactory sensory cell is highly sensitive to the toxic effects of DMA, with minor lesions being produced in rodents even at the current threshold limit value of 10 ppm.