Diethylammonium chloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientific publication that meets documentation requirements. Study was performed according to NTP standard protocol.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented report which meets basic scientific principles. Study performed according to standard NTP protocols.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CB6F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 23.3 g (mean), female: 19.8 g (mean)
- Housing: individually in stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly and rotated daily
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure periods
- Water: Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C (72°±3 °F)
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged

Details on inhalation exposure:

Diethylamine was pumped onto glass beads in a heated glass column where it was vaporized. Heated nitrogen flowed through the column and carried the vapor into a short vapor distribution manifold. Concentration in the manifold was determined by the chemical pump rate and nitrogen flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Individual Teflon® delivery lines carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to the exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. A metering valve with a flow indicator at the manifold controlled the flow rate to each chamber. To initiate exposure, the chamber exposure valves were rotated to allow the vapor to flow to each exposure chamber inlet duct where it was further diluted with HEPA®-filtered, conditioned air to achieve the desired exposure concentration.
The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concen-trations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A small particle detector was used with and without animals in the exposure chambers to ensure that diethylamine vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of diethylamine in nitrogen supplied by a standard generator. The on-line gas chromatograph was calibrated by a comparison of chamber concentration data to data from grade samples that were collected with acrylic ester adsorbent gas sampling tubes, extracted with methylene chloride containing triethylamine as an internal standard, and analyzed using an off-line gas chromatograph. Known values of chamber atmosphere were sampled at a constant flow rate ensured by a calibrated orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of diethylamine and the internal standard (triethylamine) in methylene chloride.

Duration of treatment / exposure:

93 days

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

8 ppm (analytical)

Remarks:

(corresponding to 24 mg/m3)

Dose / conc.:

16 ppm (analytical)

Remarks:

(corresponding to 49 mg/m3)

Dose / conc.:

32 ppm (analytical)

Remarks:

(corresponding to 97 mg/m3)

Dose / conc.:

62 ppm (analytical)

Remarks:

(corresponding to 188 mg/m3)

Dose / conc.:

125 ppm (analytical)

Remarks:

(corresponding to 379 mg/m3)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale:
Because exposure to 250 and 500 ppm diethylamine for 17 days caused mortality in mice and body weight losses exceeding 18%, a high concentration of 125 ppm was selected for both sexes of mice in the 3-month study. Although nasal lesions were present in mice exposed to 125 ppm for 17 days, these lesions were generally mild and were not likely to compromise the 3-month study. Diethylamine exposure concentrations of 0, 8, 16, 32, 62 and 125 ppm were selected for both sexes of mice in the 3-month study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 7 and weekly afterwards

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 0, 23 and 93
- Anaesthetic used for blood collection: Yes
- How many animals: all
- Parameters examined: Erythrocyte count, Mean corpuscular volume, Hemoglobin, Packed cell volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Erythrocyte morphologic assessment, Leukocyte count, Leukocyte differential, Reticulocyte count, Platelet count and morphologic assessment

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 0, 23 and 93
- How many animals: all
- Parameters examined: Sorbitol dehydrogenase (SDH), Alkaline Phosphatase (ALP), Creatine Kinase (CK), Creatinine, Total Protein, Albumin, Urea Nitrogen (BUN), Total Bile Acids, Alanine Aminotransferase (ALT), Glucose

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, a complete necropsy was performed on all treated and control animals that either die or are sacrificed

HISTOPATHOLOGY: Complete histopathology was performed on 0 and 125 ppm core study rats. In addition to gross lesions and tissue masses, the following tissues were examined to a no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, lymph nodes (bronchial, mandibular, mediastinal, and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Standard statistical methods have been applied for data processing.

Key result

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

16 ppm

Based on:

test mat.

Remarks:

equivalent to 49 mg/m3

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

16 ppm

Based on:

test mat.

Remarks:

equivalent to 49 mg/m3 and 0.049 mg/L ait

Sex:

male

Basis for effect level:

other: sperm motility

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

32 ppm

Based on:

test mat.

Remarks:

equivalent to 97 mg/m3 and 0.097 mg/L air

Sex:

female

Basis for effect level:

other: changes in estrous cycling

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

32 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

DEA was studied in a subchronic study to mice using the NTP standard protocol. From the data a NOAEC for systemic and local toxicity of 16 ppm could be established.

Executive summary:

Diethylamine (DEA) provides a suitable surrogate for diethylamine hydrochloride.

DEA was studied in a subchronic study in mice using the NTP standard protocol. The report is publicly avaialable on the NTP website (TR 566).

In the studies groups of 10 male and 10 female mice were exposed to diethylamine vapor at concentrations of 0, 8, 16, 32, 62, or 125 ppm (corresponding to 24, 49, 97, 1882 and 379 mg/m³, respectively)

, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All mice survived to the end of the study. The mean body weights of 125 ppm males and females were significantly less than those of the chamber controls. There were significant exposure concentration-related decreases in sperm motility in males exposed to 32, 62, or 125 ppm; the estrous cycle of 125 ppm females was significantly longer than that of the chamber controls but only by half a day. Histopathologic changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of males and females principally in the 62 or 125 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates.

From the data a local and systemic NOAEC of 16 ppm (corresponding to 49 mg/m³) could be established.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethylamine

EC Number:

203-716-3

EC Name:

Diethylamine

Cas Number:

109-89-7

Molecular formula:

C4H11N

IUPAC Name:

N-ethylethanamine

Test material form:

other: free base liquid

Details on test material:

- Name of test material (as cited in study report): diethylamine
- Physical state: colorless liquid with a strong ammonia odor
- Analytical purity: approximately 99.9%
- Lot/batch No.: BE/07/01
- Stability under test conditions: no degradation of the bulk chemical was detected
- Storage condition of test material: at controlled room temperature

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 23.3 g (mean), female: 19.8 g (mean)
- Housing: individually in stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly and rotated daily
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure periods
- Water: Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 12 days

Administration / exposure

Route of administration:

inhalation: vapour

Duration of treatment / exposure:

93 days

Frequency of treatment:

6 h/d, 5 d/w

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 ppm (analytical)

Remarks:

(corresponding to 24 mg/m3)

Doses / concentrations 2

Dose / conc.:

16 ppm (analytical)

Remarks:

(corresponding to 49 mg/m3)

Doses / concentrations 3

Dose / conc.:

32 ppm (analytical)

Remarks:

(corresponding to 97 mg/m3)

Doses / concentrations 4

Dose / conc.:

62 ppm (analytical)

Remarks:

(corresponding to 188 mg/m3)

Doses / concentrations 5

Dose / conc.:

125 ppm (analytical)

Remarks:

(corresponding to 379 mg/m3)

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Examinations

Tissues and cell types examined:

Peripheral blood

Details of tissue and slide preparation:

Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1,000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above).

Statistics:

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Any other information on results incl. tables

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment with Diethylamine by Inhalation for 13 Weeks:

Dose (ppm)

Micronucleated Normochromatic Erythrocytes/1000 cells

Male

Female

0

2.80 ± 0.30

2.60 ± 0.29

8

4.60 ± 0.60

2.50 ± 0.61

16

4.10 ± 0.48

2.20 ± 0.25

32

3.30 ± 0.34

3.50 ± 0.57

62

4.00 ± 0.52

3.80 ± 0.60

125

2.60 ± 0.33

2.20 ± 0.25

No significant increase in micronucleated NCEs was observed in males or females and all tested dose groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
No significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Executive summary:

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice exposed to 8 to 125 ppm (corresponding to 24 to 379 mg/m3) diethylamine by inhalation. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1,000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

No significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.