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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
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.

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:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 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:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 110 g (mean), female: 93 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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: not appropriate for vapors
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 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
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating vapors: 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.
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: 15 air changes per hour
- Method of particle size determination: 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
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: Samples of the test atmosphere from the distribution lines and the low and high exposure concentration chambers were collected prior to the 3-month and also at the beginning and end of one generation day during the 3-month study. The atmosphere samples were collected with adsorbent gas sampling tubes containing an acrylic ester, followed by a tube containing activated coconut charcoal, and extracted with methylene chloride. Chamber and room concentrations of diethylamine were monitored by an on-line gas chromatograph.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 30 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
93 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
8, 16, 32, 62, and 125 ppm (16.8, 33.6, 67.2, 130.2, and 262.5 mg/m3)
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
8.0 ± 0.3, 15.9 ± 0.6, 32.0 ± 1.3, 62.2 ± 2.3, 126 ± 5 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Because exposure to 250 or 500 ppm diethylamine for 16 days caused significantly decreased body weights in rats, a high concentration of 125 ppm was selected for both sexes in the 3-month study. Although nasal lesions were present in rats exposed to 125 ppm for 16 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 rats in the 3-month study.

Examinations

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 3, 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 3, 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

NEUROBEHAVIOURAL EXAMINATION: No


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

ORGAN WEIGHT: OTHER: Heart, right kidney, liver, lung, right testis, and thymus

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.

Other examinations:
Sperm Motility and Vaginal Cytology
At the end of the studies, sperm samples were collected from male animals in the 0, 32, 62, and 125 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days prior to the end of the studies from females exposed to 0, 32, 62, or 125 ppm for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
a single occurrence of a torso lateral ulcer/abscess in a 125 ppm male
Mortality:
mortality observed, treatment-related
Description (incidence):
a single occurrence of a torso lateral ulcer/abscess in a 125 ppm male
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats survived to the end of the study. The only clinical finding was a single occurrence of a torso lateral ulcer/abscess in a 125 ppm male.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights and body weight gains of all exposed groups were similar to those of the chamber control groups.
HAEMATOLOGY
There were no exposure-related changes in hematology endpoints.

CLINICAL CHEMISTRY
There were no exposure-related changes in clinical chemistry endpoints.

ORGAN WEIGHTS
The relative kidney weights of all groups of exposed females were increased and were significantly greater than those of the chamber controls, except in the 32 ppm group (Table G2). The relative liver weight of 125 ppm males was significantly increased.

HISTOPATHOLOGY: NON-NEOPLASTIC
Exposure-related histopathology findings in rats were limited to the nose and were seen primarily in rats exposed to 62 or 125 ppm. These lesions included turbinate necrosis, suppurative inflammation, respiratory epithelial hyperplasia, squamous metaplasia of the respiratory epithelium, and olfactory epithelial atrophy.

Exposure-related histopathology findings in rats were limited to the nose. Suppurative inflammation of minimal to mild severity was observed in all males and most females in the 125 ppm groups; a few 62 ppm males and females and one 16 ppm female also exhibited inflammation (Table 5). Suppurative inflam-mation was characterized by increased numbers of neutrophils in the nasal tissue and was most obvious on the tips of the nasal turbinates. Necrosis of the nasal turbinates in Level I was noted in one male and one female exposed to 125 ppm and consisted of sloughing of the respiratory epithelium with exposure and partial necrosis of the underlying bone. Respiratory epithelial hyperplasia was present in all 125 ppm males, in most 125 ppm females and 62 ppm males and females, and in a few 16 and 32 ppm males and one 16 ppm female. Hyperplastic respiratory epithelium contained three or more layers of epithelial cell nuclei with loss of the normal orderly, polarized, arrangement. Nonkera-tinizing squamous metaplasia of the respiratory epithelium occurred in all 125 ppm males, five 125 ppm females, one 62 ppm male, and one 16 ppm male. Both hyperplasia and metaplasia of the respiratory epithelium were most commonly observed and most severe on the tips of the nasal turbinates and on the lateral wall of the dorsal half of nasal Level I. Olfactory epithelial atrophy was present in all 125 ppm males and females, most 62 ppm males and females, and two 32 ppm females. Atrophy was most pronounced in the dorsal meatus of nasal Level II and only rarely affected the olfactory epithelium of nasal Level III. The atrophy was characterized by decreased height of the olfactory epithelium associated with a decreased number of olfactory cell nuclei, and was accompanied by a reduction in the number of nerves and Bowman’s glands in the underlying lamina propria.

There were no inflammatory changes of the eye as had been observed in the 2-week study at higher concentrations.

OTHER FINDINGS
There was a dose-related decrease seen in the motility of sperm from male rats with the values of those exposed to 32, 62, or 125 ppm diethylamine being significantly lower (5-26%) than those of the chamber controls (Table H1); no significant differences were observed in the estrous cyclicity of female rats administered 32, 62, or 125 ppm diethylamine when compared to the chamber controls (Table H2).

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
16 other: ppm (analytical) (33.6 mg/m3)
Sex:
male/female
Basis for effect level:
other: Exposure-related nasal lesions were seen primarily in rats exposed to 62 or 125 ppm
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
16 other: ppm (analytical) (33.6 mg/m3)
Sex:
male
Basis for effect level:
other: significant exposure concentration-related decreases in sperm motility in 32, 62, and 125 ppm males
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
>= 125 other: ppm (analytical) (262.5 mg/m3)
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

TABLE H1: Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Inhalation Study of Diethylaminea                                                                                                                                                                                                                                                     

 

Chamber Control

32 ppm

62 ppm

125 ppm

n

10

10

10

10

Weights (g)

 

 

 

 

Necropsy body wt

347 ± 6

350 ± 7

355 ± 7

338 ± 6

L. Cauda epididymis

0.1871 ± 0.0052

0.1802 ± 0.0061

0.1873 ± 0.0044

0.1736 ± 0.0043

L. Epididymis

0.4913 ± 0.0094

0.4861 ± 0.0119

0.4947 ± 0.0114

0.4662 ± 0.0069

L. Testis

1.5165 ± 0.0238

1.4772 ± 0.0350

1.5102 ± 0.0278

1.4473 ± 0.0264

Spermatid measurement

Spermatid heads (103/mg testis)

Spermatid heads (106/testis)

 

123.55 ± 5.72

 

171.00 ± 7.16

 

125.24 ± 5.86

 

168.63 ± 9.72

 

125.76 ± 3.96

 

173.38 ± 6.35

 

128.93 ± 4.48

 

169.38 ± 4.01

Epididymal spermatozoal measurements

Sperm motility (%)

 

93.01 ± 0.72

 

88.60 ± 1.45**

 

87.27 ± 1.57**

 

68.44 ± 2.78**

Sperm (103/mg cauda epididymis)

669 ± 37

660 ± 30

660 ± 22

598 ± 34

Sperm (106/cauda epididymis)     

124.3 ± 5.7

118.0 ± 4.6

123.3 ± 3.6

103.9 ± 6.3

 

** Significantly different (P≤0.01) from the chamber control group by Shirley’s test

a   Data are presented as mean ± standard error. Differences from the chamber control group are not significant by Dunnett’s test (body and tissue weights) or Dunn’s test (spermatid, sperm/mg cauda epididymis, and sperm/cauda epididymis measurements).

 

TABLE H2: Estrous Cycle Characterization for Female Rats in the 3-Month Inhalation Study of Diethylaminea                            

                  

 

Chamber Control

32 ppm

62 ppm

125 ppm

Number weighed at necropsy

10

10

10

10

Necropsy body wt (g)

204 ± 6

200 ± 4

202 ± 3

201 ± 5

Proportion of regular cycling femalesb

 

10/10

 

10/10

 

10/10

 

10/10

Estrous cycle length (days)

5.0 ± 0.05

5.0 ± 0.05

5.0 ± 0.00

5.0 ± 0.05

Estrous stages (% of cycle)

 

 

 

 

Diestrus

58.3

54.2

53.3

55.0

Proestrus

16.7

15.8

16.7

14.2

Estrus

20.0

17.5

20.0

20.8

Metestrus

5.0

12.5

10.0

10.0

 

a  Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the chamber control group are not significant by Dunnett’s test (body weight) or Dunn’s test (estrous cycle length). By multivariate analysis of variance, exposed females do not differ significantly from the chamber control females in the relative length of time spent in the estrous stages. The tests for equality of transition probability matrices among exposure groups and between the chamber control group and each exposed group indicated the exposed females did not have extended estrus or diestrus.

b  Number of females with a regular cycle/number of females cycling

Applicant's summary and conclusion

Executive summary:

 In a study conducted similar to OECD TG 413 (Subchronic Inhalation Toxicity: 90-Day), groups of ten Fischer 344 rats/sex were exposed to the test substance by whole body vapor inhalation for six hours/day, five days/week for 93 days at concentrations of 0, 8, 16, 32, 62, or 125 ppm (0, 16.8, 33.6, 67.2, 130.2, and 262.5 mg/m3) (NTP, 2011). There were no deaths. A single occurrence of a torso lateral ulcer/abscess occurred in a 0.37 mg/L male. Clonic seizures were seen in males (1/50, 2/50, 7/50, 9/50) and in females (2/50, 7/50, 11/50, 13/50) [doses not stated but the lowest numbers presumably were in controls – i.e., 1/50 males and 2/50 females]. There were no effects on body weights, clinical chemistry or hematology findings. The relative kidney weights of all groups of exposed females were increased and were significantly greater than those of the chamber controls (p< 0.05 or 0.01), except in the 0.096 mg/L group (which had an n=9). The relative heart weight was increased in females at 0.37 mg/L (p< 0.05). The relative liver weight of 0.37 mg/L males was significantly increased (p < 0.05). Exposure-related histopathology findings in rats were limited to the nose (primarily at 0.19 or 0.37 mg/L). These lesions included turbinate necrosis, suppurative inflammation, respiratory epithelial hyperplasia, squamous metaplasia of the respiratory epithelium, and olfactory epithelial atrophy. The NOAEC for the local irritation and the systemic toxicity was 16 ppm (33.6 mg/m3) based on histopathology findings.