Registration Dossier

Administrative data

Description of key information

inhalation:

NOAEC local: 16 ppm (48 mg/m3); LOAEC systemic: 32 ppm (96 mg/m3) (93 day NTP-Study, rat and mouse)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
Principles of method if other than guideline:
Study performed according to standard NTP protocols.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
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


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 (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Analytical verification of doses or concentrations:
yes
Remarks:
analysed with an on-line gas chromatograph
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
14 weeks (93 days exposure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
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 of the test substance 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.
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 is 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.

Other examinations:
At the end of the study: samples for sperm motility, densitiy and count; vaginal cytology (controls, 32, 62, 125 ppm).
Sampling of vaginal fluid and cells was performed.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Non-parametric multiple comparison methods of Shirley (1977) for hematology, clinical chemistry, spermatid, and epididymal spermatozoal data
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights and body weight gains of 125 ppm males and females were significantly less than those of the chamber controls (males: 78%, females: 84% of control).
Food efficiency:
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the liver, right kidney, and thymus of 125 ppm males; heart, liver, and right kidney of 125 ppm females; and thymus of 62 and 125 ppm females were significantly less than those of the chamber controls.
The relative weights of the heart, right kidney, lung, and right testis of 125 ppm males and the lung of 125 ppm females were significantly greater than those of the chamber controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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. Sigificant olfactory epithelial atrophy was already observed after treatment with 32 ppm.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related decrease seen in the motility of sperm from male mice with values of those exposed to 32, 62, or 125 ppm being significantly lower (7-15%) than those of the chamber controls; except for a slight (0.5 day), but statistically significant increase in estrous cycle length, no significant differences were observed in the estrous cyclicity of female mice administered 32, 62, or 125 ppm of the vapourized test substance when compared to the chamber controls.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
16 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
32 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased sperm motility
Critical effects observed:
not specified

Tab. 1 Mortality and relative weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

10/10

-

8

10/10

98/98

16

10/10

69/105

32

10/10

101/100

62

10/10

100/97

125

10/10

78/84

Tab. 2 Incidences of nonneoplastic lesions in male mice

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

8** (1.5)b

Olfactory Epithelium, Atrophy

0

0

0

4* (1.0)

9** (2.2)

10** (2.9)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

3

1 (1.0)

9** (2.2)

Turbinate, Necrosis

0

0

0

0

0

7** (2.4)

* significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of nonneoplastic lesions in female mice

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

3 (1.0)b

8** (1.0)

Olfactory Epithelium, Atrophy

0

0

0

9**(1.1)

10** (2.4)

10** (2.8)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

3

1 (1.0)

9** (1.8)

Turbinate, Necrosis

0

0

0

0

0

6** (2.7)

** significantly different (p≤ 0.01) from the chamber control group by the Fisher exact test

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
A NOAEC of 16 ppm for local effects was derived based on the observation of nonneoplastic lesions of the mouse nose.
The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male mouse exposed to vapour concentrations of 32 ppm and higher concentration levels.
Executive summary:

In the 14 week study, groups of 10 male and 10 female mice were exposed to the test substance as a vapour at concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 hours per day with a additional T90 value (time to achieve 90% of target concentration) of 12 min, 5 days per week. All animals survived to the end of the study. The final mean body weights and body weight gains of 125 ppm males and females were significantly less than those of the chamber controls (males: 78%, females: 84% of control). The absolute weights of the liver, right kidney, and thymus of 125 ppm males; heart, liver, and right kidney of 125 ppm females; and thymus of 62 and 125 ppm females were significantly less than those of the chamber controls. The relative weights of the heart, right kidney, lung, and right testis of 125 ppm males and the lung of 125 ppm females were significantly greater than those of the chamber controls. There was a dose-related decrease seen in the motility of sperm for male mice with values of those exposed to 32, 62, or 125 ppm diethylamine being significantly lower (7-15%) than those of the chamber controls (animals exposed to 8 and 16 ppm were not investigated for spem motility changes). Except for a slight (0.5 day), but statistically significant increase in estrous cycle length, no significant differences were observed in the estrous cyclicity of female mice administered 32, 62, or 125 ppm of the vapour when compared to the chamber controls.

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. Significant olfactory epithelial atrophy was already observed after treatment with 32 ppm.

A NOAEC of 16 ppm for local effects was derived based on the observed nonneoplastic lesions of the mouse nose.

The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in the sperm motility for male mouse exposed to vapour concentrations of 32 ppm and higher concentration levels.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
Principles of method if other than guideline:
Study performed according to standard NTP protocols.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
Species:
rat
Strain:
other: Fisher344/N
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 to 7 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
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h




Analytical verification of doses or concentrations:
yes
Remarks:
analysed with an on-line gas chromatograph
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
14 weeks (93 days expsoure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale:
Because exposure to 250 or 500 ppm of the test substance 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.
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 is 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.

Other examinations:
At the end of the study: samples for sperm motility, densitiy and count; vaginal cytology (controls, 32, 62, 125 ppm).
Sampling of vaginal fluid and cells was performed.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Non-parametric multiple comparison methods of Shirley (1977) for hematology, clinical chemistry, spermatid, and epididymal spermatozoal data
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1 male animal showed torso lateral abcesss/ulcer
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- the relative kidney weights of exposed females were increased and significantly gerater than for chamber control animals, except in the 32 ppm group
- the relative liver weights of males exposed to 125 ppm was significantly increased
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.

There were no inflammatory changes of the eye.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related decrease seen in the motility of sperm from male rats exposed to 32, 62, or 125 ppm being significantly lower (5-26%) than those of the chamber controls; no significant differences were observed in the estrous cyclicity of female rats administered 32, 62, or 125 ppm of the vapour when compared to the chamber controls. These parameters were not assessed in rats exposed to 8 and 16ppm.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
16 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
32 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased sperm motility
Critical effects observed:
not specified

Tab. 1 Mortality and relative final weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

10/10

-

8

10/10

99/98

16

10/10

103/97

32

10/10

101/98

62

10/10

102/99

125

10/10

97/99

Tab. 2 Incidences of nonneoplastic lesions of the nose in male rats

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

2 (2.5)b

10** (1.6)

Turbinates, Necrosis

0

0

0

0

0

1 (2.0)

Respiratory Epithelium, Hyperplasia

0

0

3 (1.0)

3

9** (1.3)

10** (2.3)

Respiratory Epithelium, Metaplasia, Squamous

0

0

1 (1.0)

0

1 (2.0)

10** (2.1)

Olfactory Epithelium, Atrophy

0

0

0

0

7** (1.1)

10** (1.9)

** significantly different (p≤ 0.01) from the chamber control group by the Fisher exact test

a Number of animals with lesions

b Average severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of nonneoplastic lesions of the nose in female rats

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

1 (2.0) b

0

3 (1.0)

7** (1.4)

Turbinates, Necrosis

0

0

0

0

0

1 (1.0)

Respiratory Epithelium, Hyperplasia

0

0

1 (1.0)

0

9** (1.2)

9** (1.9)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

0

0

5* (1.6)

Olfactory Epithelium, Atrophy

0

0

0

2 (1.0)

9** (1.2)

10** (2.2)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01)

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
A NOAEC for local effects of 16 ppm was derived. As such related observations were not detected in animals treated with 8 ppm of the vapour, the NOEC for local effects was set to be 8 ppm.
The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male rats exposed to vapour concentrations of 32 ppm and higher concentration levels.
Executive summary:

In the 14 week study, groups of 10 male and 10 female rats were exposed to the test substance as a vapour at concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 hours per day with a additional T90 value (time to achieve 90% of target concentration) of 12 min, 5 days per week. All animals survived to the end of the study. No exposure-related changes in hematology, serum chemistry indices or bodyweights were observed for exposed rats. The relative kidney weights of exposed females were increased and significantly gerater than for chamber control animals, except in the 32 ppm group. The relative liver weights of males exposed to 125 ppm was significantly increased. Examinations of the sperm motility were performed for male rats exposed to 32, 62 and 125 ppm. Significant exposure concentration-related decreases in the sperm motility were detected for all investigated vapour concentration levels. 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, and necrosis of the turbinates. Olfactory epithelial atrophy was observed in animals exposed to 32 ppm and higher concentrations.

Based on these findings, a NOAEC for local effects of 16 ppm was derived. As such related observations were not detected in animals treated with 8 ppm of the vapour, the NOEC for local effects was set to be 8 ppm. The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male rats exposed to vapour concentrations of 32 ppm and higher concentration levels.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
no data on control animals
Principles of method if other than guideline:
- Principle of test:
Rabbits in groups of six were exposed to the amine vapours in a dynamic chamber. Dry air was passed into a bottle equipped with a fritted disk and containing the liquid amines. These were thus volatilized and dispersed into the chamber. The bottle was held in a refrigerated, constant temperature bath.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from the level of animals nose and mouth
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
7 h/day; 5 days peer week
Dose / conc.:
50 ppm
Remarks:
analytical concentration 52.71 ppm
Dose / conc.:
100 ppm
Remarks:
analytical concentration 109.31 ppm
No. of animals per sex per dose:
6 (no gender stated)
Control animals:
other: no data specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 ppm one animal showed some forced movements of the neck; at 150 mg/m3 one rabbit was losing hair, and exhibited a muscular weakness of the hind legs.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
One animal at 100 ppm and one animal at 50 ppm showed slight loss of weight.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Considerable amount of cloudiness, indicative of edema; areas of punctate erosions of the corneal epithelium
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The blood sugar increased in all rabbits, but this effect could not be confirmed in a repeate experiment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: dark pink colored areas in the lungs of one animal; 150 mg/m3 dose: moderate to marked hyperemia of the lungs.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: In all but one animal, the kidneys showed changes indicating injury to the vascular apparatus; intense, diffuse hyperemia with small numbers of red blood corpuscles and protein precipitated into the capsular spaces; numerous casts in the cortical tubules, slight tubular degeneration (probably at least partly due to postmortem changes); marked parenchymatous degeneration in the liver (same comment as for tubular degeneration) . In the lungs of one animal, infiltration of polymorpho-nuclear cells in an area of atelectasis and in the interstitial tissue was found; in another animal, bronchopneumonia was present.

50 ppm: The lungs of all animals showed slight and moderate degrees of chronic irritation characterized by thickening of the blood vessels and focal collections of lymphocytes (peribronchitis); small inflammatory areas in the livers of five animals, in one case combined with
moderate parenchymatous degeneration ; the same animal showed also some inflammatory changes in the kidneys, with moderate parenchymatous and fatty degeneration.
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
50 ppm
Based on:
test mat.
Remarks:
(0.15mg/L)
Sex:
not specified
Basis for effect level:
other: effects on eyes and lungs
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
50 ppm
Sex:
not specified
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
limited parameters assessed
Critical effects observed:
not specified

All rabbits exposed at 100 and 50 ppm seven hours a day, five days a week, for six weeks, survived, but at 50 ppm still showed marked irritation of the lung tissue and cornea, with moderate peribronchitis and slight thickening of the vascular walls, and multiple punctate erosions and edema of the cornea. Changes in the liver consisted of occasional foci of moderate parenchymatous degeneration.

Executive summary:

Rabbits in groups of six were exposed to the vapour in a dynamic chamber at 50 and 100 ppm (equivalent to 150 or 300 mg/m3) for 7 h a day, 5 days a week, for 6 weeks. All animals survived. Expsoure to 100 ppm of the vapour produced parenchymatous degeneration and, occasionally, inflammatory processes in liver and kidney. Both vapour concentrations resulted in marked chronic irritation of the lungs. Animals treated with 50 ppm of the vapour showed corneal injury present as corneal erosions and edema.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: MCB Manufacturing Chemists, Inc. Norwood Ohio
- Lot/batch No.: Lot 2J20 and J8L17
- Analytical purity: 99.9 %
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 (Wilmington, Mass)
- Housing: individually in stainless, steel wire mesh cages
- Diet: Purina Laboratory Chow, ad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4.5 m3 stainless-steel and glass inhalation chambers
- Rate of air: 1.1 m3/min
- Temperature, humidity, pressure in air chamber: 23 +/- 3°C, 50+/-10%, slightly negative pressure relative to ambient (- 0.25 cm water)
- Air change rate: 12-15 air changes/h


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
infrared analyzer; Chamber atmospheres were monitored 2-4 times per hour
Duration of treatment / exposure:
24 weeks (118-123 expsoure days)
Frequency of treatment:
5 days per week, 6.5 hours daily
Dose / conc.:
25 ppm (nominal)
Remarks:
analytical concentration 26 ppm; equivalent to 0.076 mg/L
Dose / conc.:
250 ppm (nominal)
Remarks:
analytical concentration 251 ppm; equivalent to 0.76 mg/l
No. of animals per sex per dose:
10 animals per sex per dose were sacrificed after 30 and 60 days.
50 animals per sex per dose were sacrificed after 120 days.
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: the day predceding the exposure and at 2 weeks intervals thoughout the study.


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: Yes
- Parameters examined: hemoglobin, hematocrit, complete and differential counts.


CLINICAL CHEMISTRY: Yes
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphatase (CPK), blood urea nitrogen (BUN), creatinine (CRE), sorbitol dehydrogenase (SDH)


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data


OTHER: Electrocardiograms (ECGs) were recorded from 10 anesthetized rats/sex/group (35 mg/kg secobarbital, ip) at the terminal sacrifice.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes all major organs and tissue (not further specified)
HISTOPATHOLOGY: Yes, lungs, liver, kidneys, heart, spleen, tracheobronchial lymph nodes, adrenals, testes, seminal vesicles, ovaries, uterus, trachea, eye, urinary bladder, nares (nares not examined for 25 ppm rats).
Statistics:
One-way analysis of variance, Duncan's multiple range test, Kruskal-Wallis test, χ2-test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 250 ppm both sexes exhibited evidence of the strong irritant properties; animal observations included sneezing, tearing, squinting and attempted avoidance of the the test substance by burying their noses in their fur during the entire exposure period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
10 female rats (250 ppm) died due to malfunction of the automatic watering system.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight exposed at 250 ppm were significantly reduced compared to the control groups at week 2 and remained reduced at each subsequent weighing period.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Several clinical chemistry indices, e.g., SDH and CPK, were elevated in one group of rats, or in only one sex of rats, at one of the scheduled interim sacrifices. These changes were not attributed to the exposure. Creatinine levels were significantly elevated in female rats exposed at both 25 and 250 ppm for 24 weeks, while BUN levels were significantly elevated in rat of both sexes exposed at 250 ppm for 24 weeks. These changes, however, could not be correlated with any observed gross or histologic renal damage.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No consistent changes in absolute or relative organ weights were seen in rats sacrificed following 30 or 60 days of exposure. Absolute lung weights were significantly decreased and lung-to-body weight ratios were significantly increased in male rats exposed at 250 for 24 weeks. While statistically significant, these appear to reflect the statistically reduced mean body weights of the rats exposed at 250 ppm rather than indicating toxic effects on these organs.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The group distributions for squamous metaplasia, lymphoid hyperplasia, and suppurative rhinitis in the nares of both male and female rats exposed at 250 ppm for 24 weeks were significantly increased over the distributions for controls. Most of the rats exposed at 250 ppm had an acute inflammatory exudate present in the nasal cavity. Inflammation of the nasal passages was manifested by increased numbers of lymphocytes and plasmacytes in the lamina propria and hyperplasia of lymphoid tissue. The inflammatory changes were located primarily at level two, i.e., approximately one-third of the distance from the posterior aspect of the incisor teeth to the incisive papilla. The region just anterior to the incisor showed inflammatory changes, but not as pronounced. Mild to moderate squamous metaplasia of the respiratory mucosa occurred in focal areas of the middle portions of the nasal septum and lateral walls of the nasal cavity primarily at level two.
Nasal passages of rats exposed to 25ppm were not examined.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In the testes of male rats (250 ppm) slight increase in occurence of hyperspermatogenesis, degenration of seminiferous tubules, and mineralization within the tubules compared to control animals. In most cases unilateral and not considered to be related to treatment
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
25 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
local
Remarks on result:
not determinable
Remarks:
target organ not examined in lowest dose
Critical effects observed:
no

Rats exposed at 250 ppm exhibited evidence of toxicity indicated by a decrease in body weight and histopathologic changes the nares. The incidence of the bronchiola lymphoid hyperplasia in the lungs of exposed animals, however, did not increase with increasing exposure.

Conclusions:
Rats exposed at 250 ppm exhibited evidence of toxicity indicated by a decrease in body weight and histopathologic changes of the nares. Therefore, a NOAEC of 25 ppm was derived.
Executive summary:

In a repeated exposure study, male and female Fischer 344 (F-344) rats were exposed at 75 mg/m3 (25 ppm) or 750 mg/m3 (250 ppm) vapourized test substance, 6.5 hours/day, five days/week, for 24 weeks in order to assess cardiac and other organ system toxicity. Scheduled sacrifices were performed following 30, 60, and 120 days of exposure. During the first two weeks of exposure, the rats exposed at 750 mg/m3 did not gain weight. After two weeks, however, the rate of weight gain of these rats was greater than that of controls. Nevertheless, mean body weights for both sexes of rats exposed at 750 mg/m3 remained depressed compared to controls throughout the study. Sneezing, tearing, and reddened noses were seen in rats exposed at 750 mg/m3 of the vapour. Histopathologic examinations revealed lesions of the nasal mucosa of rats exposed at 750 mg/m3 (rats exposed at 75 mg/m3 were not evaluated for nasal effects). These lesions of the nasal respiratory epithelium consisted of squamous metaplasia, suppurative rhinitis, and lymphoid hyperplasia. There were no pronounced treatment-related effects on organ weights, hematology, or clinical chemistry indices except for blood urea nitrogen which was evaluated in rats of both sexes exposed at 750 mg/m3 for 24 weeks. In contrast to the high-dose animals, no treatment-related effects were observed in rats intermittently exposed at 75 mg/m3 of the test item for up to 24 weeks. No evidence of cardiotoxicity was seen in rats exposed to either concentration for up to 24 weeks.

 

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Animals were exposed via whole body inhalation to each amine at 250 or 1000 ppm for 6 hrs/day for a total of 10 exposure days .
GLP compliance:
not specified
Specific details on test material used for the study:
- Analytical purity: 99.9%
Species:
other: rats and mice
Strain:
other: Fischer 344 rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Diet: NIH 07 pellets (Ziegler Bros., Gardner, PA), ad libitum
- Water: water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
using a Wilks-Miran 1A infrared analyzer
Duration of treatment / exposure:
10 days
Frequency of treatment:
6 hours/day, on 10 days (no exposure on weekends)
Dose / conc.:
250 ppm
Remarks:
nominal
Dose / conc.:
1 000 ppm
Remarks:
nominal
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: each exposure day and on the day of scheduled sacrifice


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete gross necropsy was performed on the sacrificed animals as well as any animals dying prior to the termination of the study
HISTOPATHOLOGY: Yes, lungs, liver, kidneys, hea rt, aorta, spleen, pancreas, tracheobronchial lymph nodes, adrenals, testes, epididymides, ovaries, uterus, trachea, and nares (two sections).
Statistics:
ANOVA, Fisher's exact test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At both concentrations, rats and mice kept their eyes closed and noses buried in their fur during the entire exposure period. When compared to controls, exposed animals demonstrated a concentration-related delay in resuming normal locomotive and feeding activities following termination of the 6 hour exposures.
Mortality:
mortality observed, treatment-related
Description (incidence):
250 ppm: no mortality
1000 ppm: Four of five male and female rats and 1/5 male and female mice exposed died
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was significantly reduced compared to concurrent controls in rats and mice exposed at 1000 ppm. Animals exposed at the 1000 ppm concentration also showed signs of transient weight gains during extended periods of no exposure (weekends).
Description (incidence and severity):
Histopathology of the major organs was unremarkable except for the upper respiratory tract and nares. Treatment-related effects seen in the nasal passages of animals exposed at 1000 ppm showed squamous metaplasia and turbinate atrophy. Effects seen in the nasal passages of animals exposed at 250 ppm ranged squamous metaplasia to localized turbinate atrophy. In all cases the lesions were most prominent in the rostral section of the nasal cavity and tended to be less pronounced in the caudal section . Squamous metaplasia of the tracheal mucosa was found in rats exposed to 1000 ppm. Ulcerative tracheitis was also found in rats exposed to 1000 ppm. Squamous metaplasia of the trachea and the mainstem bronchi was found in mice exposed to 1000 ppm
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
mortality
Remarks on result:
other: limited parameters
Critical effects observed:
no
Conclusions:
With the exception of the observed nasal and tracheal histopathology and reduced body weights in the animals exposed to 1000 ppm, no other biologically significant changes attributable to the inhalation exposures were observed.
Executive summary:

Inhalation exposure of rats and mice with 250 and 1000 ppm of the test substance for 10 days, 6h/day caused mortality and significantly reduced body weight gain compared to concurrent controls for the high expsoure scenario. Moreover, treatment-related effects were seen in the upper respiratory tract of animals exposed to 250 and 1000 ppm.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Aug - 28 Aug 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
NTP standard protocol
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
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: 23.4 g (mean), female: 19.7 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
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
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: 13 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
17 days (13 exposures)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
31 ppm
Remarks:
analytical concentration 30.9 ± 0.5
Dose / conc.:
62.5 ppm
Remarks:
analytical concentration was 62.8 ± 1.2 ppm
Dose / conc.:
125 ppm
Remarks:
analytical concentration 125 ppm ± 3
Dose / conc.:
250 ppm
Remarks:
analytical concentration was 252 ± 4 ppm
Dose / conc.:
500 ppm
Remarks:
analytical concentration was 499 ± 9 ppm
No. of animals per sex per dose:
5
Control animals:
yes
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: on days 1, 6, and 13.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

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 and all tissues are saved in formalin.
HISTOPATHOLOGY: Yes, Histopathologic evaluation is done only on those organs/tissues showing gross evidence of treatment-related lesions to a no-effect level plus corresponding tissues are evaluated in control animals.

Other examinations:
Liver, thymus, right kidney, right testicle, heart, and lung weights are recorded for all animals surviving until the end of the study.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lethargy and abnormal breathing were observed in all mice exposed to 250 or 500 ppm; thinness was observed in most of these mice. Eye irritation was observed in all 500 ppm mice and eye discharge was observed in three 500 ppm males and two 500 ppm females. Nasal discharge was observed and low fecal and urine output were noted in 500 ppm mice.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males and three females exposed to 500 ppm died during the first week of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of males and females exposed to 125 ppm or greater were significantly less than those of the chamber controls. Males and females exposed to 250 or 500 ppm lost weight during the study. Mean male body weights were more severely depressed than mean female weights relative to sex-matched controls for all exposure levels at terminal sacrifice.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute heart weights were significantly less than those of the chamber controls in all exposed groups of males and 125 ppm or greater females; the relative heart weights of 31 and 250 ppm females were significantly less than those of the chamber controls. Absolute and relative liver weights of 125 ppm or greater males and 250 ppm females, and the absolute liver weight of 125 and 500 ppm females were significantly less than those of the chamber controls. Absolute and relative thymus weights were significantly less than those of the chamber controls in 250 and 500 ppm males and 125 ppm or greater females. Absolute lung weights of 250 and 500 ppm males were significantly less than those of the chamber controls; the relative lung weights of 250 and 500 ppm males and 125 ppm or greater females were significantly greater than those of the chamber controls. Absolute right kidney weights of 250 ppm males and females and 500 ppm males were significantly less than those of the chamber controls; relative right kidney weights of 31, 125, 250, and 500 ppm females were significantly greater than those of the chamber controls. Relative right testis weights of 250 and 500 ppm males were significantly greater than those of the chamber controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No exposure-related gross lesions were seen in early death or terminal sacrifice mice
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the nose, suppurative inflammation occurred in all males exposed to 250 or 500 ppm and all females exposed to 125 ppm or greater, and most males exposed to 125 ppm. Turbinate necrosis occurred in all exposed mice except one 31 ppm female. The necrosis was characterized by partial to complete loss of maxillo- and/or nasoturbinates with necrosis of respiratory epithelium and underlying bone. The nasal septum was also necrotic in some mice. Squamous metaplasia of the respiratory epithelium was seen on the nasal septum, turbinates, and/or lateral walls of most 125 and 250 ppm males and females. Incidences of olfactory epithelial atrophy were significantly increased in 125 and 250 ppm males and in 125 ppm or greater females. Necrosis of individual olfactory epithelial cells was observed in three females and one male exposed to 500 ppm. These changes primarily involved the dorsal meatus.

In the lung, minimal chronic active inflammation of mainstem bronchi at their bifurcation was noted in four males and two females in the 500 ppm groups. The inflammatory infiltrate consisted primarily of mononuclear cells with a few neutrophils and was associated with minimal necrosis of overlying individual epithelial cells in one male and one female in the 500 ppm groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
31 ppm
Basis for effect level:
other: local irritation
Critical effects observed:
no

Tab.1 Mortality and relative weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

5/5

-

31

5/5

94/101

62.5

5/5

97/102

125

5/5

87/91

250

5/5

73/81

500

3/2

59/68

Tab 2 Incidences of selected nonneoplastic lesions in male mice

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

0

3 (1.3) b

5** (2.0)

5** (2.4)

Turbinates, Necrosis

0

5** (1.0)

5** (1.0)

5** (1.8)

5** (3.8)

5** (3.6)

Olfactory Epithelium, Atrophy

0

0

0

5** (1.8)

4* (2.0)

2 (1.5)

Olfactory Epithelium, Necrosis

0

0

0

0

0

1 (2.0)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

4* (2.0)

5** (2.2)

3 (1.7)

Lung of males

 

 

 

 

 

 

Bronchus, Inflammation, Chronic, Active

0

 

 

 

0

4* (1.0)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of selected nonneoplastic lesions in female mice

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

0

5 (1.8)b

5** (1.8)

5** (2.0)

Turbinates, Necrosis

0

4** (1.0)

5** (1.0)

5** (2.2)

5** (3.4)

5** (3.8)

Olfactory Epithelium, Atrophy

0

0

1 (1.0)

5** (2.0)

5** (2.0)

4* (2.3)

Olfactory Epithelium, Necrosis

0

0

0

0

0

3 (1.7)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

5**(1.8)

5**(1.8)

 (2.0)

Lung of females

 

 

 

 

 

 

Bronchus, Inflammation, Chronic, Active

0

 

 

 

0

2 (1.0)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
Inhalative expsoure with 31 ppm of the vapourized test substance caused necrosis of the turbinates in male and female mice under the tested conditions. Therefore, no NOAEC could be derived.
Executive summary:

In a 2 week study groups of five male and five female mice were exposed to the test substance as a vapor at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days (13 exposures). Two male and three female mice exposed to 500 ppm died during the first week of the study. The mean body weights of mice exposed to 125 ppm or greater were significantly less than those of the chamber controls. Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, eye abnormalities, and discolored urine. The thymus weights of male mice of 250 and 500 ppm and females of 125 ppm or greater were significantly less than those of the chamber controls. Histopathologic changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of males and females principally in the 125, 250 or 500 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Aug - 28 Aug 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
NTP standard protocol
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
Species:
rat
Strain:
other: Fisher344/N
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: 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: 13 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: no data
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
16 days (12 days exposure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
31 ppm
Remarks:
analytical concentration 30.9 ± 0.5
Dose / conc.:
62.5 ppm
Remarks:
analytical concentration 62.9 ± 1.3
Dose / conc.:
125 ppm
Remarks:
analytical concentration 125 ± 2
Dose / conc.:
250 ppm
Remarks:
total analytical concentration was 252 ± 4 ppm
Dose / conc.:
500 ppm
Remarks:
total analytical concentration was 499 ± 9 ppm
No. of animals per sex per dose:
5
Control animals:
yes
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: on days 1, 6, and 13 and 14

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

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 and all tissues are saved in formalin.
HISTOPATHOLOGY: Yes (control and 500 ppm treatment group), Histopathologic evaluation is done only on those organs/tissues showing gross evidence of treatment-related lesions to a no-effect level plus corresponding tissues are evaluated in control animals.
Other examinations:
Liver, thymus, right kidney, right testicle, heart, and lung weights are recorded for all animals surviving until the end of the study.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, and discolored urine (250 and 500 ppm). Crusty noses were observed in most 500 ppm males and females and in two 250 ppm males. Eye abnormalities occured in 4 males and 4 females in the 500 ppm group
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of 250 and 500 ppm males and females and 125 ppm males were significantly less than those of the chamber controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute organ weights were significantly decreased compared to the chamber controls in the 250 and 500 ppm groups, except for testis (250 ppm), male lung (250 ppm), female lung (250 and 500 ppm), and female kidney (250 ppm). Relative organ weights that were significantly increased included the heart of both sexes at 500 ppm, the heart of females at 250 ppm, the kidney of both sexes (125 ppm or greater males, 62.5 ppm or greater females), the liver of females at 250 and 500 ppm, and the testis of the 250 and 500 ppm groups. Significant decreases were noted in the absolute and relative thymus weights of both sexes (125 ppm or greater males, 500 ppm females).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Focal eye lesions were noted at necropsy in four males and three females exposed to 500 ppm and one male exposed to 250 ppm. Suppurative inflammation of the cornea was present in most rats exposed to 500 ppm.
Suppurative inflammation, necrosis of the turbinates (except in one 125 ppm female), and squamous metaplasia of the respiratory epithelium of the nose were present in all rats exposed to 125 ppm or greater. Ulcer of the respiratory epithelium and atrophy of the olfactory epithelium occurred in all rats exposed to 250 or 500 ppm, and ulcer of the nasopharyngeal duct was present in all 500 ppm rats.
Minimal to mild inflammation and metaplasia of the respiratory epithelium was still present at 62.5ppm in all rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
31 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation
Critical effects observed:
no

Tab. 1 Mortality and relative bodyweight

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

5/5

-

31

5/5

99/99

62.5

5/5

99/101

125

5/5

91/96

250

5/5

74/82

500

5/5

59/69

Tab. 2 Incidences of selected nonneoplastic lesions in male rats

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

5**(1.6)b

5** (2.8)

5** (3.8)

5** (1.0)

Respiratory Epithelium, Ulcer

0

0

1 (1.0)

1 (2.0)

5** (3.6)

5** (3.6)

Nasopharyngeal Duct, Ulcer

0

0

0

0

0

5** (3.2)

Turbinates, Necrosis

0

0

0

5** (2.2)

5** (3.8)

5** (3.4)

Respiratory Epithelium, Metaplasia, Squamous

0

0

5** (1.0)

5**

5** (3.0)

5** (3.0)

Olfactory Epithelium, Atrophy

0

0

0

0

5** (3.0)

5** (2.8)

Eye of males

 

 

 

 

 

 

Cornea, Inflammation, Suppurative

0

 

 

0

1 (1.0)

3 (3.0)

** significantly different from the chamber control group by the Fisher exact test ( p 0.01)

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 2 Incidences of selected nonneoplastic lesions in female rats

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

2 (1.0)b

5** (2 .2)

5** (3.8)

5** (4.0)

Respiratory Epithelium, Ulcer

0

0

0

0

5** (4.0)

5** (3.8)

Nasopharyngeal Duct, Ulcer

0

0

0

0

0

5** (3.4)

Turbinates, Necrosis

0

0

0

4* (2.0)

5** (3.0)

5** (3.8)

Respiratory Epithelium, Metaplasia, Squamous

0

0

2 (1.0)

5** (2.2)

5** (2.8)

5** (3.8)

Olfactory Epithelium, Atrophy

0

0

0

0

5** (2.8)

5** (2.8)

Eye of females

 

 

 

 

 

 

Cornea, Inflammation, Suppurative

0

 

 

 

0

5** (1.2)

* significantly different from the chamber control group by the Fisher exact test ( p0.05)

** p0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
The test substance induced nonneoplastic lesions of the nose in rats after inhalative expsoure of 62.5 ppm and nd higher under the tested conditions.
Executive summary:

In a 2-week study, groups of five male and five female rats were exposed to the test substance as a vapour at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for16 days (12 days exposure). All rats survived to the end of the study. The mean body weights of rats aexposed to 125 ppm or greater were significantly less than those of the chamber controls. Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, eye abnormalities, crusty noses and discolored urine. The thymus weights of male rats exposed to 125 ppm or greater and female rats exposed to 500 ppm were significantly less than those of the chamber controls. Histopathologic changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of males and females principally in the 125, 250 or 500 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates. Focal eye lesions were noted at necropsy in four males and three females exposed to 500 ppm and one male exposed to 250 ppm. Suppurative inflammation of the cornea was present in most rats exposed to 500 ppm.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Principles of method if other than guideline:
no data
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 days
Frequency of treatment:
no data
Dose / conc.:
500 ppm
Remarks:
equivalent to 1.52 mg/L
No. of animals per sex per dose:
5
Control animals:
not specified
Critical effects observed:
not specified

Moderate to marked necrotic inflammation of the nasal cavity was detected in 5/5 male and 5/5 female rats exposed to 500 ppm for 10 days.

Conclusions:
Necrotizing inflammation of the nasal cavity under the tested conditions.
Executive summary:

Short-term inhalation exposure of rats to 500 ppm test substance caused moderate to marked necrotic inflammation of the nasal cavity in 5/5 male and 5/5 female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
96 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
other: rat and mouse

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
Principles of method if other than guideline:
Study performed according to standard NTP protocols.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
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


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 (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Analytical verification of doses or concentrations:
yes
Remarks:
analysed with an on-line gas chromatograph
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
14 weeks (93 days exposure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
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 of the test substance 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.
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 is 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.

Other examinations:
At the end of the study: samples for sperm motility, densitiy and count; vaginal cytology (controls, 32, 62, 125 ppm).
Sampling of vaginal fluid and cells was performed.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Non-parametric multiple comparison methods of Shirley (1977) for hematology, clinical chemistry, spermatid, and epididymal spermatozoal data
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights and body weight gains of 125 ppm males and females were significantly less than those of the chamber controls (males: 78%, females: 84% of control).
Food efficiency:
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the liver, right kidney, and thymus of 125 ppm males; heart, liver, and right kidney of 125 ppm females; and thymus of 62 and 125 ppm females were significantly less than those of the chamber controls.
The relative weights of the heart, right kidney, lung, and right testis of 125 ppm males and the lung of 125 ppm females were significantly greater than those of the chamber controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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. Sigificant olfactory epithelial atrophy was already observed after treatment with 32 ppm.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related decrease seen in the motility of sperm from male mice with values of those exposed to 32, 62, or 125 ppm being significantly lower (7-15%) than those of the chamber controls; except for a slight (0.5 day), but statistically significant increase in estrous cycle length, no significant differences were observed in the estrous cyclicity of female mice administered 32, 62, or 125 ppm of the vapourized test substance when compared to the chamber controls.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
16 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
32 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased sperm motility
Critical effects observed:
not specified

Tab. 1 Mortality and relative weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

10/10

-

8

10/10

98/98

16

10/10

69/105

32

10/10

101/100

62

10/10

100/97

125

10/10

78/84

Tab. 2 Incidences of nonneoplastic lesions in male mice

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

8** (1.5)b

Olfactory Epithelium, Atrophy

0

0

0

4* (1.0)

9** (2.2)

10** (2.9)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

3

1 (1.0)

9** (2.2)

Turbinate, Necrosis

0

0

0

0

0

7** (2.4)

* significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of nonneoplastic lesions in female mice

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

3 (1.0)b

8** (1.0)

Olfactory Epithelium, Atrophy

0

0

0

9**(1.1)

10** (2.4)

10** (2.8)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

3

1 (1.0)

9** (1.8)

Turbinate, Necrosis

0

0

0

0

0

6** (2.7)

** significantly different (p≤ 0.01) from the chamber control group by the Fisher exact test

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
A NOAEC of 16 ppm for local effects was derived based on the observation of nonneoplastic lesions of the mouse nose.
The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male mouse exposed to vapour concentrations of 32 ppm and higher concentration levels.
Executive summary:

In the 14 week study, groups of 10 male and 10 female mice were exposed to the test substance as a vapour at concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 hours per day with a additional T90 value (time to achieve 90% of target concentration) of 12 min, 5 days per week. All animals survived to the end of the study. The final mean body weights and body weight gains of 125 ppm males and females were significantly less than those of the chamber controls (males: 78%, females: 84% of control). The absolute weights of the liver, right kidney, and thymus of 125 ppm males; heart, liver, and right kidney of 125 ppm females; and thymus of 62 and 125 ppm females were significantly less than those of the chamber controls. The relative weights of the heart, right kidney, lung, and right testis of 125 ppm males and the lung of 125 ppm females were significantly greater than those of the chamber controls. There was a dose-related decrease seen in the motility of sperm for male mice with values of those exposed to 32, 62, or 125 ppm diethylamine being significantly lower (7-15%) than those of the chamber controls (animals exposed to 8 and 16 ppm were not investigated for spem motility changes). Except for a slight (0.5 day), but statistically significant increase in estrous cycle length, no significant differences were observed in the estrous cyclicity of female mice administered 32, 62, or 125 ppm of the vapour when compared to the chamber controls.

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. Significant olfactory epithelial atrophy was already observed after treatment with 32 ppm.

A NOAEC of 16 ppm for local effects was derived based on the observed nonneoplastic lesions of the mouse nose.

The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in the sperm motility for male mouse exposed to vapour concentrations of 32 ppm and higher concentration levels.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
Principles of method if other than guideline:
Study performed according to standard NTP protocols.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
Species:
rat
Strain:
other: Fisher344/N
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 to 7 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
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h




Analytical verification of doses or concentrations:
yes
Remarks:
analysed with an on-line gas chromatograph
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
14 weeks (93 days expsoure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale:
Because exposure to 250 or 500 ppm of the test substance 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.
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 is 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.

Other examinations:
At the end of the study: samples for sperm motility, densitiy and count; vaginal cytology (controls, 32, 62, 125 ppm).
Sampling of vaginal fluid and cells was performed.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Non-parametric multiple comparison methods of Shirley (1977) for hematology, clinical chemistry, spermatid, and epididymal spermatozoal data
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1 male animal showed torso lateral abcesss/ulcer
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
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 specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- the relative kidney weights of exposed females were increased and significantly gerater than for chamber control animals, except in the 32 ppm group
- the relative liver weights of males exposed to 125 ppm was significantly increased
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.

There were no inflammatory changes of the eye.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related decrease seen in the motility of sperm from male rats exposed to 32, 62, or 125 ppm being significantly lower (5-26%) than those of the chamber controls; no significant differences were observed in the estrous cyclicity of female rats administered 32, 62, or 125 ppm of the vapour when compared to the chamber controls. These parameters were not assessed in rats exposed to 8 and 16ppm.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
16 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
32 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased sperm motility
Critical effects observed:
not specified

Tab. 1 Mortality and relative final weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

10/10

-

8

10/10

99/98

16

10/10

103/97

32

10/10

101/98

62

10/10

102/99

125

10/10

97/99

Tab. 2 Incidences of nonneoplastic lesions of the nose in male rats

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

0

0

2 (2.5)b

10** (1.6)

Turbinates, Necrosis

0

0

0

0

0

1 (2.0)

Respiratory Epithelium, Hyperplasia

0

0

3 (1.0)

3

9** (1.3)

10** (2.3)

Respiratory Epithelium, Metaplasia, Squamous

0

0

1 (1.0)

0

1 (2.0)

10** (2.1)

Olfactory Epithelium, Atrophy

0

0

0

0

7** (1.1)

10** (1.9)

** significantly different (p≤ 0.01) from the chamber control group by the Fisher exact test

a Number of animals with lesions

b Average severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of nonneoplastic lesions of the nose in female rats

 

Control

8 ppm

16 ppm

32 ppm

62 ppm

125 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurativea

0

0

1 (2.0) b

0

3 (1.0)

7** (1.4)

Turbinates, Necrosis

0

0

0

0

0

1 (1.0)

Respiratory Epithelium, Hyperplasia

0

0

1 (1.0)

0

9** (1.2)

9** (1.9)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

0

0

5* (1.6)

Olfactory Epithelium, Atrophy

0

0

0

2 (1.0)

9** (1.2)

10** (2.2)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01)

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
A NOAEC for local effects of 16 ppm was derived. As such related observations were not detected in animals treated with 8 ppm of the vapour, the NOEC for local effects was set to be 8 ppm.
The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male rats exposed to vapour concentrations of 32 ppm and higher concentration levels.
Executive summary:

In the 14 week study, groups of 10 male and 10 female rats were exposed to the test substance as a vapour at concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 hours per day with a additional T90 value (time to achieve 90% of target concentration) of 12 min, 5 days per week. All animals survived to the end of the study. No exposure-related changes in hematology, serum chemistry indices or bodyweights were observed for exposed rats. The relative kidney weights of exposed females were increased and significantly gerater than for chamber control animals, except in the 32 ppm group. The relative liver weights of males exposed to 125 ppm was significantly increased. Examinations of the sperm motility were performed for male rats exposed to 32, 62 and 125 ppm. Significant exposure concentration-related decreases in the sperm motility were detected for all investigated vapour concentration levels. 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, and necrosis of the turbinates. Olfactory epithelial atrophy was observed in animals exposed to 32 ppm and higher concentrations.

Based on these findings, a NOAEC for local effects of 16 ppm was derived. As such related observations were not detected in animals treated with 8 ppm of the vapour, the NOEC for local effects was set to be 8 ppm. The derived LOAEC for systemic effects was 32 ppm taking into account the decrease in sperm motility for male rats exposed to vapour concentrations of 32 ppm and higher concentration levels.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
no data on control animals
Principles of method if other than guideline:
- Principle of test:
Rabbits in groups of six were exposed to the amine vapours in a dynamic chamber. Dry air was passed into a bottle equipped with a fritted disk and containing the liquid amines. These were thus volatilized and dispersed into the chamber. The bottle was held in a refrigerated, constant temperature bath.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from the level of animals nose and mouth
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
7 h/day; 5 days peer week
Dose / conc.:
50 ppm
Remarks:
analytical concentration 52.71 ppm
Dose / conc.:
100 ppm
Remarks:
analytical concentration 109.31 ppm
No. of animals per sex per dose:
6 (no gender stated)
Control animals:
other: no data specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 ppm one animal showed some forced movements of the neck; at 150 mg/m3 one rabbit was losing hair, and exhibited a muscular weakness of the hind legs.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
One animal at 100 ppm and one animal at 50 ppm showed slight loss of weight.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Considerable amount of cloudiness, indicative of edema; areas of punctate erosions of the corneal epithelium
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The blood sugar increased in all rabbits, but this effect could not be confirmed in a repeate experiment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: dark pink colored areas in the lungs of one animal; 150 mg/m3 dose: moderate to marked hyperemia of the lungs.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: In all but one animal, the kidneys showed changes indicating injury to the vascular apparatus; intense, diffuse hyperemia with small numbers of red blood corpuscles and protein precipitated into the capsular spaces; numerous casts in the cortical tubules, slight tubular degeneration (probably at least partly due to postmortem changes); marked parenchymatous degeneration in the liver (same comment as for tubular degeneration) . In the lungs of one animal, infiltration of polymorpho-nuclear cells in an area of atelectasis and in the interstitial tissue was found; in another animal, bronchopneumonia was present.

50 ppm: The lungs of all animals showed slight and moderate degrees of chronic irritation characterized by thickening of the blood vessels and focal collections of lymphocytes (peribronchitis); small inflammatory areas in the livers of five animals, in one case combined with
moderate parenchymatous degeneration ; the same animal showed also some inflammatory changes in the kidneys, with moderate parenchymatous and fatty degeneration.
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
50 ppm
Based on:
test mat.
Remarks:
(0.15mg/L)
Sex:
not specified
Basis for effect level:
other: effects on eyes and lungs
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
50 ppm
Sex:
not specified
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
limited parameters assessed
Critical effects observed:
not specified

All rabbits exposed at 100 and 50 ppm seven hours a day, five days a week, for six weeks, survived, but at 50 ppm still showed marked irritation of the lung tissue and cornea, with moderate peribronchitis and slight thickening of the vascular walls, and multiple punctate erosions and edema of the cornea. Changes in the liver consisted of occasional foci of moderate parenchymatous degeneration.

Executive summary:

Rabbits in groups of six were exposed to the vapour in a dynamic chamber at 50 and 100 ppm (equivalent to 150 or 300 mg/m3) for 7 h a day, 5 days a week, for 6 weeks. All animals survived. Expsoure to 100 ppm of the vapour produced parenchymatous degeneration and, occasionally, inflammatory processes in liver and kidney. Both vapour concentrations resulted in marked chronic irritation of the lungs. Animals treated with 50 ppm of the vapour showed corneal injury present as corneal erosions and edema.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: MCB Manufacturing Chemists, Inc. Norwood Ohio
- Lot/batch No.: Lot 2J20 and J8L17
- Analytical purity: 99.9 %
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 (Wilmington, Mass)
- Housing: individually in stainless, steel wire mesh cages
- Diet: Purina Laboratory Chow, ad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4.5 m3 stainless-steel and glass inhalation chambers
- Rate of air: 1.1 m3/min
- Temperature, humidity, pressure in air chamber: 23 +/- 3°C, 50+/-10%, slightly negative pressure relative to ambient (- 0.25 cm water)
- Air change rate: 12-15 air changes/h


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
infrared analyzer; Chamber atmospheres were monitored 2-4 times per hour
Duration of treatment / exposure:
24 weeks (118-123 expsoure days)
Frequency of treatment:
5 days per week, 6.5 hours daily
Dose / conc.:
25 ppm (nominal)
Remarks:
analytical concentration 26 ppm; equivalent to 0.076 mg/L
Dose / conc.:
250 ppm (nominal)
Remarks:
analytical concentration 251 ppm; equivalent to 0.76 mg/l
No. of animals per sex per dose:
10 animals per sex per dose were sacrificed after 30 and 60 days.
50 animals per sex per dose were sacrificed after 120 days.
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: the day predceding the exposure and at 2 weeks intervals thoughout the study.


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: Yes
- Parameters examined: hemoglobin, hematocrit, complete and differential counts.


CLINICAL CHEMISTRY: Yes
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphatase (CPK), blood urea nitrogen (BUN), creatinine (CRE), sorbitol dehydrogenase (SDH)


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data


OTHER: Electrocardiograms (ECGs) were recorded from 10 anesthetized rats/sex/group (35 mg/kg secobarbital, ip) at the terminal sacrifice.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes all major organs and tissue (not further specified)
HISTOPATHOLOGY: Yes, lungs, liver, kidneys, heart, spleen, tracheobronchial lymph nodes, adrenals, testes, seminal vesicles, ovaries, uterus, trachea, eye, urinary bladder, nares (nares not examined for 25 ppm rats).
Statistics:
One-way analysis of variance, Duncan's multiple range test, Kruskal-Wallis test, χ2-test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 250 ppm both sexes exhibited evidence of the strong irritant properties; animal observations included sneezing, tearing, squinting and attempted avoidance of the the test substance by burying their noses in their fur during the entire exposure period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
10 female rats (250 ppm) died due to malfunction of the automatic watering system.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight exposed at 250 ppm were significantly reduced compared to the control groups at week 2 and remained reduced at each subsequent weighing period.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Several clinical chemistry indices, e.g., SDH and CPK, were elevated in one group of rats, or in only one sex of rats, at one of the scheduled interim sacrifices. These changes were not attributed to the exposure. Creatinine levels were significantly elevated in female rats exposed at both 25 and 250 ppm for 24 weeks, while BUN levels were significantly elevated in rat of both sexes exposed at 250 ppm for 24 weeks. These changes, however, could not be correlated with any observed gross or histologic renal damage.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No consistent changes in absolute or relative organ weights were seen in rats sacrificed following 30 or 60 days of exposure. Absolute lung weights were significantly decreased and lung-to-body weight ratios were significantly increased in male rats exposed at 250 for 24 weeks. While statistically significant, these appear to reflect the statistically reduced mean body weights of the rats exposed at 250 ppm rather than indicating toxic effects on these organs.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The group distributions for squamous metaplasia, lymphoid hyperplasia, and suppurative rhinitis in the nares of both male and female rats exposed at 250 ppm for 24 weeks were significantly increased over the distributions for controls. Most of the rats exposed at 250 ppm had an acute inflammatory exudate present in the nasal cavity. Inflammation of the nasal passages was manifested by increased numbers of lymphocytes and plasmacytes in the lamina propria and hyperplasia of lymphoid tissue. The inflammatory changes were located primarily at level two, i.e., approximately one-third of the distance from the posterior aspect of the incisor teeth to the incisive papilla. The region just anterior to the incisor showed inflammatory changes, but not as pronounced. Mild to moderate squamous metaplasia of the respiratory mucosa occurred in focal areas of the middle portions of the nasal septum and lateral walls of the nasal cavity primarily at level two.
Nasal passages of rats exposed to 25ppm were not examined.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In the testes of male rats (250 ppm) slight increase in occurence of hyperspermatogenesis, degenration of seminiferous tubules, and mineralization within the tubules compared to control animals. In most cases unilateral and not considered to be related to treatment
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
25 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
local
Remarks on result:
not determinable
Remarks:
target organ not examined in lowest dose
Critical effects observed:
no

Rats exposed at 250 ppm exhibited evidence of toxicity indicated by a decrease in body weight and histopathologic changes the nares. The incidence of the bronchiola lymphoid hyperplasia in the lungs of exposed animals, however, did not increase with increasing exposure.

Conclusions:
Rats exposed at 250 ppm exhibited evidence of toxicity indicated by a decrease in body weight and histopathologic changes of the nares. Therefore, a NOAEC of 25 ppm was derived.
Executive summary:

In a repeated exposure study, male and female Fischer 344 (F-344) rats were exposed at 75 mg/m3 (25 ppm) or 750 mg/m3 (250 ppm) vapourized test substance, 6.5 hours/day, five days/week, for 24 weeks in order to assess cardiac and other organ system toxicity. Scheduled sacrifices were performed following 30, 60, and 120 days of exposure. During the first two weeks of exposure, the rats exposed at 750 mg/m3 did not gain weight. After two weeks, however, the rate of weight gain of these rats was greater than that of controls. Nevertheless, mean body weights for both sexes of rats exposed at 750 mg/m3 remained depressed compared to controls throughout the study. Sneezing, tearing, and reddened noses were seen in rats exposed at 750 mg/m3 of the vapour. Histopathologic examinations revealed lesions of the nasal mucosa of rats exposed at 750 mg/m3 (rats exposed at 75 mg/m3 were not evaluated for nasal effects). These lesions of the nasal respiratory epithelium consisted of squamous metaplasia, suppurative rhinitis, and lymphoid hyperplasia. There were no pronounced treatment-related effects on organ weights, hematology, or clinical chemistry indices except for blood urea nitrogen which was evaluated in rats of both sexes exposed at 750 mg/m3 for 24 weeks. In contrast to the high-dose animals, no treatment-related effects were observed in rats intermittently exposed at 75 mg/m3 of the test item for up to 24 weeks. No evidence of cardiotoxicity was seen in rats exposed to either concentration for up to 24 weeks.

 

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Animals were exposed via whole body inhalation to each amine at 250 or 1000 ppm for 6 hrs/day for a total of 10 exposure days .
GLP compliance:
not specified
Specific details on test material used for the study:
- Analytical purity: 99.9%
Species:
other: rats and mice
Strain:
other: Fischer 344 rats and B6C3F1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Diet: NIH 07 pellets (Ziegler Bros., Gardner, PA), ad libitum
- Water: water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
using a Wilks-Miran 1A infrared analyzer
Duration of treatment / exposure:
10 days
Frequency of treatment:
6 hours/day, on 10 days (no exposure on weekends)
Dose / conc.:
250 ppm
Remarks:
nominal
Dose / conc.:
1 000 ppm
Remarks:
nominal
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: each exposure day and on the day of scheduled sacrifice


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: No data


CLINICAL CHEMISTRY: No data


URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete gross necropsy was performed on the sacrificed animals as well as any animals dying prior to the termination of the study
HISTOPATHOLOGY: Yes, lungs, liver, kidneys, hea rt, aorta, spleen, pancreas, tracheobronchial lymph nodes, adrenals, testes, epididymides, ovaries, uterus, trachea, and nares (two sections).
Statistics:
ANOVA, Fisher's exact test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At both concentrations, rats and mice kept their eyes closed and noses buried in their fur during the entire exposure period. When compared to controls, exposed animals demonstrated a concentration-related delay in resuming normal locomotive and feeding activities following termination of the 6 hour exposures.
Mortality:
mortality observed, treatment-related
Description (incidence):
250 ppm: no mortality
1000 ppm: Four of five male and female rats and 1/5 male and female mice exposed died
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was significantly reduced compared to concurrent controls in rats and mice exposed at 1000 ppm. Animals exposed at the 1000 ppm concentration also showed signs of transient weight gains during extended periods of no exposure (weekends).
Description (incidence and severity):
Histopathology of the major organs was unremarkable except for the upper respiratory tract and nares. Treatment-related effects seen in the nasal passages of animals exposed at 1000 ppm showed squamous metaplasia and turbinate atrophy. Effects seen in the nasal passages of animals exposed at 250 ppm ranged squamous metaplasia to localized turbinate atrophy. In all cases the lesions were most prominent in the rostral section of the nasal cavity and tended to be less pronounced in the caudal section . Squamous metaplasia of the tracheal mucosa was found in rats exposed to 1000 ppm. Ulcerative tracheitis was also found in rats exposed to 1000 ppm. Squamous metaplasia of the trachea and the mainstem bronchi was found in mice exposed to 1000 ppm
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
mortality
Remarks on result:
other: limited parameters
Critical effects observed:
no
Conclusions:
With the exception of the observed nasal and tracheal histopathology and reduced body weights in the animals exposed to 1000 ppm, no other biologically significant changes attributable to the inhalation exposures were observed.
Executive summary:

Inhalation exposure of rats and mice with 250 and 1000 ppm of the test substance for 10 days, 6h/day caused mortality and significantly reduced body weight gain compared to concurrent controls for the high expsoure scenario. Moreover, treatment-related effects were seen in the upper respiratory tract of animals exposed to 250 and 1000 ppm.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Aug - 28 Aug 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
NTP standard protocol
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
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: 23.4 g (mean), female: 19.7 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
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
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: 13 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
17 days (13 exposures)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
31 ppm
Remarks:
analytical concentration 30.9 ± 0.5
Dose / conc.:
62.5 ppm
Remarks:
analytical concentration was 62.8 ± 1.2 ppm
Dose / conc.:
125 ppm
Remarks:
analytical concentration 125 ppm ± 3
Dose / conc.:
250 ppm
Remarks:
analytical concentration was 252 ± 4 ppm
Dose / conc.:
500 ppm
Remarks:
analytical concentration was 499 ± 9 ppm
No. of animals per sex per dose:
5
Control animals:
yes
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: on days 1, 6, and 13.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

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 and all tissues are saved in formalin.
HISTOPATHOLOGY: Yes, Histopathologic evaluation is done only on those organs/tissues showing gross evidence of treatment-related lesions to a no-effect level plus corresponding tissues are evaluated in control animals.

Other examinations:
Liver, thymus, right kidney, right testicle, heart, and lung weights are recorded for all animals surviving until the end of the study.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lethargy and abnormal breathing were observed in all mice exposed to 250 or 500 ppm; thinness was observed in most of these mice. Eye irritation was observed in all 500 ppm mice and eye discharge was observed in three 500 ppm males and two 500 ppm females. Nasal discharge was observed and low fecal and urine output were noted in 500 ppm mice.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males and three females exposed to 500 ppm died during the first week of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of males and females exposed to 125 ppm or greater were significantly less than those of the chamber controls. Males and females exposed to 250 or 500 ppm lost weight during the study. Mean male body weights were more severely depressed than mean female weights relative to sex-matched controls for all exposure levels at terminal sacrifice.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute heart weights were significantly less than those of the chamber controls in all exposed groups of males and 125 ppm or greater females; the relative heart weights of 31 and 250 ppm females were significantly less than those of the chamber controls. Absolute and relative liver weights of 125 ppm or greater males and 250 ppm females, and the absolute liver weight of 125 and 500 ppm females were significantly less than those of the chamber controls. Absolute and relative thymus weights were significantly less than those of the chamber controls in 250 and 500 ppm males and 125 ppm or greater females. Absolute lung weights of 250 and 500 ppm males were significantly less than those of the chamber controls; the relative lung weights of 250 and 500 ppm males and 125 ppm or greater females were significantly greater than those of the chamber controls. Absolute right kidney weights of 250 ppm males and females and 500 ppm males were significantly less than those of the chamber controls; relative right kidney weights of 31, 125, 250, and 500 ppm females were significantly greater than those of the chamber controls. Relative right testis weights of 250 and 500 ppm males were significantly greater than those of the chamber controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No exposure-related gross lesions were seen in early death or terminal sacrifice mice
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the nose, suppurative inflammation occurred in all males exposed to 250 or 500 ppm and all females exposed to 125 ppm or greater, and most males exposed to 125 ppm. Turbinate necrosis occurred in all exposed mice except one 31 ppm female. The necrosis was characterized by partial to complete loss of maxillo- and/or nasoturbinates with necrosis of respiratory epithelium and underlying bone. The nasal septum was also necrotic in some mice. Squamous metaplasia of the respiratory epithelium was seen on the nasal septum, turbinates, and/or lateral walls of most 125 and 250 ppm males and females. Incidences of olfactory epithelial atrophy were significantly increased in 125 and 250 ppm males and in 125 ppm or greater females. Necrosis of individual olfactory epithelial cells was observed in three females and one male exposed to 500 ppm. These changes primarily involved the dorsal meatus.

In the lung, minimal chronic active inflammation of mainstem bronchi at their bifurcation was noted in four males and two females in the 500 ppm groups. The inflammatory infiltrate consisted primarily of mononuclear cells with a few neutrophils and was associated with minimal necrosis of overlying individual epithelial cells in one male and one female in the 500 ppm groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
31 ppm
Basis for effect level:
other: local irritation
Critical effects observed:
no

Tab.1 Mortality and relative weights

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

5/5

-

31

5/5

94/101

62.5

5/5

97/102

125

5/5

87/91

250

5/5

73/81

500

3/2

59/68

Tab 2 Incidences of selected nonneoplastic lesions in male mice

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

0

3 (1.3) b

5** (2.0)

5** (2.4)

Turbinates, Necrosis

0

5** (1.0)

5** (1.0)

5** (1.8)

5** (3.8)

5** (3.6)

Olfactory Epithelium, Atrophy

0

0

0

5** (1.8)

4* (2.0)

2 (1.5)

Olfactory Epithelium, Necrosis

0

0

0

0

0

1 (2.0)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

4* (2.0)

5** (2.2)

3 (1.7)

Lung of males

 

 

 

 

 

 

Bronchus, Inflammation, Chronic, Active

0

 

 

 

0

4* (1.0)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 3 Incidences of selected nonneoplastic lesions in female mice

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

0

5 (1.8)b

5** (1.8)

5** (2.0)

Turbinates, Necrosis

0

4** (1.0)

5** (1.0)

5** (2.2)

5** (3.4)

5** (3.8)

Olfactory Epithelium, Atrophy

0

0

1 (1.0)

5** (2.0)

5** (2.0)

4* (2.3)

Olfactory Epithelium, Necrosis

0

0

0

0

0

3 (1.7)

Respiratory Epithelium, Metaplasia, Squamous

0

0

0

5**(1.8)

5**(1.8)

 (2.0)

Lung of females

 

 

 

 

 

 

Bronchus, Inflammation, Chronic, Active

0

 

 

 

0

2 (1.0)

*significantly different (p≤ 0.05) from the chamber control group by the Fisher exact test

** p≤ 0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
Inhalative expsoure with 31 ppm of the vapourized test substance caused necrosis of the turbinates in male and female mice under the tested conditions. Therefore, no NOAEC could be derived.
Executive summary:

In a 2 week study groups of five male and five female mice were exposed to the test substance as a vapor at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days (13 exposures). Two male and three female mice exposed to 500 ppm died during the first week of the study. The mean body weights of mice exposed to 125 ppm or greater were significantly less than those of the chamber controls. Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, eye abnormalities, and discolored urine. The thymus weights of male mice of 250 and 500 ppm and females of 125 ppm or greater were significantly less than those of the chamber controls. Histopathologic changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of males and females principally in the 125, 250 or 500 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Aug - 28 Aug 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
NTP standard protocol
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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
Species:
rat
Strain:
other: Fisher344/N
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: 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: 13 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: no data
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using stream-select and gas sampling valves in a separate heated valve oven.
Duration of treatment / exposure:
16 days (12 days exposure)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
31 ppm
Remarks:
analytical concentration 30.9 ± 0.5
Dose / conc.:
62.5 ppm
Remarks:
analytical concentration 62.9 ± 1.3
Dose / conc.:
125 ppm
Remarks:
analytical concentration 125 ± 2
Dose / conc.:
250 ppm
Remarks:
total analytical concentration was 252 ± 4 ppm
Dose / conc.:
500 ppm
Remarks:
total analytical concentration was 499 ± 9 ppm
No. of animals per sex per dose:
5
Control animals:
yes
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: on days 1, 6, and 13 and 14

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

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 and all tissues are saved in formalin.
HISTOPATHOLOGY: Yes (control and 500 ppm treatment group), Histopathologic evaluation is done only on those organs/tissues showing gross evidence of treatment-related lesions to a no-effect level plus corresponding tissues are evaluated in control animals.
Other examinations:
Liver, thymus, right kidney, right testicle, heart, and lung weights are recorded for all animals surviving until the end of the study.
Statistics:
- The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Dose related effects on survival were analysed using Cox´s method for testing 2 groups for equality and Taraone´s life table test to identify dose-realted trends.
- Organ and body weight data: multiple comparison procedure of Dunnett (1955) and Williams (1971)
- Fisher exact test and chi-square statistics
- Polyk-test for neoplastic and nonneoplastic lesions prevalence assessment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, and discolored urine (250 and 500 ppm). Crusty noses were observed in most 500 ppm males and females and in two 250 ppm males. Eye abnormalities occured in 4 males and 4 females in the 500 ppm group
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of 250 and 500 ppm males and females and 125 ppm males were significantly less than those of the chamber controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute organ weights were significantly decreased compared to the chamber controls in the 250 and 500 ppm groups, except for testis (250 ppm), male lung (250 ppm), female lung (250 and 500 ppm), and female kidney (250 ppm). Relative organ weights that were significantly increased included the heart of both sexes at 500 ppm, the heart of females at 250 ppm, the kidney of both sexes (125 ppm or greater males, 62.5 ppm or greater females), the liver of females at 250 and 500 ppm, and the testis of the 250 and 500 ppm groups. Significant decreases were noted in the absolute and relative thymus weights of both sexes (125 ppm or greater males, 500 ppm females).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Focal eye lesions were noted at necropsy in four males and three females exposed to 500 ppm and one male exposed to 250 ppm. Suppurative inflammation of the cornea was present in most rats exposed to 500 ppm.
Suppurative inflammation, necrosis of the turbinates (except in one 125 ppm female), and squamous metaplasia of the respiratory epithelium of the nose were present in all rats exposed to 125 ppm or greater. Ulcer of the respiratory epithelium and atrophy of the olfactory epithelium occurred in all rats exposed to 250 or 500 ppm, and ulcer of the nasopharyngeal duct was present in all 500 ppm rats.
Minimal to mild inflammation and metaplasia of the respiratory epithelium was still present at 62.5ppm in all rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
31 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation
Critical effects observed:
no

Tab. 1 Mortality and relative bodyweight

Concentration (ppm)

Survival (male/female)

Final weight in % relative to control (male/female)

0

5/5

-

31

5/5

99/99

62.5

5/5

99/101

125

5/5

91/96

250

5/5

74/82

500

5/5

59/69

Tab. 2 Incidences of selected nonneoplastic lesions in male rats

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of males

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

5**(1.6)b

5** (2.8)

5** (3.8)

5** (1.0)

Respiratory Epithelium, Ulcer

0

0

1 (1.0)

1 (2.0)

5** (3.6)

5** (3.6)

Nasopharyngeal Duct, Ulcer

0

0

0

0

0

5** (3.2)

Turbinates, Necrosis

0

0

0

5** (2.2)

5** (3.8)

5** (3.4)

Respiratory Epithelium, Metaplasia, Squamous

0

0

5** (1.0)

5**

5** (3.0)

5** (3.0)

Olfactory Epithelium, Atrophy

0

0

0

0

5** (3.0)

5** (2.8)

Eye of males

 

 

 

 

 

 

Cornea, Inflammation, Suppurative

0

 

 

0

1 (1.0)

3 (3.0)

** significantly different from the chamber control group by the Fisher exact test ( p 0.01)

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Tab. 2 Incidences of selected nonneoplastic lesions in female rats

 

Control

31 ppm

62.5 ppm

125 ppm

250 ppm

500 ppm

Nose of females

 

 

 

 

 

 

Inflammation, Suppurative a

0

0

2 (1.0)b

5** (2 .2)

5** (3.8)

5** (4.0)

Respiratory Epithelium, Ulcer

0

0

0

0

5** (4.0)

5** (3.8)

Nasopharyngeal Duct, Ulcer

0

0

0

0

0

5** (3.4)

Turbinates, Necrosis

0

0

0

4* (2.0)

5** (3.0)

5** (3.8)

Respiratory Epithelium, Metaplasia, Squamous

0

0

2 (1.0)

5** (2.2)

5** (2.8)

5** (3.8)

Olfactory Epithelium, Atrophy

0

0

0

0

5** (2.8)

5** (2.8)

Eye of females

 

 

 

 

 

 

Cornea, Inflammation, Suppurative

0

 

 

 

0

5** (1.2)

* significantly different from the chamber control group by the Fisher exact test ( p0.05)

** p0.01

aNumber of animals with lesions

bAverage severity grade of lesions in affected animals: 1= minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
The test substance induced nonneoplastic lesions of the nose in rats after inhalative expsoure of 62.5 ppm and nd higher under the tested conditions.
Executive summary:

In a 2-week study, groups of five male and five female rats were exposed to the test substance as a vapour at concentrations of 0, 31, 62.5, 125, 250, or 500 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for16 days (12 days exposure). All rats survived to the end of the study. The mean body weights of rats aexposed to 125 ppm or greater were significantly less than those of the chamber controls. Clinical findings included lethargy, nasal/eye discharge, abnormal breathing, thinness, eye abnormalities, crusty noses and discolored urine. The thymus weights of male rats exposed to 125 ppm or greater and female rats exposed to 500 ppm were significantly less than those of the chamber controls. Histopathologic changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of males and females principally in the 125, 250 or 500 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates. Focal eye lesions were noted at necropsy in four males and three females exposed to 500 ppm and one male exposed to 250 ppm. Suppurative inflammation of the cornea was present in most rats exposed to 500 ppm.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Principles of method if other than guideline:
no data
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 days
Frequency of treatment:
no data
Dose / conc.:
500 ppm
Remarks:
equivalent to 1.52 mg/L
No. of animals per sex per dose:
5
Control animals:
not specified
Critical effects observed:
not specified

Moderate to marked necrotic inflammation of the nasal cavity was detected in 5/5 male and 5/5 female rats exposed to 500 ppm for 10 days.

Conclusions:
Necrotizing inflammation of the nasal cavity under the tested conditions.
Executive summary:

Short-term inhalation exposure of rats to 500 ppm test substance caused moderate to marked necrotic inflammation of the nasal cavity in 5/5 male and 5/5 female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
48 mg/m³
Study duration:
subchronic
Species:
other: rat and mouse

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Inhalation:

GLP-compliant 14-week inhalation studies in rats and mice were performed according to NTP protocols.

 

In the sub-chronic study with male and female rats (NTP 2003, reliability 2) 10 animals per sex and dose were exposed to vapour concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 h plus T90 (time to achieve 90% of target concentration) of 12 minutes per day, 5 days per week for 14 weeks. All animals survived to the end of the study. No exposure-related changes in haematology, serum chemistry indices or bodyweights were observed for exposed rats. The relative kidney weights of all exposed females were increased and significantly greater than those of the chamber controls, except in the 32 ppm group. Examinations of the sperm motility were performed for male rats exposed to 32, 62 and 125 ppm. Significant exposure concentration-related decreases in the sperm motility were detected for all investigated vapour concentration levels. 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, and necrosis of the turbinates. Olfactory epithelial atrophy was observed in animals exposed to 32 ppm and higher concentrations.

Another sub-chronic study conducted with male and female mice (NTP 2003, reliability 2) is available. 10 animals per sex and dose were exposed to the test substance as a vapour at concentrations of 0, 8, 16, 32, 62, or 125 ppm, 6 hours per day with an additional T90 value of 12 min, 5 days per week. All animals survived to the end of the study.The final mean body weights and body weight gains of 125 ppm males and females were significantly less than those of the chamber controls (males: 78%, females: 84% of control). The absolute weights of the liver, right kidney, and thymus of 125 ppm males; heart, liver, and right kidney of 125 ppm females; and thymus of 62 and 125 ppm females were significantly less than those of the chamber controls. The relative weights of the heart, right kidney, lung, and right testis of 125 ppm males and the lung of 125 ppm females were significantly greater than those of the chamber controls. There was a dose-related decrease seen in the motility of sperm for male mice with values of those exposed to 32, 62, or 125 ppm diethylamine being significantly lower (7-15%) than those of the chamber controls (animals exposed to 8 and 16 ppm were not investigated for spem motility changes). Except for a slight (0.5 day), but statistically significant increase in estrous cycle length, no significant differences were observed in the estrous cyclicity of female mice administered 32, 62, or 125 ppm of the vapour when compared to the chamber controls. 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, and necrosis of the turbinates. Sigificant olfactory epithelial atrophy was already observed after treatment with 32 ppm.

 

Justification for classification or non-classification

The primary effect observed was local irritation. Systemic toxicity was mostly limited to reduced body weight (gain) at concentrations well above those leading to local toxicity and might be secondary to local inflammation. Reduced sperm motility was observed in rats and mice, and though the values were significantly different from the controls, the changes were not large enough to not be within normal biological variation. In the absence of any histopathological findings in the testis, a final decision on the relevance of these effects is postponed until data from a modified (longer pre-treatment time) OECD 422 study become available.

Based on the currently available data, classification is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008