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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylhydroxylamine
- Physical state: liquid
- Analytical purity: 99.1%
- Lot/batch No.: K-03-E
- Expiration date of the lot/batch: April 3rd, 1995

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Age: seven and one-half weeks old at initiation of exposure
- Weight at study initiation: 197 to 241 grains for the males and from 148 to 189 grams for the females
- Fasting period before study: not relevant
- Housing: individually in clean suspended wire-mesh cages suspended above cage-board
- Diet (ad libitum): Purina® Certified Rodent Chow® #5002
- Water (ad libitum): Tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70° to 77°F
- Humidity (%): 22-56
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: In-Tox Products, Inc. nose-only exposure units
- Method of holding animals in test chamber: individually in tubes
- System of generating vapors: vaporization of the neat test material, using a glass U-tube filled with 3 mm glass beads
- Temperature, humidity, pressure in air chamber: Engineering probes and transmitters were used to monitor temperature (°C) and relative humidity (% RH). Oxygen content was measured by Mine Safety Appliances Remote Sampling Systems with an oxygen sensor. Negative pressure was monitored by Dwyer Magnehelic® Indicating Transmitter pressure gauges (0-2.0 inches of water).
- Air change rate: at least 12 to 15 air changes per hour
- Method of particle size determination:
- Treatment of exhaust air: drawing the air through an acetone dry ice bath and an in-lire HEPA filter

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectroscopy using a Miran lA gas analyzer
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test atmosphere sampling for concentration analysis was performed at approximate 60 minute (minimum) intervals during the exposure periods.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours per day , 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
15 ppm
Dose / conc.:
150 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Ye
- Time schedule: twice daily for all animais, once prior to placement in the tubes and once approximately one hour after completion of exposure. The clinical condition of the animals also was monitored (to the extent possible for rats restrained in exposure tubes) during exposure periods. On nonexposure days, including the recovery period, clinical examinations were performed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning one week prior to initiation of test article exposure, and just prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet)
Differential Leukocyte Count*:
- Unsegmented Neutrophil (Unsegmented)
- Lymphocyte
- Monocyte
- Eosinophil -Basophil
- Segmented Neutrophil
(Segmented) RBC Morphology, Platelet Estimate
* Performed on the control and high dose groups at the week 4 and 6 necropsies and on the mid dose group at the week 4 necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Glucose, Urea Nitrogen, Creatinine, Total Protein, Albumin, Albumin/Globulin Ratio (A/G Ratio), Globulin, Calcium, Phosphorus, Chloride, Total Bilirubin, Gamma Glutamyltransferase, Bile Acids, Serum Aspartate Aminotransferase, Serum Alanine Aminotransferase, Serum Alkaline Phosphatase, Sodium, Potassium, Total Cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 or week 6 necropsies
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked:
Volume, Color (CLOR), Appearance (APP), Specific Gravity (SG), pH, Leukocytes (WBC), Nitrites (NIT), Protein (PRO), Glucose (GLU), Ketones (KET,) Bilirubin (BIL), Occult Blood (BLD), Urobilinogen, Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly, beginning one week prior to initiation of test article exposure, and prior to the scheduled necropsy
- Dose groups that were examined: all
- Battery of functions tested: handling observations, open-field observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.

ORGAN WEIGHTS: Yes
Adrenals Spleen
Brain Ovaries
Epididymides Testes
Heart Thymus Kidneys
Liver
Lungs

HISTOPATHOLOGY: Yes
Adrenals (2), Aorta, Bone with marrow (sternebrae), Brain (forebrain, midbrain, hindbrain), Eyes with optic nerve (2), Gastrointestinal tract ( Esophagus, Stomach, Duodenum, Jejunum, Ileum, Peyers' patches, Cecum, Colon, Rectum), Heart, Kidneys (2), Liver (sections of two lobes), Lungs [including bronchi, fixed by inflation with fixative (2)], Lymph node (bronchial, mesenteric, suprapharyngeal), Mammary gland (females only), Nasal cavities, Ovaries with oviducts (2), Pancreas, Parathyroids (if present), Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands [submaxillary (2)], Seminal vesicles (2), Skeletal muscle (vastus medialis), Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes with epididymides (2), Thymus, Thyroids, Trachea, Urinary bladder, Uterus with vagina, All gross lesions.
Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the treatment groups to the vehicle control group by sex. All means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a Digital® MicroVAX® 3400 computer with appropriate programming. Body weight, body weight change, food consumption, clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance followed by Dunnett's test.
Clinical laboratory values for cell types that occur at a low incidence (monocytes, eosinophils, basophils, unsegmented neutrophils) were not subjected to statistical analyses.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (Tables 1 through 6)
In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies.
The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level.

BODY WEIGHT AND WEIGHT GAIN (Tables 35, 36)
Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. 

FOOD CONSUMPTION (Table 37)
Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animals were similar to the control group values.

HAEMATOLOGY (Tables 38, 39, 40)
At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group mates and females, and the lymphocyte count was reduced in the females in this group.

CLINICAL CHEMISTRY (Table 41)
Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the mates in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm mates, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level.

URINALYSIS (Tables 42, 43)
Total urine volume was increased in the 1500 ppm group males and females relative to the control group values at the week 4 and week 6 evaluations; however, the increases were not statistically significant. In addition, urine volume is a highly variable parameter, and no other findings were noted in these animals that would account for this increase in volume; therefore, the changes in urine volume were not attributed to test article exposure.

NEUROBEHAVIOUR (Tables 7 through 20)
The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related findings were noted during the Functional Observational Battery evaluations

ORGAN WEIGHTS (Tables 47, 48, 49, 50)
At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the 1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy.

GROSS PATHOLOGY (Tables 44, 45, 46)
No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. 

HISTOPATHOLOGY: NON-NEOPLASTIC (Tables 51, 52, 53)
At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal nonsuppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level.

Effect levels

open allclose all
Dose descriptor:
LOAEC
Remarks:
systemic toxicity
Effect level:
1 506 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight reversible decreases in albumin and A/G ratio (females only)
Dose descriptor:
NOEC
Remarks:
systemic toxicity
Effect level:
15 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
LOAEC
Remarks:
nasal irritation
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis
Dose descriptor:
NOEC
Remarks:
nasal irritation
Effect level:
15 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
150 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
other: liver and thymus
Organ:
liver
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights-and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues.
Executive summary:

Potential subchronic toxic effects of the test article, Diethylhydroxylamine (DEHA), were evaluated in this 28-day study in rats. The test article was administered via nose-only inhalation to three groups, each comprised of 15 male and 15 female Crl:CD®BRrats, for a period of six hours per day, five days per week, for four consecutive weeks (minimum of 20 total exposures). The targeted exposure concentrations were 15, 150 and 1500 ppm. The test atmosphere concentrations were monitored by infrared absorbance and were found to be 15, 150 and 1506 ppm. A concurrent control group of identical design received only filtered air, on a comparable regimen. The animals were observed for clinical signs and effects on body weight, food consumption and clinical pathology parameters. Data from detailed physical examinations, including Functional Observational Battery data (handling and open field observations), were recorded during the pretest period and during weeks 0 through 5. After completion of exposure, 5 rats/sex/group entered an approximate two-week (nonexposure) recovery period, after which they were euthanized; necropsies were performed, and selected organs were weighed. The remaining rats in each group were euthanized immediately following the exposure period and necropsied as described above. A microscopic examination was conducted on selected tissues from all groups.

In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies. The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level. The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related fmdings were noted during the Functional Observational Battery evaluations.Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animais were similar to the control group values. At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group males and females, and the lymphocyte count was reduced in the females in this group. Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the males in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm males, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level. No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy. At the necropsy of animals that died during the study, no test article-related microscopic observations were noted. At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal non suppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level. In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues. No toxicity was noted at a dose level of 15 ppm. Based on these results, exposure levels of 150 and 15 ppm were considered to be the NOAEC (no observed adverse effect concentration) and NOEC (no observed effect concentration), respectively, for systemic toxicity and the exposure level of 15 ppm was considered to be the NOEC for nasal irritation, under the conditions of this study.