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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well docuemented, realised according to a protocol similar to an OECD guideline, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Effect of Inhaled Azodicarbonamide on F344/N Rats and B6C3F Mice with 2-Week and 13-Week Inhalation Exposures
Author:
Medinsky, M. A., Bechtold, W.E., Birnbaum, L.S., et al.
Year:
1990
Bibliographic source:
Fundamental and Applied Toxicology 15, 308-319
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
lower number of animals, no evaluation of water consumption, no ophtalmological examination
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Azodicarbonamide
- Molecular formula (if other than submission substance):H2NOC-N=N-CONH2
- Substance type:organic
- Physical state:solid (powder)
- Analytical purity:98%
- Impurities (identity and concentrations): biurea (0.4-0.7%)
- Source: Midwest Research Institute

Test animals

Species:
rat
Strain:
other: F344/N
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 2week study: Frederick Cancer Research Facility (Frederick, MD); 13 week study: Simonsen laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 7weeks (at the beginning of exposure)
- Weight at study initiation:no data
- Fasting period before study:no
- Housing:2/cage during acclimatization, 1/cage during main study.
- Diet (e.g. ad libitum): Zeigler NIH-07 Open Formula Rat Ration (Zeigler Brothers, Inc., Gardners, PA)
- Water (e.g. ad libitum): not mentioned but "automatic watering system" suppose ad libitum
- Acclimation period: 2 week study: 20-21 days, 13week study: 18-22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.2-25.4
- Humidity (%):70-85
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:no data

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: 2-week: The overall, mean ADA concentration for each chamber was within 8% or less of target concentration. The relative standard deviation of daily means was within 16%. The aerosol had an average mass median aerodynamic diameter (MMAD) of 2.13 µm (range 1.89 to 2.45) with a mean geometric standard deviation of 1.9.

13-week: The overall mean ADA concentration for each chamber was within 3% or less of target concentration. The relative standard deviations of daily means were within 10%. The aerosol had an average MMAD of 2.38 µm (range 2.33 to 2.45), with a mean standard deviation of 1.7.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Stainless-steel, multitiered, whole-body exposure chambers (H1000, Hazelton Systems, Aberdeen, MD)
- Method of holding animals in test chamber: cages
- Source and rate of air:7±1 ft^3/min
- Method of conditioning air:
- System of generating particulates/aerosols:Jet-O-Mizer/screw feed method
- Temperature, humidity, pressure in air chamber:23.6°C, 70-85%, pressure not specified
- Air flow rate:7±1 ft^3/min
- Air change rate:12±2 air changes per hour
- Method of particle size determination:Lovelace multijet cascade impactor
- Treatment of exhaust air:not specified

TEST ATMOSPHERE
- Brief description of analytical method used:The aerosol concentration in the exposure chamber was monitored by sampling at a flow rate of 0.5 liter/min for three, 2-hr periods during the 6-hr exposure. Samples were collected on 25-mm fiberglass filters (Type AE, Gelman, Ann Arbor, MI). Each expsosure day, aerosol generation was started and sampling began after 12 min of rise time, when the chamber concentration had reached 90% of equilibrium concentration (T90). Therefore, the total exposure was 6 hr plus a T90 of 12 min. The control chamber was also sampled daily.

A RAM-S continuous aerolsol monitor (GCA, Bedford, MA) was used to monitor the stability of the aerosol concetration and to adjust the aerosol generator during exposure. It was operated on each chamber at the beginning, middle and end of the filter sampling period.
- Samples taken from breathing zone: not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration in the exposure chamber was monitored by sampling at a flow rate of 0.5 liter/min for three, 2-hr periods during the 6-hr exposure.
Samples were collected on 25-mm fiberglass filters (Type AE, Gelman, Ann Arbor, MI).
Each expsosure day, aerosol generation was started and sampling began after 12 min of rise time, when the chamber concentration had reached 90% of equilibrium concentration (T90).
Therefore, the total exposure was 6 hr plus a T90 of 12 min.
The control chamber was also sampled daily.
Duration of treatment / exposure:
2 weeks and 13 weeks
Frequency of treatment:
5d/w for 2 or 13 weeks
Doses / concentrationsopen allclose all
Remarks:
2 weeks
Basis: nominal conc.
Remarks:
13 weeks
Basis: nominal conc.
No. of animals per sex per dose:
2 week study: 5 animals
13 week study: 10 animals
Control animals:
yes
Details on study design:
- Dose selection rationale: 10 times higher than expected exposure to workers
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: morbidity/mortality checks

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: initiation, 1 week, and termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: termination
- Anaesthetic used for blood collection: Yes
- Parameters checked
For the 13-week study, blood was collected at necropsy from anesthetized rats and mice from the basic study group by cardiac puncture and placed in glass vials containing EDTA. A Coulter Electronics Model S-550 was used for analysis of erythrocyte count, mean corpuscular volume, haemoglobin concentration, hematocrit (calculated), and leukocyte count. Smears were made from the blood, stained with Wright’s stain, and examined under a light microscope to obtain differential leukocyte counts and counts of nucleated erythrocytes. Additional blood smears were stained with new methylene blue and examined for the presence of reticulocytes.

URINALYSIS: Yes
- Time schedule for collection of urine: 1 week prior to termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked:URINARY ENZYMES- In the 13-week study, 1 week prior to termination, all rats in the basic study group were placed in metabolism cages for overnight urine collection. Urine was collected on ice and analyzed for total amounts of lactate dehydrogenase (LDH), β-galactosidase (β-G), N-acetylglucosamididase (NAG), and alkaline phosphatise (AP). Lactate dehydrogenase was quantitated by using pyruvate as a substrate; 4-nitrophenylphophate was the substrate for alkaline phosphatise; p-nitrophenol was the substrate for both N-acetyl-β-D-glucosaminidase and β-galactosidase.
Sacrifice and pathology:
NECROPSY AND HISTOPATHOLOGY: Yes
All rats and mice in the basic study group, for both the 2-week and the 13-week studies, were given complete gross necropsy examinations. Rats and mice were killed by cardiac puncture exsanguination while under halothane anesthesia and were necropsied immediately. Weights of liver, thymus, right kidney, right testicle, brain, heart, and lungs (including trachea) were taken. Tissues were fixed in 10% neutral-buffered formalin. Tissues for microscopic examination were embedded in paraffin, sectioned at 5µm, and stained with hematoxylin and eosin.

The following tissues were trimmed and sectioned for histopathology: adrenals, bone (vertebra, with bone marrow and spinal cord; femur; rib), brain, epididymus or oviduct, esophagus, heart, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), both kidneys, larynx, liver, lung (4 lobes), lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary glands, nose (3 levels), pancrease (including islets), parathyroid gland, pituitary gland, prostate or uterus, salivary glands, seminal vesicles.
Other examinations:
SPERM MORPHOLOGY-VAGINAL CYTOLOGY. In the 13-week study, vaginal cytology samples were taken on a daily basis from basic study females for 1 week before final termination. Samples were obtained with sterile saline, placed on duplicate Dakin slides, fixed with Spray Cyte (Clay Adams 7180), and stained with toluidine blue (0.5% in 20% ethanol). Live sperm were obtained from the cauda of the right epididymis of male rats and mice at necropsy. Sperm were incubated at 37°C in Tyrode buffer, and viability was quantitated as the percentage of motile sperm in the sample. Sperm density (number of sperm per gram of caudal tissue) was quantified on a hemocytometer. The number of sperm in the sample was divided by the weight of the cauda of the epididymis to obtain the value for sperm density. Evaluations of sperm morphology were done using preparations of sperm fixed in ethanol and stained with eosin Y.

SPECIAL STUDY ENDPOINTS—Determinations of methemoglobin in whole blood of rats and mice exposed to ADA for 2 weeks were made using the method of Evelyn and Malloy (1938). Acetylcholinesterase concentrations in whole blood of rats only in the 13-week study and both rats and mice in the 2-week study were determined using the method of Ellman et al (1961), in which acetylthiocholine is used as the stubstrate. Serum thyroxine (T4 or tetraiodothyronine) and triiodothyronine (T3) levels for rats exposed to ADA for 13 weeks were measured by radioimmunoassay (Fietz, 1986).
Statistics:
All data were analyzed seperately for each sex.
Organ and terminal body weights on animals found dead or euthanized because of a moribund condition were not included in the statistical analysis.
Analysis of variance techniques were used for statistical evaluation.
Provided Bartlett's test of homogenetiy of variance was not significant, exposure groups were compared to controls by using Dunnett's multiple range test.
When Bartlett's test was significant, comparisons with the control group were made by a modified Student's t test, making allowance for unequal variance.
All statistical tests were conducted at a 5%, two-sided risk level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase lung weight male 50 mg/m^3
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
2-week study
CLINICAL SIGNS AND MORTALITY
There were no abnormal clinical observations during the 2-week exposures for rats. No rats died.
BODY WEIGHT AND WEIGHT GAIN
The mean terminal body weights for the basic study male rats exposed to 200 mg/m3 ADA were significantly less than those of controls (95% of control value).

HAEMATOLOGY-CLINICAL CHEMISTRY
There were no significant differences in methemoglobin levels or acetylcholinesterase activities in whole blood of male and female rats exposed to any concentration of ADA, when compared to controls.

ORGAN WEIGHTS
The mean liver weights of male rats were significantly less at the 200 mg/m3 exposure than those of controls (83% of control values). Other organ weights of rats were not influenced by ADA exposure.
GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC
A complete set of tissues from rats exposed to 200 and 0 mg ADA/m3 was examined histologically. The two exposure groups could not be distinguished on the basis of histological findings. Because no effect was seen at the highest exposure concentration, we did not examine tissues from rats exposed to lower concentrations.


13- Week Study
CLINICAL SIGNS AND MORTALITY
There were no abnormal clinical observations during the 13-week exposures for rats that could be attributed to exposure to ADA. No deaths occurred that could be attributed to ADA. One female rat (50 mg/m3 exposure group) was euthanized because of weight loss and dehydration.

BODY WEIGHT AND WEIGHT GAIN
The mean terminal body weights for the basic study rats exposed to ADA were not significantly different from those of the controls.

HAEMATOLOGY
Results obtained for hematology in rats exposed to ADA for 13 weeks indicated that there were no exposure-related alterations in blood parameters. There were no significant, exposure-related changes, in either male or female rats, in the amounts of the four urinary enzymes (LDH, AP, beta-G, NAG) excreted.

ORGAN WEIGHTS
Lung weights of male and female rats were increased (111% of control) at the 50 mg/m3 exposure level.

HISTOPATHOLOGY: NON-NEOPLASTIC
A complete set of tissues from rats exposed to 200 and 0 mg ADA/m3 was examined histologically. Although several incidental lesions were present in both groups, lesions attributable to the ADA exposure were not present.
Five male and six female rats exposed to 50 mg/m3 ADA were described as having enlarged bronchial or bronchial and mediastinal lymph nodes at gross necropsy. Histological examination of these nodes showed a moderate-to-severe lymphoid hyperplasia. Subsequently, the lungs of all rats and mice in the 50 mg/m3 exposure group were examined histologically. All male and female rats in the 50 mg/m3 exposure group euthanized at the end of the 13-week exposure period had a spectrum of lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia that was associated with a moderate number of mixed inflammatory cells.
The extent of lung involvement varied from mild to moderate among animals. In some animals, the perivascular cuffs were predominant, while other animals showed both the perivascular cuffs and the epithelial hyperplasia. The lungs from all rats and mice exposed to 100 mg ADA/m3, as well as the lungs of mice exposed to 50 mg/m3, were examined.
There were no significant lesions on histologic examination.

Samples of lung and kidney of male rats exposed to ADA for 13 weeks were analyzed for the presence of ADA and biurea. These tissues were chosen, because they are the most likely tissues to contain significant quantities of one or both compounds, lung being the organ of exposure and kidney, because biurea had been detected in kidney.
No ADA was detected in either lung or kidney of male rats exposed to ADA for 13 weeks. Biurea, however, was detected in lungs, but not kidney, of rats exposed to 50, 100, and 200 mg/m3 ADA. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration. The percentage of biurea retained in lungs was calculated as a percentage of ADA deposited on the last day of exposure. Although 66% of the amount of ADA expected to be deposited in rats exposed to 200 mg/m3 on the last exposure day is retained in the lungs as biurea, this is a small percentage (1%) of the total amount of ADA deposited over the entire study.

OTHER FINDINGS
There were no adverse effects from inhalation exposure to ADA for 13 weeks, with respect to right caudal weight, right epididymal weight, right testicular weight, sperm motility, sperm count per gram caudal tissue, or incidence of abnormal sperm. However, there was a small, but significant, increase in sperm count in male rats exposed to 50 or 100 mg ADA/m3.

There were no apparent adverse effects on estrual cyclicity or on estrous cycle length in any of the dose groups, except in 2 out of the 10 animals in the 200 mg/m3 dose group. For these animals, estrous cycle length was >7 days or was not precisely determined.

There were no significant differences in acetylcholinesterase activities in whole blood of male and female rats exposed to any concentration of ADA when compared to controls. T3 and T4 levels in serum from male rats increased with increasing ADA exposure concentration in male rats. T3 and T4 levels in the highest exposure group were significantly elevated, relative to control. T3 was increased approximately 50%,while T4 was increased approximately 40%. T3 and T4 were unchanged in female rats exposed to ADA.

Effect levels

Dose descriptor:
NOAEC
Effect level:
200 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: 13 -weeks microscopic observations in lungs and lymph nodes of rats.

 

 

Males

Males

Males

Males

Females

Females

Females

Females

 

Exposure group (mg/m^3)

0

50

100

200

0

50

100

200

 

No. in Group

10

10

10

10

10

9

10

10

 

Microscopic observations

 

 

 

 

 

 

 

 

Lungs

 

10

10

10

10

10

9

10

10

 

inflammation; nonsuppurative; alveolus

0

10

0

0

0

8

0

0

 

Hyperplasia; alveolar epithelium

0

10

0

0

0

8

1

0

 

Infiltrating cell; lymphocytic plasmacytic; perivascular

0

10

0

1

0

9

1

0

 

Hemorrhage; multifocal, acute; alveolus, right apical

0

0

0

1

0

0

0

0

Tracheobronchial lymph nodes (number examined)

8

10

0

7

9

9

0

7

 

Hemorrhage; acute; sinus

0

0

0

0

1

0

0

0

 

Hyperplasia; lymphoid

0

7

0

0

0

7

0

0

Mandibular lymph nodes (number examied)

9

0

0

9

9

0

0

10

 

Hemorrhage; acute; sinus

0

0

0

0

1

0

0

0

 

Hyperplasia; lymphoid; follicular

0

0

0

0

1

0

0

0

Mediastinal LN(number examined)

7

10

0

7

7

9

0

7

 

Hemorrhage; acute; sinus

2

0

0

1

2

0

0

6

 

Hyperplasia; lymphoid

0

5

0

0

0

4

0

0

Table 2: air concentrations

 SUMMARY OF AIR CONCENTRATIONS AND PARTICLE SIZES IN EXPOSURE CHAMBERS FOR RATS AND MICE EXPOSED TO AZODlCARBONAMIDE
  2-week repeated   13-week subchronic 
Target exposure concentration  Rats  Mice  Particle size  Rats  Mice  Particle size
mg/m3 mg/m3 mg/m3 MMAD SD mg/m3 mg/m3 MMAD SD
 Control   0   0   -  0   0   -
 2  2.0 (13)   2.1 (12) 1.89 2.1   -  -  -
 10  9.4 (11)   9.6 (10) 1.95 1.8   -  -  -
 50  52 (10)  52 (7)  2.15 1.8  50(10)   50 (10) 2.33 1.8  
 100   102 (16)  102 (15)  2.22 1.7  100(7)   100 (7)  2.45 1.7  
 200   207 (10)  217 (11)  2.43 1.9  204(5)   204 (5)  2.37 1.8  

Table 3:

 LEVELS OF ADA AND BlUREA IN LUNGS AND KIDNEYS OF MALE RATS EXPOSED TO AZODICARBONAMIDE (ADA) FOR 13 WEEKS
Exposure level(mg/m3)  ADA   Biurea 
Kidney Lungs andbronchi  Kidney  Lungs andbronchi
 0   NP b  NP   NP   NP 
 50   ND e  ND   ND   79± 15 d
 100   ND   ND   ND   303± 79 
 200   NP   NP   NP   948 ±238 
b NP, no peak was observed at the expected retention time. Limit of quantitation is 100µg per sample.
e ND, not determined.
d Mean µg/g tissue ± standard deviation; n = 5

Applicant's summary and conclusion

Conclusions:
In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations up to 200 mg/m^3. Exposure to ADA for up ot 13 weeks did not appear to be toxic to rodents.
Executive summary:

Two-week repeated and 13-week sub-chronic inhalation exposures of F344/N rats to Azodicarbonamide (named ADA) were conducted to determine the toxicity of inhaled. No exposure-related mortality or abnormal clinical signs were observed in rats during or after exposure. The terminal body weights were slightly depressed in the highest exposure group. Liver weights were lower in male rats exposed to 200 mg ADA/m3. No significant lesions were noted on either gross or histologic evaluation of rats or mice. In the 13-week sub-chronic study, the mean air concentrations of ADA were 204, 100, or 50 mg/m3. No mortality or clinical signs related to exposure were observed. The terminal body weights of exposed rats were not significantly different from those of control rats but were significantly depressed in mice exposed to 100 or 200 mg ADA/m3. No histopathological lesions were noted in mice. Lung weights were increased and enlarged mediastinal and/or tracheobronchial lymph nodes were noted in rats exposed to 50 mg ADA/m3. No exposure-related lesions were observed microscopically in rats exposed to 100 or 200 mg ADA/m3. All rats in the 50 mg ADA/m3 exposure group only had lung lesions that consisted of perivascular cuffing with lymphocytes and a multifocal type II cell hyperplasia, suggesting a possible immune reaction to an antigen in the lung. The possibility of an unknown viral antigen causing this lesion cannot be eliminated. Lung tissue from male rats was analyzed for ADA and biurea, the major metabolite of ADA. No ADA was detected. The amount of biurea in the lungs increased nonlinearly with increasing exposure concentration, suggesting that clearance was somewhat impaired with repeated exposures. However, even at the highest exposure concentration, this amount of biurea was less than 1% of the estimated total deposited over the exposure period. In summary, ADA is rapidly cleared from the lungs, even when inhaled at concentrations 1% to 200 mg/m3. Exposure tofor up to 13 weeks did not appear to be toxic to rodents. The NOAEC was 200mg/m3 in rats.