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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no data on test substance purity)

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
no sacrifice at the end of treatment, no data about lung perfusion and levels of nose sections, no data on adrenal weight, clotting parameters, histopathology from selected tissues, incomplete documentation of all summary group or individual results
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): nitrobenzene
- Analytical purity: analytical grade obtained from Fisher Scientific Products

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory, Kingston, NY
- Age at study initiation: 8 - 9 weeks
- Housing: acclimation period: rats were placed three per cage in suspended wire-bottomed stainless steel cages; exposure period: rats housed individually in suspended wire-bottomed stainless stell cages in 8 m3 glass and stainless steel inhalation chambers
- Diet (e.g. ad libitum): NIH-07, an open-formula cereal-based diet; ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 50 ± 10
- Air changes (per hr): 8 - 16 (in inhalation chamber)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: clean pressurized air
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 8 m3 inhalation chamber
- Method of holding animals in test chamber: animals were in cages in the inhalation chamber
- Source and rate of air: clean pressurized air delivered at a rate of 50 L/min carried the vaporized nitrobenzene; the airstream containing nitrobenzene was diluted at the entrance of the inhalation chamber by approximately 2000 L/min of room air
- System of generating vapour: heated pyrex J-tube; rates of delivery were 0.09, 0.3 and 1.04 mL/min for 10, 35 and 125 ppm
- Temperature, humidity, pressure in air chamber: 21 ± 3 °C; 50 ± 10% humidity
- Air change rate: 8 - 16 per hour


TEST ATMOSPHERE
- Brief description of analytical method used: Infrared analysis of samples using a Miran 80 infrared analyzer (S. Wilks, Norfolk, CT)
- Samples taken from breathing zone: yes, online samples were taken hourly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis by infrared analysis of samples of chamber atmosphere. Online samples were taken hourly during the 6-h exposure and analyzed by absorption at 11.8 µm using a Miran 80 infrared analyzer
Duration of treatment / exposure:
2 w
Frequency of treatment:
6 h/d, 5 d/w
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.050, 0.175 and 0.625 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10, 35 and 125 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 9.1 ± 1.2, 35.8 ± 1.5 and 124.5 ± 9.1 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 3 or 14 d

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to and at the end of each exposure. Dead or moribund animals were removed from the chambers at the end of exposure periods.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: at necropsy; 3 or 14 days following the last exposure


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy; 3 or 14 days following the last exposure by heart puncture
- Anaesthetic used for blood collection: Methoxyfluran
- Animals fasted: No data
- How many animals: up to five animals of each sex for each dose
- Parameters checked: packed cell volume, erythrocyte count, hemoglobin concentration and total and differential leucocyte count. Methemoglobin concentrations were spectrophotometrically determined according to Evelyn and Malloy, 1938.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy; 3 or 14 days following the last exposure by heart puncture
- Animals fasted: No data
- How many animals: up to five animals of each sex for each dose
- Parameters checked: SGPT, SGOT, total bilirubin, blood urea nitrogen, calcium, glucose, GGTP and inorganic phosphorous,


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: spleen, left kidney, liver, testes and brain including their weights
HISTOPATHOLOGY: Yes: adrenals, bone marrow, brain, sternum, colon, duodenum, testes with epididymis, heart ileum, left kidney, liver, mesenteric lymph nodes, lungs, nose and turbinates, ovaries, pancreas, spleen, stomach, thyroid glands, thymus, trachea, urinary bladder, uterus and any gross lesion
Statistics:
Dunnet's multiple range test

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
10, 35, 125 ppm: all animals showed no signs of clinical toxicity; no mortality observed


HAEMATOLOGY (see table)
- A concentration-dependent increase in blood methemoglobin was noted in rats sacrificed 3 days after exposure; however, in animals sacrificed 14 days after exposure methemoglobin concentrations were not significantly elevated above controls.
- No significant alterations in mean white blood cell counts were noted
- a dose-dependent reduction in red blood cell counts at day 3 after exposure but recovered by day 14. Values for males ranged from 8.7 +/- 0.2 in the control group to 5.8 +/- 0.4 in the 125 ppm exposure group on day 3. Similar minor shifts were noted in mean hemoglobin and hematocrit from males and females exposed to 125 ppm.
- no other effects reported


ORGAN WEIGHTS (see table)
- Concentration-dependent increases in relative liver, spleen, and kidney weights were observed at necropsy
- Relative spleen weights were increased as much as three times compared to those of control animals sacrificed 3 days after final exposure and were still greater than controls in animals necropsied 14 days after the last exposure
- animals sacrificed 3 days after the last exposure exhibited a concentration-dependent increase in relative liver weight and in relative kidney weight that was not observed in animals allowed to recover for 14 days
- A decrease in testicular size and weight was evident in animals exposed to 125 ppm that persisted in animals necropsied 14 days after the last exposure


GROSS PATHOLOGY
125 ppm:
- a concentration-dependent increase in the number of hemosiderin-laden macrophages infiltrating the red pulp, increased extramedullary hematopoiesis relative to controls and acute sinusoidal congestion (10/10 in both sexes)
- A capsular hyperplastic lesion of possible mesenchymal origin was noted in males (7/10)

35 ppm:
- a concentration-dependent increase in the number of hemosiderin-laden macrophages infiltrating the red pulp, increased extramedullary hematopoiesis relative to controls. Acute sinusoidal congestion persisted but was diminished in severity in rats allowed to recover 14 days
- A capsular hyperplastic lesion of possible mesenchymal origin was noted in males. It was encountered in only one female sacrificed 3 days after
the final exposure. This lesion was present in animals allowed to recover for 14 days as well as in those sacrificed 3 days after the last exposure.

.

HISTOPATHOLOGY: NON-NEOPLASTIC
125 ppm:
- Kidney: irreversible hyaline nephrosis (10/10 male, 2/10 female)
- Spleen: hemosiderosis, extramedullary hematopoiesis, acute congestion and focal hyperplasia (10/10, both sexes),
- Testes: testicular lesions consisting of an increased number of multinuclear giant cells (7/10 males), sertoli cell hyperplasia (8/10 males) and severe dysspermiogenesis (10/10). Seminiferous tubules contained few sperm with maturation arrested at the level of primary and secondary spermatocytes. The lumen of the ductus epididymus contained reduced numbers of mature sperm. This lesion persisted throughout the 2-week recovery period; however, sertoli cell hyperplasia and the increased numbers of multinucleated giant cells were less severe in rats sacrificed at the end of the 2-week recovery period
- No brain lesions were found
- No significant histological findings were observed in livers
- No significant histopathology was found in lungs from males or females

35 ppm:
- Alterations of the spleen, consisting mainly of hemosiderosis, extramedullary hematopoiesis and capsular hyperplasia (10/10, both sexes)
- No brain lesions were found
- No significant histological findings were observed in livers
- No significant histopathology was found in lungs from males or females

10 ppm:
- a concentration-dependent increase in the number of hemosiderin-laden macrophages infiltrating the red pulp, increased extramedullary hematopoiesis relative to controls and acute sinusoidal congestion. No significant differences from controls were seen 14 days after exposure

Effect levels

open allclose all
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
0.05 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: various splenic lesions
Dose descriptor:
NOAEC
Remarks:
local (respiratory system)
Effect level:
0.625 mg/L air
Sex:
male/female
Basis for effect level:
other: No adverse effects related to the respiratory system were noted at any dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Relative organ weights:

    Dose Liver Kidney Spleen Testes
3 day post-exposure period Male 125 ppm 5.52* 0.42* 0.58* 0.73*
35 ppm 5.02* 0.39* 0.34* 1.41
10 ppm 4.28* 0.39* 0.22 1.33
Control 3.78 0.34 0.18 1.31
           
Female 125 ppm 4.88* 0.43* 0.68*  
35 ppm 4.22* 0.43* 0.38*  
10 ppm 3.42* 0.37* 0.24  
Control 3.32 0.35 0.18  
             
14 day post-exposure period Male 125 ppm 3.62 0.35 0.27* 0.73*
35 ppm 3.50 0.36 0.23 1.52
10 ppm 3.51 0.35 0.21 1.39
Control 3.69 0.36 0.22 1.39
           
Female 125 ppm 3.33 0.37 0.32  
35 ppm 3.27 0.38 0.25  
10 ppm 3.06 0.36 0.24  
Control 3.13 0.38 0.23  

*p<0.05 (n = 5)

Percent Methemoglobin:

    Dose % Methemoglobin
3 day post-exposure period Male 125 ppm 11.7 ± 1.2
35 ppm 6.6 ± 0.2
10 ppm 1.9 ± 0.7
Control 0
     
Female 125 ppm 13.4 ± 2.1
35 ppm 6.6 ± 0.8
10 ppm 4.8 ± 0.8
Control 3.6 ± 2.2
       
14 day post-exposure period Male 125 ppm 4.8 ± 1.9
35 ppm 5.6 ± 2.2
10 ppm 4.1 ± 0.1
Control 4.5 ± 0.3
     
Female 125 ppm 4.5 ± 1.5
35 ppm 5.1 ± 1.9
10 ppm 3.1 ± 0.3
Control 4.1 ± 0.5

Applicant's summary and conclusion