N,N-diethylaniline

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Subacute Inhalation Toxicity of Aniline in Rats: Analysis of Time-Dependence and Concentration-Dependence of Hematotoxic and Splenic Effects

Author:

Jurgen Pauluhn

Year:

2004

Bibliographic source:

TOXICOLOGICAL SCIENCES 81, 198–215 (2004)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aniline

EC Number:

200-539-3

EC Name:

Aniline

Cas Number:

62-53-3

Molecular formula:

C6H7N

IUPAC Name:

Aniline

Details on test material:

- Name of test material: Aniline
- Molecular formula: C6H7N
- Molecular weight: 93.13 g/mol
- Substance type: Organic
- Physical state: liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Specific-pathogen-free.
- Age at study initiation: approximately 2-3 months old
- Weight at study initiation: average body weight was 240 g
- Fasting period before study: 5 days
- Housing: polycarbonate cages containing bedding material (low-dust wood shavings)
- Diet (e.g. ad libitum): KLIBA 3883 pellets maintenance diet; PROVIMI KLIBA SA; ad libitum
- Water (e.g. ad libitum): municipality tap water in drinking bottles; ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ≈22°C
- Humidity (%): 40%–60%
- Air changes (per hr): Not available.
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:No data
- Method of holding animals in test chamber:No data
- Source and rate of air:No data
- Method of conditioning air: No data
- System of generating particulates/aerosols: digitally controlledmassflowcontrollers
- Temperature, humidity, pressure in air chamber:pproximately 23°C & Humidity 55%
- Air flow rate: The total flow rate directed into the nose-only exposure chamber was 30 l/min.
- Method of particle size determination: liquid aniline measures of bubbler: diameter: ≈2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level: ≈5 cm

TEST ATMOSPHERE
- Brief description of analytical method used:bubbler containing liquid aniline measures of bubbler: diameter: ≈2 cm in the 10, 30, and 90 mg/m3 groups and 4.5 cm in the 270 mg/m3, height of liquid level: ≈5 cm)These bubblers were maintained at 25°C with a digitally controlled thermostat Air flows passed through the liquid aniline were controlled by a calibrated gas-metering device (digitally controlled mass flow controllers) and ranged from 0.12 l/min to 3.4 l/min in the low and high group, respectively. This atmosphere was subsequently diluted by conditioned, dry air. The total flow rate directed into the nose-only exposure chamber was 30 l/min. Air flows passed through the liquid aniline were controlled digitally by calibrated mass flow controllers
Rats of the control group were exposed nose-only to dry, filtered air only.
The inhalation chamber had a volume of 7.6 l and was suitable to accommodate 40 rats. The air flow rate supplied into and extracted from the chamber provided a slight positive balance of air flow toward the rats’ breathing zone, and was maintained at 0.75 l/min/exposure port.:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure atmospheres were characterized by using a gas chromatographic technique (Hewlett-Packard GC 5890 equipped with a flame ionization
detector, split/splitless injector capillary column HPUltra 50+, length: 30 m, ID: 0.32 mm, film thickness 0.17 mm, autosampler HP 7673, and an HP 3365 Work-Station for data processing). For GC analyses, samples were taken from the vicinity of the rats’ breathing zone three times per exposure day using two impingers (in-line) filled with toluene. Between the glass bubblers and the gas-metering device (digital flow controller) a cool-trap was used to scrub volatile constituents from the sampled air. For calibrations, the test substance served as reference material. The sampling flow rate was 0.5 l/min. The sample volumes were 50, 20, 10, and 10 l/sample in the 10, 30, 90, and 270 mg/m3 group, respectively. In the last two groups, the temporal stability of concentrations of aniline in the chambers was monitored continuously using a Compur Total Hydrocarbon Analyzer (Compur, Munich, Germany). Chamber temperature and humidity were measured electronically, and mean values were approximately 23°C and 55%, respectively.

Duration of treatment / exposure:

28 days
6h/day head-only to aniline vapor

Frequency of treatment:

5 days/week for 14 days followed by a post-exposure period up to 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10, 30, 90, and 270 mg/m3
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
9.2, 32.4, 96.5, and 274.9 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

30 Male

Control animals:

yes

Details on study design:

Although female rats are often shown to be more susceptible to the formation of MetHb and the development of anemia than male rats, this study utilized male rats only to allow a better comparison with existing mechanistic studies.

Male Wistar rats were exposed nose only to aniline vapor in targeted concentrations of 10, 30, 90, and270 mg/m3 for 6h/day, 5days/week for 2 weeks (days 0–11), followed by a 2-week post-exposure period (up to day 28).
The control group was exposed to conditioned, dry air under otherwise identical conditions.
Serial sacrifices for specialized examinations were performed on days 0, 4, 11, 14, and 28 to address the time-course and reversibility of changes.
The selection of specific endpoints considered publications of previous studies with aniline and its structural analogs.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
Data generated on the clinical chemistry focused on potential hepatotoxicity and erythrocyte (hemoglobin) catabolism by the end of the exposure and post-exposure periods. Statistical comparisons did not reveal any consistent concentration–response relationship considered to be of pathodiagnostic relevance except slightly altered bilirubin serum concentrations at 90 and 270 mg/m3. Bilirubin concentration was increased dose dependent on day 14 (up to 1.4 fold over control) and dose dependently decreased by the end of the post-exposure period (up to 40%).
Some electrolytes (calcium, magnesium) were statistically significantly decreased at 30 mg/m3 and above, especially at the end of the post-exposure period. Because of the lack of a clear dose-dependence from 30 to 270 mg/m3 - in comparison to the control and 10 mg/m3 groups - these changes are considered without biological significance. They are considered to be secondary to changes in the concentrations of negatively charged
counterions (anion gap), such as plasma proteins or bicarbonate, rather than reflecting any specific, aniline-induced disturbance in electrolyte homeostasis.
A concentration-dependent and time-dependent increase in the total splenic iron content were observed in rats exposed to 90 and 270 mg/m3, when compared to the controls. Rhe increase in total iron and splenomegaly are related. In contrast, the maximum iron content in liver tissue homogenates was transient, and a slight increase was observed at 270 mg/m3 only. Based on a gram tissue level comparison, the maximum accumulation of iron in the liver and spleen exceeded the control levels by approx. 60% and approx. 500%, respectively.
Lipid peroxidation, measured as the sum of malondialdehyde and 4-hydroxy-2(E)-nonenal, was significantly increased at 90 and 270 mg/m3 three to four-fold over the control group. Despite the marked increase in lipid peroxidationfrom exposure day 0 to exposure day 4, a time-dependent progression between the later exposure days was not apparent. Changes subsided toward the level of the control group at the end of the post-exposure period, i.e., the aniline exposure groups were indistinguishable from the control. During the exposure period, the increase of splenic lipid peroxidation and total iron was highly correlated (r2 = 0.93), whereas this high degree of correlation ceased to exist at the end of the post-exposure period.
The determination of ferritin and glutathione peroxidase (GPx) in the tissue homogenates of the spleen did not attain statistical significance, when compared with the respective controls. However, despite the lack of any consistent concentration-dependence or time-dependence, GPx activity showed a tendency toward increase at the high exposure level.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly; Mondays and
Fridays and once weekly during the post-exposure period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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 AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 4,11 & 14.
- Anaesthetic used for blood collection: No data
- Animals fasted: no data
- How many animals:
- Parameters checked in table [No.?] were examined.
Hematological parameters were significantly affected at 90 and 270 mg/m3 starting on day 4, with maximum effects on days 11 and 14. They were characterized by a decrease up to 16% in hemoglobin (Hb), red blood cell count (RBC, up to 24%), and hematocrit (HCT, up to 20%), and an increase in reticulocyte counts (RC, up to 6.3fold over control) and erythrocytes containing Heinz bodies (day 4: high dose. 513/1000 cells vs 0/1000 in the control group; day 14: high dose. 266/1000 cells vs 0/1000 in the control group).
The RC observed in controls on day 0 (56/1000) were twice as high as those observed at the subsequent time points. This change might be attributable to the immobilization stress caused by restraint of non-acclimatized rats to exposure tubes and the associated increased discharge of mature reticulocytes from the bone marrow.
On day 11 only rats exposed to 270 mg/m3 showed a significantly increased mean corpuscular hemoglobin (MCH, 1.1 fold over control) and mean corpuscular volume (MCV, 1.2 fold). Furthermore, on day 11 the red blood cells in some rats exhibited hypochromia and anisocytosis.
With regard to the time-course, the hematological changes were maximal on the last exposure day (day 11) and showed some decrease in magnitude on the third post-exposure day (day 14); then, with the exception of the RBC, MCH, and MCHC, hematological changes subsided to the levels of the control group after the 2 week post-exposure.
Thrombocytes were mildly, although significantly increased at the end of the 2-week exposure period (data not shown). Conclusive changes in the total leukocyte counts and leukocyte differentials did not occur at any time point (data not shown).
In the blood sampled and determined immediately after cessation of exposure, MetHb was concentration-dependent, and then was significantly increased at 90 mg/m3 (days 4 and 11, up to approx. 2.5% vs. approx 1% in the control group) and at 270 mg/m3 (days 0, 4, 11, up to approx. 14% vs. approx. 1% in the control group). The MetHb concentrations from blood samples taken in the post-exposure period were indistinguishable from the control.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No data
- How many animals:
- Parameters checked in table [No.?] were examined.
Data generated on the clinical chemistry focused on potential hepatotoxicity and erythrocyte (hemoglobin) catabolism by the end of the exposure and post-exposure periods. Statistical comparisons did not reveal any consistent concentration–response relationship considered to be of pathodiagnostic relevance except slightly altered bilirubin serum concentrations at 90 and 270 mg/m3. Bilirubin concentration was increased dose dependent on day 14 (up to 1.4 fold over control) and dose dependently decreased by the end of the post-exposure period (up to 40%).
Some electrolytes (calcium, magnesium) were statistically significantly decreased at 30 mg/m3 and above, especially at the end of the post-exposure period. Because of the lack of a clear dose-dependence from 30 to 270 mg/m3 - in comparison to the control and 10 mg/m3 groups - these changes are considered without biological significance. They are considered to be secondary to changes in the concentrations of negatively charged
counterions (anion gap), such as plasma proteins or bicarbonate, rather than reflecting any specific, aniline-induced disturbance in electrolyte homeostasis.
A concentration-dependent and time-dependent increase in the total splenic iron content were observed in rats exposed to 90 and 270 mg/m3, when compared to the controls. Rhe increase in total iron and splenomegaly are related. In contrast, the maximum iron content in liver tissue homogenates was transient, and a slight increase was observed at 270 mg/m3 only. Based on a gram tissue level comparison, the maximum accumulation of iron in the liver and spleen exceeded the control levels by approx. 60% and approx. 500%, respectively.
Lipid peroxidation, measured as the sum of malondialdehyde and 4-hydroxy-2(E)-nonenal, was significantly increased at 90 and
270 mg/m3 three to four-fold over the control group. Despite the marked increase in lipid peroxidationfrom exposure day 0 to exposure day 4, a time-dependent progression between the later exposure days was not apparent. Changes subsided toward the level of the control group at the end of the post-exposure period, i.e., the aniline exposure groups were indistinguishable from the control. During the exposure period, the increase of splenic lipid peroxidation and total iron was highly correlated (r2 = 0.93), whereas this high degree of correlation ceased to exist at the end of the post-exposure period.
The determination of ferritin and glutathione peroxidase (GPx) in the tissue homogenates of the spleen did not attain statistical significance, when compared with the respective controls. However, despite the lack of any consistent concentration-dependence or time-dependence, GPx activity showed a tendency toward increase at the high exposure level

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were euthanized by complete exsanguination (heart puncture) after intraperitoneal sodium-pentobarbital injection (Narcoren, Merial GmbH, Hallbergmoos, Germany). All rats were given a gross-pathological examination.
Weights were recorded for brain, liver, lung, spleen, and testes at interim sacrifices (days 0, 4, and 11) and, in addition, heart, kidneys, and thymus at post-exposure sacrifices (days 14/15 and 28). For the heart, kidneys, and thymus no appreciable time-related effects were expected to occur. Therefore, the weights of these organs were determined only at the time points where the maximum effect, including reversibility, can be evaluated

HISTOPATHOLOGY: Yes
From the additional 5 rats/group that were sacrificed on day 15, selected organs and tissues (spleen, femur, sternum, liver, lung) were preserved in 10% neutral-buffered formalin or Davidson’s solution (testes) for histopathology. The lung was intratracheally instilled with the fixative under 20 cm H2O pressure. Osseous tissues were first decalcified and then, as for all other organs, embedded in Paraplast. All slides were stained with hematoxylin and eosin (H&E).
For a better appreciation of the degree of hemosiderosis, a specific iron stain (Prussian blue stain according to Perls) was prepared for formol-fixed sections of the liver and spleen.

Other examinations:

All animals survived the duration of study. Concentrations up to and including 30 mg/m3 were tolerated without clinical signs.
Rats exposed to 90 mg/m3 and above were cyanotic (blue discoloration of the skin in areas easily observed), whereas those exposed to 270 mg/m3 also displayed tachypnea, labored breathing patterns, increased salivation, and an ungroomed hair-coat. All signs disappeared toward the following day and did not show any exacerbation during the course of the study. Rectal temperatures measured shortly after cessation of exposure on days 0, 4, and 11 were at all-time points indistinguishable among the groups (range of means in the control group: 37.3°–38.6°C, aniline exposure groups: 36.6°–38.4°C).
Body weights were not statistically significantly affected in any group during the course of the study.
The weights of liver (Table 3), brain, lung, testes, heart, kidneys, and thymus did not show treatment-related effects (data not shown). Absolute and relative spleen weights were significantly increased in a time and dose dependent matter. In the group treated with 270 mg aniline/m3 maximum increase of 2.2 fold over the spleen weights of the control (day 11) was observed.
Gross necropsy findings of rats exposed to 90 mg/m3 and above provided evidence of dark discolorations and enlargement of spleens at all sacrifices beyond the first exposure week.
Some discoloration of lungs was found; however, no consistent time dependence or concentration-dependence existed. An increased incidence of discoloration of the thymus, including involution, and of the testes occurred at 270 mg/m3 (days 11 and 14).

Statistics:

Body weights, hematology, and clinical pathology data were compared using either
(1) the Kruskal-Wallis test and the adjusted U-test or adjusted Welsh-test as the post hoc test or
(2) One-way analysis of variance (ANOVA) and the Dunnett test as the post hoc tests for nonparametric and parametric analyses, respectively (SAS 6.12-routines).
Specialized endpoints (GPx, ferritin, iron) were analyzed with one-way ANOVA and the Tukey-Kramer post hoc test (BCTIC).
Histopathological findings were compared with the concurrent control using Fisher’s exact test.
The proportion of erythrocytes with Heinz bodies was transformed prior to ANOVA analysis using the arcsine square root function. This is appropriate for percentages and proportions, because the transformed data more closely approximate a normal distribution than the non-transformed proportions.
Transformed data were analyzed separately for normality of distribution. For all tests, the criterion for statistical significance was set at p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant change in body weight

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Subsequent increases in erythrocyte damage and turnover, including anemia, RBC morphological alterations.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

9.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slight increase in extra medullary hematopoiesis; this concentration is considered NOAEC for erythro toxicity, iron accumulation and lipid peroxidation

2

Dose descriptor:

LOAEC

Effect level:

32.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slight increase in extra medullary hematopoiesis

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated administration of the test chemical at doses of 9.2 mg/m³ air (analytical) shows no observed adverse effect concentration (NOAEC) and the low observed adverse effect concentration (LOAEC) was considered to be at 32.6 mg/m³ air (analytical) Wistar rats in 28 days study.

Executive summary:

A subacute inhalation toxicity study of the test chemical was conducted inmale Wistar rats for 28 days. Clinical signs of toxicity, body weights, hematology, and clinical chemistry tests, including total iron in liver and spleen, splenic lipid peroxidation, organ weights, gross and histological changes in target organs were recorded. No mortality was observed during the study. The changes observed included anemia, red blood cell morphological alterations (e.g., Heinz bodies), decreased hemoglobin and hematocrit, reticulocytosis, and effects on the spleen (splenomegaly, hemosiderin accumulation, and increased hematopoietic cell proliferation). The total content of iron in spleen homogenates increased in a concentration-dependent and time-dependent manner with increasing duration of exposure. The maximum accumulation of iron in the liver and spleen exceeded the respective control levels by ≈60% and ≈500%, respectively. The Lowest observed adverse effect cocentration (LOAEC) was observed in rat at 32.6 mg/m³ air (analytical) and the No observed adverse effect concentration (NOAEC) wasconsidered to be at 9.2 mg/m³ air (analytical) respectively.