Aniline

Direct observations: clinical cases, poisoning incidents and other

Administrative data

Endpoint:

direct observations: clinical cases, poisoning incidents and other

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

publication

Title:

Exposure to Airborne Aniline and Formation of Methemoglobin in Humans: a contribution to occupational exposure limits

Author:

Käfferlein HU et al

Year:

2015

Bibliographic source:

Archives of Toxicology 88, 1419-1426, 2015

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Study type:

study with volunteers

Endpoint addressed:

basic toxicokinetics

acute toxicity: inhalation

other: dermal contact to airborne aniline

Principles of method if other than guideline:

Human volunteer study following state-of-the-art requirements regarding testing protocol, analytics and ethical considerations.
Study to determine the formation of methemoglobin in human volunteers after an 8/6 hour exposure to the official workplace occupational exposure limit of 2ppm aniline. The exposure laboratory, its technical set-up, safety settings and various capabilities of generating both, quality-controlled aerosols and particle-containing atmospheres of hazardous substances, has been previously described in detail (Monsé et al., Inhal. Toxicol. 24: 99-108, 2012).
This study was approved by the Ethic Commission of the Faculty of Medicine (Ruhr University, Bochum in 2012).

GLP compliance:

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 (as cited in study report): aniline

Method

Subjects:

Two male (age: 30 and 58) and two female volunteers (age: 29 and 42) in the pilot study and 10 male (age 26-59 years) and 9 female volunteers (age 23-59 years), proved to be healthy non-smokers. Inclusion criteria for the study were good physical health and a differential blood count without pathological findings. Individuals with slow and fast NAT2 phenotype (slow and fast acetylators) were included.

Ethical approval:

confirmed and informed consent free of coercion received

Remarks:

This study was approved by the Ethic Commission of the Faculty of Medicine (Ruhr University, Bochum in 2012).

Route of exposure:

inhalation

other: dermal contact to airborne aniline

Reason of exposure:

intentional

Exposure assessment:

measured

Details on exposure:

TYPE OF EXPOSURE:
A constant aniline vapour atmosphere of 2 ppm was generated in the exposure chamber. The volunteers wore standardized cloth typical for aniline manufacturing industry in terms of regular pants (e.g. jeans), cotton T-shirt, cotton blended fabric overalls and closed footwear. No skin or respiratory protection measures in terms of gloves or breathing masks were used, thus exposure occurred via inhalation and dermal contact.

GENERATION OF EXPOSURE:
In brief, an atmosphere containing 2 ppm aniline vapour was generated by the use of a test gas generator, first preparing a pre-mixture of high aniline concentrations in a stirred tank followed by dilution to the desired concentration in the air stream of the air conditioning system. This procedure allows a reproducible dosage. The flow direction of the inlet air containing aniline was forced from the floor to the ceiling of the laboratory thus yielding atmospheres with very high temporal and spatial stability. The concentration in the 300-L stirred tank was assessed by discrete measurements using a two channel process gas chromatograph. Calibration was carried out using a calibration gas generator with mass flow controllers which control the required amount of aniline and carrier gas.

TYPE OF EXPOSURE MEASUREMENT:
The final aniline concentration in the chamber was monitored online by chemical ionization mass spectrometry to continuously guarantee the safety of the volunteers. For this purpose, 2-second intervals were chosen. The aniline concentrations were supervised at the four available seats for the volunteers in the exposure laboratory and at the height of their heads. All four places can be individually monitored. The climatic parameter within the laboratory consisted of an aerial exchange rate of 12 per hour (360 m³/h), whereas the temperature range was set to 23-24°C and relative humidity was adjusted in the range of 40-50%. All climatic parameters were monitored online.
EXPOSURE LEVELS:
2ppm aniline

EXPOSURE PERIOD:
single exposure for 8 hours (pilot study) and 6 hours (main study)

POSTEXPOSURE PERIOD:
16 hours (pilot study) and 42 hours (main study; for N-acetyl-4-aminophenol in urine: 66h)

Examinations:

Methemoglobin levels in blood were assessed shortly before the exposure started (time point 0 h), during exposure (time points 2, 4, 6 and 8 h in the pilot study, time points 1, 2, 3, 4, 5, 6 h in the main study) and during a post-exposure examination period (time points 10, 12 and 24 h in the pilot study; time points 7, 8, 10, 12, 24, and 48 h in the main study).
In addition, the background levels of Met-Hb were studied in 8 volunteers (4 female, 4 male, all non-smokers) with no exposure to aniline for five days in order to study the range of normal Met-Hb saturation in humans. For this purpose, Met-Hb levels of these individuals were measured in the morning, at lunch and in the afternoon on day 1 followed by measurements in the morning on days 2-5. The results allow for the interpretation of the intraday and interday variation of Met-Hb levels in non-exposed individuals.
Method: The blood flow in an earlobe was stimulated by the use of capsicum cream and a capillary blood sample was taken from the earlobe by lancet puncture a few minutes later. Three capillary blood samples were analyzed atevery single time point and analysis was carried out in <5 min immediately after blood sampling using an automated ABL 850 benchtop blood gas analyzer.
 
Aniline excretion in urine was measured for time periods between 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-20, 20-24, and 24-28 hours after start of exposure in the pilot study and between 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-24, and 24-48 hours in the main study.
Method: The urine samples were hydrolyzed followed by a liquid/liquid extraction step of aniline from the urinary matrix at pH 6.0-6.4. After derivatization, aniline was analyzed by gas chromatography and tandem mass spectrometry (GC/MS-MS). Standardisation and quantification was carried out by isotope dilution using aniline-d7 as internal standard. Relative recovery was 100%. No losses occurred during sample preparation. The within-series and the between-day imprecision was <10%. The limit of detection was 50 ng/L based on a signal to noise ratio of 3:1 thus capable of analyzing aniline in the environmental range. The ananalysis covers exposure to free aniline and acetanilide after N-acetylation of aniline (‘total aniline’).
 
N-Acetyl-4-aminophenol in urine, a conjugated product of p-aminophenol in humans, was only measured in the main study. It was assessed shortly before the exposure started (time point 0 h), during exposure (time points 2, 4, and 6 h) and during a post-exposure examination period (time points 8, 10, 12, 20, 24, 48, and 72 h).
Method: The urine samples were mixed with ammonium acetate puffer and glucuronidase/ arylsulfatase. After incubation and freeze-precipitation of proteins samples were thawed and centrifuged. N-Acetyl-4-aminophenol was analyzed by 2-dimensional high performance liquid chromatography and tandem mass spectrometry (2D-HPLC-MS/MS). Standardisation and quantification was carried out by isotope dilution using N-acetyl-4-aminophenol-d4 as internal standard. Relative recovery was 100%. No losses occurred during sample preparation. The within-series and the between-day imprecision was <5%. The limit of detection was 0.25 μg/L based on a signal to noise ratio of 3:1 thus capable of analyzing N-acetyl-4-aminophenol in the environmental range. The analysis covers exposure to N-acetyl-4-aminophenol and its phenolic conjugates (glucuronates, sulfates).
 
Hemoglobin adducts of aniline (Hb-adduct, sulfinic acid anilide) were measured only in the main study at time points 0, 6, 12, 24, and 48 h.
Method: Intact erythrocytes were isolated from EDTA blood samples by centrifugation and multiple washed with 0.9% saline solution to remove all plasma components (e.g.glutathione). The erythrocytes were lysed, homogenized and cell debris was removed by centrifugation. Precipitation of hemoglobin (Hb) was achieved by the addition of ethanol to the supernatant. Hb was purified by suction using a solid phase extraction station and further washed with ethanol and diethylether and finally dried. 200 mg Hb were hydrolyzed by the addition of sodium hydroxide solution thus freeing aniline which was initially bound as sulfinic acid anilide atβ-cysteinyl groups of Hb. Aniline was subsequently extracted byn-hexane, derivatizedand finally analyzed by gas chromatography and mass spectrometry with negative chemical ionization (GC/NCI-MS). Standardisation and quantification was carried out by isotope dilution using aniline-d7 as internal standard. Relative recovery was 98%. The within-series and the between-day imprecision was <15%. The limit of detection was 0.5 ng/L based on a signal to noise ratio of 3:1 thus capable of analyzing aniline in the environmental range.

Medical treatment:

not required

Results and discussion

Clinical signs:

No clinical adverse health effects in terms of irritative effects on skin, eyes, respiratory tract or cyanosis could be observed in the volunteers.

Results of examinations:

Exposure to aniline at 2 ppm resulted in a clear increase in Met-Hb levels in all volunteers. The individual maximum concentration observed was 1.57% saturation in the pilot study and 2.07% saturation in the main study thus being clearly below the threshold limit of 5% Met-Hb saturation in Germany (DFG 2007b). Mean Met-Hb levels prior start of exposure were 0.21±0.09% in the pilot study and 0.72±0.19% in the main study. The maximum of the mean Met-Hb saturation level (1.21%) was reached after 6 hours of exposure and no further incline could be observed at 8 hours of exposure (1.15±0.21%) in the pilot study. No clinical adverse health effects in terms of irritative effects on skin, eyes, respiratory tract or cyanosis could be observed in the pilot or main stuy. These results are in line with the definition of the OEL in Germany that aniline levels of 2 ppm represents the maximum permissible airborne concentration at the workplace which generally does not have known adverse effects on the health of employees even when the employees are repeatedly exposed during long times (8 h daily and assuming a 40-h working week).
A plateau of Met-Hb was achieved after approximately 6 hours of exposure most likely based on an equilibrium between Met-Hb formation due to ongoing exposure to aniline and its degradation via the intraerythrocytic MetHb reductase. Intraindividual and interindividual standard deviations were low (< 40%) although individual persons were able to respond quite differently to aniline exposure (Figure 3b). In example, one out of the 19 persons in the main study showed a weak response to aniline only (‘non-responder’) in terms of a baseline level of 0.93 prior to exposure and a maximum observed saturation level of 1.0% at the end of exposure thus resulting in a 0.7-fold change only. In contrast, the highest observed fold-change in a volunteer was 3.0 ( ‘responder’) with a baseline level of 0.47 at start and a maximum saturation level of 1.40% at the end of exposure.
Met-Hb concentrations consistently dropped after end of exposure to baseline levels and were generally below 1% saturation 16/18 and 42 h after the end of exposure (1st and 2nd-day post exposure) with the exception of two single individuals in the main study who had – 18 h after end of exposure – a Met-Hb level of 1.10%. The results were directly compared to those observed in eight non-exposed volunteers. The mean Met-Hb saturation in non-exposed controls was 0.58±0.15% (range: 0.20-1.00) with a 95th percentile of 0.80%. The intra- and interday variation were 21.0 and 29.1%. Overall, the results are in line with those reported by ECB (2004) that the physiological background of Met-Hb in humans is about 1%. Fifteen out of 19 exposed volunteers (79%) showed a Met-Hb saturation ≤0.80% after 24 hours (18 h post-exposure). The Met-Hb levels of the remaining four volunteers were 0.85, 1.10, 0.87, and 1.10%. These levels further decreased after 24 h (42 h after end of exposure) to 0.83, 0.77, 0.63, and 0.80%, respectively. Overall, Met-Hb saturation in exposed individuals rapidly decreased after the end of exposure and after about 24 h the levels were in the range of those observed for non-exposed individuals.
In contrast to the pilot study, the slight differences in the formation of Met-Hb between men and women could not be confirmed in the main study (Fig. 4a). No studies on gender differences in Met-Hb formation in humans could be traced in the literature in order to compare the results with previously published data. An animal study in Beagle dogs with 4-aminopropiophenone, likewise a Met-Hb producing chemical after metabolic N-oxidation, showed higher formation of Met-Hb in female dogs (Bright et al. 1987). This result, however, could not be confirmed in our human study using aniline.
No differences in Met-Hb formation were observed between slow and fast acetylators after exposure to aniline (Fig. 4b) although a previous study in humans at the workplace suggested higher Met-Hb levels in “slow” acetylators compared to “fast” acetylators (1.4 vs. 0.9%, Lewalter and Korallus 1985). The acetylation phenotype of the workers in the latter study, however, remains uncertain because it was deduced only based upon the ratio of N-acetylaniline vs. unconjugated (free) aniline in post-shift urine samples rather than determining the phenotype of the workers in a controlled and standardized manner (Blaszkewicz 2004).
In general, the kinetic pattern observed for Met-Hb was also found on the level of aniline excretion in urine (Fig. 5a). Mean post-shift excretion of aniline in all volunteers was 168.0±51.8 µg/L (range: 79.5-418.3 µg/L). The maximum individual concentration observed was 418.3 µg/L thus being below the binding current threshold limit of 1,000 µg/L in Germany (BMAS 2013). A similar elimination pattern was also observed when creatinine-related aniline concentrations rather than volume-related data were evaluated (not shown). Aniline concentrations dropped to the individual baseline levels at the start of exposure within 24 hours thus indicating no accumulation of aniline in the body or during the work week. Mean baseline (5.7±3.8 µg/L), 24 h-post-exposure (17.0±17.1 µg/L) and 48 h post exposure levels of aniline (10.2±12.4 µg/L) were in the range of persons who are exposed to aniline via the environment only (Kütting et al. 2009; Weiss and Angerer 2002). In general, the results are in line with the short half-life of aniline (about 3.5 h, Piotrowski 1972) thus its fast metabolism and elimination in humans.
By analyzing aniline in urine it was seen that the person who showed no increase in Met-Hb (‘non-responder’) nevertheless was exposed to aniline thus the fact that the ‘non-responder’ did not show an increase in Met-Hb could not be attributed to a missing airborne exposure of aniline (Fig. 5b). No differences in the elimination pattern of aniline in urine could be observed between males and females as well as between slow and fast acetylators in the main study (Fig. 6a, b) and in accordance with the outcome on Met-Hb.

Applicant's summary and conclusion

Conclusions:

The results on controlled exposure of 4 volunteers in the pilot study and 19 volunteers in the main study to aniline vapour and the formation of Met-Hb at current permissible exposure levels in Germany (2 ppm) show maximal individual increases of Met-Hb up to 2.07% and of aniline in urine up to 418.3 µg/L. These values are well below the current German guidance levels of Met-Hb (5%, DFG 2007) and the current German threshold limit of aniline in urine (1,000 µg/L, BMAS 2013). The chamber exposure of human volunteers in the experimental setup is taking into account the worst-case consideration of a possible uptake via the skin and/or the respiratory tract under physical exertion, including potentially sensitive volunteers with slow-acetylator phenotype. Overall, the results directly contribute to the risk assessment of occupational exposure to aniline vapors.

Executive summary:

Human volunteers (4 in the pilot study, 19 in the main study) were exposed to the maximum workplace concentration (MAK) of aniline in Germany (2 ppm) for a complete work shift (8 h in the pilot study, 6 h in the main study).The chamber exposure in the experimental setup followed the worst-case consideration of a possible uptake via the skin and/or the respiratory tract under physical exertion.All volunteers were non-smokers only thus allowing the sensitive detection of even small aniline-induced increases of Met-Hb in blood. Potentially sensitive individuals with slow-acetylator phenotype were included.

The exposure resulted in a time-dependent increase of Met-Hb in blood. Maximal individual levels of up to 2.07% Met-Hb and of 418.3 µg/L total aniline in urine were observed, which were thus well below the current German guidance levels of Met-Hb (5%, DFG 2007) and the current German threshold limit of aniline in urine (1,000 µg/L, BMAS 2013). Maximal individual Hb-adduct levels of aniline in blood of up to 18.04 ng/L were recorded. All biomarkers of exposure (Met-Hb saturation in blood, aniline in urine, Hb adduct of aniline in blood) were well below the corresponding current threshold limit values of aniline in biological material in Germany. A plateau of Met-Hb was reached after 6 hours of exposure, with a mean saturation of 1.21±0.29% (range 0.80-2.07).

The main study showed that Met-Hb saturation in blood and aniline in urine rapidly decreased after the end of exposure and about 24 h post exposure the levels were in the range of those observed prior exposure of non-exposed individuals. In line with the prolonged half-life of Hb adducts and the life span of erythrocytes in humans (120 days) the Hb adduct levels of aniline decreased more slowly. Overall, the results indicated no differences between males and females as well as between slow and fast acetylators for any of the biomarkers with the exception of a minor difference in Hb adduct levels of aniline between slow and fast acetylators with about 30% higher levels in slow acetylators in post-shift blood samples.