Aniline

Endpoint summary

Additional information

For acute oral toxicty LD50 in rat ranges between 442 -930 mg/kg (Bio-Fax, 1969; Bier and Oliveira, 1980). Clinical signs observed during the study included cyanosis, tremors, hyperpnea, tachypnea, salivation, prostration, and lethargy. At necropsy among others inflamation and hemorrhage of gastrointestinal tract were described. Data on methemoglobin (MetHb) formation in the rat from studies providing an LD50 is not available. However, when anilin was applied by gavage to not-fasted male rats in doses up to 1000 mg/kg bw maximum MetHb formation was observed within 1 - 4 hours after treatment. For the highest dose the highest MetHb concentration (48%) was observed (Jenkins et al., 1972).

Old and limited studies showed that cats reacted more sensitive to MetHB inducing substances. In two out of three studies with cats death was observed following oral application of 100 mg/kg but not 50 mg/kg bw. Because of the small dose groups no LD50 could be derived from these studies (BASF 1970 & 1971). Clinical signs observed included cyanosis, atony, vomiting and salivation. Gross pathology was not performed. Blood analysis of methemoglobin showed a rapid increase within 24 hours after aniline application and reached maximum concentrations higher than 70%.

In dogs orally dosed with of 15 mg/kg no mortality was observed. All dogs started to show a cyanotic appearance of the visible mucous membranes shortly after administration of aniline. On the first postexposure day all dogs appeared to be normal. Maximum MetHb concentration was observed three hours after administration was in the range of 27% and returned to normal within one day after administration (Pauluhn, 2002).

For the assessment of the acute dermal toxicity only limited data or peer-reviewed literature (MAK, 1992) is available. Because of some limitation in the test design, limited reporting and the insensitivity of rabbit and rodents against methemoglobinemia associated acute toxicity none of the three cited studies is considered as key study but all together are considered in a weight of evidence approach. In accordance with the results from studies addressing acute oral toxicity cat (LD50: 254 mg/kg bw; Kondrashov, 1969) was the most sensitive species after dermal exposure compared with rat (LD50: 670 mg/kg bw, Czaikowska 1977, cited in MAK 1992). Rabbit (LD50: 1540 mg/kg bw; Bio-Fax, 1969) and guinea pig (LD50: 1316 mg/kg bw, Roudabush, 1965) were less sensitive. Clinical signs and gross pathology are reported scarcely and indicate that they were similar to observations made after oral application. Amongst the available LD50 the ones established in rat and cat are considered the most relevant for the purpose of hazard assessment, even though there is only little information on the study performance.

Studies which provide an LC50 for acute inhalation toxicity are available only for rats. Head only exposure of rats to an aniline vapor/aerosol atmosphere for 4 hours lead to an LC50 of 839 ppm/4 h, which is approximately 3.3 mg/l/4 h. In contrast, whole-body exposure of rats to an aniline vapor/aerosol atmosphere for 4 hours resulted in a much lower LC50 of 479 ppm/4 h (~1.9 mg/l/4 h). Clinical signs reported were in accordance with observations from acute oral studies (cyanosis, tremors, prostration and salivation) plus those resulting from inhalation exposure, i.e. corneal clouding, hair loss, colored discharge and chromodachryorrhea.

Information on acute inhalation toxicity of aniline in non-rodent are available from dog studies addressing route-to-route extrapolation (Pauluhn, 2002) and derivation of an acute reference concentration based on MetHb formation (Pauluhn, 2005). Dogs were exposed nose-only to aniline vapor in concentrations ranging from 0.016 to 0.494 mg/l for a total exposure time of 4 h. No mortality was observed in both studies. Clinical signs reported were reversible cyanosis of mucous membranes and salivation. Based on these two studies, the LC50 in dogs for aniline vapor is >0.49 mg/l for a total exposure time of 4 h. Maximum MetHb concentration did not exceed 30% except in one hyperventilating dog after exposure to 0.243 mg/l where a maximum MetHb concentration of about 44% was observed. For head-only exposed dogs the estimated threshold concentrations to cause 0.8% MetHb following a single exposure to aniline for 4 h and 8 h exposure were 23.6 and 20.6 mg/m3, respectively.

Whole body exposed dogs (0.24 mg/l/4 h) displayed at least a three-fold higher MetHb concentration as well as a somewhat delayed maximum in MetHb concentration when compared to head-only exposed dogs (0.24 mg/l/4 h), indicating that aniline can be absorbed to a significant amount through the skin causing a slightly protracted systemic availability (Pauluhn 2005).

Under normal conditions MetHb reductase is the rate limiting enzyme controlling the toxicokinetics of MetHb reduction. Species specific differences with a five and ten time higher activity of erythrocyte MetHb reductase from rats and mice than in human have been observed. On the other hand, MetHb reductase in cats and dogs is lower than in humans. Since Beagle dogs also lack arylamine N-acetyltransferase, they are much more susceptible to methemoglobinemia than even slow-acetylation humans.

In a study of Pauluhn (2002) beagle dogs were exposed head-only to aniline vapor in concentrations to attain a targeted total exposure dose of approximately15mg aniline/kg bw. One group of dogs received this calculated dose by gavage. Maximal MetHb levels at the end of the inhalation exposure attained approximately5%,whereas administration by gavage produced a maximum MetHb response of26%.This study demonstrates that for aniline, an agent known to be bioactivated by a hepatic first-pass metabolism, the conversion of MetHb formation after oral exposure to inhalation exposure concentrations is subject to overestimating dramatically the magnitude of MetHb formation. As to whether the 5-fold lower potency by inhalation is solely related to the hepatic first-pass bioactivation, to the rate of delivery, or to a less than 100% retention of the inhaled vapor within the respiratory tract remains to be elucidated.

EVALUATION OF HUMAN INFORMATION

The dose-response relationship between oral aniline uptake and MetHb formation was studied in 20 volunteers which received a bolus dose of 5, 15 or 25 mg/day on three consecutive days (Jenkins et al., 1972). Some volunteers were given higher doses (35, 45 or 65 mg) on subsequent days. The mean maximum increase in MetHb-formation occurred in less than 4 hours. After intake of 5 and 15 mg the increase of MetHb was not significant (1.2 or 1.8%, respectively). Significant increases were seen at doses >= 25 mg/day (2.5%) and more. Uptake of 35 mg anline led to a MetHb formation of 3.7%. Since MetHb concentrations of up to 5% were considered as uncritical and tolerable (Henschler and Lehnert, 1986) this dose can be considered as NOAEL for MetHb formation in humans.

In a pilot study to determine the formation of MetHb after exposure of human volunteers to airbone concentrations of aniline under work place conditions (IPA, 2012) 2 male and 2 female volunteers, proved to be healthy non-smokers and carriers of the slow-acetylator phenotype, were exposed to the official occupational exposure limit of 2 ppm aniline for a complete work shift duration of 8 hours. This study was approved by the Ethic Commission of the Faculty of Medicine (Ruhr University, Bochum in 2012). The volunteers wore standardized clothes typical for aniline manufacturing industry. To mimic worst-case workplace conditions no skin or respiratory protection measures in terms of gloves or breathing masks were used to allow dermal contact with the aniline airflow. Additionally the volunteers were exercising 4 x 20 min on a cycle ergometer to mimick physical acitivity at work. Blood methemoglobin concentrations were assessed shortly before the exposure started (0 h), during exposure (2 h, 4 h, 6 h and 8 h) and during a post-exposure examination period (10 h, 12 h and 24 h).

The basic MetHb level before exposure ranged between 0.03 to 0.27%. Exposure to aniline at 2 ppm resulted in a slight increase in MetHb levels in all 4 volunteers, reaching maximal levels of 0.97 to 1.57%. Thus, all maximal levels reached were below or only slightly above the level of 1.5%, which is considered as the upper limit of the physiological background and thereby as biomarker of exposure and these levels are also clearly below the threshold limit of adversity of 5% MebHb (BAT, 2007). Thus, no clinical adverse effects could be observed in any of the volunteers. A plateau of MetHb was achieved after approximately 6 hours of exposure most likely based on an equilibrium between MetHb formation and its degradation via MetHb reductase. Met-Hb concentrations consistently dropped after end of exposure. The preliminary results on the small number of 4 volunteers indicate that the individual baseline levels at the start of exposure may not have fully reached 16 h after the end of the exposure, the end levels being between 0.03 and 0.3% higher than the start levels. Therefore, a slight increase of Met-Hb levels throughout a work-week at permanent exposures of 2 ppm cannot be excluded. No relevant differences in the baseline levels of Met-Hb could be observed between male and female volunteers. The maximum concentrations of Met-Hb at exposures of 2 ppm aniline for 8 hours were slightly higher in the two female (1.57 and 1.33%) compared to the male volunteers (1.03 and 0.97%). However, the low number of individuals in this pilot study does not allow a conclusion with regard to possible differences in gender susceptibility.

Based on the results of the human inhalation study of Käfferlein (2013, 2014) the OEL of 2 ppm (7.7 mg/m³; 8 hour exposure) has been proven as no adverse effect concentration (NOAEC) under workplace conditions.

Justification for classification or non-classification

After a single oral dose of aniline lethal doses or concentrations observed in acute animal studies are species dependent. It is commonly agreed on that the mode of action of aniline induced acute toxicity is determined by methemoglobinemia, i.e. species differences in acute aniline toxicity result from both the formation of MetHb and the regenerative capacity of the test species to restore hemoglobin.

In general, the rat, mouse, rabbit and guinea pig seem significantly less sensitive to the formation of MetHb than humans and dogs. The cat is known to be particularly sensitive to the formation of MetHb and thus may over-estimate the hazard to humans. The species difference can be attributed to the different activities of methemoglobin reducing reductase in erythrocytes which is much higher in rodents than in human, whereas enzyme activities in cats or dogs are similar to human.

Classification criteria for acute toxicity outlined in Annex VI to 67/548/EEC as well as Annex I to CLP regulation (EC) 1272/2008 refer to the rat as relevant species for classification. However it stated that "when the classification is to be established from experimental results obtained in animal tests the results should have validity for man in that the tests reflect, in an appropriate way, the risks to man (67/548/EEC Annex VI, § 3.1.4).

Similarily, in the CLP regulation (EC) 1272/2008 it is stated that "the preferred test species for evaluation of acute toxicity by the oral and inhalation routes is the rat, while the rat or rabbit are preferred for evaluation of acute dermal toxicity. When experimental data for acute toxicity are available in several animal species, scientific judgement shall be used in selecting the most appropriate LD50 value from among valid, well-performed tests" (EC 1272/2008 Annex I, § 3.1.2.2.1).

For aniline data from studies in rats are not considered solely relevant for classification purposes because they may lead to an underestimation of aniline toxicity in human. Available data from cats, dogs and humans are considered more relevant because of the similarity in sensitivity to MetHb forming substances and the MetHb reductase activities. Taking into account all available data on animals and humans aniline is classified according to criteria in Annex VI of 67/548/EEC as “Toxic” and labelled as “R 23/24/25". According to the criteria in the CLP regulation (EC) 1272/2008 aniline is classified in acute toxicity category 3 for the oral and dermal route.

For inhalation of aniline an LC₅₀ of 3.27 mg/l (839 ml/m³, head-only exposure to a mixture of vapour and aerosol) was determined for rats (Du Pont, 1982). This would lead to Cat. 3 (for vapour) or Cat. 4 (for aerosol). In an old and poorly documented study of Carpenter (1949) a median lethal concentration (2-4 of 6 animals died, no further information) of 250 ppm was given for a 4 hour exposure via anilin vapour. Since the concentration was not measured analytically but ’based on empirical calculation’, this study will not be taken for classification. After whole body exposure an LC₅₀rat of 1.84 mg/l (478 ml/m³) was determined considering inhalative and dermal uptake (Du Pont, 1082). Since dermal uptake is already classified with Cat. 3 the head-only value of 3.27 mg/l is taken for classification. Focussing on exposure via anline vapour (worst case assumption) Cat. 3 for acute inhalation toxicity is justified.