Benzenamine, 4-fluoro-N-(1-methylethyl)-

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Sep 2005 - 11 Oct 2005 (exposure period); on 08 Nov 2005 last necropsy of post-exposure animals

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The present study was conducted according to OECD TG 412 (adopted in 1981) under GLP conditions and was checked by quality assurance. However, there are some inconsistencies in the final study report. The guideline followed (e.g. on the coverpage OECD TG 413 is mentioned instead of OECD TG 412), the exposure duration, the animal number per dose group, and the number of dose groups of the post-exposure period are not correctly reported at several points. Nevertheless, the toxicological results and final outcome of the study are not affected by this discrepancies.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in 1981

Deviations:

yes

Remarks:

Food consumption was not measured. Thyroid and uterus weights were not assessed. Bronchoalveolar lavage was not performed. Relative humidity values were below guideline values due to the use of conditioned dry air for dispersion of the test substance.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-100-7

EC Name:

-

Cas Number:

70441-63-3

Molecular formula:

C9H12FN

IUPAC Name:

4-fluoro-N-(propan-2-yl)aniline

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The study was carried out in rats, a rodent species recommended in test guidelines for inhalation toxicity studies.
Healthy young adult SPF bred Wistar rats, strain Hsd Cpb:WU (SPF), were used. Animals of this strain have been used at Bayer AG in toxicological studies for years. Historical data on their physiology, diseases and spontaneous alterations are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Nederland, Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Animals were assumed to be approximately 2 months old, based on body weight data.
- Range of body weight at study initiation: males: 201.97 - 207.91 g; females: 152.68 - 155.73 g (variation did not exceed ±10 % of the mean)
- Fasting period before study: no
- Housing: Animals were conventionally housed in Makrolon cages type IIIh on BK 8/15 low-dust wooden granulate (Source: Ssniff, Soest/Westfalen, Germany). Animals were single housed; 1 animal per cage.
- Diet: fixed-formula standard diet KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 1.5 weeks

DETAILS OF FOOD AND WATER QUALITY
The nutritive composition and contaminant content of the standard diet were routinely checked and analyzed. The tap water complied with the current version of the German Drinking Water Decree (TrinkwV). The available data provided no indication of any effect on the study objective.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Sep 2005 To: 11 Oct 2005 (end of exposure period), 08 Nov 2005 (end of post-exposure period)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

The test substance was either dissolved in paraffin oil DAB (low and mid concentrations, 10 and 40 % solution concentration, respectively) or used neat (high concentration).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminium inhalation chamber with two-chamber system (inner diameter = 14 cm, outer diameter = 35 cm), height = 25 cm, internal volume = 3.8 L
- Source and rate of air: Compressed air (supplied by Boge compressors) was conditioned (i.e. freed from water, dust, and oil) automatically. Inlet air flow was 10 L/min for all treatment groups; dilution air flow was 30 L/min for the control group and 20 L/min for the treatment groups.
- Method of conditioning air: automatically by a VIA compressed air dryer.
- Temperature and humidity in air chamber: 20.3 - 22.0 °C, 0.1 - 3.3 %
- Air flow rate: Air flows were monitored and controlled continuously by calibrated mass flow meters. Air flow rate was 30 L/min.
- Treatment of exhaust air: The exhaust air was purified using gas scrubbing devices (bubblers with NaOH), followed by exhaust air filters (activated char coal, filter mat, Makropur F-Filter), and HEPA-filters. Exhaust air was discarded via a digitally controlled vacuum.

TEST ATMOSPHERE
- Brief description of analytical method used: The test substance was sampled using two in-line connected gas bubblers filled with methanol (at least two samples during each exposure period). The sampling flow rate was 0.5 L/min. The sample volume was 10 L/sample. The concentration of the airborne test-substance was analyzed and quantified by reversed phase high performance liquid chromatography with DAD-detection.
- Samples taken from breathing zone: Yes.

VEHICLE
- Justification for use and choice of vehicle: Dry conditioned air was used to evaporate the test substance. Before, the test substance was either dissolved in paraffin oil DAB (group 2 and 3) or metered as neat substance (group 4).
- Concentration of test material in vehicle: group 2: 10 %, group 3: 40 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was sampled using two in-line connected gas bubblers filled with methanol. The concentration of the airborne test-substance was analyzed and quantified by reversed phase high performance liquid chromatography with DAD-detection (wavelength: 238 nm).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Duration of exposure per day: 6 hours /day
Dosing regime: 5 days/week, for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.03 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

0.1 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.3 mg/L air (nominal)

Doses / concentrations 4

Dose / conc.:

0.032 mg/L air (analytical)

Doses / concentrations 5

Dose / conc.:

0.114 mg/L air (analytical)

Doses / concentrations 6

Dose / conc.:

0.266 mg/L air (analytical)

No. of animals per sex per dose:

(Note: The study report is very inconsistent regarding animal numbers per group and regarding concentration and group size of post-exposure groups.)
According to the single animal data tables of the Appendix, each test group including the control consisted of 15 animals/sex.
Whereby, 10 animals/sex/concentration were sacrificed after the 4 weeks exposure period and the remaining 5 animals/sex/concentration were sacrificed after a 4 weeks post-exposure period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the structural similarity to aniline, the inhalation toxicity of 4-Fluor-N-isopropylaniline was assumed to be likely related to hematotoxicity. However, based on the findings of the acute inhalation study (OECD TG 403, Bayer, 1999) it appeared that respiratory tract irritation might occur at potential hematotoxic concentrations. Therefore, the concentrations used in the present study were assessed based on the findings obtained with the structurally related compound aniline and a irritation pre-test performed with 4-Fluor-N-isopropylaniline in rats and mice.
An exploratory subacute inhalation toxicity study (OECD TG 412) with aniline vapour was performed. Five groups, each of 30 male Wistar rats, were nose-only exposed to actual concentrations of 0, 0.0092, 0.0326, 0.0965, and 0.2749 mg/L aniline, under dynamic conditions for 6 hours/day on at least five days per week, for two consecutive weeks. This was followed by a maximum post-exposure period of approximately two weeks. Serial sacrifices were performed after the first exposure and on study days 4, 11, 14/15 and 28. Body weights, clinical signs, clinical pathology with extended hematological examinations, and organ weights as well as histopathology of selected organs were assessed. Rats exposed to 0.0092 and 0.0326 mg aniline/L tolerated the exposures without specific clinical signs. At 0.0965 and 0.2749 mg aniline/L rats were cyanotic. Changes in breathing patterns occurred at 0.2749 mg aniline/L. Changes in body weights were not observed in any exposure group at any time point. At 0.0965 and 0.2749 mg/L, aniline produced toxicity to the hematopoietic system, i.e., the red blood cell in circulation. Methemoglobinemia was the primary toxic response. Other changes appeared to have occurred secondary to methemoglobin formation and included subsequent erythrocyte injury and increased turnover. These abnormalities included red blood cell morphological alterations (e.g, Heinz bodies) and compensatory, macrocytic anemia. In regard to erythrocytotoxicity, 0.0326 mg aniline/L were tolerated without an adverse effect at any time point. Moreover, at 0.0965 and 0.2749 mg/L, a concentration- and time-related increase in spleen weight and in spleen size (splenomegaly) was apparent especially towards the end of the exposure period (secondary to erythrocyte toxicity). The development of hemolytic anemia produced by a compensatory erythropoiesis, which was observed in the spleen (0.0326 mg/L and above) and liver (0.2749 mg/L) was associated with an increased production of reticulocytes (0.0965 and 0.2749 mg/L). After the 2-week post-exposure period, the indices of increased red blood cell turnover turned back to normal, as well as the spleen weights, whilst at 0.274.9 mg/L some of the erythrocyte indices were still minimally affected.
In summary, the no-observed-adverse-effect concentration (NOAEC) was 0.0326 mg aniline/L because in the circulating blood was no evidence of direct or indirect erythrocytotoxicity. In regard to the erythroclastic effects, evidence of adversity occurred at doses ≥0.0965 mg aniline/L (slightly increased compensatory extramedullary hematopoiesis is considered to be homeostatic and non-adverse).
A study on the acute inhalation toxicity of 4-Fluor-N-isopropylaniline on rats has been conducted in accordance with OECD TG 403. Several groups of rats were nose-only exposed to vapor (0.510 mg/L) or liquid aerosol concentrations (1.925 and 3.076 mg/L) of 4-Fluor-N-isopropylaniline. Attempts were made so that aerosol generated was respirable to rats. The exposure to concentrations ≤1.925 mg/L were tolerated without mortality but with clinical signs. Mortality occurred at an exposure concentration of 3.076 mg/L. The following clinical signs were observed: piloerection, ungroomed hair-coat, reduced motility, high­legged gait, limp, bradypnea, labored breathing pattern, cyanosis, palpebral closure, nostrils with red encrustations, emaciation, prostration, apathy, hypothermia, reduced reflexes, and decreased body weights. The duration of signs appeared to be governed by changes in breathing patterns and cyanosis and resolved within the first post-exposure week. Rats exposed to the vapour phase experienced sings on the exposure day only. Necropsy findings were unobtrusive in surviving rats. Rats that succumbed showed a lung edema (OECD TG 403, Bayer, 1999).
In addition, the breathing behaviour of rats and mice was investigated in a sensory irritation potency study. After inhalation exposure to nominal concentrations of 0.030, 0.100, or 0.300 mg/L 4-Fluor-N-isopropylaniline for 45 min, data demonstrates that the respiratory decrease fifty percent (RD50) in mice was about 0.298 mg/L whilst at 0.022 mg/L minimal effect still existed. In rats, in turn, this lower concentration was without appreciable changes in breathing patterns. From this it was concluded that based on acute sensory irritation, approximately 0.300 mg/L are in the range of the maximum tolerable concentration (approximately RD40) whereas at approximately 0.020 - 0.030 mg/L sensory irritant effects are unlikely to occur. Thus, undue suffering of animals as a result of irritant effects on the respiratory tract is precluded in this range of concentration.
Because the volatilized test substance proved to have moderate upper respiratory tract irritation potency in rats, 0.300 mg/L were considered to be the maximum tolerated concentration. With regard to this endpoint in rats, 0.030 mg/L was believed to be in the range of the threshold concentration causing sensory irritation. With regard to hematotoxicity a somewhat comparable or lower potency to aniline was assumed (due to the sterical hindrance of the phenyl­amino group it was assumed that the oxidation and activation of the amino moiety is less efficacious when compared to aniline). Therefore, the following target concentrations were selected for 4-Fluor-N-isopropylaniline in the present study: 0.030, 0.100, and 0.300 mg/L.

- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: twice on exposure days (before and after exposure), once on exposure-free days
- Cage side observations included but were not limited to: changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The time of death was recorded as precisely as possible, if applicable.

BODY WEIGHT: Yes.
- Time schedule for examinations: twice per week during exposure and once per week during post-exposure period

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

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.

WATER CONSUMPTION: No.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: once before start of the exposure period (study day -3), once towards the end of the exposure period (study day 22)
- Dose groups that were examined: 10 animals of control group, 5 animals of high dose group

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: study days 28 - 30 and study days 56 - 57
- Anaesthetic used for blood collection: Yes. Pentobarbital narcosis.
- Animals fasted: Not specified.
- How many animals: 10 animals/group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: study days 28 - 30
- Animals fasted: Not specified .
- How many animals: 10 animals/group
- Parameters checked in table 1 were examined.

URINALYSIS: Yes.
- Time schedule for collection of urine: once on study days 24 and 25
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: No.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: once on study day 22
- Dose groups that were examined: 5 animals/sex/concentration, all test groups were examined
- Battery of functions tested: Visual placing response, grip strength (vertical, horizontal), tonus, cornea reflex, light reflex, pinna reflex, startle reflex (sound, touch), tail-pinch response, righting response (open field, drop method)

IMMUNOLOGY: No.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No.

LUNG BURDEN: No.

OTHER:
RECTAL TEMPERATURE
- Time schedule for examinations: study day 0, 4, and 23

METHEMOBLOBIN-DETERMINATION
- Time schedule for examinations: study days 14 - 17

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All rats were subjected to gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.
HISTOPATHOLOGY: Yes (table 3).

Other examinations:

Clinical Pathology - Liver
N-DEM (N-Demethylase, Aminopyrin-N-demethylase), O-DEM (O-Demethylase, p-Nitroanisol-N-demethylase), Cytochrome P450

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical tests are used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variate in question can be considered approximately normally distributed with equal variances across treatments, the Dunnett test is used, if heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA are carried out, i. e. the Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures. Within such a family, the experiment­ wise error is controlled. If not otherwise noted, all pair-wise tests are two-sided comparisons.
The specific findings from the respiratory tract are evaluated statistically using the pair-wise Fisher test after the R x C chi-squared test. The Fisher test was only performed if differences occurred between groups in the R x C chi-squared test or if a frequency value of <5 was calculated. For calculation of the unilateral p value a symmetrical distribution was assumed (p unilateral = (p bilateral)/2).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Low concentration: mild cyanotic appearance was observed only on study day 25 in all animals of both sexes
Mid and high concentration: cyanosis (after exposure) was observed on several study days throughout the study in all animals of both sexes
The onset and intensity of the occurrence of signs was clearly concentration- as well as time-dependent.
Prior to the following exposure (day after exposure) all rats appeared normal.

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The results of the hematological examinations revealed toxic effects on the haematopoietic system. A range of hematological parameters was statistically significantly affected at 0.0316 mg/L and above. The effects were largely dose-related in frequency, intensity and severity. The test substance induced a compensatory macrocytic hemolytic anaemia evidenced by statistically significant increases in reticulocyte count at 0.0316 mg/L and above and decreased red blood cell count, hematocrit and haemoglobin values in males and females, statistically significant at 0.1142 mg/L and above, and additionally increased MCV and MCH values. With regard to atypical erythrocytes, anisocytosis, anisochromasia, and hypochromasia were evident in rats exposed to 0.1142 mg/L and above. The presence of reticulocytes and Heinz' bodies were concentration dependently increased at 0.0316 mg/L and above. The latter being consistent with oxidative damage to the red blood cells. The analysis of the differential counts of the peripheral blood revealed increased evidence of directly damaged erythrocytes.
Methemoglobin values (samples taken retroorbitally in the third exposure week) were concentration dependently increased (statistically significant) in all animals of both sexes. Statistical significance was not reached for male animals of the low concentration group.
All hematological endpoints were indistinguishable from the control at the end of the 4-week post-exposure period. The evaluation of hemostasis did not reveal any specific effects.
No conclusive changes emerged from the analysis of the differential leukocyte count.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Statistical comparisons of clinical biochemistry parameters did not reveal any concentration-dependent changes considered to be of pathodiagnostic relevance. Some endpoints (especially those associated with the erythrotoxicity) appeared to be affected. Total bilirubin levels were statistically significant and dose-related increased in females exposed to 0.0316 mg/L and above, and in males exposed to 0.1142 mg/L and above. Some other endpoints showed some changes, however, are considered to be secondary due to the respiratory irritation, rather than specific disturbances caused by the test substance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The statistical analysis of data shows that absolute and relative spleen weights were significantly increased at 0.1142 mg/L and above at the end of the exposure period. A tendency of these changes was already observed at 0.0316 mg/L. All changes were reversible following the post-exposure period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, dark and enlarged spleens were found in all treated groups.
After four weeks post-exposure, black discolouration occurred in one female exposed to 0.2662 mg/L.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology of the spleen revealed congestion and increased extramedullary hemopoiesis. Prussian blue stained sections of the spleen showed increased pigmentation in almost all treated animals. In the liver, a Prussian blue positive pigmentation was observed at 0.1142 mg/L and above. In the mid and high concentration groups, an increased erythropoiesis was observed in the bone marrow of femur and sternum.
At four weeks post exposure, evidence of partial recovery ws noticed. In the spleen, a Prussian blue positive pigmentation was still seen at 0.0316 mg/L and above. However, a slightly increased incidence of Prussian blue positive hepatic pigmentation was only evident at 0.2662 mg/L. Further, at the end of the post-exposure period a slightly increased erythropoiesis was still present in the sternum of some animals of the mid and high dose groups.
In the kidneys of males, an increased grading of AZAN-positive tubular droplets - corresponding to alpha-2-microglobulin - was detected at 0.1142 and 0.2662 mg/L. The tubular pigmentation, seen in two males at 0.2662 mg/L was identified to be Prussian blue negative. All findings in the kidneys disappeared at the end of post-exposure.
In the thyroid gland, a slightly increased incidence and grading of follicular cell hypertrophy occurred in treated males at dose levels of 0.0316 mg/L and above. However, these changes were not strictly dose-dependent. No such finding was seen at the end of the post-exposure period.
Local effects were observed in the distal nasal cavity (level 3 and 4) at the end of exposure period. In both sexes, a minimal increased subepithelial, focal vacuolation was detected at 0.1142 and 0.2662 mg/L. However, the epithelium was completely unaffected. At the end of recovery period, all findings in the nasal cavity were fully reversible.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Rectal Temperatures: There was no evidence of a conclusive, significant effect on body (rectal) temperature in any treatment group, compared to control.
Clinical pathology in liver tissue: Statistical comparisons did not reveal any conclusive concentration-dependent changes considered as being of pathodiagnostic relevance.

Details on results:

Exposure to the test substance resulted in adverse systemic toxic effects at all tested concentrations. Repeated exposure to mean concentrations of ≥0.0316 mg/L led to dose-related increases in the incidence and severity of hematotoxicity and organ lesions resulting from hematotoxicity. Methemoglobinemia was the primary toxic response. Further changes resulting in a regenerative hemolytic anaemia were accompanied by alterations in the erythrocyte-related parameters (decrease in red blood cell count, hemoglobin concentrations, and hematocrit) and abnormalities in red blood cell morphology (e.g., Heinz bodies, anisocytosis), hyperbilirubinemia, medullary and extramedullary hematopoiesis in the spleen and liver, and increased incidence of Prussian blue positive pigmentation (hemosiderin) in the spleen and liver, and in addition, significant increases in spleen weight and spleen size (splenomegaly). A no-observed-adverse-effect-concentration (NOAEC) for systemic effects could not be established in this study, since the lowest tested concentration of 0.0316 mg/L air refers to the lowest-observed-adverse-effect-concentration (LOAEC). In fact, the findings are indicative of functional disturbances and morphological changes, of relevance for the human health. So, it was considered that the lesions identified at ≥0.0316 mg/L in rats of both sexes represent serious adverse effects on the health as defined by the criteria given in Commission Directive 2001/59/EWG.
Adverse local toxic effects were not observed in this study. Findings in the respiratory tract were observed as a minimal increased subepithelial, focal vacuolation, however without signs of inflammation or degeneration, detected in some animals exposed to 0.1142 or 0.266.2 mg/L, which were fully reversible at the end of post-exposure period of four weeks. Therefore, the NOAEC for local effects in the respiratory tract is 0.2662 mg/L for male and female rats, and the local NOEC is 0.0316 mg/L.

Effect levels

Key result

Dose descriptor:

LOAEC

Effect level:

0.032 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.032 mg/L air (analytical)

System:

other: red blood cells/erythrocytes

Organ:

other: erythrotoxicity (methemoglobin formation leading to compensatory macrocytic hemolytic anaemia)

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Based on the erythrotoxicity and the resulting anaemia observed at 0.0316 mg/L in this 28 d inhalation toxicity study, the substance is classified STOT RE Cat. 1, H372, based on GHS criteria.