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

Endpoint summary

Description of key information

Oral: EU method B.1, rat (male/female), 1993: LD50 356 mg/kg bw

Inhalation: OECD 403, rat (male/female), 1999: LC50 2.58 mg/L

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jun - 22 Jul 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF-bred Wistar rats (Wsd/Win:WU strain, formerly named: Bor: WISW (SPF Cpb)) from Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at delivery on 25 Jun and 02 Jul 1993: males were 7 - 8 weeks of age, females were 9 - 10 weeks of age
- Mean body weight at study initiation: males: 169 g; females 156 g
- Fasting period before study: approximately 16 hours before and 4 hours after test substance administration feed was withdrawn from animals
- Housing: Animals were conventionally housed in Makrolon cages type III on low-dust wooden granulate (Source: Ssniff, Soest/Westfalen, Germany). Animals were group housed; 5 animals per cage.
- Diet: "fixed-formula" standard diet, Altromin® 1324 pellets (Source: Altromin GmbH und Co KG, Lage, Germany), ad libitum. Composition and potential contamination of diet was analysed on a routine basis.
- Water: tap water of drinking water quality, ad libitum.
- Acclimation period: at least 5 days
- Microbiological status: SPF-bred rats were used
- Method of randomisation in assigning animals to test and control groups: Animals were assigned to study groups based on random numbers from a randomlist.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 Jun To: 22 Jul 1993

Route of administration:

oral: gavage

Vehicle:

physiological saline

Remarks:

plus 2 % Cremophor EL (v/v)

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

DOSAGE PREPARATION
Test substance was formulated in physiological saline with 2 % Cremophor EL under continuous stirring. Detailed analyses of test substance identity, stability and homogeneity were conducted.

Doses:

200, 633, and 2000 mg/kg bw

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: On the day of test substance administration animals were repeatedly observed for clinical signs of toxicity. During the 14-day observation period animals were observed twice daily. On weekends and public holidays animals were observed once daily. Animals were weighed directly before, one week after and two weeks after (end of observation period) test substance administration.
- Clinical signs including body weight : type, start, duration, and intensity of clinical signs was recorded.

Statistics:

Calculation of the median lethal dose.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

356 mg/kg bw

Based on:

test mat.

Mortality:

All animals of the lowest dose group (200 mg/kg bw) survived until scheduled sacrifice.
All animals of both, the mid and the high dose group (633 and 2000 mg/kg bw) died within 8 hours following single administration of the test substance by gavage.

Clinical signs:

other: Clinical signs of toxicity were reported for the mid and high dose groups (663 and 2000 mg/kg bw) whereas no such signs were seen at the lowest tested dose of 200 mg/kg bw. The clinical signs observed included sedation, paleness and ruffled fur. Signs of

Gross pathology:

Findings at necropsy:
2000 mg/kg bw: stomac filled with white liquid
633 mg/kg bw: inflated stomac filled with liquid, reddened lungs. Additionally, the liver of the females of this dose group was pale and spotted.
200 mg/ kg bw: No gross pathological abnormalities were found in the animals sacrificed at study termination.

Table 1. Mortality and incidence of clinical signs

Dose [mg/kg bw]	Mortality	Clinical signs
	N*	N*
Males
200	0/5	0/5
633	5/5	5/5
2000	5/5	5/5
Females
200	0/5	0/5
633	5/5	5/5
2000	5/5	5/5

*N= Number of animals/ number of animals used

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Examining the test substance for acute oral toxicity in male and female Wistar rats resulted in a LD50 value of 356 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

356 mg/kg bw

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Sep - 22 Sep 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Adopted in 1981

Deviations:

yes

Remarks:

Humidity values were below guideline values due to use of conditioned dry air for dispersion of test substance. Particle size distribution was measured once (not twice) during the 4 h exposure. These minor deviations have no impact on the study outcome.

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF bred Wistar rats (strain: Hsd Cpb:WU (SPF)) were obtained from Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2 - 3 months
- Mean body weight at study initiation: males: 203 g; females: 182 g
- Housing: Animals were housed singly in conventional Makrolon® Type II cages. Bedding consisted of type S 8/15 low-dust wood granulate from Ssniff, Soest/Westfalen, Germany.
- Historical data: 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.
- Diet: standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Lage, Germany), ad libitum. The nutritive composition and contaminant content of the standard diet was checked regularly by random sampling by the Laboratory Animal Services, Bayer AG.
- Water: drinking quality tap water, ad libitum
- Acclimation period: Animals were acclimatized to the animal room conditions for at least 5 days before use.
- Microbiological status: SPF bred rats
- Method of randomisation in assigning animals to test and control groups: A computerized list of random numbers served the purpose to assign animals at random to the treatment groups.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Sep 1998 To: 22 Sep 1998

Route of administration:

inhalation: mixture of vapour and aerosol / mist

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.57 - <= 4.13 µm

Geometric standard deviation (GSD):

>= 1.9 - <= 2.13

Remark on MMAD/GSD:

1925 mg/m³: aerosol with MMAD = 4.13 µm and GSD = 2.13
3076 mg/m³: aerosol with MMAD = 3.57 µm and GSD = 1.90

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminum inhalation chamber
- Exposure chamber volume: 3.8 L
- Method of holding animals in test chamber: plexiglas exposure tubes (nose-only exposure)
- Source and rate of air (airflow): dry conditioned air with an inlet air flow of 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- System of generating vapor atmosphere: Under dynamic conditions the vapor phase of the test substance was conveyed into the intake of the cylindrical inhalation chamber. Dry conditioned air was fed through the liquid of the test substance contained in two glass bubblers (content: 100 g/bubbler). Without further dilution the saturated vapor atmosphere was conveyed into the inhalation chamber.
- System of generating particulates/aerosols: Under dynamic conditions the test substance was nebulized into a baffle (pre-separator) from which the substance was conveyed into the intake of the cylindrical inhalation chamber. The test substance was nebulized using a binary nozzle and conditioned compressed air (15 L/min; dispersion pressure 600 kPa). The test substance was fed into the nozzle by an infusion pump equipped with 50 mL glass syringe (Braun Melsungen).
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS critical orifice, low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria). The individual impactor stages had been covered by an aluminum foil and glass fiber filter which were subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was therefore not used to prevent particle bounce and re-entrainment. Gravimetric analyses were made using a digital balance.
- Treatment of exhaust air: The exhaust air was purified via cotton-wool/HEPA/char coal filters. These filters were disposed of by Bayer AG.
- Temperature, humidity in air chamber: 21.1 - 23.9 °C, 3.3 - 13.5 %

TEST ATMOSPHERE
- Brief description of analytical method and equipment used:
Vapor (low dose group): The test substance was sampled using gas bubblers filled with ethanol and were quantified analytically by gas chromatography (GC) using an flame ionization detector (FID). Between the glass bubblers and gas metering device a cool-trap was used to scrub volatile organic constituents from the sampling train to prevent sampling errors.
Aerosol (mid and high dose group): The test atmosphere consisting an equilibrium of vapor and aerosol phases was determined by gas chromatography (GC). Samples were taken by using Florisil filled tubes and/or additional gas bubblers (to check for quantitative absorption in one adsorbent or the other). For reference/calibration purposes the test substance was used.
- Samples taken from breathing zone: yes

VEHICLE
The test substance was evaporated or aerosolized neat without any vehicle. For aerosol generation, the test substance was nebulized using a binary nozzle and conditioned compressed air.

TEST ATMOSPHERE
- Particle size distribution: Mid concentration group (1.925 mg/L): 3.1 % particel mass <1.0 µm; 33.7 % particle mass <3.0 µm; 60.1 % particle mass <5.0 µm. High concentration group (3.076 mg/L): 2.5 % particel mass <1.0 µm; 39.3 % particle mass <3.0 µm; 69.9 % particle mass <5.0 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Mid concentration group (1.925 mg/L): MMAD = 4.13 µm and GSD = 2.13. High concentration group (3.076 mg/L): MMAD = 3.57 µm and GSD = 1.90

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

nominal: 0.800, 1.500, 5.000 mg/L
analytical: 0.510, 1.925, 3.076 mg/L

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The appearance and behavior of each rat were examined several times on the day of exposure and at least once daily thereafter. Body weights were measured before exposure, on days 3 and 7, and weekly thereafter. Individual weights were also recorded at death, if applicable.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The following reflexes were tested: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).
- Other examinations performed: The rectal temperatures were measured directly after cessation of exposure.

Statistics:

Necropsy: Respiratory tract findings of survivors were evaluated statistically using pair-wise Fisher test after the RxC chi-squared test. The Fisher test was only performed if differences occurred between groups in the RxC chi-squared test or if the frequency value was <5 (Gad and Weil, 1982). For calculation of the unilateral p value a symmetrical distribution was assumed.
Body weight (bw): Means and single standard deviations of bw were calculated. Since in acute studies individual group means may differ prior to the first exposure, the bw gain was statistically evaluated for each group using one-way ANOVA.
Physiological data: Data of rectal temperature measurements were evaluated using the ANOVA procedure.
Calculation of LC50: The LC50 calculation was performed by computer according to Rosiello et al. (1977) and Pauluhn (1983). If only two pairs of values with >0 % lethality and <100 % are available then the first linear approximation is based on these values and a χ²-homogeneity test is not performed. In this case the interpolated concentration at 50 % lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-α) = 95 % (p = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than two study groups were compared with each other. If the F-test shows that intra-group variability > inter-group variability, this is indicated as "no statistical difference between the groups". If a difference was found then a pairwise post-hoc comparison was conducted (one- and two-sided) (Games and Howell modification of Tukey-Kramer test).

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

2.58 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Control group: All animals survived until scheduled sacrifice.
Low concentration group (0.510 mg/L air): No mortality was noticed.
Mid concentration group (1.925 mg/L air): No mortality was noticed.
High concentration group (3.076 mg/L air): All males died within 1 - 2 days, whereas 3/5 females died wiithin 2 - 4 days.

Clinical signs:

other: Starting from the lowest tested concentration (0.510 mg/L): piloerection, reduced motility, and bradypnea

Remarks:

At higher concentrations, additionally: ungroomed hair-coat, high-legged gait, limp, labored breathing pattern, cyanosis, palpebral closure, nostrils with red encrustations, emaciation, prostration, and apathy.

Body weight:

As compared to the control group, the animals of the groups exposed to the test substance showed a transient decrease of body weight and body weight gain regarding in particular the females of the mid and the males and females of the high concentration groups.

Gross pathology:

- Animals sacrificed at the end of the observation period:
In rats exposed to the test substance a conclusive, concentration-dependent increased incidence of macroscopic findings could not be ascertained. Therefore, the discolorations of lungs observed are not considered to be causally related to the exposure to the test substance.
Findings such as mild discolorations of lungs and other parenchymatous organs are often observed in control animals euthanized with pentobarbital.
- Animals that died intercurrently: Lungs slightly collapsed, dark-red discolorations; white foamy content in trachea; black content (mucus) in gastrointestinal tract.

Other findings:

Reflex measurements
A battery of reflex measurements was made on the first post-exposure day. Some rats of the mid and high concentration group experienced decreased reflexes (grip strength, tonus, startle reflex, light reflex, tail pinch and righting response). The animals of the remaining groups were inconspicuous.

Rectal temperature
Statistical comparisons between control animals with those in the exposure groups revealed statistically significant differences, i.e., the rats exposed to the test substance experienced a decrease in body temperature (down to 27.6 °C in high concentration animals, versus approximately 38 °C for the control group).
According to the study report, the difference in body temperature may reflect a more labile thermoregulatory physiology among rodents, as a reaction toward inhalative exposure to respiratory irritating substances.

Table 1. Results of acute inhalation toxicity testing with Wistar rats exposed to N-Isopropyl-4-fluoroaniline

Test substance concentration [mg/L air]	Toxicological results*	Onset and duration of clinical signs	Time of death	Mortality (%)	Mean rectal temperature (°C)
Males
0	0/0/5	---	---	0	38.1
0.51	0/5/5	Day 0	---	0	34.9**
1.925	0/5/5	Day 0 - 7	---	0	32.4**
3.076	5/5/5	Day 0 - 2	Day 1 - 2	100	27.6**
Females
0	0/0/5	---	---	0	38.3
0.51	0/4/5	Day 0	---	0	35.1**
1.925	0/5/5	Day 0 - 4	---	0	33.0**
3.076	3/5/5	Day 0 - 5	Day 2 - 4	60	28.7**

* first number = number of dead animals; second number = number of animals with clinical signs; third number = number of animals exposed

** = statistically significantly different from control at p <0.05

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Examining the test substance for acute inhalation toxicity in male and female Wistar rats resulted in a LC50 value of 2.58 mg/L air for the test substance as aerosol.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

2 580 mg/m³ air

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The acute toxicity potential of the test substance was evaluated in one acute oral toxicity study (Bayer, 1993) and one acute inhalation toxicity study (Bayer, 1999). Both studies were performed on rats as test species. The results of both animal studies warrant classification and labelling at least as Acute Tox. Cat. 4 according to Regulation (EC) 1272/2008. However, repeated dose toxicity studies performed with the test substance revealed the mode of action/toxicity being methaemoglobin (MetHb) formation. This haematotoxicity lead to anaemia and secondary organ lesions. Because humans are more susceptible to MetHb formation compared to rodents, the human-relevant classification for acute toxicity of MetHb-forming substances requires a more severe category than that derived of rodent studies. In Regulation (EC) 1272/2008 (CLP) it is stated that "The preferred test species for evaluation of acute toxicity by the oral and inhalation routes is the rat, […]. 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" (Regulation (EC) 1272/2008 Annex I, § 3.1.2.2.1). In general, it is well known that humans are more susceptible to MetHb formation compared to rodents, therefore scientific judgement is necessary in addition to the available acute oral and inhalation toxicity studies in rodents. According to ECHA guidance document (GD) Guidance on the Application of the CLP Criteria (Version 5.0, July 2017) Section 3.1.5.1.2., many case reports demonstrating death from methaemoglobinaemia following human exposure to aromatic amines at low dose levels are available. Further, it is pointed out in the ECHA GD that “The extensive and consistent human experience is considered to be sufficiently robust by expert judgement to be used for classification [considerations].” and that rodent data are generally not considered to be adequate for human-relevant acute toxicity classification of MetHb forming substances. The ECHA GD on CLP criteria further underlines in Section 3.9.2.5.2., if lethality is observed in humans or animals based on MetHb formation, the substance should be classified for acute toxicity. However, since observation of lethality following MetHb formation in rodents is not usual, because rodents are more tolerant to it, extrapolation to the human situation must be the critical decision key and expert judgement should be used.

In addition to the example of N,N-Dimethylaniline provided in ECHA GD on CLP criteria Section 3.1.5.1.2., current examples of a more severe acute toxicity classification may be found in the ECHA registration dossiers of nitrobenzene and aniline:

- The RAC Opinion on nitrobenzene concludes: Taking into account that formation of MetHb, in response to a single exposure to nitrobenzene, may cause secondary toxic effects in internal organs such as spleen, liver, kidney, brain and lungs, the high difference between humans and rats in susceptibility to induction of MetHb should be taken into consideration in the hazard classification. Therefore, in the opinion of RAC the classification derived solely on animal data - Acute Tox. 4 with hazard statement H302 Harmful if swallowed – is not relevant for humans because of the higher sensitivity of humans to MetHb forming action of nitrobenzene, thus acute oral toxicity of nitrobenzene should by classified Acute Tox. 3, H301 Toxic if swallowed.

- The registration dossier of aniline states: 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 methaemoglobinaemia, 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 haemoglobin. 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 methaemoglobin reducing reductase in erythrocytes, which is much higher in rodents than in human, whereas enzyme activities in cats or dogs are similar to human.

 

For the general MetHb-forming toxicity the German DFG MAK assessment may be taken into account: https://onlinelibrary.wiley.com/doi/10.1002/3527600418.bb6253e1516

 

 

In conclusion, based on the acute and repeated dose toxicity data for the test substance and since humans are more susceptible to MetHb formation compared to rodents, the human-relevant classification for acute toxicity of the MetHb-forming test substance requires a more severe category than that derived from the available acute rodent studies alone (Bayer, 1993 oral route, 1999 inhalation route). Based on the aforementioned considerations and examples, the following more stringent classification for acute toxicity is proposed: classification as Acute Tox Cat. 3, H301 Toxic if swallowed, for the oral route, and Acute Tox Cat. 3, H331 Toxic if inhaled, for the inhalation route.

Justification for classification or non-classification

The available data on acute oral and inhalation toxicity of the test substance, together with the findings of repeated dose toxicity studies (haematotoxicity caused by MetHb-formation), meet the criteria for classification as Acute Tox Cat. 3 (H301 Toxic if swallowed) and Acute Tox Cat. 3 (H331 Toxic if inhaled) according to Regulation (EC) 1272/2008. For MetHb-forming substances classification of acute toxicity results in a more severe category than that derived from rodent studies, since humans are more susceptible to MetHb formation compared to rodents.