3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 October 2020 - 22 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

7 September 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage conditions: Room temperature

Specific details on test material used for the study:

- The original test material (as supplied) could not be used to produce an appropriate atmosphere because during technical tests it had been proven that it is not suitable for atmosphere generation due to the physical properties of the test material (specifically - its sticky nature). Therefore, the test material was micronized by a ball mill (Pulverisette 6, Fritsch GmbH Germany) in order to increase the number of particles under 4 μm.
- The test material was milled in two phases. In the first phase, the test material (approximately 30 g) was loaded in stainless steel grinding bowl of size 250 mL with stainless steel grinding balls (Ø 10 mm / 100 pieces) and with appropriate milling settings it was milled, rotational speed of 600 rpm for 3 mins with 5 mins break, this cycle was performed four times.
- In the second phase, the previously ground test material (approximately 30 g) was loaded in the agate grinding bowl of size 250 mL with agate grinding balls (Ø 5 mm / 390 pieces) and with appropriate milling settings it was milled, rotational speed of 400 rpm for 1 min with 5 mins break, this cycle was performed two times.
- For the technical trials and animal exposure the test material which was milled according to the milling settings mentioned above was used.
- Prior to the animal exposure a range of technical trials with original milled test material were performed in order to prove that the most optimal MMAD was selected for the main study (ie that which would attain the highest possible test concentration).
- Different milling tests and technical trials with the original and/or milled test material were performed in order to achieve the target concentration of 5 mg/L, but none of them was proved to be effective, as due to the sticky nature of the milled test material the target concentration of 5 mg/L could not be reached, therefore the main study was performed by the exposure of animals to the maximum attainable concentration (~3 mg/L) with optimum particle size distribution around 4 μm MMAD.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 8-9 weeks old
- Weight at study initiation: 315 - 341 g (males), 175 - 185 (females). The weight variation did not exceed ± 20 % of the mean weight for either sex.
- Housing: Group caging (up to 3 animals, by sex, per cage), in Polypropylene/polycarbonate (type III) cages with stainless steel mesh lids. Rats were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 6 days. (Animals were also acclimatised to the test apparatus (restrain procedures) for a short period (1 hour) prior to testing in order to lessen the stress during exposure.)
- Method of randomisation in assigning animals to test and control groups: The animals were set in order of their bodyweight. The animals were randomly assigned to test groups using a randomization scheme. The randomization was checked according to the actual bodyweights verifying the homogeneity and deviations between the groups.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: Above 10 air exchanges/hour by central air-conditioning system.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.72 µm

Geometric standard deviation (GSD):

2.18

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Prior to animal exposure, test material atmospheres were generated within the exposure chamber. During this period, airflow settings, test material input and the atmosphere generation system was varied to achieve the required atmospheric concentrations and constancy.

- Exposure apparatus: Anodised aluminium Flow Past Exposure Chamber (TSE Systems GmbH, Bad Homburg, Germany). The test material was aerosolised using a Rotating Brush Generator, RBG 1000 (Palas GmbH, Greschbachstraße 3 b, 76229 Karlsruhe, Germany) located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port.
- Source and rate of air (airflow): Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.7 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere as it is about twice the respiratory minute volume of a rat.
- Method of particle size determination: The particle size analysis of the generated test atmosphere was performed three times during the exposure period using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). During the exposure period samples were collected three times with one sample taken during the first hour of exposure, next in the second hour and last one during the last hour. Samples were collected from vacant animal exposure ports (representing the animal’s breathing zone) and were analysed. The collection substrates and the backup filter were weighed before and after sampling and the weight of test material, collected at each stage, calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 0.55, 0.91, 1.55, 2.11, 3.51, 6.61 and 10.5 μm was calculated.
- Treatment of exhaust air: After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic.
- Temperature and humidity in air chamber: 21.1 °C and 1.2 % (mean values.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: The actual concentration of generated test atmosphere was measured gravimetrically at regular intervals during exposure by pulling a suitable, known volume of test atmosphere from the exposure chamber, through GF10 glass fibre filters. Sampling was performed shortly after chamber equilibration and then uniformly distributed, at approximately regular intervals. Samples were collected from a vacant animal exposure port (animals breathing zone). The amount of material collected on glass fibre filters was determined gravimetrically. The filter holder was opened before sampling and filter was weighed right before exposure. Immediately after sampling the filters were weighed again. Nominal concentration was calculated by dividing the total weight of test item disseminated into the test chamber by the total volume of air used during the same period.
- Particle size distribution and MMAD: An aerosol atmosphere was generated containing particles with a mass median aerodynamic diameter (MMAD) between 3 to 4 μm with a geometric standard deviation (GSD) in the range of 1.5 to 3.
- Time needed for equilibrium of exposure concentration before animal exposure: 11 minutes

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Measured gravimetrically at regular intervals during exposure by pulling a suitable, known volume of test atmosphere from the exposure chamber, through GF10 glass fibre filters (GE Healthcare Life Science, Whatman GmbH, Germany).

Duration of exposure:

4 h

Remarks on duration:

14-day observation period after 4 h exposure.

Concentrations:

3.38 mg/L (highest attainable concentration)

No. of animals per sex per dose:

10 animals, single dose (5 males / 5 females).

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing
1) Morbidity/mortality: Checks were made twice daily, early and late during the normal working day, for mortality and/or morbidity amongst the test animals.
2) Clinical Signs: As a minimum, individual, clinical observations were performed after one, two and three hours exposure whilst the animals were still restrained. Following exposure clinical observations were performed twice on the day of exposure (as soon as practicable after removal from restraint, and 1 hour after completion of the exposure), and then once daily for fourteen days. Cage-side observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. All observations were recorded.
3) Bodyweight: Individual bodyweights were recorded on the day of exposure day 0 (prior to exposure) and on days 1, 3, 7, 14.
- Necropsy of survivors performed: Yes. A gross necropsy was performed on all animals euthanised by exsanguination following intra-peritoneal injection of pentobarbital solution at the end of the 14-day observation period. A complete examination of the abdominal and thoracic cavities was made and special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity. Any abnormalities were recorded.

Statistics:

The mean of the bodyweight and bodyweight gain were calculated by Excel spreadsheet software.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3.38 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There was no mortality in the study.

Clinical signs:

other: Slight dyspnoea was noted during and/or after exposure in all exposed animals. In addition, red staining on the snout was noted in three animals shortly after exposure. All signs ceased from the day following exposure until the end of the observation.

Body weight:

Slight bodyweight loss/stagnation was observed in nine exposed animals on the day following exposure, which comes from the restrain procedure and has no toxicological relevance. Normal bodyweight gain was noted for all exposed animals from Day 1 until the end of observation period.

Gross pathology:

In the current study, no macroscopic abnormalities were found during the necropsy.

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

Under the conditions of this study the 4-hour LC50 of the test material in rats was considered to be > 3.38 mg/L since no adverse effectd were noted at the highest attainable test concentration.

Executive summary:

The acute inhalation toxicity of the test material was assessed according to the standardised guidelines OECD 403, EC B.2 and OPPTS 870.1300, under GLP conditions. 

During the study, groups of 5 males and 5 females were exposed to the maximum attainable concentration of the test material of 3.38 mg/L. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14-day observation period. The day of exposure was designated Day 0. Aerosol concentration was measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and bodyweights were recorded throughout the experiment and at the end of the scheduled period the surviving animals were euthanised and subjected to a gross examination post mortem.

The mean achieved concentration in the study was 3.38 mg/L. The mean mass median aerodynamic diameter (MMAD) was 3.72 μm with a geometric standard deviation (GSD) 2.84.

Under the conditions of the study, no mortality was observed. Slight dyspnoea was noted during and/or after exposure in all exposed animals. In addition, red staining on the snout was noted in three animals shortly after exposure, however all clinical signs ceased and all animals were symptom free from the day following exposure until the end of observation period. Slight bodyweight loss/stagnation was observed in nine exposed animals on the day following exposure, which comes from the restrain procedure and has no toxicological relevance. Normal bodyweight gain was noted for all exposed animals from Day 1 until the end of observation period. In the current study, no macroscopic abnormalities were found.

The 4-hour LC50 of the test material in rats was therefore considered to be > 3.38 mg/L since no adverse effectd were noted at the highest attainable test concentration.