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Diss Factsheets

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 October 2006 to 25 October 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
according to guideline
EU Method B.2 (Acute Toxicity (Inhalation))
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
EC Number:
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
Cas Number:
Molecular formula:
This is a UVCB substance. See section 1.2 for individual components.
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: approximately eight to twelve weeks old
- Weight at study initiation: within the weight range of 200g to 350g
- Housing: housed in groups of five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard "fun tunnels" (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): With the exception of the exposure period, free access to food (EU Rodent Diet 5LF2, BCM IPS Limited, London, UK) was allowed throughout the study.
- Water (e.g. ad libitum): free access to mains drinking water
- Acclimation period: at least 5 days

- Temperature (°C): 19 - 25 °C
- Humidity (%):30 - 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Details on inhalation exposure:
Atmosphere Generation
A dust atmosphere was produced from the test material using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.
The cylindrical exposure chamber had a volume of approximately 30 litres (dimensions: 28 cm diameter x 50 cm high). The concentration within the chamber was controlled by adjusting the test material feed rate from the SAG 410. The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals' breathing zone with a wide variety of test materials (Green J D et al, 1984).
Prior to the start of the study, test material atmospheres were generated within the exposure chamber. During this characterisation period air flow settings and test material input rates were varied to achieve the required atmospheric concentrations.

Exposure Procedure
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber '0' ring. Only the nose of each animal was exposed to the test atmosphere.
Following an appropriate equilibration period a single group of ten rats (five males and five females) was exposed to an atmosphere of the test material for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 100% of target and no deaths occurred, no further levels were required.

Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals' breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period.

Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough, East Sussex) located in a sampling port in the animals breathing zone during each exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fibre filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.
Each filter was weighed before and after sampling in order to calculate the weight of collected test material. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
The nominal chamber concentration was calculated by dividing the mass of test material used by the total volume of air passed through the chamber.

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (9.6, 6.6, 3.5, 1.8, 0.87 and 0.33 µm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test material, collected at each stage, calculated by difference.
The mean amount 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 9.6, 6.6, 3.5, 1.8, 0.87 and 0.33 µm was calculated.
The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.
Analytical verification of test atmosphere concentrations:
gravimetric method
Duration of exposure:
4 h
The mean achieved atmosphere concentration was 5.00 ± 0.20 mg/L. The mean mass median aerodynamic diameter was 3.07 µm, and the inhalable fraction (% < 4 µm) was 60.3 while the geometric standard deviation was 2.78.
No. of animals per sex per dose:
Five males and five females
Control animals:
Details on study design:

Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for fourteen days. Any evidence of overt toxicity was recorded at each observation.

Individual bodyweights were recorded prior to treatment on the day of exposure and on Days 7 and 14.

At the end of the fourteen day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Data evaluations included the relationship, if any, between the animals' exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, necropsy findings, bodyweight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test material was made.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: No deaths occurred
No deaths occurred
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. Fur staining by the
Body weight:
Variations in bodyweight gain are frequently seen for female animals of this strain and age during this type of study and, in isolation, are considered not to be significant.
Apart from one female animal that showed slight bodyweight loss during Week 2, normal bodyweight development was noted during the study.
Gross pathology:
The following macroscopic abnormalities were detected amongst animals at necropsy:
Lungs — fluid filled, abnormally dark, dark patches.

Any other information on results incl. tables

Exposure Chamber Concentration

The test atmosphere was sampled at approximately fifteen minute intervals during the exposure period and the actual concentration of the test material calculated. The mean values obtained were:


Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)






The chamber flow rate was maintained at 55 L/min providing 110 air changes per hour.

The theoretical chamber equilibration time (T99) was 3* minutes (Silver, 1946).

* = Atmospheres of the test material were generated prior to animal insertion, therefore, the equilibration period was carried out for a total of 16 minutes.


Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals' breathing zone was as follows:

Mean Achieved

Atmosphere Concentration (mg/L)

Mean Mass Median

Aerodynamic Diameter


Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation







Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to EU criteria.
No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.00 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of the test material, in the Sprague-Dawley Crl:CD (SD) IGS BR strain rat, was greater than 5.00 mg/L.
Executive summary:

A study was performed to assess the acute inhalation toxicity of the test material. The method used followed that described in the OECD Guidelines for Testing of Chemicals No. 403 "Acute Inhalation Toxicity". A group of ten Sprague-Dawley strain rats (five male and five female) were exposed to a dust atmosphere for four hours using a nose only exposure system, followed by a fourteen day observation period. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection, fur staining by the test material and wet fur. There were frequent instances of laboured respiration. Animals recovered to appear normal from Days 6 to 8 post-exposure. One female rat showed slight bodyweight loss during Week 2 while normal bodyweight development was noted in the remaining 9 rats. Macroscopic abnormalities noting during the study were fluid filled lungs, as well as abnormally dark and dark patched lungs.

No deaths occurred, therefore the acute inhalation median lethal concentration (4h LC50) of the test material in the Sprague-Dawley strain rat was greater than 5.00 mg/L.