Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection: 19-21 July 2011, Date of signature: 31 August 2011
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid
Details on test material:
Sponsor's identification: trisodium trimetaphosphate
Description: White solid
CAS number: 7785-84-4
EC number: 232-088-3
Batch number: 1-10671-56
Total phosphate: 69.4 %
Date received: 09 March 2012
Expiry Date: 01 February 2015
Storage conditions: Room temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Further details on strain: RccHanTM : WIST strain rats
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 200-350 g
- Fasting period before study: none
- Housing: The animals were housed in groups of up to three 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 and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): with the exception of the exposure period diet (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was available ad libitum
- Water (e.g. ad libitum): with the exception of the exposure period water was available ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A dust atmosphere was produced from the test item 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.
- Exposure chamber volume: 30 litres (dimensions: 28 cm diameter x 50 cm high)
-Chamber flow rate: The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of holding animals in test chamber: 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 ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Method of conditioning air: The concentration within the chamber was controlled by adjusting the test item 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.
- System of generating particulates/aerosols: In order to facilitate aerosolisation and reduce particle size, the test item was ground using a small amount of diethyl ether in a Retsch Planetary Ball Mill (Retsch (UK) Ltd, Leeds, UK) all of the solvent was removed via evaporation prior to use. The absorption of the test item was not determined. See 'exposure apparatus' for further details.
- Method of particle size determination: 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 (9.7, 6.7, 3.8, 1.8, 0.94 and 0.46 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 item, 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 9.7, 6.7, 3.8, 1.8, 0.94 and 0.46 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.
- Temperature, humidity, pressure in air chamber: 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. Individual values are given in Attachment 1.
-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 port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen. Individual values are given in Attachment 2.

TEST ATMOSPHERE
- Brief description of analytical method used: 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 items (Green J D et al, 1984).
Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterisation period test item input rates, grinding techniques and generation systems were varied in order to achieve the required atmospheric conditions.
- Samples taken from breathing zone: yes
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 item. 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 item used by the total volume of air passed through the chamber.
The nominal concentration is 356% of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.

VEHICLE
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable):
- Justification of choice of vehicle:
- Lot/batch no. (if required):
- Purity:

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
5.09 mg/L (target concentration 5 mg/L)
No. of animals per sex per dose:
3 males, 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 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 up to fourteen days. Any evidence of overt toxicity was recorded at each observation.
- Bodyweight: Individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
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.
Statistics:
Evaluation of Data
Data evaluations included the relationship, if any, between the animals’ exposure to the test item 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 item was made.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.09 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There was no mortality.
Clinical signs:
other: Individual clinical observations are given in Attachment 5. 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
Body weight:
Individual bodyweights, together with bodyweight changes, are given in Attachment 6.
Two males and all female animals exhibited bodyweight losses or showed no bodyweight gains on the first day post-exposure. All animals exhibited reasonable bodyweight gains during the remainder of the recovery period.
Gross pathology:
Individual necropsy findings are given in Appendix 7.
With the exception of one instance of dark patches on the lungs, no macroscopic abnormalities were detected amongst animals at necropsy.

Any other information on results incl. tables

It is noted that the mean mass median aerodynamic diameter (MMAD) exceeds the range given in test guidelines (1-4 μm). This deviation is due to the physical characteristics of the test item. During the characterisation phase of the study various techniques were employed in order to try and reduce the achievable MMAD, these included grinding the test item in a planetary ball mill and adding particle separation devices into the generation system in order to try and remove larger particles. During the characterisation phase of the study the test atmosphere concentration was reduced to 2 mg/L in an attempt to achieve a particle size as close to 4 μm as possible. This did reduce the particle size slightly but it wasn’t considered to be significant. It was, therefore, preferable to expose the animals to a higher concentration of test item, even though this also increased the MMAD, as this resulted in the animals being exposed to the highest possible concentration of particles.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.09 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of trisodium trimetaphosphate, in the RccHanTM : WIST strain rat, was greater than 5.09 mg/L (Globally Harmonised Classification System – unclassified).
This study is considered to be acceptable for use as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Further testing is scientifically unjustified.