Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 15 November 2010 and 20 December 2010.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted 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
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
(the achieved particle size (9.4 µm) and GSD range (5.3) were larger than generally acceptable for this type of study (MMAD: 1 -4 µm, GSD: 1.5-3.0, respectively)).
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
yes
Remarks:
(only six animals (three males and three females) were used)
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 20 July 2010, date of signature: 29 October 2010
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
435-740-7
EC Name:
-
Cas Number:
94317-64-3
Molecular formula:
C4H14N3PS
IUPAC Name:
butyl[diamino(sulfanylidene)-lambda5-phosphanyl]amine
Constituent 2
Reference substance name:
N-(n-butyl) thiophosphoric triamide (NBPT)
IUPAC Name:
N-(n-butyl) thiophosphoric triamide (NBPT)
Details on test material:
Sponsor's identification: NBPT
Description: White powder
Batch number: 100111
Purity: >97%
Date received: 04 August 2010
Expiry Date: 01 July 2011
Storage conditions: Stored cold at approximately 4°C, in the dark

Test animals

Species:
rat
Strain:
other: HsdHan™ : WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 200 g to 350 g
- Fasting period before study: Not reported
- Housing: 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: Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK
- Water: free access to mains drinking water
- Acclimation period: at least five days

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

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) plus cylindrical exposure chamber
- Exposure chamber volume: approximately 30 litres
- 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.
- Source and rate of air: 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. 35 L/min providing 70 air changes per hour.
- Method of conditioning air: water trap and respiratory quality filters
- System of generating particulates: SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany)
- Method of particle size determination: Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK)
- Treatment of exhaust air: filtered
- Temperature, humidity, pressure in air chamber: 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.

TEST ATMOSPHERE
- Brief description of analytical method used: glass fibre filters (Gelman type A/E 25 mm) 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 (Gravimetric).
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): Not applicable
- Concentration of test material in vehicle: Not applicable
- Justification of choice of vehicle: Not applicable
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Inhalable fraction (% <4 µm) 30.3
- MMAD (Mass median aerodynamic diameter: 9.40 µm; Geometric Standard Deviation (GSD):
5.30

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Not applicable
Analytical verification of test atmosphere concentrations:
no
Remarks:
Gravimetric only
Duration of exposure:
4 h
Concentrations:
Mean Achieved (mg/L) = 2.10
Mean Mass Median Aerodynamic Diameter (µm) = 9.40
Inhalable Fraction (% <4 µm) = 30.3
Geometric Standard Deviation (GSD) = 5.30
No. of animals per sex per dose:
3 males and 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 fourteen days. Individual bodyweights were recorded prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.
- Necropsy of survivors performed: yes
Statistics:
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

Preliminary study:
Not applicable
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2.1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 2.10 mg/L for four hours.

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 during exposure. These observations are considered to be associated wit
Body weight:
All animals exhibited a slight bodyweight loss or no bodyweight gain on the first day postexposure. Normal bodyweight development was noted for all animals during the remainder of the recovery period.
Gross pathology:
With the exception of one instance of dark patches on the lungs, no macroscopic abnormalities were detected amongst animals at necropsy.
Other findings:
Not applicable.

Any other information on results incl. tables

DISCUSSION

It is recognized that the achieved particle size and GSD range are larger than would generally be acceptable for this type of study (1 -4 µm and 1.5-3.0, respectively). During the method development phase of the study, changes were made to the generation system (addition of a particle size separator in an attempt to remove larger particles and thereby increasing the percentage of particles <4 µm) and variations in grinding techniques were implemented in an attempt to increase the inhalable portion of the test item. However, changing the generation system markedly reduced the achieved atmosphere concentration (maximum attainable concentration with the particle separator added was approximately 0.26 mg/Lwith a particle size distribution of ~ 5.5 µm and therefore, also reduced the actual concentration of particles <4 µm. It was, therefore, preferable to expose the animals to a higher concentration of test item (target concentration of 2 mg/L) as this resulted in the animals being exposed to the highest possible concentration of particles <4 µm.

 

During the method development phase of the study it proved impossible to achieve consistent particle size distributions. These could be described as being random at best. A target concentration of 2 mg/L was therefore set for the formal exposure. This target concentration was considered to be the most likely concentration that would allow the largest concentration of particles < 4 µm to be generated. It is considered that running at this concentration (2 mg/L) provided a much smaller particle size than generating at a target concentration of 5mg/L(the original target concentration for this study) where data from the method development phase showed that the achieved particle size was much greater than 13 µm. The large particle sizes achieved are considered to be due to the physical characteristics of the test item. It appears to clump together making it extremely difficult to reduce the achieved particle size. It is therefore considered that the particle size achieved during this study is the optimum attainable and provided approximately 0.64 mg/L of test item at <4 µm (30.3% of the mean achieved atmosphere concentration).

 

It is also worth mentioning that the atmospheres has been generated by specialist equipment which has been proven over many years to generate test atmospheres from the majority of test items at <4 µm. It can, therefore, be concluded that it would be highly unlikely in a real world situation that anyone would be exposed to atmospheres of this test item containing particle size' distributions quoted in this test report. The nominal concentration also shows that the test item would be very unlikely to form an atmosphere in the real world as it is shown to be 775% of the actual achieved concentration.

Exposure Chamber Concentration

The test atmosphere was sampled eighteen times 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)

2.10

0.16

16.3

The chamber flow rate was maintained at 35 L/min providing 70 air changes per hour.The theoretical chamber equilibration time (T99) was 4 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 (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

2.10

9.40

30.3

5.30

Mortality Data

The mortality data are summarised as follows:

Mean Achieved Atmosphere Concentration
(mg/L)

Deaths

Male

Female

Total

2.10

0/3

0/3

0/6

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No deaths occurred in a group of six rats (3 males /3 females) exposed to a mean achieved atmosphere concentration of 2.10 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of NBPT, in the HsdHan™ : WIST strain rat, was greater than 2.10 mg/L.
Executive summary:

Introduction.

A study was performed to assess the acute inhalation toxicity of the test item. The method used was compatible with that described in the OECD Guidelines for Testing of Chemicals (2009) No. 403 "Acute Inhalation Toxicity" referenced as Method 82 (Inhalation) of Commission Regulation (EC) No. 440/2008. The method was also designed to be compatible with the US Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, August 1998, with the exception that only six animals (three males and three females) were utilized during the "limit test".

Methods.

A group of six HsdHan™ : WIST strain rats (three males and three females) was exposed to a dust atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

Results. The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

2.10

0.16

16.3

The characteristics of the achieved atmosphere were as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

2.10

9.40

30.3

5.30

The mortality data were summarised as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

2.10

0/3

0/3

0/6

Clinical Observations.

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered quickly to appear normal on Day 3 post-exposure.

Bodyweight.

All animals exhibited a slight bodyweight loss or no bodyweight gain on the first day post-exposure. Normal bodyweight development was noted for all animals during the remainder of the recovery period.

Necropsy.

With the exception of one instance of dark patches on the lungs no macroscopic abnormalities were detected amongst animals at necropsy.

Conclusion.

No deaths occurred in a group of six rats (3 males / 3 females) exposed to a mean achieved atmosphere concentration of 2.10 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of NBPT, in the HsdHan™ : WIST strain rat, was greater than 2.10 mg/L.