Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January to 31 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
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:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(piperidinothiocarbonyl) hexasulphide
EC Number:
213-537-2
EC Name:
Bis(piperidinothiocarbonyl) hexasulphide
Cas Number:
971-15-3
Molecular formula:
C12H20N2S8
IUPAC Name:
1,1'-(hexasulfane-1,6-diyldicarbonothioyl)dipiperidine
Constituent 2
Reference substance name:
Dipentamethylenethiuram hexasulfide
IUPAC Name:
Dipentamethylenethiuram hexasulfide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: CRL:(WI) rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: CRL:(WI) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: Recognized by international guidelines as a recommended test system
Number of animals: 10
Sex: Male and Female rats, nulliparous and non-pregnant
Age when treated: Young adult rats, 9 weeks old
Body weight (at dosing): 194 to 351 g (M: 311-351g; F: 194-214g)
Randomization: Selected based on the bodyweight prior to the exposure
Acclimatization time: 14 days.

Husbandry
Animal health: Only healthy animals were used for the test. The health status was certified by the veterinarian
Animal room: 245/7
Housing: Group of 5 (by sex)
Cage type: Type III solid floor cages with stainless steel mesh lids
Bedding: Lignocel Bedding for Laboratory Animals was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3°C
Relative humidity: 30 – 70 %
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.

Diet and Water : The animals were provided with ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany; Batch: 445 8440, Expiry: May 2013) and tap water fit for human consumption, ad libitum.
The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are retained in the archive of CiToxLAB Hungary Ltd.
Water quality control analysis is performed once every 3 months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archive of CiToxLAB Hungary Ltd.

Identification : Each animal was identified by a unique number marked on the tail. The animal number was assigned on the basis of the CiToxLAB Hungary Ltd. master file.
Cages were identified by cage card, giving details of study code, sex, dose-group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Technical Trials: Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings and test material input rates were varied to achieve the required atmospheric characteristics.

Atmosphere Generation: The test item was aerosolised using two Wright’s Dust Feed Systems (TSE Systems GmbH, Bad Homburg, 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.

Animal Exposure System: The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two, concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
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. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port. 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.
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.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones (Pauluhn, 1994).

Exposure Procedure: Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99) (Silver, 1946), a group of ten rats (five male and five female) was exposed to an atmosphere of the test material for a period of four hours. The maximum attainable concentration was used for the exposure as a target. As no deaths occurred at the maximum attainable concentration, no further data were required.
Analytical verification of test atmosphere concentrations:
no
Remarks:
nominal
Duration of exposure:
4 h
Concentrations:
2.83 mg/l
No. of animals per sex per dose:
10 animals (5 males/5 females).
Control animals:
no
Details on study design:
Test Atmosphere Concentrations: The test atmosphere was sampled at regular intervals during each exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume of test atmosphere through weighed GF10 glass fibre filters (Whatman GmbH, Hahnestraße 3 – D-37586 Dassel, Germany). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that went through the chamber during the same period.

Particle Size Analysis: The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone).
The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, 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, 0.96, 1.55, 2.11, 3.56, 6.66 and 10.55 µm was calculated.
From these data, using software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4µm (considered to be the inhalable portion) was determined.

Chamber Environmental Conditions: The following variables were monitored continuously and recorded every minute during each exposure period by the TSE-DACO monitoring system integrated into the exposure system:
Chamber airflow rates
Test Atmosphere temperature
Test atmosphere relative humidity
Test atmosphere carbon dioxide concentration
Test atmosphere oxygen concentration

OBSERVATIONS
Morbidity/Mortality: Animals were checked hourly during exposure, one hour after exposure and twice daily (early and late in the working day) during the 14-day observation period for morbidity and/or mortality.
Clinical Signs: All animals were observed for clinical signs at hourly intervals during exposure, as soon as practically possible following removal from restraint at the end of exposure, one hour after exposure and subsequently once daily for fourteen days.
Bodyweight: Individual bodyweights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
Necropsy: At the end of the fourteen day observation period, the animals were euthanised by exsanguination under anaesthesia (RELEASE 300mg/ml injekció A.U.V.; Lot No.: 063012; Expiry: 01-2015; Produced by Wirtschaftsgenossenschaft deutscher Tierartze eG, Siemensstr. 14, 30827 Garbsen, Germany) and gross macroscopic examination was performed. All animals were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Statistics:
No

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC0
Effect level:
> 2.83 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No animals died during the study.
Clinical signs:
other: Wet fur and fur staining were commonly recorded on the day of exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be biologically significant. Laboured respiration w
Body weight:
Normal bodyweight gain was noted during the observation period for all animals.
Gross pathology:
A single four hours nose-only exposure of Dipentamethylenethiuram hexasulfide to Wistar CRL: (WI) rats dosed at the maximum attainable concentration of 2.83 mg/L followed by a 14 day of observation period, was not associated with any macroscopic findings.
Other findings:
no

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, no death occurred in a group of ten rats exposed to the maximum attainable concentration of 2.83 mg/L for four hours. The acute inhalation median lethal concentration (4hr LC50) of Dipentamethylenethiuram hexasulfide, in CRL: (WI) Wistar strain rats, was therefore considered to be greater than 2.83 mg/L.
Executive summary:

Introduction: This study was performed to assess the acute inhalation toxicity of Dipentamethylenethiuram hexasulfide. The method used followed that described in the US Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.1300, Acute Inhalation Toxicity, August 1998. The method was also designed to meet OECD guideline 403 (07 September 2009), Council Regulation (EC) No 440/2008, Annex Part B, B.2: "Acute Toxicity (Inhalation)", Official Journal of the European Union No. L 142, dated May 31st, 2008, in line with the Sponsor requirements.

 

Study Design: A group of ten CRL: (WI) Wistar strain rats (five male and five female) was exposed to the maximum attainable concentration of Dipentamethylenethiuram hexasulfide. The animals were exposed for four hours using a nose-only exposure system, followed by a fourteen day observation period. The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically 17 times during the animal exposure. The particle size distribution of the test aerosol was determined regularly (3 times) during the exposure period.

Clinical observations and bodyweights were recorded throughout the study. At the end of the scheduled period the animals were sacrificed and subjected to a gross examination post mortem.

No control group was used in this study.

 

Results

The mean achieved atmosphere concentration was as follows:

Mean Achieved Concentration (Maximum attainable)

(mg/l)

Standard Deviation

Nominal Concentration

(mg/l)

2.83

0.40

5.32

 

The characteristics of the test atmosphere were as follows:

Mean Achieved

(mg/l)

Mean Mass Median Aerodynamic Diameter

(MMAD) (µm)

Geometric Standard Deviation

Inhalable Fraction

(% <4µm)

2.83

3.96

2.34

50.4

 

The mortality data were summarised as follows:

Mean Achieved

 (mg/l)

Male Deaths

Female Deaths

Total Deaths

2.83

0/5

0/5

0/10

 

Clinical Observations: Wet fur and fur staining were commonly recorded on the day of exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be biologically significant.

Laboured respiration was only noted for the exposed animals on day of exposure. No abnormalities were detected in any animal from Day 2 of the observation period.

Bodyweights: Normal bodyweight gain was noted during the observation period for all animals.

Necropsy: A single four hours nose-only exposure of Dipentamethylenethiuram hexasulfide to Wistar CRL: (WI) rats dosed at the maximum attainable concentration of 2.83 mg/L followed by a 14 day of observation period, was not associated with any macroscopic findings.

 

Conclusion: Under the experimental conditions of this study, no death occurred in a group of ten rats exposed to the maximum attainable concentration of 2.83 mg/L for four hours. The acute inhalation median lethal concentration (4hr LC50) of Dipentamethylenethiuram hexasulfide, in CRL: (WI) Wistar strain rats, was therefore considered to be greater than 2.83 mg/L.