Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 09 May 2016 Experimental completion date: 26 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: Macrolex Rotviolett R
Physical state/appearance: Violet solid
CAS Number: 6408-72-6
Expiry: 28 July 2017
Storage/Usage Conditions: Stored in darkness at room temperature; may be used/formulated in light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.
Animal Care and Husbandry
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. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.48 µm
Geometric standard deviation (GSD):
3.12
Remark on MMAD/GSD:
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 (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum 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 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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.
Details on inhalation exposure:
Atmosphere Generation
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.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). 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.

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 characterization period test item input rates were varied in an attempt to achieve the required atmospheric conditions.

Exposure Procedure
One day prior to the day of exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, 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.
Following an appropriate equilibration period a single group of six rats (three males and three females) was exposed to an atmosphere of the test item for a period of 4 hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 101 % 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 30 minutes throughout the 4 Hour exposure period.

Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour 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 each exposure period. The gravimetric method used glass fiber 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 disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.

The nominal concentration was 242 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric method
Duration of exposure:
4 h
Concentrations:
5.00 mg/L
No. of animals per sex per dose:
3 males at 5.00 mg/L
3 females at 5.00 mg/L
Control animals:
no
Details on study design:
Serial Observations
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, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.
Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
Terminal Investigations
Necropsy
At the end of the 14 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.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.04 mg/L air
Based on:
test mat.
Mortality:
There was no mortality at Mean Achieved Atmosphere Concentration 5.04 (mg/L)
Clinical signs:
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. Generalized fur staining caused by the test item is commonly recorded during exposure, on removal from the chamber and for several days post-exposure. These observations are considered to be associated with the restraint procedure and, in isolation, are not indicative of toxicity.

In addition to the observations considered to be due to the restraint procedure, the following abnormalities were detected:

During exposure, there were occasional instances of decreased respiratory rate. On removal from the chamber and one hour post-exposure, all animals exhibited decreased respiratory rate and there was an isolated instance of noisy respiration.

One day after exposure, significant observations noted in all animals included; decreased respiratory rate and hunched posture. All animals recovered such that no significant observations were apparent on Day 2 post-exposure.
Body weight:
All animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, two female animals showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.

Any other information on results incl. tables

Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were as follows:

The mean values obtained were as follows:

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.04

0.12

12.2

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

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

 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

5.04

3.48

54.9

3.12


*= Test atmospheres were generated for a total of 17 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

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 5.04 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of Macrolex Rotviolett R in the Wistar strain rat was greater than 5.04 mg/L (Globally Harmonized Classification System – Un-classified).
Executive summary:

Summary

Introduction

A study was performed to assess the acute inhalation toxicity of Macrolex Rotviolett R. The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals (2009) No. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method” and Method B.52. Acute Inhalation Toxicity – Acute Toxic Class Method, 2014, of Commission Regulation (EC) No. 440/2008.

Methods

A group of six RccHanTM: WIST strain rats (three males and three females) was exposed to a dust atmosphere of the test item. The animals were exposed for 4 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)

5.04

0.12

12.2

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

5.04

3.48

54.9

3.12

The mortality data were summarized as follows:

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

5.04

0/3

0/3

0/6

Clinical Observations. Common abnormalities noted during the study included decreased respiratory rate, hunched posture, pilo-erection, generalized purple fur staining caused by the test item and wet fur. An isolated occurrence of noisy respiration was also noted. All animals recovered such that no significant observations were apparent on Day 2 post-exposure.

Body Weight. All animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period.  In contrast, two female animals showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.

 

Necropsy. No macroscopic abnormalities were detected amongst animals at necropsy.

 

Conclusion

No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.04 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of Macrolex Rotviolett R in the Wistar strain rat was greater than5.04 mg/L (Globally Harmonized Classification System – Un-classified).