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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 28-Jul-2010, Experimental CCCCompletion Date: 11-Aug-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological defiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Details on test material:
Identification: di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide
Batch Number: 0905137109
CAS Number: 6731-36-8
Description: colorless liquid
Purity: 98.16%
Expiry (Retest) Date: 01-Jun-2011

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd, Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 290.1 to 295.4 g, females: 191.1 to 192.7 g
- Fasting period before study: none
- Housing: Animals were housed in groups of 3 of the same sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel" J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet (e.g. ad libitum): Animals had ad libitum access to a pelleted standard Harlan Teklad 2914C rat maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst, Switzerland) batch no. 20/10 except during the period when the animals were restrained in exposure tubes.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum in water bottles, except during the period when they were restrained in exposure tubes.
- Acclimation period: for 13 days under laboratory conditions, after a clinical health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Animal Welfare: This study was performed in an AAALAC-accredited laboratory in accordance with the Swiss Animal Protection Law under license no. 397

IN-LIFE DATES: From: To: 28-Jul-2010 to 11-Aug-2010

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test atmosphere was generated using a Hospitak N°950 nebulizer connected to a syringe pump. The polyethylene injector inside the nebulizer was replaced by a stainless steel injector
- Exposure chamber volume: not applicable
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
- Source and rate of air: compressed air was supplied by means of an oil free compressor and passed respiratory quality filters before it was introduced into the exposure system
- Method of conditioning air: respiratory quality filters
- System of generating particulates/aerosols: The test atmosphere was generated using a Hospitak N°950 nebulizer connected to a syringe pump. The polyethylene injector inside the nebulizer was replaced by a stainless steel injector
- Method of particle size determination: Mercer impactor
- Treatment of exhaust air: filtered
- Temperature, humidity, pressure in air chamber: 22.8 °C, 3.5 % rel. humidity, 20.4 % oxygen

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric determinations of aerosol concentration were performed four times during exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
Chemical determinations of aerosol concentration were performed four times during exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The filters were transferred into appropriate labeled vials containing 5 mL of Acetonitrile, forwarded on dry ice to the scientist responsible for formulation analysis and stored at -20 ± 5 °C until analysis. The samples were analyzed using a GC method provided by the Sponsor and implemented at Harlan Laboratories Ltd.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): no vehicle used
- 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.): MMAD: 1.98/2.17/2.10 µm, GSD: 1.96/1.97/2.08

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4
Concentrations:
gravimetric: 5.3 mg/L air
chemical: 5.6 mg/L air
nominal: 6.1 mg/L air
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for viability were recorded once before exposure on the day of exposure (test day 1), three times during exposure, immediately and 1 h after exposure on test day 1 and twice daily during the observation period. Each animal was examined three times during exposure, immediately and 1 h after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal signs were detectable during exposure as the animals were restrained in the exposure tubes. The body weight of each animal was recorded on test days 1 (before exposure), 2, 4, 8 and 15 (before necropsy).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
no

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.6 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
none
Clinical signs:
other: (See Individual Tables in attachement) Salivation was observed during and immediately after exposure in all animals. Decreased activity, hunched posture, bradypnea and ruffled fur were recorded in all animals after the end of exposure on day 1. Ruffled f
Body weight:
(See Individual Tables in attachement).

From test day 1 to test day 2, slight body weight loss was noted in all animals. Thereafter normal body weight development was recorded.
Gross pathology:
(See Individual Tables in attachement).

There were no macroscopic findings.
Other findings:
none

Any other information on results incl. tables

    Test Atmosphere Conditions

Temperature, relative humidity and oxygen concentration during exposure were considered to be satisfactory for this type of study.

 

Data on temperature, relative humidity and oxygen concentration are presented in the following table:

 

Recording Time

[hours:min]

O2Concentration

[Vol %]

Temperature

[°C]

Relative Humidity

[% RH]

07:30

20.3

22.7

7.6

08:00

20.4

22.8

5.8

08:30

20.4

22.9

2.3

09:00

20.4

22.9

2.2

09:30

20.4

22.8

2.2

10:00

20.3

22.8

4.1

10:30

20.4

22.8

2.4

11:00

20.4

22.8

2.4

11:30

20.4

22.8

2.2

Mean

20.4

22.8

3.5

St. Dev.

0.0

0.0

2.0

N

9

9

9

 

 

   Determination of Nominal Aerosol Concentration

The nominal aerosol concentration was 6.1 mg/L air.

 

 

  Gravimetric Determination of Aerosol Concentrations

The mean gravimetric aerosol concentration determined was 5.3 mg/L air as targeted. The aerosol concentration was stable during the exposure period. Data on gravimetric aerosol concentrations are presented in the following table:

 

Sampling Time

[hours:min]

Sampling Volume

[L]

Amount of Test Item on the Filter

[mg]

Gravimetric Aerosol Concentration

[mg/L air]

8:00 – 08:04

4.0

20.847

5.5

9:00 – 09:03

3.0

14.620

5.1

10:00 – 10:03

3.0

14.978

5.2

11:00 – 11:03

3.0

15.404

5.4

Mean

 

 

5.3

St. Dev.

 

 

0.2

N

 

 

4

 

  Chemical Determination of Aerosol Concentrations

The mean chemical aerosol concentration determined was 5.6 mg/L air as targeted. The aerosol concentration was stable during the exposure period. The chemical aerosol concentration compared favourably with the gravimetrically determined concentration. This was in accordance with the high purity of the test item. Details on chemically determined aerosol concentrations are presented in the following table:

Sampling Time

[hours:min]

Sampling Volume

[L]

Amount of Test Item on the Filter

[mg]

Chemical Aerosol Concentration

[mg/L air]

8:00 – 08:04

4.0

22.72

5.9

9:00 – 09:03

3.0

15.04

5.2

10:00 – 10:03

3.0

15.48

5.4

11:00 – 11:03

3.0

16.78

5.9

Mean

 

 

5.6

St. Dev.

 

 

0.3

N

 

 

4

Particle Size Distribution:

The Mass Median Aerodynamic Diameters (MMAD) obtained from three gravimetric measurements of particle size distribution during the exposure were similar (MMAD between 1.98µmand 2.17µm). This led to the conclusion that the particle size of the generated aerosol was stable during the whole exposure period. The MMADs were well within the target range of 1 to 4mm, thus deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. In addition, the Geometric Standard Deviations (GSD) were within the target range of 1.5 to 3. Hence, the particle size distributions obtained were considered to be appropriate for acute inhalation toxicity testing.

 

Data on particle size distribution are presented in the following table:

 

Time

Total Amount Collected
[µg]

Percentages:
Stage No.
Effective Cut-Off Diameter [µm]

MMAD
[µm]

GSD

Corr. Coeff.
(R)

% <
4 µm

1
--

2
4.6

3
3.0

4
2.13

5
1.6

6
1.06

7
0.715

8
0.325

08:01

947

9.3

17.3

17.4

21.1

22.1

10.6

0.1

2.1

1.98

1.96

0.965

85.3%

09:45

954

11.2

18.0

22.3

23.5

16.1

6.8

0.0

2.0

2.17

1.97

0.957

81.6%

11:10

820

11.7

17.4

20.6

24.4

14.8

7.6

0.0

3.5

2.10

2.08

0.952

81.0%

 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, the LC50 of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide obtained in this study was estimated to be greater than 5.6 mg/L air (chemically determined mean aerosol concentration). There was no indication of relevant sex-related differences in toxicity of the test item.


Executive summary:

A group of three male and three female albino rats [RccHanTM:WIST(SPF)] was exposed by nose-only, flow-past inhalation for four hours to the test item at a chemically determined mean concentration of 5.6 mg/L air. All animals were observed for clinical signs and mortality during the inhalation exposure and the subsequent 14-day observation period. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On test day 15 all animals were sacrificed and necropsied. The ranges of aerosol concentration, temperature, relative humidity, oxygen content and airflow rate measured during the exposure were considered to be satisfactory for a study of this type. In addition, the test item was considered to be respirable to rats. All animals survived the scheduled observation period. Salivation was observed during exposure in all animals. Decreased activity, hunched posture, bradypnea, ruffled fur and salivation were recorded in all animals after exposure. There were no clinical signs from day 3 onwards. There were no effects on body weight that were considered to be related to treatment with the test item. There were no macroscopic findings in any animal.

In conclusion, the LC50 of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide obtained in this study was estimated to be greater than 5.6 mg/L air (chemically determined mean aerosol concentration). There was no indication of relevant sex-related differences in toxicity of the test item.