Docosane

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental Starting Date: 06 January 2014. Experimental Completion Date: 06 februry 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study on supporting substance (predominantly ≤C26) 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. Read-across is considered to be reliability 2.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

Experimental Starting Date: 06 January 2014. Experimental Completion Date: 06 februry 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study on supporting substance (predominantly ≤C26) 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. Read-across is considered to be reliability 2.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female RccHan™ : WIST strain rats were supplied by Harlan Laboratories UK Ltd, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five 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 eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

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). With the exception of the exposure period, free access to mains drinking water and food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding and chew blocks are routinely analyzed and are considered not to contain any contaminants that could 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 twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Unchanged (no vehicle)

Details on inhalation exposure:

Atmosphere Generation
The test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber. The nebulizer was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and 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 nebulizer.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. 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. A diagram of the dynamic (continuous flow) system employed is shown in Figure 1.

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 to achieve the required atmospheric conditions.


Exposure Procedure
Prior to the day of each 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 two groups, each of six rats (three males and three females), were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. The second concentration was selected after consideration of the results of the first exposure.


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 thirty minutes throughout the four-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 four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Analytical verification of test atmosphere concentrations:

no

Remarks:

(Gravimetric Only)

Duration of exposure:

4 h

Concentrations:

Group 1:
Mean Achieved (mg/L): 5.09
Mean Mass Median Aerodynamic Diameter (um): 1.92
Inhalable Fraction (%<4um): 77.3
Geometric Standard Deviation: 2.69

Group 2:
Mean Achieved (mg/L): 1.05
Mean Mass Median Aerodynamic Diameter (um): 1.64
Inhalable Fraction (%<4um): 89.4
Geometric Standard Deviation:2.05

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

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, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.

Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.


Necropsy
At the end of the fourteen day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died during the course of the study 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. As deaths were noted, the following tissues were preserved; Lungs (inflated to approximately normal inspiratory volume, with buffered formalin) and the upper respiratory tract in buffered formalin.

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 behavioral and clinical observations, necropsy findings, body weight 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.

Preliminary study:

Not applicable

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 - 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

1 male (Day 2) and 3 females (Day 1) animals were found dead post-exposure
1 male (Day 2) and 1 female (Day 1) animal were found dead post-exposure

Clinical signs:

other: Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. There were occasional instances of tip-toe gait, isolated occurrences of labored respiration and ataxia were also noted. Survivi

Body weight:

Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. None of the female animals survived past Day 1 post-exposure, however, reasonable body weight development was noted in both of the surviving male animals during the remainder of the recovery period.

Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. Reasonable body weight development was noted in all surviving male animals during the remainder of the recovery period. In contrast, two surviving female animals exhibited a slight body weight loss or showed no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight gains were noted in these two animals during the remainder of the recovery period.

Gross pathology:

Pale patches on the lungs were noted in one of two surviving animals from Group 1 at necropsy. Dark patches on the lungs or pale lungs were noted in all four surviving animals from Group 2 at necropsy.

Abnormally dark lungs were detected in the animals that died during the course of the study at necropsy.

From consideration of results of Histopathological examinations of tissues retained from a study conducted on a similar test item (Harlan Study Number: 41300142) the deaths noted during this study may be attributable to hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis).

Other findings:

Not Applicable

Exposure Chamber Atmosphere Concentrations – Dose Group 1

Duration of Exposure (minutes)	Net Weight of Sample (mg)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
5	10.16	2	60	5.08
15	10.51	2	60	5.26
30	10.15	2	60	5.08
45	10.03	2	60	5.02
60	10.22	2	60	5.11
75	10.14	2	60	5.07
90	10.11	2	60	5.06
105	10.05	2	60	5.03
120	10.18	2	60	5.09
135	9.94	2	60	4.97
150	10.38	2	60	5.19
165	10.41	2	60	5.21
180	10.00	2	60	5.00
195	10.17	2	60	5.09
210	10.12	2	60	5.06
225	10.24	2	60	5.12
238	10.14	2	60	5.07

 

Exposure Chamber Atmosphere Concentrations – Dose Group 2

Duration of Exposure (minutes)	Net Weight of Sample (mg)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
5	4.85	4	60	1.21
15	4.87	4	60	1.22
30	4.21	4	60	1.05
45	4.08	4	60	1.02
60	4.01	4	60	1.00
75	4.13	4	60	1.03
90	4.03	4	60	1.01
105	4.08	4	60	1.02
120	4.04	4	60	1.01
135	4.03	4	60	1.01
150	4.17	4	60	1.04
165	4.09	4	60	1.02
180	4.15	4	60	1.04
195	4.11	4	60	1.03
210	4.15	4	60	1.04
225	4.10	4	60	1.03
238	4.29	4	60	1.07

 

Particle Size Distribution – Dose Group 1

Cascade Impactor Data

 

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.9	0.12	0.14	0.06	0.11
4	6.2	0.17	0.11	0.09	0.12
5	3.6	0.48	0.36	0.27	0.37
6	1.6	1.09	0.71	0.66	0.82
7	0.93	0.15	0.13	0.11	0.13
8	0.37	0.57	0.38	0.40	0.45
Back-up Filter	<0.37	0.16	0.05	0.14	0.12
Total Mean Amount of Test Item Collected					2.12

 

Calculation

 

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.9	0.949	2.01	94.8	6.63
6.2	0.792	1.89	89.2	6.24
3.6	0.556	1.52	71.7	5.57
1.6	0.204	0.70	33.0	4.56
0.93	-0.032	0.57	26.9	4.38
0.37	-0.432	0.12	5.66	3.42

 

Results

 

Mean Mass Median Aerodynamic Diameter (MMAD) =1.92µm

Geometric Standard Deviation (GSD) =2.69

Predicted amount less than 4 µm =77.3%

Particle Size Distribution – Dose Group 2

Cascade Impactor Data

 

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.9	0.00	0.00	0.02	0.01
4	6.2	0.00	0.02	0.02	0.01
5	3.6	0.05	0.09	0.11	0.08
6	1.6	0.26	0.59	0.58	0.48
7	0.93	0.00	0.09	0.00	0.03
8	0.37	0.11	0.28	0.26	0.22
Back-up Filter	<0.37	0.00	0.04	0.03	0.02
Total Mean Amount of Test Item Collected					0.85

 

Calculation

 

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.9	0.949	0.84	98.8	7.27
6.2	0.792	0.83	97.6	6.99
3.6	0.556	0.75	88.2	6.19
1.6	0.204	0.27	31.8	4.53
0.93	-0.032	0.24	28.2	4.42
0.37	-0.432	0.02	2.35	3.01

 

Results

 

Mean Mass Median Aerodynamic Diameter (MMAD) = 1.64 µm

Geometric Standard Deviation (GSD) = 2.05

Predicted amount less than 4 µm = 89.4 %

 

KEY TO CLINICAL OBSERVATIONS

A	=	ataxia
H	=	hunched posture
P	=	pilo-erection
Ri	=	increased respiratory rate
Rl	=	labored respiration
Wf	=	wet fur
Wt	=	tip-toe gait
0	=	no abnormalities detected
X	=	animal dead

 

Individual Clinical Observations (Day of Exposure) – Dose Group 1

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Hours During Exposure			On Removal From Chamber	One Hour Post-Exposure
		1	2	3
5.09	1 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri Wt	H P Ri Wt
	2 Male	Wf	Wf Ri	Wf Ri	Wf H P Ri	H P Ri
	3 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	4 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri Wt	Wf H P Ri Wt
	5 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri Wt	Wf H P Ri Wt
	6 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri	H P Ri

 

Individual Clinical Observations (Recovery Period) – Dose Group 1

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		1	2	3	4	5	6	7	8 - 14
5.09	1 Male	H P Ri	X
	2 Male	H P Ri	H P Ri	H Ri	H Ri	H Ri	Ri	Ri	0
	3 Male	H P Ri	H P Ri	H Ri	H Ri	Ri	Ri	Ri	0
	4 Female	X
	5 Female	X
	6 Female	H P Ri Rl A Xlater in the day

 

Individual Clinical Observations (Day of Exposure) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Hours During Exposure			On Removal From Chamber	One Hour Post-Exposure
		1	2	3
1.05	7 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	8 Male	Wf	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	9 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	10 Female	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	11 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	12 Female	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri

 

Individual Clinical Observations (Recovery Period) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		1	2	3	4	5	6	7
1.05	7 Male	H P Ri	H P Ri	P Ri	P Ri	Ri	Ri	0
	8 Male	H P Ri	X
	9 Male	H P Ri	H P Ri	P Ri	P Ri	Ri	Ri	0
	10 Female	H P Ri	H P Ri	H P Ri	H P Ri	Ri	Ri	Ri
	11 Female	H P Ri	H P Ri	H P Ri Rl	H P Ri	Ri	Ri	Ri
	12 Female	H P Ri A XLater in the day

 

(Continued)   Individual Clinical Observations (Recovery Period) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		8	9	10	11	12	13	14
1.05	7 Male	0	0	0	0	0	0	0
	8 Male
	9 Male	0	0	0	0	0	0	0
	10 Female	Ri	Ri	Ri	Ri	Ri	0	0
	11 Female	Ri	Ri	Ri	Ri	Ri	0	0
	12 Female

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Criteria used for interpretation of results: other: Globally Harmonized Classification System

Conclusions:

Four out of six animals died at a mean achieved atmosphere concentration of 5.09 mg/L, whereas, two deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 1.05 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of GTL Base oil 3, in the RccHanTM : WIST strain rat, was in the range >1 mg/L to 5 mg/L (Globally Harmonized Classification System – Category 4).

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. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method”.

 

Methods.......

Two groups of six RccHan™ : WIST strain rats (three males and three females) were exposed to an aerosol 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:

 

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	5.09	0.07	12.7
2	1.05	0.06	2.14

 

The characteristics of the achieved atmosphere were as follows:

 

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	5.09	1.92	77.3	2.69
2	1.05	1.64	89.4	2.05

 

The mortality data were summarized as follows:

 

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Deaths
		Male	Female	Total
1	5.09	1/3	3/3	4/6
2	1.05	1/3	1/3	2/6

 

 

Clinical Observations

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. There were occasional instances of tip-toe gait, isolated occurrences of labored respiration and ataxia were also noted. Surviving Group 1 animals recovered to appear normal on Day 8 post-exposure. Surviving Group 2 animals recovered to appear normal from Days 7 to 13 post-exposure. 

Body Weight

Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. None of the female animals survived past Day 1 post-exposure, however, reasonable body weight development was noted in both of the surviving male animals during the remainder of the recovery period. 

 

Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. Reasonable body weight development was noted in all surviving male animals during the remainder of the recovery period. In contrast, two surviving female animals exhibited a slight body weight loss or showed no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight gains were noted in these two animals during the remainder of the recovery period.

 

 

Necropsy

Pale patches on the lungs were noted in one of two surviving animals from Group 1 at necropsy. Dark patches on the lungs or pale lungs were noted in all four surviving animals from Group 2 at necropsy.

 

Abnormally dark lungs were detected in the animals that died during the course of the study at necropsy.

 

From consideration of results of Histopathological examinations of tissues retained from a study conducted on a similar test item (Harlan Study Number: 41300142) the deaths noted during this study may be attributable to hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis).

 

 

Conclusion

Four out of six animals died at a mean achieved atmosphere concentration of 5.09 mg/L, whereas, two deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 1.05 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC₅₀) ofGTL Base oil3, in the RccHan^TM: WIST strain rat, was in the range >1 mg/L to 5 mg/L (Globally Harmonized Classification System – Category 4).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

GTL base oil 3

IUPAC Name:

GTL base oil 3

Test material form:

other: Liquid

Details on test material:

- Description: Clear colorless liquid
- Batch: Not supplied
- Purity: 100.00 %
- Expiry date: 23 December 2015
- Storage conditions: Room temperature, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female RccHan™ : WIST strain rats were supplied by Harlan Laboratories UK Ltd, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five 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 eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

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). With the exception of the exposure period, free access to mains drinking water and food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding and chew blocks are routinely analyzed and are considered not to contain any contaminants that could 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 twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Unchanged (no vehicle)

Details on inhalation exposure:

Atmosphere Generation
The test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber. The nebulizer was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and 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 nebulizer.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. 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. A diagram of the dynamic (continuous flow) system employed is shown in Figure 1.

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 to achieve the required atmospheric conditions.


Exposure Procedure
Prior to the day of each 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 two groups, each of six rats (three males and three females), were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. The second concentration was selected after consideration of the results of the first exposure.


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 thirty minutes throughout the four-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 four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Analytical verification of test atmosphere concentrations:

no

Remarks:

(Gravimetric Only)

Duration of exposure:

4 h

Concentrations:

Group 1:
Mean Achieved (mg/L): 5.09
Mean Mass Median Aerodynamic Diameter (um): 1.92
Inhalable Fraction (%<4um): 77.3
Geometric Standard Deviation: 2.69

Group 2:
Mean Achieved (mg/L): 1.05
Mean Mass Median Aerodynamic Diameter (um): 1.64
Inhalable Fraction (%<4um): 89.4
Geometric Standard Deviation:2.05

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

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, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.

Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.


Necropsy
At the end of the fourteen day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died during the course of the study 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. As deaths were noted, the following tissues were preserved; Lungs (inflated to approximately normal inspiratory volume, with buffered formalin) and the upper respiratory tract in buffered formalin.

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 behavioral and clinical observations, necropsy findings, body weight 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

Preliminary study:

Not applicable

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 - 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

1 male (Day 2) and 3 females (Day 1) animals were found dead post-exposure
1 male (Day 2) and 1 female (Day 1) animal were found dead post-exposure

Clinical signs:

other: Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. There were occasional instances of tip-toe gait, isolated occurrences of labored respiration and ataxia were also noted. Survivi

Body weight:

Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. None of the female animals survived past Day 1 post-exposure, however, reasonable body weight development was noted in both of the surviving male animals during the remainder of the recovery period.

Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. Reasonable body weight development was noted in all surviving male animals during the remainder of the recovery period. In contrast, two surviving female animals exhibited a slight body weight loss or showed no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight gains were noted in these two animals during the remainder of the recovery period.

Gross pathology:

Pale patches on the lungs were noted in one of two surviving animals from Group 1 at necropsy. Dark patches on the lungs or pale lungs were noted in all four surviving animals from Group 2 at necropsy.

Abnormally dark lungs were detected in the animals that died during the course of the study at necropsy.

From consideration of results of Histopathological examinations of tissues retained from a study conducted on a similar test item (Harlan Study Number: 41300142) the deaths noted during this study may be attributable to hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis).

Other findings:

Not Applicable

Any other information on results incl. tables

Exposure Chamber Atmosphere Concentrations – Dose Group 1

Duration of Exposure (minutes)	Net Weight of Sample (mg)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
5	10.16	2	60	5.08
15	10.51	2	60	5.26
30	10.15	2	60	5.08
45	10.03	2	60	5.02
60	10.22	2	60	5.11
75	10.14	2	60	5.07
90	10.11	2	60	5.06
105	10.05	2	60	5.03
120	10.18	2	60	5.09
135	9.94	2	60	4.97
150	10.38	2	60	5.19
165	10.41	2	60	5.21
180	10.00	2	60	5.00
195	10.17	2	60	5.09
210	10.12	2	60	5.06
225	10.24	2	60	5.12
238	10.14	2	60	5.07

 

Exposure Chamber Atmosphere Concentrations – Dose Group 2

Duration of Exposure (minutes)	Net Weight of Sample (mg)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
5	4.85	4	60	1.21
15	4.87	4	60	1.22
30	4.21	4	60	1.05
45	4.08	4	60	1.02
60	4.01	4	60	1.00
75	4.13	4	60	1.03
90	4.03	4	60	1.01
105	4.08	4	60	1.02
120	4.04	4	60	1.01
135	4.03	4	60	1.01
150	4.17	4	60	1.04
165	4.09	4	60	1.02
180	4.15	4	60	1.04
195	4.11	4	60	1.03
210	4.15	4	60	1.04
225	4.10	4	60	1.03
238	4.29	4	60	1.07

 

Particle Size Distribution – Dose Group 1

Cascade Impactor Data

 

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.9	0.12	0.14	0.06	0.11
4	6.2	0.17	0.11	0.09	0.12
5	3.6	0.48	0.36	0.27	0.37
6	1.6	1.09	0.71	0.66	0.82
7	0.93	0.15	0.13	0.11	0.13
8	0.37	0.57	0.38	0.40	0.45
Back-up Filter	<0.37	0.16	0.05	0.14	0.12
Total Mean Amount of Test Item Collected					2.12

 

Calculation

 

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.9	0.949	2.01	94.8	6.63
6.2	0.792	1.89	89.2	6.24
3.6	0.556	1.52	71.7	5.57
1.6	0.204	0.70	33.0	4.56
0.93	-0.032	0.57	26.9	4.38
0.37	-0.432	0.12	5.66	3.42

 

Results

 

Mean Mass Median Aerodynamic Diameter (MMAD) =1.92µm

Geometric Standard Deviation (GSD) =2.69

Predicted amount less than 4 µm =77.3%

Particle Size Distribution – Dose Group 2

Cascade Impactor Data

 

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.9	0.00	0.00	0.02	0.01
4	6.2	0.00	0.02	0.02	0.01
5	3.6	0.05	0.09	0.11	0.08
6	1.6	0.26	0.59	0.58	0.48
7	0.93	0.00	0.09	0.00	0.03
8	0.37	0.11	0.28	0.26	0.22
Back-up Filter	<0.37	0.00	0.04	0.03	0.02
Total Mean Amount of Test Item Collected					0.85

 

Calculation

 

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.9	0.949	0.84	98.8	7.27
6.2	0.792	0.83	97.6	6.99
3.6	0.556	0.75	88.2	6.19
1.6	0.204	0.27	31.8	4.53
0.93	-0.032	0.24	28.2	4.42
0.37	-0.432	0.02	2.35	3.01

 

Results

 

Mean Mass Median Aerodynamic Diameter (MMAD) = 1.64 µm

Geometric Standard Deviation (GSD) = 2.05

Predicted amount less than 4 µm = 89.4 %

 

KEY TO CLINICAL OBSERVATIONS

A	=	ataxia
H	=	hunched posture
P	=	pilo-erection
Ri	=	increased respiratory rate
Rl	=	labored respiration
Wf	=	wet fur
Wt	=	tip-toe gait
0	=	no abnormalities detected
X	=	animal dead

 

Individual Clinical Observations (Day of Exposure) – Dose Group 1

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Hours During Exposure			On Removal From Chamber	One Hour Post-Exposure
		1	2	3
5.09	1 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri Wt	H P Ri Wt
	2 Male	Wf	Wf Ri	Wf Ri	Wf H P Ri	H P Ri
	3 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	4 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri Wt	Wf H P Ri Wt
	5 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri Wt	Wf H P Ri Wt
	6 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri	H P Ri

 

Individual Clinical Observations (Recovery Period) – Dose Group 1

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		1	2	3	4	5	6	7	8 - 14
5.09	1 Male	H P Ri	X
	2 Male	H P Ri	H P Ri	H Ri	H Ri	H Ri	Ri	Ri	0
	3 Male	H P Ri	H P Ri	H Ri	H Ri	Ri	Ri	Ri	0
	4 Female	X
	5 Female	X
	6 Female	H P Ri Rl A Xlater in the day

 

Individual Clinical Observations (Day of Exposure) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Hours During Exposure			On Removal From Chamber	One Hour Post-Exposure
		1	2	3
1.05	7 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	8 Male	Wf	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	9 Male	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	10 Female	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	11 Female	Wf	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri
	12 Female	Wf Ri	Wf Ri	Wf Ri	Wf H P Ri	Wf H P Ri

 

Individual Clinical Observations (Recovery Period) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		1	2	3	4	5	6	7
1.05	7 Male	H P Ri	H P Ri	P Ri	P Ri	Ri	Ri	0
	8 Male	H P Ri	X
	9 Male	H P Ri	H P Ri	P Ri	P Ri	Ri	Ri	0
	10 Female	H P Ri	H P Ri	H P Ri	H P Ri	Ri	Ri	Ri
	11 Female	H P Ri	H P Ri	H P Ri Rl	H P Ri	Ri	Ri	Ri
	12 Female	H P Ri A XLater in the day

 

(Continued)   Individual Clinical Observations (Recovery Period) – Dose Group 2

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Days Post Exposure
		8	9	10	11	12	13	14
1.05	7 Male	0	0	0	0	0	0	0
	8 Male
	9 Male	0	0	0	0	0	0	0
	10 Female	Ri	Ri	Ri	Ri	Ri	0	0
	11 Female	Ri	Ri	Ri	Ri	Ri	0	0
	12 Female

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Criteria used for interpretation of results: other: Globally Harmonized Classification System

Conclusions:

Four out of six animals died at a mean achieved atmosphere concentration of 5.09 mg/L, whereas, two deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 1.05 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of GTL Base oil 3, in the RccHanTM : WIST strain rat, was in the range >1 mg/L to 5 mg/L (Globally Harmonized Classification System – Category 4).

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. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method”.

 

Methods.......

Two groups of six RccHan™ : WIST strain rats (three males and three females) were exposed to an aerosol 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:

 

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	5.09	0.07	12.7
2	1.05	0.06	2.14

 

The characteristics of the achieved atmosphere were as follows:

 

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	5.09	1.92	77.3	2.69
2	1.05	1.64	89.4	2.05

 

The mortality data were summarized as follows:

 

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Deaths
		Male	Female	Total
1	5.09	1/3	3/3	4/6
2	1.05	1/3	1/3	2/6

 

 

Clinical Observations

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. There were occasional instances of tip-toe gait, isolated occurrences of labored respiration and ataxia were also noted. Surviving Group 1 animals recovered to appear normal on Day 8 post-exposure. Surviving Group 2 animals recovered to appear normal from Days 7 to 13 post-exposure. 

Body Weight

Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. None of the female animals survived past Day 1 post-exposure, however, reasonable body weight development was noted in both of the surviving male animals during the remainder of the recovery period. 

 

Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. Reasonable body weight development was noted in all surviving male animals during the remainder of the recovery period. In contrast, two surviving female animals exhibited a slight body weight loss or showed no body weight gain from Days 1 to 3 post-exposure. Reasonable body weight gains were noted in these two animals during the remainder of the recovery period.

 

 

Necropsy

Pale patches on the lungs were noted in one of two surviving animals from Group 1 at necropsy. Dark patches on the lungs or pale lungs were noted in all four surviving animals from Group 2 at necropsy.

 

Abnormally dark lungs were detected in the animals that died during the course of the study at necropsy.

 

From consideration of results of Histopathological examinations of tissues retained from a study conducted on a similar test item (Harlan Study Number: 41300142) the deaths noted during this study may be attributable to hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis).

 

 

Conclusion

Four out of six animals died at a mean achieved atmosphere concentration of 5.09 mg/L, whereas, two deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 1.05 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC₅₀) ofGTL Base oil3, in the RccHan^TM: WIST strain rat, was in the range >1 mg/L to 5 mg/L (Globally Harmonized Classification System – Category 4).