Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2016 to 26 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD and EU test guidelines and conducted in an accredited GLP laboratory

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for more details.
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for more details.
Principles of method if other than guideline:
Deviations:Deviation No 1 After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching. Deviation No 2 The Archiving statement has been amended to reflect the fact that no Specimens were retained during this study. Therefore, the following sentence has been removed: Specimens that no longer afford evaluation will be discarded in accordance with Standard Operating Procedures and without further notice. These deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Test type:
traditional method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Water white liquid
Details on test material:
Name: NovaSpec base oilBatch/Lot No.: TS13732CAS number: 1472010-43-7Chemical name: Alkenes, C-10-16a-, mixed with (6E)-7,11-dimethyl-3-methylene-116,10-dodecatriene, dimers, tetramers and trimers, hydrogenatedPurity: 100%Manufacture date: 06 June 2014Expiry date: 06 June 2016Description: Water white liquidStorage condition: Controlled room temperature (15-25 oC below 70 RH%)Safety Precautions: Routine safety precautions for unknown materials (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.No correction for purity of the test item was applied.
Specific details on test material used for the study:
Identification: NovaSpec Base OilPhysical state/appearance: Clear colorless, slightly viscous liquidBatch: TS17605Purity: 100 %Expiry date: 01 June 2018Storage conditions: Room temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animal InformationMale 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 HusbandryThe animals were housed in groups of up to five 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: mist
Type of inhalation exposure:
nose only
Vehicle:
not specified
Mass median aerodynamic diameter (MMAD):
1.37 µm
Geometric standard deviation (GSD):
3
Remark on MMAD/GSD:
Inhalable Fraction (% <4 µm) = 83.7Mean Achieved Atmosphere Concentration (mg/L) =5.09
Details on inhalation exposure:
Atmosphere GenerationThe test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The nebulizer was connected to a glass 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 in an attempt to achieve the required atmospheric conditions.Exposure ProcedureOne 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 ten rats (five males and five 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 102 % of target and no deaths occurred, no further levels were required. Exposure Chamber Temperature and Relative HumidityThe 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 ConcentrationOxygen 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 ConcentrationPrior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator at room temperature for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the formal exposure was considered to be 100 % (n=10). As the test item did not show signs of containing a volatile component it was considered acceptable to determine test atmosphere concentrations within the test chamber by using wet filter weights only.The actual chamber concentration was measured at regular intervals during the 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 283 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. Mean atmospheric concentration 5.09 mg/L
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
not specified
Details on study design:
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.

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.
Statistics:
Data EvaluationData 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
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.09 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occured
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 both during and for a short period after exposure. These observations a
Body weight:
All male animals and three females exhibited body weight losses on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, four female animals exhibited body weight losses or 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.
Other findings:
No other information available.

Any other information on results incl. tables

Individual Clinical Observations – (Day of Exposure)

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Effects Noted During Exposure
(Hours)

Effects Noted on Removal from Chamber

Effects Noted 1 Hour Post Exposure

1

2

3

5.09

1 Male

WfRd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

2 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

3 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

4 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

5 Male

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

6 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

7 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

8 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

9 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

10 Female

Wf Rd

Wf Rd

Wf Rd

Wf H P Ri

Wf H P Ri

 Key:

Wf= Wet Fur         Rd = Decreased respiratory rate      H = Hunched posture        P = Pilo-erection        Ri = Increased respiratory rate        

Individual Clinical Observations – Recovery Period

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Effects Noted Post Exposure
(Days)

1

2

3

4

5

6

7

8-14

5.09

1 Male

H

0

0

0

0

0

0

0

2 Male

H

0

0

0

0

0

0

0

3 Male

H

0

0

0

0

0

0

0

4 Male

H

0

0

0

0

0

0

0

5 Male

H

0

0

0

0

0

0

0

6 Female

H

0

0

0

0

0

0

0

7 Female

H

0

0

0

0

0

0

0

8 Female

H

0

0

0

0

0

0

0

9 Female

H

0

0

0

0

0

0

0

10 Female

H

0

0

0

0

0

0

0

 Key:

H= Hunched posture         0 = No abnormalities detected

Individual Body Weights and Body Weight Changes

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number and Sex

Body Weight (g) at Day

Body Weight Change (g) During Days

-6

0

1

3

7

14

-6 to 0

0 to 1

1 to 3

3 to 7

7 to 14

5.09

1 Male

200

240

239

248

271

299

40

-1

9

23

28

2 Male

200

228

226

233

246

259

28

-2

7

13

13

3 Male

208

240

239

249

267

281

32

-1

10

18

14

4 Male

202

237

236

245

268

290

35

-1

9

23

22

5 Male

210

243

242

250

270

291

33

-1

8

20

21

6 Female

203

215

214

215

215

219

12

-1

1

0

4

7 Female

201

206

205

209

205

207

5

-1

4

-4

2

8 Female

200

209

201

206

213

220

9

-8

5

7

7

9 Female

200

212

213

215

211

216

12

1

2

-4

5

10 Female

196

204

205

207

205

208

8

1

2

-2

3

 

 Individual Necropsy Findings

Mean Achieved Atmosphere Concentration (mg/L)

Animal Number
and Sex

Time of Death

Macroscopic Observations

5.09

1 Male

Terminal Kill Day 14

No abnormalities detected

2 Male

Terminal Kill Day 14

No abnormalities detected

3 Male

Terminal Kill Day 14

No abnormalities detected

4 Male

Terminal Kill Day 14

No abnormalities detected

5 Male

Terminal Kill Day 14

No abnormalities detected

6 Female

Terminal Kill Day 14

No abnormalities detected

7 Female

Terminal Kill Day 14

No abnormalities detected

8 Female

Terminal Kill Day 14

No abnormalities detected

9 Female

Terminal Kill Day 14

No abnormalities detected

10 Female

Terminal Kill Day 14

No abnormalities detected

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.09 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.09 mg/L and as such is not classified for Acute Inhalation Toxicity.
Executive summary:

A study was performed to assess the acute inhalation toxicity of the test item.The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals No. 403 “Acute Inhalation Toxicity” (2009) and Method B.2. (Inhalation) of Commission Regulation (EC) No. 440/2008.

A group of ten RccHanTM: WIST strain rats (five males and five females) was exposed to an aerosol atmosphere. The animals were exposed for 4 hours using a nose only exposure system, followed by a fourteen day observation period.

Atmospheric conditions were as follows:

Mean Achieved Atmosphere Concentration (mg/L) 5.09

Mean Mass Median Aerodynamic Diameter (µm) 1.37

Inhalable Fraction (% <4 µm) 83.7

Geometric Standard Deviation 3.00

Clinical Observations: Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal on Day 2 post-exposure.

Body Weight: All male animals and three females exhibited body weight losses on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period.  In contrast, four female animals exhibited body weight losses or 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.

 

In conclusion acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.09 mg/L and as such does not meet the criteria to classify for Acute Inhalation Toxicity.