Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 February 2015 and 16 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 403 using a standard acute method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (5R)-octahydro-1H-4,7-methanoinden-5-ylacetaldehyde and (5S)-octahydro-1H-4,7-methanoinden-5-ylacetaldehyde
EC Number:
940-300-7
Cas Number:
1339119-15-1
Molecular formula:
C12H18O
IUPAC Name:
Reaction mass of (5R)-octahydro-1H-4,7-methanoinden-5-ylacetaldehyde and (5S)-octahydro-1H-4,7-methanoinden-5-ylacetaldehyde
Test material form:
other: Liquid
Details on test material:
Identification (trade name): IFF TM 11-213 (FRET 09-0425)
Chemical (IUPAC) name of multi constituent: Reaction mass of 4,7-Methano-1H-indene-5-acetaldehyde, octahydro- and 4,7-Methano-1H-indene-5-carboxaldehyde, octahydro-6-methyl-
IPC number: 134635
Physical state / Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark under nitrogen

Constituent 1
IUPAC names of constituents: 4,7-Methano-1H-indene-5-acetaldehyde, octahydro-
CAS number: 1339119-15-1
Concentration of tested sample: 92.6%
Concentration range: 90-100%
Molecular formula: C12H18O
Molecular weight: 178.28

Constituent 2
IUPAC names of constituents: 4,7-Methano-1H-indene-5-carboxaldehyde, octahydro-6-methyl-
CAS number: 1338815-87-4
Concentration of tested sample: 5.7%
Concentration range: 5-15%
Molecular formula: C12H18O
Molecular weight: 178.28

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 196g to 350g. The females were nulliparous and non-pregnant.

The animals were housed in groups of up to five 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:
air
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 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.

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
One day prior to the day of exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During each 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 three groups, each of ten rats (five males and five 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. Further concentrations were selected after consideration of the results of the previous exposure.


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.

Exposure Chamber Atmosphere Concentration
Prior to the start 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 between 18 and 20 °C 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 non-volatile component of the batch used during the study was found to be approximately 64.43 % (n=9). However, during preliminary generation trials it was apparent that much more test item was being lost than was expected (possibly due to the large air flows used to generate the test item causing an increase in evaporation) and as such it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations for both groups.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 to 10 liters of test atmosphere (dependent on concentration) being drawn through a glass impinger containing Acetonitrile (each made up to 80mL). The samples were then submitted for chemical analysis.

The nominal chamber concentrations were calculated by dividing the mass of test item used by the total volume of air passed through the chamber during each exposure.

The nominal concentrations were 794 %, 365 % and 524 % of the actual mean achieved atmosphere concentration for Groups 1, 2 and 3 respectively, these show that keeping the aerosol airborne was moderately difficult for Groups 1 and 3 but was relatively straightforward when generating at the lowest dose level (Group 2). This is not an unexpected occurrence as generating and maintaining test atmospheres at progressively higher concentrations becomes more difficult. For example, doubling the syringe pump rate will not necessarily result in a two fold increase of the test atmosphere concentration. For the exposures performed during this study the syringe pump rates were set as follows; 125 mL/hr, 15 mL/hr and 70 mL/hr for Groups 1, 2 and 3 respectively.


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 back up 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.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Mean achieved atmosphere concentration: 4.64, 1.30, 3.99 mg/L
No. of animals per sex per dose:
Five per sex per dose
Control animals:
no
Details on study design:
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.

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, one hour after termination of exposure and subsequently once daily for up to fourteen or twenty one 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, 14, 21* or at death.
* = Group 3 only

Necropsy
At the end of each fourteen or twenty one* day observation period, the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died or were humanely killed during 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.

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
3.49 mg/L air
Based on:
test mat.
95% CL:
2.33 - 4.64
Exp. duration:
4 h
Sex:
male
Dose descriptor:
LC50
Effect level:
4.3 mg/L air
Based on:
test mat.
95% CL:
2.62 - 5.99
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
2.73 mg/L air
Based on:
test mat.
95% CL:
1.23 - 4.23
Exp. duration:
4 h
Mortality:
Group 1 - Mean achieved atmosphere concentration 4.64 mg/L
Deaths: 5/5 males, 5/5 females

Group 2 - Mean achieved atmosphere concentration 1.30 mg/L
Deaths: 0/5 males, 1/5 females

Group 3 - Mean achieved atmosphere concentration 3.99 mg/L
Deaths: 0/5 males, 2/5 females
Clinical signs:
other: See below
Body weight:
Group 1 – As none of the animals survived past the day of exposure, no significant body weight data was collected for these animals.

Group 2 – All males and three female animals exhibited body weight losses on the first day post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. Four female animals exhibited body weight gains and the remaining female showed no body weight gain from Days 1 to 3 post-exposure, however, four females exhibited a slight body weight loss or showed no body weight gains from Days 3 to 7 post-exposure. Body weight gains were noted in the four surviving female animals during the final week of the recovery period.

Group 3 – All surviving animals exhibited body weight losses on Day 1 post-exposure. One male and one surviving female animal exhibited body weight losses from Days 1 to 3 post-exposure and a further male and female exhibited body weight loss from Days 3 to 7 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods.

The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study:

Lungs –abnormally dark or dark patches;
Stomach – gaseous distension;

Due to the observations noted during the study and at necropsy it is considered that deaths noted during the study may have been mainly attributable to systemic toxicity.

Any other information on results incl. tables

Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by high performance gas chromatography (GC). The test atmosphereswere sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during each exposure period.

 

The concentration of the test item was shown to be stable and the mean values obtained were:

 

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

1

4.64

0.21

36.9

2

1.30

0.03

4.74

3

3.99

0.29

20.9

 

Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour during each exposure.

The theoretical chamber equilibration time (T99) was 3 minutes[1](Silver, 1946).

 

 

Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone, was as follows:

 

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

1

4.64

2.28

75.2

2.30

2

1.30

2.23

78.1

2.14

3

3.99

2.86

66.8

2.16

[1]= Test atmospheres were generated for a total of 30, 25 and 31 minutes for Groups 1, 2 and 3 respectively prior to animal insertion to ensure test item concentrations were being achieved.

Clinical Observations

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 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:

 

Dose Group 1 – 4.64 mg/L

During exposure all animals exhibited decreased respiratory rate and labored respiration. On removal from the chamber all animals exhibited decreased respiratory rate and labored respiration, frequent occurrences of coma and occasional instances of prostration were also noted. By one hour post-exposure all animals were comatose. One female animal was found dead approximately 70 minutes post-exposure and another female was humanely killed at the same time as it was considered unlikely to survive. The remaining animals were all humanely killed at approximately 120 minutes post-exposure as they were considered unlikely to survive. 

 

 

Dose Group 2 – 1.30 mg/L

Isolated occurrences of increased respiratory rate were noted in two males during exposure. On removal from the chamber and one hour post-exposure all animals exhibited increased respiratory rate.

 

One day after exposure, all animals exhibited increased respiratory rate and hunched posture. Animals recovered to appear normal on Day 2 or Day 3 post-exposure. However, one female animal was found dead on Day 12 after showing no abnormal clinical signs over the previous 10 days.

 

 

Dose Group 3 – 3.99 mg/L

Decreased respiratory rate was noted in all animals during exposure. On removal from the chamber, all animals exhibited decreased respiratory rate and ataxia, one female animal also exhibited lethargy and ptosis. One hour post-exposure a deterioration in the condition of the animals was noted as they all now appeared lethargic and one female was humanely killed approximately 90 minutes post-exposure as it was considered unlikely to survive.

 

One day after exposure, one female animal was found dead. All surviving animals exhibited increased or decreased respiratory rate and hunched posture. There were frequent instances of pilo-erection and isolated occurrences of lethargy.  Slight signs of improvement were noted over the next two days, however, on Days 4 and 5 noisy respiration and sneezing became prevalent. One female animal exhibited fur loss on the right side of the head on Day 7 which persisted for eight days, however, this was considered not to be treatment related. Surviving animals recovered slowly such that they appeared normal Day 18 post-exposure.

Exposure Chamber Atmosphere Concentrations– Group 1

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

2

60

4.52

30

2

60

4.54

60

2

60

4.61

90

2

60

4.49

120

2

60

4.61

150

2

60

5.17

180

2

60

4.52

210

2

06

4.62

230

2

60

4.69

Mean achieved atmosphere concentration (mg/L) = 4.64

Standard deviation = 0.21

 

Nominal concentration:

Test item used (g)

570

Air Flow (L/min)

60

Total Generation Time (mins)

270[1]

Nominal Concentration (mg/L)

36.9

 [1]

= Test atmospheres were generated for a total of 30 minutes prior to animal insertion to ensure test item concentration was being achieved.

Exposure Chamber Atmosphere Concentrations – Group 2

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

10

10

60

1.27

30

10

60

1.32

60

10

60

1.28

90

10

60

1.29

120

10

60

1.36

151

10

60

1.35

180

10

60

1.30

210

10

60

1.27

230

10

60

1.30

Mean achieved atmosphere concentration (mg/L) = 1.30

Standard deviation = 0.03

 

Nominal concentration:

Test item used (g)

75.3

Air Flow (L/min)

60

Total Generation Time (mins)

265[1]

Nominal Concentration (mg/L)

4.74

[1]= Test atmospheres were generated for a total of 25 minutes prior to animal insertion to ensure test item concentration was being achieved.

Exposure Chamber Atmosphere Concentrations – Group 3

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

10

3

60

3.57

30

3

60

3.82

60

3

60

3.92

90

3

60

3.91

120

3

60

3.96

150

3

60

4.06

180

3

60

4.24

210

3

60

4.61

230

3

60

3.84

Mean achieved atmosphere concentration (mg/L) = 3.99

Standard deviation = 0.29

 

Nominal concentration:

Test item used (g)

340

Air Flow (L/min)

60

Total Generation Time (mins)

271[1]

Nominal Concentration (mg/L)

20.9

[1]= Test atmospheres were generated for a total of 31 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution – 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

8.4

0.04

0.16

0.07

0.09

4

7.3

0.15

0.24

0.14

0.18

5

3.6

0.36

0.52

0.41

0.43

6

1.3

0.46

0.64

0.52

0.54

7

0.94

0.65

0.83

0.67

0.72

8

0.43

0.07

0.08

0.04

0.06

Back-up Filter

<0.43

0.04

0.13

0.07

0.08

Total Mean Amount of Test Item Collected

2.10

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.01

95.7

6.72

7.3

0.863

1.83

87.1

6.13

3.6

0.556

1.40

66.7

5.43

1.3

0.114

0.86

41.0

4.77

0.94

-0.027

0.14

6.67

3.50

0.43

0.367

0.08

3.81

3.23

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) =2.28µm

Geometric Standard Deviation (GSD) =2.30

Predicted amount less than 4 µm =75.2%

Particle Size Distribution – 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

8.4

0.02

0.03

0.07

0.04

4

7.3

0.09

0.12

0.13

0.11

5

3.6

0.41

0.34

0.43

0.39

6

1.3

0.41

0.51

0.55

0.49

7

0.94

0.46

0.54

0.52

0.51

8

0.43

0.03

0.03

0.01

0.02

Back-up Filter

<0.43

0.05

0.04

0.10

0.06

Total Mean Amount of Test Item Collected

1.62

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

1.58

97.5

6.97

7.3

0.863

1.47

90.7

6.33

3.6

0.556

1.08

66.7

5.43

1.3

0.114

0.59

36.4

4.65

0.94

-0.027

0.08

4.94

3.35

0.43

0.367

0.06

3.70

3.21

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 2.23 µm

Geometric Standard Deviation (GSD) = 2.14

Predicted amount less than 4 µm = 78.1 %

Particle Size Distribution – Group 3

Cascade Impactor Data

 

Impactor Stage Number

Cut Point

(µm)

Amount Collected (mg) per Sample Number

Mean Amount Collected (mg)

1

2

3

3

8.4

0.18

0.25

0.21

0.21

4

7.3

0.29

0.40

0.35

0.35

5

3.6

0.68

0.76

0.79

0.74

6

1.3

0.83

0.83

0.79

0.82

7

0.94

0.86

0.92

0.91

0.90

8

0.43

0.04

0.09

0.09

0.07

Back-up Filter

<0.43

0.00

0.05

0.03

0.03

Total Mean Amount of Test Item Collected

3.12

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.91

93.3

6.50

7.3

0.863

2.56

82.1

5.92

3.6

0.556

1.82

58.3

5.21

1.3

0.114

1.00

32.1

4.53

0.94

-0.027

0.10

3.21

3.15

0.43

0.367

0.03

0.962

2.66

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 2.86 µm

Geometric Standard Deviation (GSD) = 2.16

Predicted amount less than 4 µm = 66.8 %

 

Mortality Data

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Sex

Deaths During Exposure

Deaths Post Exposure
(1 Hour)

Deaths During Day of Observation

Total Deaths

1

2

3

4

5

6

7

8-14

1

4.64

Male

0

5[1]

-

-

-

-

-

-

-

-

10/10

Female

0

1[2]1[3]

 3*

-

-

-

-

-

-

-

-

2

1.30

Male

0

0

0

0

0

0

0

0

0

0

1/10

Female

0

0

0

0

0

0

0

0

0

1[4]

3

3.99

Male

0

0

0

0

0

0

0

0

0

0

2/10

Female

0

1[5]

1

0

0

0

0

0

0

0

[1]= Humanely killed Approximately 120 minutes post-exposure as considered unlikely to survive

[2]= Found dead Approximately 70 minutes post-exposure

[3]= Humanely killed Approximately 70 minutes post-exposure as considered unlikely to survive

[4]= Found dead on Day 12 post-exposure

[5]= Humanely killed Approximately 90 minutes post-exposure as considered unlikely to surviv

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute inhalation median lethal concentrations (LC50) and 95% confidence limits of IFF TM 11-213 (FRET 09-0425) in the RccHanTM : WIST strain rat, were calculated to be:

All animals : 3.49 (2.33 – 4.64) mg/L
Males only: 4.30 (2.62 – 5.99) mg/L
Females only: 2.73 (1.23 – 4.23) mg/L
(Globally Harmonized Classification System – Category 4).
Executive summary:

The acute inhalation toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 403 using a standard acute method. The LD50 of all animals was 3.49 (2.33 – 4.64) mg/L; the LD50 for males only was 4.30 (2.62 – 5.99) mg/L and for females only was 2.73 (1.23 – 4.23) mg/L.