Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-04-2019 to 08-05-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
2009
Deviations:
yes
Remarks:
Clinical observation and body weight were performed for the 1 mg/L exposure group on Day 8 and for the 5 mg/L exposure group on Day 2 but a day later.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
August 1998
Deviations:
yes
Remarks:
Clinical observation and body weight were performed for the 1 mg/L exposure group on Day 8 and for the 5 mg/L exposure group on Day 2 but a day later
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
May 2008
Deviations:
yes
Remarks:
Clinical observation and body weight were performed for the 1 mg/L exposure group on Day 8 and for the 5 mg/L exposure group on Day 2 but a day later
Qualifier:
according to guideline
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF).
Version / remarks:
2000
Deviations:
yes
Remarks:
Clinical observation and body weight were performed for the 1 mg/L exposure group on Day 8 and for the 5 mg/L exposure group on Day 2 but a day later
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-160-5
EC Name:
-
Cas Number:
130786-09-3
Molecular formula:
C12H13N
IUPAC Name:
(2Z)-2-phenylhex-2-enenitrile
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light, container flushed with nitrogen
- Other: colourless to very pale yellow liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: [yes/no]: Yes
- Age at study initiation: Approximately 9-10 weeks old
- Weight at study initiation:Males: 248 to 305 g. Females: 169 to 210 g.
- Fasting period before study:
- Housing:On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same exposure group together) in polycarbonate cages (Makrolon MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet (e.g. ad libitum):Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
- Water (e.g. ad libitum):Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS 
 - Temperature (°C): 20 to 21°C
- Humidity (%): 47 to 51%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle

 IN-LIFE DATES: From: To: Not stated

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
not specified
Mass median aerodynamic diameter (MMAD):
>= 3.4 - <= 3.9 µm
Geometric standard deviation (GSD):
>= 1.9 - <= 2
Remark on MMAD/GSD:
At 1 mg/L, the Mass Median Aerodynamic Diameter (MMAD) was 3.4 μm (geometric standard deviation (gsd) 2.0) and 3.4 μm (gsd 1.9). At 5 mg/L, the MMAD was 3.9 μm (gsd 2.0) and 3.6 μm (gsd 1.9).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose only exposure apparatus.
- Exposure chamber volume: The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal.
- Method of holding animals in test chamber: Each animal port has its own test atmosphere inlet and exhaust outlet.
- Source and rate of air: The mean total airflows were 66 and 24 L/min for the 1 mg/L and 5 mg/L exposure groups, respectively.
- Method of conditioning air: It was considered that the opacity of the test atmosphere could not be reliably monitored by means of an aerosol monitoring system. An indication of stability of the test atmosphere was obtained from the concentration measurements equally distributed over time.
- System of generating particulates/aerosols: Metal concentric jet nebulizer located at the top of the exposure chamber.
- Method of particle size determination: The particle size distribution was characterized twice during each exposure period. The samples were drawn with a flow of 2 L/min. from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (TE- 290-GF. Tisch Environmental, Cleves, Ohio, USA) and a fiber glass back-up filter (SEC-290- F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test item collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined based on OECD guidance document No 39. Graphs of the cumulative mass of test item collected (percentage of total collected) against the cut points of the impactor stages were drawn on log-normal paper. When drawing the graphs more weight was given to the cut points where the cumulative mass sampled was within the range of 5 to 95%. The Mass Median Aerodynamic Diameter (MMAD), i.e. the particle size where 50% of the particle mass was borne by particles smaller than the MMAD and the sigma-84%, (the particle size where 84% of the particle mass was borne by particles smaller than the MMAD was read from the graph. The geometric standard deviation (gsd) was calculated as sigma-84% / MMAD. The performance characteristics of the test atmosphere generation and exposure system to be used was assessed during trial generations. These trials were performed according to test facilities SOP’s and since these trials were performed prior to preparation of the study plan no GLP was claimed for these trials. This was considered as no exception of the GLP guidelines since the measurements and observations were not used for interpretation for the outcome of this study. For the same reason, these results are not reported and kept in the raw data of this study. These investigations were undertaken to establish:
Aerosol concentration assessment
Temporal variation in chamber concentration
Test item utilization
Particle size distribution measurements (within the target MMAD range 1-4 μm)
Trial generation results showed that the test item was not volatile and the test atmosphere consisted mainly of aerosol with a negligibly small (if any) vapor part.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. - Temperature, humidity, pressure in air chamber: The temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and recorded after the animals were connected to the exposure chamber and at 30 minute intervals after initiation of exposure. The probe was inserted in a tube mounted in one of the free animal ports of the exposure chamber. The temperature of the atmosphere during the exposures was between 21.1 and 21.6oC. The relative humidity was between 4 and 46% which was considered appropriate for this relatively short 4 hours exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method used: A total of 15 and 19 representative samples were taken for determination of the actual concentration during exposure at 1 and 5 mg/L, respectively. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. Samples were drawn through a glass fiber filter (type APFC04700, Millipore, Billerica, MA, USA). Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands). The collected amount of test item in the air sample was measured gravimetrically. Subsequently the time-weighted mean concentrations with the standard deviations were calculated.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): n/a
- Concentration of test material in vehicle (if applicable): n/a
- Justification of choice of vehicle: n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Tabulated
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Tabulated

CLASS METHOD (if applicable): n/a
- Rationale for the selection of the starting concentration:
Analytical verification of test atmosphere concentrations:
no
Remarks:
t was considered that the opacity of the test atmosphere could not be reliably monitored by meansofanaerosolmonitoringsystem. Anindicationofstabilityofthetestatmospherewas obtained from the concentration measurements equally distributed over time.
Remarks on duration:
4 hours
Concentrations:
1 mg/L and 5 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
The test item was administered once via the inhalation route on Day 1, by nose only directed flow exposure for four hours.
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were checked for mortality, behavioral signs of distress and effects on respiration at least three times during exposure. Post exposure observations were performed at periodic intervals on the day of exposure (at least two times) and once daily thereafter, except for Day 2 (5 mg/L exposure group) and Day 8 (1 mg/L exposure group), see deviations in Appendix 3. The observation period was 14 days. All the animals were examined for reaction to exposure. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals for the first hours after exposure. Animals were weighed individually on Day 1 (pre exposure), 2 (Day 3 for the 5 mg/L exposure group), 4 and 8 (Day 9 for the 1 mg/L exposure group) and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology: yes
Statistics:
No statistical analysis was carried out in the study.

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
> 1 - <= 5 other: mg/L
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
>= 5 other: mg/L
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At 5 mg/L, two females were found dead and one female was killed in extremis on Day 3.
Clinical signs:
other:
Body weight:
At 1 mg/L, overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.
At 5 mg/L, body weight loss was within the range expected for rats of this strain and age used in this type of study. The regaining of weight during the study was slightly delayed compared to what is normally seen in this type of study.
Gross pathology:
At 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the animals.
At 5 mg/L macroscopic post mortem examination revealed abnormalities of the thymus (dark red/black-brown foci and/or pale/black-brown discoloration), jejunum, ileum, caecum and colon (contents black-brown discoloration), liver (pale discoloration) and/or spleen (reduced in size) for animals that died or were sacrificed in moribund condition during the study. Macroscopic examination of the other animals revealed abnormalities of the thymus (right side and general many dark red foci) for two males. The remaining animals did not reveal any abnormalities at termination.

Any other information on results incl. tables

Table. 1. Gravimetrical Concentration of test Item.

Time (hh:mm)

Action

Sample Volume (L)

Mass Sampled (mg)

Concentration (mg/L)

% of total exposure time

Weight Concentration (mg/L)

8.52

Start of Generation

n.a.

n.a.

n.a.

0.0

n.a.

8.56

Start of sampling

5

8.12

1.624

1.7

0.027

9.02

Start of sampling

5

7.56

1.512

2.5

0.038

9.08

Start of sampling

5

7.16

1.432

2.1

0.036

9.13

Start of sampling

5

5.50

1.100

2.9

0.023

9.20

Start of sampling

5

5.43

1.086

6.7

0.032

9.36

Start of sampling

5

5.63

1.126

7.9

0.075

9.55

Start of sampling

5

5.59

1.118

8.3

0.089

10.15

Start of sampling

5

5.54

1.108

12.1

0.092

10.44

Start of sampling

5

5.20

1.040

9.2

0.026

11.06

Start of sampling

6

6.63

1.105

7.9

0.101

11.25

Start of sampling

5

5.44

1.088

8.2

0.086

11.45

Start of sampling

5

5.33

1.066

8.3

0.089

12.06

Start of sampling

5

5.52

1.104

8.8

0.097

12.26

Start of sampling

5

5.38

1.076

8.3

0.090

12.45

Start of sampling

5

5.62

1.124

7.9

0.089

12.52

End of Generation

n.a.

n.a.

1.124 I)

2.9

0.033

Time-weighted mean concentration

1.121

Standard deviation

0.033

Number of samples

15

1) Assumed concentration, based on the last sample. n.a.= not applicable

Test Atmosphere Characterization Concentration:

At 1 mg/L, the time-weighted mean actual concentration was 1.1 ± 0.0 mg/L. The nominal concentration (amount of test item used divided by the volume of pressurized air used) was 1.9 mg/L. This resulted in a generation efficiency (ratio of actual and nominal concentration) of 59%.

At 5 mg/L, the time-weighted mean actual concentration was 5.2 ± 0.2 mg/L. The nominal concentration was 7.0 mg/L. This resulted in a generation efficiency of 74%.

The concentration was measured at time points (n=15 and 19) that were equally distributed over the exposure period, the results of which demonstrated that the item was sufficiently stable. The variation in concentration was caused by adjustments to the generation equipment. By calculating the time-weighted mean concentration, effects of variations were taken into account resulting in an actual reflection of the mean exposure concentration over.

Table. 2 Aerodynamic Particle Size Distribution in the Test Atmosphere

Exposure group

 

Stage

Cut point (mm)

Mass sampled (mg)

Relative mass (%)

Cumulative mass (% of total sampled)

 

 

 

 

 

 

 

1 mg/L

 

Measurement 1:

1

21.0

0.13

1.45

98.55

2

15.0

0.00

0.00

98.55

3

10.0

0.25

2.80

95.75

4

6.0

1.12

12.53

83.22

5

3.5

1.92

32.66

50.56

6

2.0

3.99

40.94

9.62

7

0.9

0.47

5.26

4.36

8

0.5

0.35

3.91

0.45

Back up

0.25

0.04

0.45

0.00

MMAD1(mm): 3.4

gsd2: 2.0

Measurement 2

1

21.0

0.11

1.15

98.55

2

15.0

0.00

0.00

98.55

3

10.0

0.20

2.08

96.77

4

6.0

1.17

12.19

84.58

5

3.5

3.25

33.85

50.73

6

2.0

3.77

39.27

11.46

7

0.9

0.74

7.71

3.75

8

0.5

0.33

3.44

0.31

Back up

0.25

0.03

0.31

0.00

MMAD1(mm): 3.4

                                                                  gsd2: 1.9

 

 

 

 

 

 

 

 

5 mg/L

 

Measurement 1

1

21.0

0.06

0.76

99.24

2

15.0

0.07

0.89

98.35

3

10.0

0.48

6.08

92.28

4

6.0

1.25

15.82

76.46

5

3.5

2.90

36.71

39.75

6

2.0

2.68

33.92

5.82

7

0.9

0.28

3.54

2.28

8

0.5

0.17

2.15

0.13

Back up

0.25

0.01

0.13

0.00

MMAD1(mm): 3.4

                                                                  gsd2: 2.0

Measurement 2

1

21.0

0.07

1.12

99.88

2

15.0

0.05

0.80

98.09

3

10.0

0.15

2.39

95.69

4

6.0

0.31

4.94

90.75

5

3.5

2.75

43.86

46.89

6

2.0

2.52

40.19

6.70

7

0.9

0.25

3.99

2.71

8

0.5

0.15

2.39

0.32

Back up

0.25

0.02

0.32

0.00

MMAD1(mm): 3.6

                                                                  gsd2: 1.9

1  Mass Median Aerodynamic Diamete, 2  Geometric standard deviation

Particla Size:

At 1 mg/L, the MMAD was 3.4 μm (gsd 2.0) and 3.4 μm (gsd 1.9).

At 5 mg/L, the MMAD was 3.9 μm (gsd 2.0) and 3.6 μm (gsd 1.9).

Table. 3 Mortality Data

Test Day

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Hours after treatement

1

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Males 1 Mg/L

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Females 1 Mg/L

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Males 5 Mg/L

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Females 5 Mg/L

-

-

-

3

-

-

-

-

-

-

-

-

-

-

-

-

Tabel 4. Clinical signs

Treatment groups/sex

Test Day

Max Grade

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Hours after treatment

1

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Males Mg/L

Animal 1

Behavior Lethargy Posture

Hunched posture

 

 

 

(3)

 

 

 

(1)

 

 

 

1

 

 

 

1

 

 

 

1

 

 

 

1

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

Animal 2

Behavior Lethargy Posture


Hunched posture

 

 

 

(3)

 

 

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

Animal 3

Behavior Lethargy Posture


Hunched posture

Skin / fur Piloerection

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

Animal 4

Behavior Lethargy

Posture
Hunched posture

Skin / fur Piloerection

 

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

Animal 5

Behavior Lethargy Posture


Hunched posture

 

 

 

(3)

 

 

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Females Mg/L

Animal 6Behavior Lethargy Posture

Hunched posture

 

 

(3)

 

 

 

(1)

 

 

1

 

 

 

1

 

 

1

 

 

 

1

 

 

-

 

 

 

1

 

 

-

 

 

 

1

 

 

-

 

 

 

1

 

 

-

 

 

 

1

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

 

 

-

 

 

 

-

Animal 7

Behavior Lethargy Posture


Hunched posture

Skin / fur Piloerection

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

 

2

 

 

 

 

1

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

-

 

 

 

-

 

 

 

 

1

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

Animal 8Behavior Lethargy Posture

Hunched posture

 

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

-

 

 

 

 

1

 

 

 

-

 

 

 

 

1

 

 

 

-

 

 

 

 

1

 

 

 

-

 

 

 

 

1

 

 

 

-

 

 

 

 

1

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

Animal 9

Behavior Lethargy Posture


Hunched posture

Skin / fur Piloerection

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

Animal 10

Behavior Lethargy Posture


Hunched posture

 

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

Males 5 Mg/L

Animal 11

Behavior Lethargy Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection Various

Ptosis

 

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

 

(3)

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

3

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

 

 

 

.

 

 

 

 

.

 

 

.

 

 

.

 

 

 

.

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

Animal 12

Behavior Lethargy Posture


Hunched posture

Breathing
Slow breathing

Skin / fur Piloerection Various Ptosis

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

(3)

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

2

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

-

 

 

.

 

 

 

 

.

 

 

.

 

 

.

 

 

.

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

-

Animal 13

Behavior Lethargy Posture


Hunched posture

Breathing
Slow breathing

Skin / fur Piloerection

Various Ptosis

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

 

 

(3)

 

 

 

3

 

 

 

 

1

 

 

1

 

 

1

 

 

 

 

2

 

 

 

2

 

 

 

 

1

 

 

1

 

 

1

 

 

 

 

-

 

 

 

.

 

 

 

 

.

 

 

.

 

 

.

 

 

 

 

.

 

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

 

-

Animal 14

Behavior Lethargy Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection
Various

 

(3)

 

 

 

 

(1)

 

 

 

 

(1)

 

 

(1)

 

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

.

 

 

 

 

.

 

 

 

 

 

.

 

.

 

 

 

1

 

 

 

 

1

 

 

 

 

1

 

 

1

 

 

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

 

-

 

 

 

 

-

 

 

 

 

-

 

 

-

Animal 15
Behavior Lethargy
Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection
Various

 

(3)

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

 

(3)

 

2

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

2

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

.

 

 

 

.

 

 

.

 

 

.

 

 

 

.

 

1

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

-

 

 

 

-

 

 

-

 

 

-

 

 

 

-

Females 5 Mg/L

Animal 16
Behavior Lethargy
Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection
Various

 

(3)

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

(3)

 

1

 

 

 

1

 

 

1

 

 

1

 

 

2

 

1

 

 

 

1

 

 

1

 

 

1

 

 

1

 

.

 

 

 

.

 

 

.

 

 

.

 

 

.

 

1

 

 

 

1

 

 

1

 

 

1

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

ANIMAL 17
Behavior Lethargy
Posture

Hunched posture Breathing

Slow breathing

Skin / fur Piloerection

Various Pitosis

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

 

(1)

 

 

 

(3)

 

1

 

 

 

 

1

 

 

1

 

 

 

1

 

 

 

1

 

1

 

 

 

 

1

 

 

1

 

 

 

1

 

 

 

-

 

.

 

 

 

 

.

 

 

.

 

 

 

.

 

 

 

.

 

1

 

 

 

 

1

 

 

1

 

 

 

1

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

-

 

 

 

 

-

 

 

-

 

 

 

-

 

 

 

-

Animal 18
Behavior Lethargy
Posture

Flat
Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection
Various

 

 

(3)

 

 

(1)

 

(1)

 

 

(1)

 

 

(1)

 

 

(3)

 

 

2

 

 

-

 

1

 

 

1

 

 

1

 

 

 

1

 

 

2

 

 

1

 

1

 

 

1

 

 

1

 

 

 

2

 

 

.

 

 

.

 

.

 

 

.

 

 

.

 

 

 

.

 

 

1

 

 

1

 

1

 

 

1

 

 

1

 

 

 

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Animal 19Behavior Lethargy Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection Various

Ptosis

 

 

(3)

 

 

 

(1)

 

 

(1)

 

 

 

(1)

 

 

 

(3)

 

 

2

 

 

 

 

1

 

 

1

 

 

1

 

 

 

2

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

-

 

 

.

 

 

 

 

.

 

 

.

 

 

.

 

 

 

.

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

-

 

 

 

 

 

 

 

 

 

 

 

 

Animal 20

Behavior Lethargy Posture

Hunched posture Breathing

Slow breathing Skin / fur

Piloerection Various

Ptosis

 

 

 

(3)

 

 

 

 

(1)

 

 

(1)

 

 

(1)

 

 

 

(3)

 

 

 

2

 

 

 

 

1

 

 

1

 

 

1

 

 

 

2

 

 

 

2

 

 

 

 

1

 

 

1

 

 

1

 

 

 

2

 

 

 

.

 

 

 

 

.

 

 

.

 

 

.

 

 

 

.

 

 

 

1

 

 

 

 

1

 

 

1

 

 

1

 

 

 

1

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

-

 

 

-

 

 

-

 

 

 

-

- = Sign not observed
. = Observation not recorded

Table 5. Body weight (g)

SEX/DOSE LEVEL

DAY 1

DAY 2

DAY 4

DAY 8

DAY 15

Male 1 Mg/L

263

253

264

284

304

Female 1 Mg/L

182

169

176

191

205

Male 5 Mg/L

293

261

269

292

320

Female 5 Mg/L

200

178

180

190

210

Note: No body weights were obtained for the 1 mg/L exposure group on Day 8 and for the 5 mg/L exposure group on Day 2. These body weights were recorded 1 day later, on Day 9 and Day 3, for the 1 mg/L and 5 mg/L exposure groups, respectively (see deviations in appendix 3).

* Animal was killed in extremis on Day 3.

** Animal was found dead on Day 3.

Table 6. Macroscopic Findings

Sex/Dose Level

Animal no

Organ

Finding

Day of death

Male 1 Mg/L

1

 

No findings noted

Scheduled necropsy.Day 15 after treatment

2

 

No findings noted

Scheduled necropsy.Day 15 after treatment

3

 

No findings noted

Scheduled necropsy.Day 15 after treatment

4

 

No findings noted

Scheduled necropsy.Day 15 after treatment

5

 

No findings noted

Scheduled necropsy.Day 15 after treatment

Female 1 Mg/L

6

 

No findings noted

Scheduled necropsy.Day 15 after treatment

7

182

No findings noted

Scheduled necropsy.Day 15 after treatment

8

 

No findings noted

Scheduled necropsy.Day 15 after treatment

9

 

No findings noted

Scheduled necropsy.Day 15 after treatment

10

 

No findings noted

Scheduled necropsy.Day 15 after treatment

Male 5 Mg/L

11

 

No findings noted

Scheduled necropsy.Day 15 after treatment

12

Thymus

Focus/foci, many, dark red.

Scheduled necropsy.Day 15 after treatment

13

 

No findings noted

Scheduled necropsy.Day 15 after treatment

14

Thymus

Right side: focus/foci, isolated, dark Red.

Scheduled necropsy.Day 15 after treatment

15

 

No findings noted

Scheduled necropsy.Day 15 after treatment

Female 5 Mg/L

16

Jejunum

Ileum

Caecum

Colon

Liver Spleen Thymus

Contents: discolouration, Black-brown.
Contents: discolouration, Black-brown.

Contents: discolouration, Black-brown.
Contents: discolouration, Black-brown. Discolouration, pale. Reduced in size.

Right side: focus/foci, isolated, dark Red.
Discolouration, pale.

Killed in extremis.Day 3 after treatment..

17

 

No findings noted

Scheduled necropsy.Day 15 after treatment

18

General observation Thymus

Beginning autolysis.
Focus/foci, several, black-brown. Discolouration, black-brown.

Spontaneous death.Day 3 after treatment.

19

General observation Thymus

Beginning autolysis.
Focus/foci, several, black-brown. Discolouration, black-brown.

Spontaneous death.Day 3 after treatment.

20

 

No findings noted

Scheduled necropsy.Day 15 after treatment

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Based on the conditions of the study, the LC50 was concluded to be >1 - ≤5 mg/L for female rats and above 5 mg/L for male rats. Therefore, test item meets the criteria for acute toxicity via inhalation (Acute Toxicity Category 4) in accordance with Regulation (EC) No 1272/2008.
Executive summary:

OECD 403 (2019): The acute inhalation toxicity potential of the test item  was assessed in Wistar Han 
 rats of both sexes following a single 4 hour nose-only exposure to two groups of five male and five female  rats at target concentrations of 1 and 5 mg/L. Animals were retained for a 14 day post exposure observation period.

At 1 mg/L, no mortality occurred. At 5 mg/L, two females were found dead and one female was killed in extremis on Day 3. No further mortality occurred. 
At 1 mg/L, no clinical signs were seen during exposure. After exposure, lethargy, hunched posture and piloerection were seen for the animals. The males had recovered from the clinical signs on Day 2 and the females on Day 7. At 5 mg/L, slow breathing was seen for all animals during exposure. After exposure, lethargy, hunched posture, slow breathing, piloerection and ptosis were seen for the animals. In addition, one animal that died showed flat posture. The female that was killed in extremis was not expected to recover from these signs. The surviving animals had recovered from the clinical signs on Day 4. 


At 1 mg/L, overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity. At 5 mg/L, body weight loss was noted for all surviving animals. This continued for all males and three females up to Day 8; these animals regained weight during the second week.

At 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the animals. At 5 mg/L macroscopic post mortem examination revealed abnormalities of the thymus (dark red/black-brown foci and/or pale/black-brown discoloration), jejunum, ileum, caecum and colon (contents black-brown discoloration), liver (pale discoloration) and/or spleen (reduced in size) for animals that died or were sacrificed in moribund condition during the study. Macroscopic examination of the other animals revealed abnormalities of the thymus (right side and general many dark red foci) for two males. The remaining animals did not reveal any abnormalities at termination.

A difference in sensitivity between the sexes was noted as mortality occurred only for the females at 5 mg/mL. Therefore, both a combined and sex specific LC50, 4h value was determined. The combined inhalation LC50, 4h value of the test item  in Wistar Han rats of both sexes was established to exceed 5 mg/L.

The sex specific inhalation LC50, 4h value of the test item  in male Wistar Han rats was established to exceed 5 mg/L. The sex specific inhalation LC50, 4h value of the test item in female Wistar Han rats was established to be within the range of >1 - ≤5 mg/L.

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