Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Oral: LD50 > 300 - < 2000 mg/kg bw, female rat, OECD TG 420, 2007

Inhalation: LC50 >1 -≤ 5 mg/L, female rat, OECD TG 403, 2019

Dermal: measured LD50 > 2000 mg/kg bw and the estimated LD50 cut-off value was considered to be > 5000 mg/kg bw, male/female rat, OECD TG 402, 2008

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-05-2007 to 30-05-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: August 2005; signature: November 2005
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Strain: Crl : CD (SD) IGS BR
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 171-179 g (300 mg/kg including sighting test; sentinel); 179 g (2000 mg/kg sighting test; sentinel); The weight variation did not exceed ±20% of the mean weight in the definitive test (300 mg/kg bw).
- Fasting period before study: Overnight before dosing and three to four hours after dosing.
- Housing: Group housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for fasting period).
- Water (e.g. ad libitum): ad libitum (except for fasting period)
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: To: 2015-12-16 to 2016-01-06
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level.
For the purpose of the 300 mg/kg dose level the test item was freshly prepared, as required, as a solution in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water. For the purpose of the 2000 mg/kg dose level, the test item was used as supplied. The 300 mg/kg bw and 2000 mg/kg bw dose levels were treated stepwise. Singuarly and in the absence of mortality or evident toxicity a further group of 4 was tested in the appropriate dose level.
Doses:
300 mg/kg bw (initial sighting test and main study)
2000 mg/kg bw (initial sighting test)
No. of animals per sex per dose:
1 (sighting study) and 4 (main study) as applicable; total 5 per dose (in definitive test).
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were made 0.5, 1, 2, and 4 hours after dosing and then daily for fourteen days. Morbidity and mortality checks were made twice daily. Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
300 mg/kg bw (sentinel and definitive test): No mortality
2000 mg/kg bw (sentinel): Mortality (humane termination one day after dosing).
Clinical signs:
300 mg/kg bw (sentinel and definitive test): No signs of systemic toxicity were noted.
2000 mg/kg bw (sentinel): Hunched posture, lethargy, pilo-erection, ataxia, tiptoe gait, dehydration, decreased respiratory rate and laboured respiration. Hypothermia was also noted prior to termination.
Body weight:
300 mg/kg bw (sentinel and definitive test): All animals showed expected bodyweight gains over the study period.
2000 mg/kg bw (sentinel): Not applicable.
Gross pathology:
300 mg/kg bw (sentinel and definitive test): No abnormalities were noted.
2000 mg/kg bw (sentinel): Abnormally red lungs and haemorrhage of the gastric mucosa, non-glandular region of the stomach and small and large intestines.
Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in female Sprague-Dawley Crl : CD (SD) IGS BR strain rats.
Executive summary:

The study was performed according to OECD TG 420 and EU Method B.1 bis Acute Toxicity (Oral) and in accordance with GLP to assess the acute oral toxicity of the test material following a single oral administration in the female Sprague-Dawley Crl : CD (SD) IGS BR strain rat by the fixed dose method. The test item was administered by oral gavage in a solution in arachis oil BP in an initial sighting study at 300 mg/kg bw and following an absence of toxicity then at 2000 mg/kg bw. Subsequently, a further group of four fasted females was given a single oral dose of test item, at a dose level of 300 mg/kg body weight. The animal treated at a dose level of 2000 mg/kg was humanely terminated on 1 day after dosing. Clinical signs were noted during the day of dosing in the animal treated at a dose level of 2000 mg/kg. There was no mortality at a dose level of 300 mg/kg. There were no clinical signs of systemic toxicity and all animals showed expected gains in bodyweight over the study period at a dose level of 300 mg/kg. Abnormalities noted at necropsy of the animal treated at a dose level of 2000 mg/kg, that was humanely terminated, were abnormally red lungs and haemorrhage of the gastric mucosa, non-glandular region of the stomach and small and large intestines. No abnormalities were noted at a dose level of 300 mg/kg. Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in the female Sprague-Dawley CD strain rat.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
discriminating dose
Value:
300 mg/kg bw
Quality of whole database:
The available information as a whole meets the tonnage driven information requirements of REACH.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
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.
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
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
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.
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
1 000 mg/m³
Quality of whole database:
The available information as a whole meets the tonnage driven information requirements of REACH

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-09-2008 to 23-09-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met; note that for local effects only: the reshaving at day 8 is a deviation is suggestive of effects that are non-test item related, which limits the reliability for assessment.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
HanRcc: WIST (SPF) strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: 9 - 11 weeks.
- Weight at study initiation: 197.3 - 264.0 g; the weight variation did not exceed ±20% of the mean weight for each sex.
- Fasting period before study: Not applicable
- Housing: during acclimation: group housed by sex; during study: individually housed in Makrolon type-4 cages furnished with softwood bedding.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: At least 5 days.

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

IN-LIFE DATES: From: To: 2008-09-02 to 2008-09-23
Type of coverage:
semiocclusive
Vehicle:
polyethylene glycol
Remarks:
PEG 300 (details available in full study report)
Details on dermal exposure:
TEST SITE
- Area of exposure: the day before treatment the back and flanks were clipped free of hair. Dorsal area application. Note: that the skin was re-shaved on day 8 to facilitate the reading of local reactions.
- % coverage: Approximately 10% of total body surface
- Type of wrap if used: The area of application was covered by a semi-occlusive dressing and wrapped with a piece of elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated skin and surrounding hair wiped with luke warm water and dried with disposable towl to remove any residual test item
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg
- Concentration (if solution): See below.
- Constant volume or concentration used: 4 mL volume ; at a dose level of 2000 mg/kg bw test item.
Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 per sex per dose (5 male/5 female)
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations and mortality checks were conducted at approximately 0.5, 1, 3, and 5 hours and subsequently twice daily for days 2 to 15. Local effects were examined once daily days 2 to 15 after the completion of the 24-hour exposure period. Full details on the scoring and criteria (appears consistent with Draize for Erythema) are given in the full study report. Individual bodyweights were recorded prior to application of the test item on Day 1 and on Days 8 and 15.
- Necropsy of survivors performed: yes
Statistics:
No statistical analyses were performed.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
- Clinical observations: No signs of systemic toxicity were noted during the observation period.
- Dermal reactions: All indicated slight (score = 1) to moderate (score =2) erythema from day 2 to day 6. The sites were re-shaved on day 8 after which one indication of slight erythema on days 8 to 12 and 9/10 indications of 'scaling'. See 'other information on results incl. tables' for further information.
Body weight:
Animals showed expected gains in body weight.
Gross pathology:
No abnormalities were noted at necropsy.

Applicant assessment indicates: within local effects the test item caused only mild transient irritation (score = 2) on day 2 which was then slight (score 1) on days 3, 4 and fully reversed by day 7. When the sites were re-shaved on day 8 this led to score = 1 slight irritation and scaling that persisted to the end of the observation period. This was clearly not a test item related effect, as the effects had already reversed on day 7 in all males/females (information provided in the full study report). The OECD TG 404 test guideline for skin irritation specifies that clipping of the fur is done at the start of the test and to attempts are to minimise abrasion of the skin which can interfere with assessment. It appears that the effects post day 8 were the result of a deviation for the reading of local effects which made fully reversed effects re-appear at the treated sites. Expert judgement indicates these were non-test item related effects.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar rat. Under the conditions of this study and under the Globally Harmonized Classification System of Classification and Labelling of Chemicals (GHS), the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.
Executive summary:

The study was performed according to OECD TG 402 and EU Method B.3 Acute Toxicity (Dermal) and in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar HanRcc: WIST (SPF) strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the diluted test item in PEG 300 vehicle to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There was no mortality during the study. There were no signs of system toxicity or abnormalities on necropsy. Animals showed expected gains in body weight. Minimal signs of dermal irritation were noted (score 2) at day 2 and (score 1) days 4 to 5 or 6, which had fully reversed at day 7. The dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar HanRcc: WIST (SPF) rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw
Quality of whole database:
The available information as a whole meets the tonnage driven information requirements of REACH.

Additional information

ORAL:

Key study : OECD TG 420, 2007: The study was performed according to OECD TG 420 and EU Method B.1 bis Acute Toxicity (Oral) and in accordance with GLP to assess the acute oral toxicity of the test material following a single oral administration in the female Sprague-Dawley Crl : CD (SD) IGS BR strain rat by the fixed dose method. The test item was administered by oral gavage in a solution in arachis oil BP in an initial sighting study at 300 mg/kg bw and following an absence of toxicity then at 2000 mg/kg bw. Subsequently, a further group of four fasted females was given a single oral dose of test item, at a dose level of 300 mg/kg body weight. The animal treated at a dose level of 2000 mg/kg was humanely terminated on 1 day after dosing. Clinical signs were noted during the day of dosing in the animal treated at a dose level of 2000 mg/kg. There was no mortality at a dose level of 300 mg/kg. There were no clinical signs of systemic toxicity and all animals showed expected gains in bodyweight over the study period at a dose level of 300 mg/kg. Abnormalities noted at necropsy of the animal treated at a dose level of 2000 mg/kg, that was humanely terminated, were abnormally red lungs and haemorrhage of the gastric mucosa, non-glandular region of the stomach and small and large intestines. No abnormalities were noted at a dose level of 300 mg/kg. Under the conditions of this study, the oral LD50 was estimated to be in the range of 300 - 2000 mg/kg bw in the female Sprague-Dawley CD strain rat.

INHALATION:

Key study: OECD TG 403, 2019: The study was performed according to OECD TG 403 and EU Method B.2: Acute Toxicity (inhalation) and in accordance with GLP to assess the acute inhalation toxicity of the test item following a single nose only exposure to rats at target concentrations of 1 and 5 mg/L for 4 hours. There was no male mortality at a dose level of 1 and 5 mg/L. However, female mortality was at 5 mg/L. Overall bodyweight gain at 1 mg/L were within the expected range for both sexes except, body weight loss was noted for all surviving animals at 5 mg/L up to Day 8 but recovery was noted in the second week. Similarly, no clinical or abnormal observation were noted in all animals at 1 mg/L. 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 the aforementioned signs. The surviving animals had recovered from the clinical signs on Day 4.  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. Under the conditions of this study, the LC50 was estimated to be in the range of >1 - ≤5 mg/L in the female Waster Han train rat.

DERMAL:

Key study : OECD TG 402, 2008 : The study was performed according to OECD TG 402 and EU Method B.3 Acute Toxicity (Dermal) and in accordance with GLP to assess the acute dermal toxicity of the test substance in the Wistar HanRcc: WIST (SPF) strain rat. A group of ten animals (five males and five females) was given a single, 24 hour, semi-occluded dermal application of the diluted test item in PEG 300 vehicle to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There was no mortality during the study. There were no signs of system toxicity or abnormalities on necropsy. Animals showed expected gains in body weight. Minimal signs of dermal irritation were noted (score 2) at day 2 and (score 1) days 4 to 5 or 6, which had fully reversed at day 7. The dermal LD50 was established to exceed 2000 mg/kg bw in male/female Wistar HanRcc: WIST (SPF) rat. Under the conditions of this study, and according to the GHS criteria, the LD50 cut-off value was considered to be greater than 5000 mg/kg body weight.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: oral Category 4

The substance meets classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: inhalation Category 4.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for acute toxicity: dermal

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