Registration Dossier

Administrative data

Description of key information

Oral LD50 (Females) > 2000 mg/kg bw (OECD 423, K, Rel.1, class method in rats)

Dermal LD50 (Combined) > 2000 mg/kg bw (OECD 402, K, Rel.1, limit test in rats)

Inhalation LC50 (Females) = 4.84 mg/L (= 4840 mg/m3) (OECD 403, K, Rel.1, nose-only in rats)

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:
20 November to 23 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 423 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau, November 24, 2000.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 01-03 July 2014 / signed on 15 September 2014)
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Date of receipt: 06 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
Species:
rat
Strain:
Wistar
Remarks:
RccHan®:WIST
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan (UK) Ltd
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: ca. 8-12 weeks
- Weight at study initiation: 154-185 g
- Fasting period before study: Animals were fasted overnight prior to and approximately four hours after dosing.
- Housing: Animals were housed in groups of three rats of the same sex. The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid.
- Diet: Animals were allowed free access to a standard rodent diet (Harlan Teklad 2014C Diet), except for overnight prior to and approximately four hours after dosing.
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 20 November to 23 December 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: The test substance was formulated at concentrations of 30 or 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg bw. The test substance formulations were prepared on the day of dosing.

CLASS METHOD
- Rationale for the selection of the starting dose: The dose levels for the study were chosen in compliance with the study guidelines. As no previous toxicological information was available the initial dose level was 300 mg/kg bw.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
6 females/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations:
Mortality: Cages of rats were checked at least twice daily for any mortalities.
Clinical signs: Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation. All animals were observed for 14 days after dosing.
- Frequency of weighing: The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15. Individual weekly body weight changes were calculated.
- Necropsy of survivors performed: Yes; All animals were humanely killed on Day 15 by carbon dioxide asphyxiation and subjected to gross necropsy.
Statistics:
None
Preliminary study:
Not applicable
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality was observed
Mortality:
There were no deaths during the study.
Clinical signs:
- Clinical signs of reaction to treatment were seen in one or more animals dosed at 2000 mg/kg bw comprised of piloerection, decreased activity, unsteady gait and hunched posture. These signs were first noted on Day 1. Recovery of all animals, as judged by external appearance and behaviour, was complete by Day 3.
- No clinical signs were seen in any animal dosed at 300 mg/kg bw.
Body weight:
All animals were considered to have achieved satisfactory body weight gains throughout the study.
Gross pathology:
No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.
Other findings:
None

None

Interpretation of results:
GHS criteria not met
Conclusions:
Oral LD50 (females) >2000 mg/kg bw
Executive summary:

In an acute oral toxicity study performed according to OECD Guideline 423 and in compliance with GLP, two groups of three fasted female rats received a single oral gavage dose of the test substance, formulated in corn oil, at a dose level of 300 mg/kg bw. As results at this dose level indicated the acute (median) lethal oral dose of the test substance to be greater than 300 mg/kg bw, in compliance with the study guidelines a further two groups of three fasted females were similarly dosed at 2000 mg/kg bw to complete the study. Animals were then observed for mortality, clinical signs and bodyweights for 14 days and were all sacrificed for macroscopic examination.

 

There were no deaths during the study. Clinical signs of reaction to treatment were seen in one or more animals dosed at 2000 mg/kg bw comprised of piloerection, decreased activity, unsteady gait and hunched posture. These signs were first noted on Day 1. Recovery of all animals, as judged by external appearance and behaviour, was complete by Day 3. No clinical signs were seen in any animal dosed at 300 mg/kg bw. All animals were considered to have achieved satisfactory body weight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Oral LD50 (females) >2000 mg/kg bw

 

Under the experimental conditions of this study, the acute median lethal oral dose (LD50) was demonstrated to be greater than 2000 mg/kg bw. Thus the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.

This study is considered as acceptable and satisfies the requirement for acute oral toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
The key study is GLP compliant and of high quality (Klimisch score = 1)

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:
23 March to 25 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 403 without any deviation.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 12-14 March 2014 / signed on 12 May 2014)
Test type:
traditional method
Limit test:
no
Specific details on test material used for the study:
- Purity test date: 02 September 2014
- Storage condition of test material: Stored cold under nitrogen at approximately 4°C, in the dark
Species:
rat
Strain:
Wistar
Remarks:
RccHan™ : WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 200-350 g
- Housing: Animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet: Food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

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

N-LIFE DATES: 23 March to 25 June 2015
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.26 - <= 3.43 µm
Geometric standard deviation (GSD):
>= 2.06 - <= 2.26
Remark on MMAD/GSD:
Mean Mass Median Aerodynamic Diameter (MMAD) = 3.29, 3.26 and 3.43 μm at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively
Geometric Standard Deviation (GSD) = 2.18, 2.06 and 2.26 at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber.
- Exposure chamber volume: Cylindrical exposure chamber had a volume of approximately 30 L (dimensions: 28 cm diameter x 50 cm high).
- One day prior to the day of exposure each rat was acclimatized (for approximately 2 h) to a tapered polycarbonate restraining tube.
- Method of holding animals in test chamber: During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- 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 and humidity in air chamber: Temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds.
, UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Temperature and humidity in air chamber were 19-20 °C and 26-41%, respectively.
- Exposure chamber oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.
- Exposure Chamber Atmosphere Concentration: Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator between 18 and 20 °C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The non-volatile component of the batch used during the study was found to be approximately 8.62 % (n=2). Due to the very low levels of non-volatiles contained within this test item it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations for all exposure groups.
The test atmosphere was sampled nine times during each exposure period. The sampling procedure involved 2 to 3 liters of test atmosphere (dependent on concentration) being drawn through a glass impinger or a series of two glass impingers (dependent on concentration) containing Methanol (each made up to 80 mL). The samples were then submitted for chemical analysis.
The nominal chamber concentrations were calculated by dividing the mass of test item used by the total volume of air passed through the chamber during each exposure.
The nominal concentrations were 434 %, 366 % and 372 % of the actual mean achieved atmosphere concentration for Groups 1, 2 and 3 respectively and show that keeping the aerosol airborne was relatively straightforward for all dose groups.
- Pretreatment: Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied to achieve the required atmospheric conditions.
- Following an appropriate equilibration period three groups, each of five or ten rats (five males and/or five females), were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test item was measured off-line by high performance gas chromatography (GC).
- Samples taken from breathing zone: Yes
- Particle size distribution: See table 7.2.2/2
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Mean Mass Median Aerodynamic Diameter (MMAD) = 3.29, 3.26 and 3.43 μm at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively; Geometric Standard Deviation (GSD) = 2.18, 2.06 and 2.26 at mean achieved concentrations of 5.05, 3.25 and 4.62 mg/L, respectively.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Nominal concentration: 21.9, 11.9 and 17.2 mg/L
Mean achieved atmosphere concentration: 5.05, 3.25 and 4.62 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Duration of observation period following administration: 14 days
- Frequency of observations: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.
- Frequency of weighing: Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.
- Necropsy of survivors performed: Yes, at the end of the 14 day observation period all animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died or were humanely killed during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) was calculated using validated data analysis software (R Environment, (The R Foundation for Statistical Computing, Vienna, Austria.)). This software utilized Log-Normal (Probit) regression models in order to calculate the LC50 values. LC50 values and 95% confidence limits were calculated for males and females separately.
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
5.23 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 4.45 - <= 6.01
Exp. duration:
4 h
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
4.84 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 4.29 - <= 5.38
Exp. duration:
4 h
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
5.18 mg/L air (analytical)
Based on:
test mat.
95% CL:
>= 3.98 - <= 6.38
Exp. duration:
4 h
Mortality:
At 5.05 mg/L, 1/5 male and 3/5 females died
At 3.25 mg/L, 0/5 female died (males not exposed)
At 4.62 mg/L, 2/5 females died (males not exposed)
Clinical signs:
Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection, and wet fur. There were frequent instances of labored respiration, occasional instances of noisy respiration, ataxia, coma and red/brown staining around the eyes and snout and isolated occurrences of exophthalmos, dehydration, pallor of the extremities, lethargy, ptosis and prostration. Surviving Group 1 animals appeared normal on Day 7 post-exposure. All Group 2 animals recovered to appear normal on Day 3 post-exposure and surviving animals from Group 3 recovered to appear normal on Day 6 post-exposure.
Body weight:
Group 1 – All surviving animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were then noted in all surviving animals during the remainder of the recovery period.
Group 2 – All animals exhibited body weight losses on the first day post-exposure. With the exception of one animal, which showed no body weight gain during the final week of the recovery period, body weight gains were noted in all animals during the remainder of the recovery period.
Group 3 – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.
Gross pathology:
With the exception of one male animal from Group 1 which exhibited dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods.
The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study: Lungs – dark patches; Small intestine – contained dark substance.
Due to the observations noted during the study and at necropsy it is considered that deaths noted during the study may have been mainly attributable to systemic toxicity.
Other findings:
None

Table 7.2.2/1: Exposure Chamber Concentration

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

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

Group Number

 

Mean Achieved (mg/L)

 

Standard Deviation

 

Nominal (mg/L)

 

1

5.05

0.12

21.9

2

3.25

0.23

11.9

3

4.62

0.08

17.2

 

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

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

Table 7.2.2/2: Particle Size Distribution

Group Number

 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (μm)

Inhalable Fraction

(% <4 μm)

Geometric Standard Deviation

1

5.05

3.29

60.0

2.18

2

3.25

3.26

61.2

2.06

3

4.62

3.43

57.6

2.26

 

Table 7.2.2/3: Mortality Data

Group Number

 

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

 

Male

 

Female

Total

1

5.05

1/5

3/5

4/10

2

3.25

NOT EXPOSED

0/5

0/5

3

4.62

NOT EXPOSED 

2/5

2/5

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The inhalation LC50 and 95% confidence limits of test item were calculated to be
All animals: 5.23 (4.45 – 6.01) mg/L; Males only: 5.18 (3.98 – 6.38) mg/L; Females only: 4.84 (4.29 – 5.38) mg/L
Under the test conditions, the test item is classified as Category 4 according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.
Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 403 and in compliance with GLP, three groups of five or ten RccHan™ : WIST strain rats (five males and/or five females) were exposed to an aerosol atmosphere of the test item for 4 hours.

Nominal concentration: 21.9, 11.9 and 17.2 mg/L (corresponding to mean achieved atmosphere concentrations: 5.05, 3.25 and 4.62 mg/L, respectively)

Animals were observed for mortality, clinical signs and body weight for 14 days and at the end of the study the animals were sacrificed for macroscopic examination.

 

At 5.05 mg/L, 1/5 male and 3/5 females died;

At 3.25 mg/L, 0/5 female died (males not exposed);

At 4.62 mg/L, 2/5 females died (males not exposed).

 

Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection, and wet fur. There were frequent instances of labored respiration, occasional instances of noisy respiration, ataxia, coma and red/brown staining around the eyes and snout and isolated occurrences of exophthalmos, dehydration, pallor of the extremities, lethargy, ptosis and prostration. Surviving Group 1 animals appeared normal on Day 7 post-exposure. All Group 2 animals recovered to appear normal on Day 3 post-exposure and surviving animals from Group 3 recovered to appear normal on Day 6 post-exposure.

 

Group 1 (5.05 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were then noted in all surviving animals during the remainder of the recovery period.

Group 2 (3.25 mg/L) – All animals exhibited body weight losses on the first day post-exposure. With the exception of one animal, which showed no body weight gain during the final week of the recovery period, body weight gains were noted in all animals during the remainder of the recovery period.

Group 3 (4.62 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.

 

With the exception of one male animal from Group 1 which exhibited dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods. The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study: Lungs – dark patches; Small intestine – contained dark substance.

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

 

The inhalation LC50 and 95% confidence limits of test item were calculated to be

All animals: 5.23 (4.45 – 6.01) mg/L; Males only: 5.18 (3.98 – 6.38) mg/L; Females only: 4.84 (4.29 – 5.38) mg/L

Under the test conditions, the test item is classified as Category 4 according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
4 840 mg/m³
Quality of whole database:
The key study is GLP compliant and of high quality (Klimisch score = 1)

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:
23 September to 21 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 402 without any deviation.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015/ signed on 24 September 2015)
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen
Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 264-293 g (males); 203-236 g (females)
- Housing: Animals were housed in suspended solid-floor polypropylene cages furnished with woodflakes.The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24-hour exposure period and in groups of four, by sex, for the remainder of the study.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 23 September to 21 October 2015
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Pretreatment: On the day before treatment the back and flanks of each animal were clipped free of hair.
- Area of exposure: Back and flank area
- In the absence of data suggesting the test item was toxic, a single group of animals was initially treated.
- % coverage: Approximately 10 % of the total body surface area.
- Application of test item: Test item was used as supplied. The test item was applied as evenly as possible to an area of shorn skin (approximately 10 % of the total body surface area) using a graduated syringe.
- Type of wrap if used: A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage. The animals were caged individually throughout the study. Shortly after dosing the dressings were examined to ensure that they were securely in place.
- As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg bw to give a total of five males and five females. The animals were caged individually for the 24-hour exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: After the 24-hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item. These animals were returned to group housing for the remainder of the test period.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Specific gravity of test item: 0.865
- Constant volume or concentration used: Yes; 2.32 mL/kg bw
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: Animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days. After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored according to Draize scale.
- Frequency of weighing: Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: Yes; at the end of the study animals were killed by cervical dislocation and subjected to gross necropsy.
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality was observed
Mortality:
No mortality was observed.
Clinical signs:
No signs of systemic toxicity were noted during the observation period.
Body weight:
Animals showed expected gains in body weight, except for two females which showed body weight loss during the first week but expected gain in body weight during the second week.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
None

None

Interpretation of results:
GHS criteria not met
Conclusions:
Dermal LD50 Combined > 2000 mg/kg bw
Executive summary:

An acute dermal toxicity study (limit test) was performed according to OECD Guideline No. 402 and in compliance with GLP. Initially, two rats (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg bw. Based on the results of the initial test, a further group of eight rats (four males and four females) was similarly treated. Test sites were covered with a semi-occlusive dressing for 24 h. Animals were observed for mortality, clinical signs and bodyweights for 14 days and at the end of the study the surviving animals were sacrificed for macroscopic examination. Skin irritation was assessed and scored according to the Draize scale at 24 h after removal of the dressings and then daily for 14 days.

 

No mortality was observed. No signs of systemic toxicity and dermal irritation were noted during the observation period. Animals showed expected gains in body weight, except for two females which showed body weight loss during the first week but expected gain in body weight during the second week. No abnormalities were noted at necropsy.

 

Dermal LD50 Combined > 2000 mg/kg bw

 

Under the experimental conditions of this study, the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.

This study is considered as acceptable and satisfies the requirement for acute dermal toxicity endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
The key study is GLP compliant and of high quality (Klimisch score = 1)

Additional information

Acute toxicity: oral

A key study was identified (HLS, 2015, rel. 1). In this acute oral toxic class method study performed according to the OECD guideline No. 423 and in compliance with GLP, two groups of three fasted female rats received a single oral gavage dose of the test substance, formulated in corn oil, at a dose level of 300 mg/kg bw. As results at this dose level indicated the acute (median) lethal oral dose of the test substance to be greater than 300 mg/kg bw, in compliance with the study guidelines a further two groups of three fasted females were similarly dosed at 2000 mg/kg bw to complete the study. There were no deaths during the study. Clinical signs of reaction to treatment were seen in one or more animals dosed at 2000 mg/kg bw comprised of piloerection, decreased activity, unsteady gait and hunched posture. These signs were first noted on Day 1. Recovery of all animals, as judged by external appearance and behaviour, was complete by Day 3. No clinical signs were seen in any animal dosed at 300 mg/kg bw. All animals were considered to have achieved satisfactory body weight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Oral LD50 (females) > 2000 mg/kg bw

 

Acute toxicity: dermal

A key study was identified (Envigo, 2015, rel. 1). In this limit acute dermal toxicity study performed according to the OECD guideline No. 402 and in compliance with GLP, initially two rats (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg bw. Based on the results of the initial test, a further group of eight rats (four males and four females) was similarly treated. Test sites were covered with a semi-occlusive dressing for 24 h. No mortality was observed. No signs of systemic toxicity and dermal irritation were noted during the observation period. Animals showed expected gains in body weight, except for two females which showed body weight loss during the first week but expected gain in body weight during the second week. No abnormalities were noted at necropsy.

Dermal LD50 (Combined) > 2000 mg/kg bw

 

Acute toxicity: inhalation

A key study was identified (Harlan, 2015, rel. 1). In this acute inhalation toxicity study performed according to the OECD Guideline No. 403 and in compliance with GLP, three groups of five or ten Wistar (RccHan™:WIST) strain rats (five males and/or five females) were exposed to an aerosol atmosphere of the test item for four hours followed by a fourteen day observation period. The nominal concentrations were 21.9, 11.9 and 17.2 mg/L, corresponding to mean achieved atmosphere concentrations: 5.05, 3.25 and 4.62 mg/L, respectively.

At 5.05 mg/L, 1/5 male and 3/5 females died; At 3.25 mg/L, 0/5 female died (males not exposed);

At 4.62 mg/L, 2/5 females died (males not exposed).

 

Common abnormalities noted during the study included decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection, and wet fur. There were frequent instances of labored respiration, occasional instances of noisy respiration, ataxia, coma and red/brown staining around the eyes and snout and isolated occurrences of exophthalmos, dehydration, pallor of the extremities, lethargy, ptosis and prostration. Surviving Group 1 animals appeared normal on Day 7 post-exposure. All Group 2 animals recovered to appear normal on Day 3 post-exposure and surviving animals from Group 3 recovered to appear normal on Day 6 post-exposure.

 

Group 1 (5.05 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were then noted in all surviving animals during the remainder of the recovery period.

Group 2 (3.25 mg/L) – All animals exhibited body weight losses on the first day post-exposure. With the exception of one animal, which showed no body weight gain during the final week of the recovery period, body weight gains were noted in all animals during the remainder of the recovery period.

Group 3 (4.62 mg/L) – All surviving animals exhibited body weight losses on Day 1 post-exposure and from Days 1 to 3 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.

 

With the exception of one male animal from Group 1 which exhibited dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods. The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study: Lungs – dark patches; Small intestine – contained dark substance.

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

The inhalation LC50 and 95% confidence limits of test item were calculated to be

All animals: 5.23 (4.45 – 6.01) mg/L; Males only: 5.18 (3.98 – 6.38) mg/L; Females only: 4.84 (4.29 – 5.38) mg/L

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Acute toxicity (Oral):

Based on the available information, the substance is:

- not classified according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) as the oral LD50 is higher than 2000 mg/kg bw

- not classified according to the GHS since there is no reliable evidence that indicates the LD50 to be in the range of Category 5 values (GHS criteria not met).

 

Acute toxicity (Dermal):

Based on the available information, the substance is:

- not classified according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) as the dermal LD50 is higher than 2000 mg/kg bw

- not classified according to the GHS since there is no reliable evidence that indicates the LD50 to be in the range of Category 5 values (GHS criteria not met)

 

Acute toxicity (Inhalation):

Based on the available information, the substance is :

- classified in Category 4 according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) as the inhalation LC50 (Females) is 4.84 mg/L.

- classified in Category 4 according to the GHS as the LC50 is 4.84 mg/L.

 

Specific target organ toxicity: single exposure (Oral):

The classification criteria according to the Annex VI of the Regulation (EC) No. 1272/2008 as specific target organ toxicant (STOT) – single exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value, oral for a Category 1 classification (C≤ 300 mg/kg bw) and at the guidance value, oral for a Category 2 classification (2000 mg/kg bw ≥C > 300 mg/kg bw). No classification is required.

The criteria for Transient Organ effects (STOT-SE Category 3) according to Annex VI of the Regulation (EC) No. 1272/2008 are not met since narcotic effects were not observed in the acute oral toxicity study.

 

Specific target organ toxicity: single exposure (Dermal):

The classification criteria according to the Annex VI of the Regulation (EC) No 1272/2008 as specific target organ toxicant (STOT) – single exposure, dermal are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value, dermal for a Category 1 classification (C≤ 1000 mg/kg bw) and at the guidance value, dermal for a Category 2 classification (2000 mg/kg bw ≥C > 1000 mg/kg bw). No classification is required.

The criteria for Transient Organ effects (STOT-SE Category 3) according to Annex VI of the Regulation (EC) No. 1272/2008 are not met since narcotic effects were not observed in the acute dermal toxicity study.

 

Specific target organ toxicity: single exposure (Inhalation):

The classification criteria according to the Annex VI of the Regulation (EC) No 1272/2008 as specific target organ toxicant (STOT) – single exposure, inhalation are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value (inhalation, dust/mist) for a Category 1 classification (C≤ 1 mg/L) and at the guidance value (inhalation, dust/mist) for a Category 2 classification (5 mg/L ≥C > 1 mg/L). No classification is required.

The criteria for Transient Organ effects (STOT-SE Category 3 / H336) according to Annex VI of the Regulation (EC) No. 1272/2008 are not met since narcotic effects were not observed in the acute inhalation toxicity study.