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Description of key information

The acute oral toxicity of the test substance, Fret 10-0367 was assessed according to OECD Test Guideline 420 using a fixed dose method. The LD50 was estimated to be greater than 2000 mg/kg body weight.


 


The acute dermal toxicity of the test substance, Fret 10-0367 was assessed according to OECD Test Guideline 402. The LD50 was greater than 2000 mg/kg body weight.


 


The acute inhalation toxicity of the test substance, Fret 10-0367 was assessed according to OECD Test Guideline 403. The 4 hour LC50 was greater than 4.97 mg/L, the mean achieved atmosphere concentration.

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:
The study was conducted between 21 September 2016 and 02 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST strain
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: [yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 154 - 178g
- Fasting period before study: overnight immediately before dosing
- Housing: Groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days

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

IN-LIFE DATES: From: 21 September 2016 To: 02 November 2016
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
For 300mg/kg dose only: 2000 mg/kg dose test material used as supplied
Details on oral exposure:
Test Item Preparation and Analysis
For the purpose of the 2000 mg/kg dose level, the test item was used as supplied. 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.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Study Design
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated at a dose level of 2000 mg/kg
In the absence of mortality at a dose level of 2000 mg/kg, an additional group of animals (4) was treated at a dose level of 2000 mg/kg.

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
300 mg/kg n = 1
2000 mg/kg n = 5
Control animals:
no
Details on study design:
Study Design
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
A single animal was treated as follows:
Dose Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Number of Rats (Female)
300 30 10 1

In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume (mL/kg) Number of Rats (Female)
2000 0.888 2.26 1

In the absence of mortality at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume(mL/kg) Number of Rats (Female)
2000 0.888 2.26 4

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.
Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily, early and late during normal working days, and once daily at weekends and public holidays.
Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained

Preliminary study:
Dose Level - 300 mg/kg:
Mortality
There was no mortality.

Clinical Observations
No signs of systemic toxicity were noted during the observation period.

Body Weight
The animal showed expected gains in body weight over the observation period.

Necropsy
No abnormalities were noted at necropsy.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
There were no deaths
Clinical signs:
Signs of systemic toxicity noted in three animals were hunched posture and lethargy. Ataxia was also noted in two animals and pilo erection was noted in one animal. These three animals appeared normal 1 or 2 days after dosing.
No signs of systemic toxicity were noted in two animals during the observation period.
Body weight:
All animals showed expected gains in body weight over the observation period.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
No other findings reported
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Category 5).
The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures
Executive summary:

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

Methods

Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item 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.

Results

Mortality.  There were no deaths.

Clinical Observations.  

Signs of systemic toxicity noted in three animals treated at a dose level of 2000 mg/kg were hunched posture, lethargy and ataxia or pilo erection.  There were no signs of systemic toxicity noted in two animals treated at a dose level of 2000 mg/kg or the animal treated at a dose level of 300 mg/kg.

Body Weight.  

All animals showed expected gains in body weight.

Necropsy.  

No abnormalities were noted at necropsy.

Conclusion

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Category 5).

The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Good

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 August 2016 to 15 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 to 12 weeks
- Weight at study initiation: within the range of 200 g to 350 g
- Fasting period before study: Not fasted
- Housing: The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet: free access, with the exception of the exposure period
- Water: free access, with the exception of the exposure period
- Acclimation period: at least 5 days
- Method of randomisation in assigning animals to test and control groups: On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.09 µm
Geometric standard deviation (GSD):
2.42
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure chamber volume: approximately 30 liters (dimensions: 28 cm diameter x 50 cm high)
- Method of holding animals in test chamber: 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 (airflow): 50 L/minute
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- 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). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 μm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (percentage) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 μm was calculated.
The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (percentage) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- 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: 20 ºC, 38-41%, negative pressure

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: ZB-5 (30 m x 0.53 mm id x 5 μm film)
- Samples taken from breathing zone: yes
- Time needed for equilibrium of exposure concentration before animal exposure: The theoretical chamber equilibration time (T99) was 3 minutes (Silver, 1946). The test atmosphere was generated for a total of 16 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.09 um/ 2.42
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
4.97 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 and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.97 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
0
Clinical signs:
bodyweight loss
Body weight:
All animals exhibited body weight losses on the first day post-exposure. With the exception of two male and two female animals which exhibited body weight losses or showed no body weight gain from Days 1 to 3 post-exposure and one female which showed no body weight gain from Days 7 to 14 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period.
Gross pathology:
With the exception of four instances of dark and/or pale patches on the lungs, no macroscopic abnormalities were detected amongst animals at necropsy.
Interpretation of results:
Category 4 based on GHS criteria
Executive summary:

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


No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 4.97 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 4.97 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
> 4.97 mg/L
Physical form:
inhalation: aerosol
Quality of whole database:
Good

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:
This study was conducted between 18 October 2016 and 01 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
24 February 1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: >200g
- Fasting period before study: none
- Housing: In suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of five, by sex, for the remainder of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days

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

IN-LIFE DATES: From: 18 October 2016 To: 01 November 2016
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, a single group of animals was treated.
The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. 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 for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
Duration of exposure:
24H
Doses:
1
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. 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 for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
The 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 the following scale:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to
slight eschar formation (injuries in depth) 4

Edema Formation Value
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter
and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

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:
no indication of skin irritation up to the relevant limit dose level
Mortality:
There were no deaths.
Clinical signs:
No signs of systemic toxicity were noted during the observation period.
Body weight:
All animals showed expected gains in body weight over the observation period.
Gross pathology:
No abnormalities were noted at necropsy
Other findings:
Dermal Reactions
Very slight erythema was noted at the test sites of three females 1 day after dosing. No other signs of dermal irritation were noted.
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight. (Globally Harmonized Classification System - Category 5).
The test item does not meet the criteria for classification according to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.

Methods

A group of ten animals (five males and five females) was 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 body weight.  Clinical signs and body weight development were monitored during the study.  All animals were subjected to gross necropsy.

Results

Mortality.  There were no deaths.

Clinical Observations.  There were no signs of systemic toxicity.

Dermal Irritation.  Very slight erythema was noted at the test sites of three females 1 day after dosing.  No other signs of dermal irritation were noted.

Body Weight.  All animals showed expected gains in body weight.

Necropsy.  No abnormalities were noted at necropsy.

Conclusion

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals or to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Good

Additional information

Justification for classification or non-classification

Substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. Acute toxicity values are expressed as approximate LD50 or LC50 (inhalation) values.

 

A test substance is classified according to one of these four toxicity categories when the acute LD50 value is ≤ 2000 mg/kg for exposure via the oral and dermal routes, or where the acute LC50 value is ≤ 5 mg/L for exposure via the inhalation route (dusts and mists).

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an

acute oral LD50 of > 2000 mg/kg and therefore the test substance, Fret 10 -0367, is not classified for acute oral toxicity.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute dermal LD50 of > 2000 mg/kg and therefore the test substance, Fret 10 -0367, is not classified for acute dermal toxicity.