[No public or meaningful name is available]

Endpoint summary

Description of key information

Acute oral toxicity: OECD TG 420: LD50 > 2000 mg/kg bw.

Acute inhalation toxicity: OECD TG 403: LC50 > 5.11 mg/L

Acute dermal toxicity: OECD TG 402: LD 50 > 2000 mg/kg bw.

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:

Experimental start date: 20 April 2017 Experimental completion date: 10 May 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

Identification: FRET 13-0460
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animal Information
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight at the start of treatment.
Animal Care and Husbandry
The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

arachis oil

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.

Doses:

300 mg/kg and 2000 mg/kg

No. of animals per sex per dose:

at 300 mg/kg 1 female
at 2000 mg/kg 5 females

Control animals:

no

Details on 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 2000 mg/kg.

In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated at 2000 mg/kg.

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.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There was no mortality, at 300 mg/kg or 2000 mg/kg.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period, at 300 mg/kg or 2000 mg/kg.

Gross pathology:

No abnormalities were noted at necropsy at 300 mg/kg or 2000 mg/kg.

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The acute oral toxicity test showed an LD50 of greater than 2000 mg/kg bw and is therefore not classified according to CLP criteria.

Executive summary:

FRET 13 -0460 was tested according to OECD Guideline for Testing of Chemicals No 420 “Acute Oral Toxicity - Fixed Dose Procedure” (2001)

Acute oral toxicity: In this study, 6 rats (0 males and 6 females) were administered the substance at dose levels of 300 mg/kg bw and 2000 mg/kg. The rats showed no mortality, clinical signs or treatment related effects at necropsy. All animals showed expected increases in bodyweight over the observation period, except for one animal treated at 2000 mg/kg, which showed no gain in body weight during the first week but expected gain in body weight during the second week.The acute oral LD50 for the substance in male and female rats was determined to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The acute oral toxicity result is of sufficient quality and adequate for this dossier.

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:

The study was conducted between 29 June 2017 and 05 October 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

The deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

Identification: FRET 13-0460
Physical state/appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. 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. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.

Animal Care and Husbandry
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. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.65 µm

Geometric standard deviation (GSD):

2.28

Details on inhalation exposure:

Atmosphere Generation
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.
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).
Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied to achieve the required atmospheric conditions.

Exposure Procedure
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.
Following an appropriate equilibration period a single group of ten rats (five males and five females), was subjected to a single exposure to the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 102% of target and no deaths occurred, no further levels were required.

Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4 Hour exposure period.

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 4 Hour exposure period. The test atmospheres were generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
Prior to the inhalation phase 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 at room temperature 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 mean non-volatile component of the batch used during the formal exposure was found to be 69.78 % (n=8). A significant difference between the expected and achieved concentrations was noted during generation trials therefore it was considered that gravimetric analysis would not be accurate and that chemical analysis would be required.
The test atmosphere was sampled nine times during the exposure period. A weighed glass fiber filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump. The samples were then submitted for chemical analysis.
The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.
The nominal concentration was 373% of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straightforward.

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (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.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

A target concentration of 5.0 mg/L was used for the exposure.

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

Serial Observations
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.

Body Weight
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.

Terminal Investigations
Necropsy
All animals 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.

Data Evaluation
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.11 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality was observed.

Clinical signs:

other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations

Body weight:

All animals exhibited body weight loss or no body weight gain on the first day post-exposure. With the exception of one female from Days 1 to 3 post-exposure and three females from Days 3 to 7 post-exposure which exhibited body weight loss, body weight gains were noted for all animals during the remainder of the recovery period.

Gross pathology:

No macroscopic abnormalities were detected amongst animals at necropsy.

 Exposure Chamber Concentration

The actual concentration of the test item was determined by gas chromatography (GC) using an external standard technique. The test atmosphere was sampled nine times and the actual concentration of the test item calculated. The mean values obtained were as follows:

Group Number	Atmosphere Concentration
	Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
1	5.11	0.29	19.04

The chamber flow rate was maintained at 40 L/min providing 80 air changes per hour.

The theoretical chamber equilibration time (T₉₉) was 4 minutes[1](Silver, 1946).

Particle Size Distribution

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

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
1	5.11	2.65	69.2	2.28

[1]= The test atmosphere was generated for a total of 11 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

Temperature and Relative Humidity in Exposure Chamber

Time (minutes)	Chamber Temperature (ºC) During Exposure	Chamber Relative Humidity (%) During Exposure
0	21	41
30	21	40
60	21	43
90	21	41
120	21	41
150	21	44
180	21	44
210	21	42
240	21	42

Air Flow and Oxygen Concentration in Exposure Chamber

Time (minutes)	Air Flow (L/minute) During Exposure	Oxygen Concentration During Exposure
-11*	40	-
0	40	20.9
30	40	-
60	40	-
90	40	-
120	40	20.9
150	40	-
180	40	-
210	40	-
240	40	20.9

*= Test atmospheres were generated for a total of 11 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

-=    Not determined

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)	Volume of Air Sampled (L)	Chamber Flow Rate (L/min)	Atmosphere Concentration (mg/L)
10	2	40	4.62
30	2	40	4.92
60	2	40	4.99
90	2	40	4.99
120	2	40	5.14
150	2	40	5.17
180	2	40	5.35
210	2	40	5.20
230	2	40	5.65

Mean achieved atmosphere concentration (mg/L)		=5.11
Standard deviation		=0.29
Nominal concentration:
Test item used (g)	191.2
Air flow (L/minute)	40
Total generation time (minutes)	251[1]
Nominal concentration (mg/L)	19.04

[1]= Test atmospheres were generated for a total of 11 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution

Cascade Impactor Data

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	10.4	0.05	0.12	0.09	0.09
4	7.7	0.13	0.18	0.15	0.15
5	4.1	0.35	0.43	0.35	0.38
6	1.3	0.49	0.60	0.55	0.55
7	0.90	0.49	0.54	0.41	0.48
8	0.56	0.18	0.16	0.10	0.15
Back-up Filter	<0.56	0.00	0.08	0.00	0.03
Total Mean Amount of Test Item Collected					1.83

Calculation

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
10.4	1.017	1.74	95.1	6.65
7.7	0.887	1.59	86.9	6.12
4.1	0.613	1.21	66.1	5.42
1.3	0.114	0.66	36.1	4.64
0.90	-0.046	0.18	9.84	3.71
0.56	-0.252	0.03	1.64	2.87

 

Results
Mean Mass Median Aerodynamic Diameter	=2.65µm
Geometric Standard Deviation	=2.28
Predicted amount less than 4 µm	=69.2%

Mortality Data

Group Number	Mean Achieved Atmosphere Concentration (mg/L)	Sex	Deaths During Exposure	Deaths Post Exposure (1 Hour)	Deaths During Day of Observation								Total Deaths
					1	2	3	4	5	6	7	8-14
1	5.11	Male	0	0	0	0	0	0	0	0	0	0	0/10
		Female	0	0	0	0	0	0	0	0	0	0

Individual Clinical Observations – (Day of Exposure)

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Effects Noted During Exposure (Hours)			Effects Noted on Removal from Chamber	Effects Noted 1 Hour Post Exposure
		1	2	3
5.11	1 Male	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	2 Male	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	3 Male	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	4 Male	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	5 Male	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	6 Female	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	7 Female	WfRd	WfRd	WfRd	WfHPRd	WfHPRd
	8 Female	WfRd	WfRd	WfRd	WfHPRdRs	WfHPRd
	9 Female	WfRd	WfRd	WfRd	WfHPRdRs	WfHPRd
	10 Female	WfRd	WfRd	WfRd	WfHPRd	WfHPRd

Wf = Wet fur                        

Rd = Decreased respiratory rate                      

H=Hunched posture                          

P = Pilo-erection                                 

Rs = Sneezing

Individual Clinical Observations – Recovery Period

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Effects Noted Post Exposure (Days)
		1	2	3	4	5	6	7	8-14
5.11	1 Male	HP	0	0	0	0	0	0	0
	2 Male	HP	0	0	0	0	0	0	0
	3 Male	HP	H	0	0	0	0	0	0
	4 Male	HP	0	Rn	Rn	Rn	0	0	0
	5 Male	HPRnRs	HRnRs	0	0	0	0	0	0
	6 Female	HP	H	0	0	0	0	0	0
	7 Female	HP	0	0	0	0	0	0	0
	8 Female	HPRs	HRs	0	0	0	0	0	0
	9 Female	HP	H	0	0	0	0	0	0
	10 Female	HP	H	0	0	0	0	0	0

H=Hunched posture                          

P = Pilo-erection                                 

Rn = Noisy respiration                      

Rs = Sneezing                      

0 = No abnormalities detected

Individual Body Weights and Body Weight Changes

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Body Weight (g) at Day						Body Weight Change (g) During Days
		-8	0	1	3	7	14	-8 to 0	0 to 1	1 to 3	3 to 7	7 to 14
5.11	1 Male	202	248	234	245	259	291	46	-14	11	14	32
	2 Male	204	247	239	253	271	304	43	-8	14	18	33
	3 Male	206	250	237	245	263	303	44	-13	8	18	40
	4 Male	198	231	220	225	235	260	33	-11	5	10	25
	5 Male	202	245	237	241	264	295	43	-8	4	23	31
	6 Female	196	215	208	215	220	224	19	-7	7	5	4
	7 Female	204	210	207	213	211	219	6	-3	6	-2	8
	8 Female	207	222	222	221	226	243	15	0	-1	5	17
	9 Female	201	218	212	229	224	235	17	-6	17	-5	11
	10 Female	192	200	200	204	203	209	8	0	4	-1	6

 

 Individual Necropsy Findings

Mean Achieved Atmosphere Concentration (mg/L)	Animal Number and Sex	Time of Death	Macroscopic Observations
5.11	1 Male	Terminal Kill Day 14	No abnormalities detected
	2 Male	Terminal Kill Day 14	No abnormalities detected
	3 Male	Terminal Kill Day 14	No abnormalities detected
	4 Male	Terminal Kill Day 14	No abnormalities detected
	5 Male	Terminal Kill Day 14	No abnormalities detected
	6 Female	Terminal Kill Day 14	No abnormalities detected
	7 Female	Terminal Kill Day 14	No abnormalities detected
	8 Female	Terminal Kill Day 14	No abnormalities detected
	9 Female	Terminal Kill Day 14	No abnormalities detected
	10 Female	Terminal Kill Day 14	No abnormalities detected

Interpretation of results:

GHS criteria not met

Remarks:

4 hour LD50 is >5.11 mg/L

Conclusions:

The acute inhalation median lethal concentration (4 hour LC50) of FRET 13-0460 was greater than 5.11 mg/L.

Executive summary:

FRET 13-0460 was assessed for acute toxicity via inhalation using testing guideline OECD 403. In this study, 10 rats (5 males and 5 females) were exposed to an aerosol atmosphere of the test item at 5.11 mg/L. One day after exposure, all animals exhibited hunched posture and pilo-erection, with occasional instances of sneezing. Noisy respiration was noted in one male. Animals recovered to appear normal from Days 2 to 3 post-exposure, however one male subsequently exhibited noisy respiration from Days 3 to 5 post-exposure and appeared normal from Day 6 post-exposure. All animals exhibited body weight losses or no body weight gain on the first day post-exposure. With the exception of one female from Days 1 to 3 post-exposure and three females from Days 3 to 7 post-exposure which exhibited body weight loss, body weight gains were noted for all animals during the remainder of the recovery period. No macroscopic abnormalities were detected amongst animals at necropsy. The inhalatory LD50 for the substance in male and female rats was determined to be > 5.11 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 110 mg/m³ air

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:

The study was conducted between 17 May 2017 and 31 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Identification: FRET 13-0460
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.

Animal Care and Husbandry
The animals were housed 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. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

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 at 2000 mg/kg.

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 arachis oil BP to remove any residual test item. The animals were returned to group housing for the remainder of the study period.

Duration of exposure:

24 hours

Doses:

2000 mg/kg

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

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.

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.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period.

Gross pathology:

No abnormalities were noted at necropsy.

Interpretation of results:

GHS criteria not met

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 and is therefore not classified according to CLP criteria.

Executive summary:

FRET 13 -0460 was tested according to OECD Guideline for Testing of Chemicals No 402 “Acute Dermal Toxicity”

Acute dermal toxicity: In this study, 10 rats (5 males and 5 females) were administered the substance at a dose level of 2000 mg/kg. The rats showed no mortality, clinical signs or treatment related effects at necropsy. All animals showed expected increases in bodyweight over the observation period. The acute dermal LD50 for the substance in male and female rats was determined to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Acute oral toxicity:

FRET 13 -0460 was tested according to OECD Guideline for Testing of Chemicals No 420 “Acute Oral Toxicity - Fixed Dose Procedure” (2001)

In this study, 6 rats (0 males and 6 females) were administered the substance at dose levels of 300 mg/kg bw and 2000 mg/kg. The rats showed no mortality, clinical signs or treatment related effects at necropsy. All animals showed expected increases in bodyweight over the observation period,except for one animal treated at 2000 mg/kg, which showed no gain in body weight during the first week but expected gain in body weight during the second week.The acute oral LD50 for the substance in male and female rats was determined to be > 2000 mg/kg bw.

Acute inhalation toxicity:

FRET 13-0460 was assessed for acute toxicity via inhalation using testing guideline OECD 403. In this study, 10 rats (5 males and 5 females) were exposed to an aerosol atmosphere of the test item at 5.11 mg/L. One day after exposure, all animals exhibited hunched posture and pilo-erection, with occasional instances of sneezing. Noisy respiration was noted in one male. Animals recovered to appear normal from Days 2 to 3 post-exposure, however one male subsequently exhibited noisy respiration from Days 3 to 5 post-exposure and appeared normal from Day 6 post-exposure. All animals exhibited body weight losses or no body weight gain on the first day post-exposure. With the exception of one female from Days 1 to 3 post-exposure and three females from Days 3 to 7 post-exposure which exhibited body weight loss, body weight gains were noted for all animals during the remainder of the recovery period. No macroscopic abnormalities were detected amongst animals at necropsy. The inhalatory LD50 for the substance in male and female rats was determined to be > 5.11 mg/L.

Acute dermal toxicity:

FRET 13 -0460 was tested according to OECD Guideline for Testing of Chemicals No 402 “Acute Dermal Toxicity”

Acute dermal toxicity: In this study, 10 rats (5 males and 5 females) were administered the substance at a dose level of 2000 mg/kg. The rats showed no mortality, clinical signs or treatment related effects at necropsy. All animals showed expected increases in bodyweight over the observation period. The acute dermal LD50 for the substance in male and female rats was determined to be > 2000 mg/kg bw.

Justification for classification or non-classification

According to the criteria outlined in Regulation 1272/2008/EC (CLP), the substance does not have to be classified as acute toxic by the oral, inhalation, and dermal routes.