Registration Dossier

Administrative data

Description of key information

The acute oral toxicity of the test substance, IFF 215 (Floriane) 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 inhalation toxicity of the test substance, IFF 215 (Floriane) was assessed according to OECD Test Guideline 403 using a standard acute method. The 4 hr LC50 was greater than 5.33 mg/L.
The acute dermal toxicity of the test substance, IFF 215 (Floriane) was assessed according to OECD Test Guideline 402. The LD50 of greater than 2000 mg/kg body weight.

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 14 January 2016 and 10 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 420 using a fixed dose method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Justification
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Animal Welfare
The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo - Shardlow policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo - Shardlow Ethical Review Process.
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.

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.
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.
A single animal was treated as follows:
Dose level: 300 mg/kg
Concentration: 30 mg/mL
Dose volume: 10 mL/kg
Number of rats: 1 female

In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated as follows:
Dose level: 2000 mg/kg
CSpecific gravity: 0.922
Dose volume: 2.17 mL/kg
Number of rats: 1 female

In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose level: 2000 mg/kg
CSpecific gravity: 0.922
Dose volume: 2.17 mL/kg
Number of rats: 4 females

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.
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
A single animal was treated at 300 mg/kg.
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 four animals were treated at 2000 mg/kg.
A total of five animals were therefore treated at a dose level of 2000 mg/kg.
Control animals:
no
Details on study design:
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.
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.
Remarks on result:
other: There was no mortality during the study.
Mortality:
Dose Level - 300 mg/kg
There was no mortality.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
There were no deaths.
Clinical signs:
Dose Level - 300 mg/kg
No signs of systemic toxicity were noted during the observation period.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
No signs of systemic toxicity were noted in the initial treated animal during the observation period.
Signs of systemic toxicity noted in the additional treated animals were hunched posture, ataxia, pilo erection, tiptoe gait, increased salivation, noisy respiration, sneezing and red/brown staining around the eyes. These four animals appeared normal 2 days after dosing.
Body weight:
Dose Level - 300 mg/kg
The animal showed expected gains in body weight over the observation period.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
Animals showed expected gains in body weight over the observation period, except for one animal which showed expected gain in body weight during the first week but no gain in body weight during the second week.
Gross pathology:
Dose Level - 300 mg/kg
No abnormalities were noted at necropsy.

Dose Level - 2000 mg/kg
Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated.
No abnormalities were noted at necropsy.

Individual Clinical Observations and Mortality Data -300mg/kg

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

300

1-0
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0=   No signs of systemic toxicity

Individual Body Weights and Body Weight Changes -300mg/kg

Dose Level

mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Gain (g)
During Week

0

7

14

1

2

300

1-0 Female

179

191

193

12

2

Individual Necropsy Findings -300 mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

300

1-0 Female

Killed Day 14

No abnormalities detected

Individual Clinical Observations and Mortality Data -2000mg/kg

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

2-0
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0
Female

0

0

H

HA

HAPWtSe

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1
Female

0

0

H

HAWt

HAWt

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2
Female

0

0

H

HAWt

HWt

0

0

0

0

0

0

0

0

0

0

0

0

0

3-3
Female

0

RnS

HSRnRs

HARnRs

HRs

0

0

0

0

0

0

0

0

0

0

0

0

0

0=   No signs of systemic toxicity

H = Hunched posture,

A =  Ataxia

P =   Pilo‑erection

Wt =Tiptoe gait

S =   Increased salivation

Rn =Noisy respiration,

Rs =Sneezing

Se = red/brown staining around the eyes

Individual Body Weights and Body Weight Changes -2000mg/kg

Dose Level

mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Gain (g) During Week

0

7

14

1

2

2000

2-0 Female

155

166

179

11

13

3-0 Female

173

186

201

13

15

3-1 Female

178

190

202

12

12

3-2 Female

163

171

186

8

15

3-3 Female

176

187

187

11

0

Individual Necropsy Findings -2000mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

2-0 Female

Killed Day 14

No abnormalities detected

3-0 Female

Killed Day 14

No abnormalities detected

3-1 Female

Killed Day 14

No abnormalities detected

3-2 Female

Killed Day 14

No abnormalities detected

3-3 Female

Killed Day 14

No abnormalities detected

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
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).
Executive summary:

The acute oral toxicity of the test substance, IFF 215 (Floriane), 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

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 03 November 2015 and 11 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 403 using a standard acute method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals and Animal Husbandry
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 five 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 eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

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 (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK). With the exception of the exposure period, free access to mains drinking water and food (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding, chew blocks and cardboard “fun tunnels” are routinely analyzed and are 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 °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 twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Inhalation Exposure
Atmosphere Generation
The test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, 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.

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
One day prior to the day of exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the day of exposure, 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 exposed to an atmosphere of the test item for a period of four hours. After the results of the sighting study were available a target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 107 % of target and only one death occurred, no further levels were required.


Sighting Exposure
During characterization, a group of two rats (one male, one female) were exposed to an atmosphere of the test item at a mean achieved atmosphere concentration of 2.43 mg/L for approximately four hours. No significant effects were noted for either animal.


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 thirty minutes throughout the four-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 four-hour exposure period. The test atmosphere was 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 non-volatile component of the batch used during the study was found to be approximately 0.73 % (n=2). Due to the 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 the exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing Hexane (made up to 80mL). The samples were then submitted for chemical analysis.

The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.
The nominal concentration was 595 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.


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 (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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 (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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 (%) 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:
Mean achieved atmosphere concentration was 5.33 mg/L (0.14 standard deviation); nominal concentration 31.7 mg/L.
No. of animals per sex per dose:
Five per sex per dose
Control animals:
no
Details on study design:
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, one hour after termination of exposure and subsequently once daily for fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.


Body Weight
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
At the end of the fourteen day observation period the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including the one that was humanely killed during the course of 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.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.33 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: One animal died in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.33 mg/L for four hours
Mortality:
One male died during the study. There was no female mortality.
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 a
Body weight:
All male animals and four females exhibited body weight losses on the first day post-exposure. With the exception of one female animal which exhibited a slight body weight loss from Days 3 to 7 post-exposure, body weight gains were noted for all surviving animals during the final week of the recovery period.
Gross pathology:
Dark patches on the lungs were noted amongst two animals that survived until the end of the recovery period at necropsy, no macroscopic abnormalities were detected amongst the other surviving seven animals.

Dark patches on the lungs were noted at necropsy in the animal that was humanely killed during the course of the study:

Due to the clinical observations noted and macroscopic abnormalities detected, the contributing factor to the death noted during the study may have been mainly attributable to systemic toxicity.

Exposure Chamber Concentration

The actual concentration of the test item wasdeterminedby gaschromatography (GC). The test atmosphereswere sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during the exposure period. The mean values obtained were:

 

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.33

0.14

31.7

 

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[1](Silver, 1946).

 

Particle Size Distribution

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

 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

5.33

3.40

57.0

2.50

[1] = Test atmospheres were generated for a total of 8 minutes prior to animal insertion to ensure test item concentration was being achieved.

Mortality Data

The mortality data are summarized as follows:

 

Mean Achieved Atmosphere Concentration
(mg/L)

Deaths

Male

Female

Total

5.33

1/5

0/5

1/10

 

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

10

2

60

5.14

30

2

60

5.15

60

2

60

5.34

90

2

60

5.23

120

2

60

5.43

150

2

60

5.46

180

2

60

5.41

210

2

60

5.27

230

2

60

5.52

Mean achieved atmosphere concentration (mg/L) = 5.33

Standard deviation = 0.14

 

Nominal concentration:

Test item used (g)

472

Air Flow (L/min)

60

Total Generation Time (mins)

248[1]

Nominal Concentration (mg/L)

31.7

[1] = Test atmospheres were generated for a total of 8 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

8.4

0.28

0.29

0.25

0.27

4

7.3

0.53

0.56

0.52

0.54

5

3.6

0.53

0.64

0.70

0.62

6

1.3

0.76

0.81

0.92

0.83

7

0.94

0.31

0.36

0.38

0.35

8

0.43

0.03

0.02

0.05

0.03

Back-up Filter

<0.43

0.07

0.08

0.09

0.08

Total Mean Amount of Test Item Collected

2.72

 

Calculation 

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.45

90.1

6.29

7.3

0.863

1.91

70.2

5.53

3.6

0.556

1.29

47.4

4.94

1.3

0.114

0.46

16.9

4.04

0.94

-0.027

0.11

4.04

3.25

0.43

-0.367

0.08

2.94

3.11

 

Results 

Mean Mass Median Aerodynamic Diameter (MMAD) = 3.40µm

Geometric Standard Deviation (GSD) = 2.50

Predicted amount less than 4 µm = 57.0%

Mortality Data

MeanAchievedAtmosphere 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

5.33

Male

0

0

1[1]

0

0

0

0

0

0

0

1/10

Female

0

0

0

0

0

0

0

0

0

0

[1]= Humanely killed as considered unlikely to survive

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
One animal died in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.33 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of IFF 215 (Floriane), in the RccHanTM : WIST strain rat, was greater than 5.33 mg/L.
Executive summary:

The acute toxicity of the test substance, IFF 215 (Floriane), via the inhalation route was assessed according to OECD Test Guideline 403 using a standard acute method. The 4 hr LC50 was greater than 5.33 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5.3 mg/m³

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 21 January 2016 and 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 402 using a limit test method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animal Welfare
The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo - Shardlow policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo - Shardlow Ethical Review Process.
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.

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 °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.
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Test Item Preparation and Analysis
For the purpose of the study 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.
The absorption of the test item was not determined.

Study Design
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 as follows:

Dose level: 2000 mg/kg
Specific gravity: 0.922
Dose volume: 2.17 mg/kg
Number of rats: 5 male and 5 female

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 male and 5 female
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.
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.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths.
Clinical signs:
No signs of systemic toxicity were noted during the observation period.
Body weight:
Animals showed expected gains in body weight over the observation period, except for one female which showed no gain in body weight during the first week but expected gain in body weight during the second week.
Gross pathology:
No abnormalities were noted at necropsy.

Individual Clinical Observations and Mortality Data

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-1
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-2
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-3
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-4
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-0
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-1
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-2
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-3
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-4

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 = No signs of systemic toxicity

Individual Dermal Reactions - Males

Dose Level mg/kg

Animal Number and Sex

Observation

Effects Noted After Initiation of Exposure (Days)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0
Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-1
Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-2
Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-3
Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-4
Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 = No reactions

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.
Executive summary:

The acute dermal toxicity of the test substance, IFF 215 (Floriane) was assessed according to OECD Test Guideline 402 using a limit test method. The LD50 was found to be greater than 2000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Additional information

Justification for selection of acute toxicity – oral endpoint
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

Justification for selection of acute toxicity – inhalation endpoint
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

Justification for selection of acute toxicity – dermal endpoint
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

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, IFF 215 (Floriane), 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 inhalation LC50 of >5.33 mg/L and therefore the test substance, IFF 215 (Floriane), is not classified for acute inhalation 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, IFF 215 (Floriane), is not classified for acute dermal toxicity.