Registration Dossier

Administrative data

Description of key information

The acute oral toxicity of the test substance, TM 12-209, was estimated to be greater than 2000 mg/kg body weight according to OECD Test Guideline 423 using the Acute Toxic Class Method. 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. 
The acute dermal toxicity of the test substance, TM 12-209, was greater than 2000mg/kg body weight according to OECD Test Guideline 402 using the fixed dose method. The test item does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals.
The acute inhalation toxicity of the test substance, TM 12-209, was assessed according to OECD Test Guideline 403 using a standard acute method and gave an acute inhalation median lethal concentration (4 hr LC50) of greater than 5.42 mg/L according to OECD Test Guideline 403 using a standard acute method.

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 27 May 2014 and 17 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 423 using the Acute Toxic Class Method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Female Wister (RccHanTM: WIST) strain rats were supplied by Harlan Laboratories UK Ltd., 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 five 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 n a cage card. At the start of the study the animals were eight to twelve weeks of age. The body weight variation did not exceed ± 20 % of the mean body weight of the initially dosed group.
The animals were housed in groups of three in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed 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 (06:00 to 18:00) and twelve 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:
DMSO
Details on oral exposure:
For the purpose of the 2000 mg/kg dose level, the test item was 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 dimethyl sulphpoxide. Dimethyl sulphpoxide was used because the test item did not dissolve / suspend in distilled water or arachis oil BP.
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.
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
3 females / dose
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.
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 group to confirm the survival of the previously dosed animals.
The animals were observed for deaths or overt signs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days. Due to a technician error, the Day 10 clinical observations for the animals treated at a dose level of 300 mg/kg body weight were not performed. This deviation was considered not to affect the purpose or integrity of the study as no signs of systemic toxicity were observed pre- or post Day 10.
Individual body weights were recorded prior to dosing and seven and fourteen days after treatment.
At the end of the observation period, the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Evaluation of Data
Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.
The results were also evaluated according to Regulation (EC) No 1272/2008, relating to the Classification, labelling and Packaging of Substances and Mixtures.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths
Clinical signs:
Hunched posture and ataxia were noted during the day of dosing in the second group of animals treated at a dose level of 2000 mg/kg. There were no signs of systemic toxicity noted in animals treated at a dose level of 300 mg/kg and the initial group treated at a dose level of 2000 mg/kg.
Body weight:
All animals showed expected gains in body weight over the observation period.
Gross pathology:
No abnormalities were noted at necropsy.

Mortality Data

Dose Level mg/kg

Sex

Number of Animals Treated

Deaths During Day of Dosing
(Hours)

Deaths During Period After Dosing
(Days)

Deaths

½

1

2

4

1

2

3

4

5

6

7

8-14

300

Female

3

0

0

0

0

0

0

0

0

0

0

0

0

0/3

2000

Female

3

0

0

0

0

0

0

0

0

0

0

0

0

0/3

Female

3

0

0

0

0

0

0

0

0

0

0

0

0

0/3

 

Individual Clinical Observations - 300 mg/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

1-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

*

0

0

0

0

1-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

*

0

0

0

0

0=   No signs of systemic toxicity

*=  Observation not performed due to technician error

Individual Clinical Observations - 2000 mg/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

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

3-0

Female

H

H

H

HA

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1

Female

0

H

H

HA

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2

Female

0

H

H

HA

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0= No signs of systemic toxicity                    

H = Hunched posture                        

A = Ataxia

Individual Body Weights and Body Weight Changes - 300 mg/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

173

184

188

11

4

1-1 Female

185

196

200

11

4

1-2 Female

171

178

187

7

9

Individual Body Weights and Body Weight Changes - 2000 mg/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

174

184

204

10

20

2-1 Female

169

176

185

7

9

2-2 Female

180

187

196

7

9

3-0 Female

172

203

212

31

9

3-1 Female

169

183

196

14

13

3-2 Female

164

180

190

16

10

 

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

1-1 Female

Killed Day 14

No abnormalities detected

1-2 Female

Killed Day 14

No abnormalities detected

 Individual Necropsy Findings - 2000 mg/kg

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

2-0 Female

Killed Day 14

No abnormalities detected

2-1 Female

Killed Day 14

No abnormalities detected

2-2 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


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 > 2000 – 5000 mg/kg body weight).
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 acute oral toxicity of the test substance, TM 12-209, was estimated to be greater than 2000 mg/kg body weight according to OECD Test Guideline 423 using the Acute Toxic Class Method.

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 15 May 2014 and 14 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
yes
Species:
rat
Strain:
other: RccHanTM : WIST
Sex:
male/female
Details on test animals and environmental conditions:
Male and female RccHan™ : WIST strain rats were supplied by Harlan Laboratories UK Ltd, 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 (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding and chew blocks 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.

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.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on 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. 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
Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. 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 exposed to an atmosphere of 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 108% of target and no deaths 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 2.71 mg/L for approximately four hours. Increased respiratory rate was the only significant observation noted in both animals on removal from the chamber and two hours after the four hour exposure period had been completed. One day post-exposure both animals exhibited increased respiratory rate, hunched posture and pilo-erection. Both animals recovered to appear normal on either Day 4 or Day 5 post-exposure.

Exposure Chamber Atmosphere Concentration
Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator between 19 and 21 C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The non-volatile component of the batch used during the study was found to be approximately 0.49% (n=10). Due to the very low proportions of non-volatiles in the test item it was considered that chemical analysis should be employed in order to determine test atmosphere concentrations.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved two liters of test atmosphere being drawn through a glass impinger containing Methanol (each made up to 80 mL). 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 223 % 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 (9.6, 6.9, 4.2, 1.7, 0.93 and 0.43 µm cut points) with stainless steel collection substrates and a back up 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 9.6, 6.9, 4.2, 1.7, 0.93 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:
5.42 mg/L
No. of animals per sex per dose:
Five / sex / dose
Control animals:
no
Details on study design:
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 up to fourteen 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 and on Days 1, 3, 7 and 14.

Necropsy
At the end of the observation period, all animals were killed by intravenous overdose of sodium pentobarbitone. 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.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.42 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no deaths during the study.
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 animals exhibited body weight losses or showed no body weight gains on the first day post-exposure. Reasonable body weight gains were noted in all male animals during the remainder of the recovery period. In contrast, two females exhibited a slight body weight loss or no body weight gain from Days 1 to 3 post-exposure. Three females either showed a slight body weight loss or showed no body weight gain from Days 3 to 7 post-exposure, two females exhibited slight body weight losses during the final week of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.

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

5.42

3.89

51.4

2.22

 

Exposure Chamber Concentration

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

 

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

 

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.42

0.09

12.1

 

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).

[1]= Test atmospheres were generated for a total of 12 minutes prior to animal insertion to ensure test item

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

2

60

5.27

31

2

60

5.27

60

2

60

5.48

93

2

60

5.44

120

2

60

5.54

150

2

60

5.43

179

2

60

5.44

210

2

60

5.43

235

2

60

5.46

 

Mean achieved atmosphere concentration (mg/L) =5.42

Standard deviation =0.09

 

Nominal concentration:

Test item used (g)

183

Air Flow (L/min)

60

Total Generation Time (mins)

252[1]

Nominal Concentration (mg/L)

12.1

 

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

9.6

0.05

0.03

0.10

0.06

4

6.9

0.27

0.15

0.35

0.26

5

4.2

0.56

0.49

0.56

0.54

6

1.7

0.20

0.02

0.21

0.14

7

0.93

0.01

0.00

0.10

0.04

8

0.43

0.01

0.00

0.05

0.02

Back-up Filter

<0.43

0.02

0.03

0.03

0.03

Total Mean Amount of Test Item Collected

1.09

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

9.6

0.982

1.03

94.5

6.60

6.9

0.839

0.77

70.6

5.54

4.2

0.623

0.23

21.1

4.20

1.7

0.230

0.09

8.26

3.61

0.93

-0.032

0.05

4.59

3.31

0.43

-0.367

0.03

2.75

3.08

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) =3.89µm

Geometric Standard Deviation (GSD) =2.22

Predicted amount less than 4 µm =51.4%


[1]= Test atmospheres were generated for a total of 12 minutes prior to animal insertion to ensure test item concentration was being achieved.

Mortality Data

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

5.42

Male

0

0

0

0

0

0

0

0

0

0

0/10

Female

0

0

0

0

0

0

0

0

0

0

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No deaths occurred in a group of ten rats (five males and five females) exposed to a mean achieved atmosphere concentration of 5.42 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of IFF TM 12-209, in the RccHanTM : WIST strain rat, was greater than 5.42 mg/L.
Executive summary:

The acute inhalation toxicity of the test substance, TM 12-209, was assessed according to OECD Test Guideline 403 using a standard acute method and gave an acute inhalation median lethal concentration (4 hr LC50) of greater than 5.42 mg/L according to OECD Test Guideline 403 using a standard acute method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5.42 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 29 May 2014 and 12 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 402 using the fixed dose method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Five male and five female Wistar (RccTM: WIST) strain rats were supplied by Harlan Laboratories UK Ltd., 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 five 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 eight to twelve weeks of age. The weight variation did not exceed ± 20 % of the mean weight for sex.
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 Harlan Laboratories UK, Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed 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 twelve hours continuous light (06:00 to 18:00) and twelve 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 formulation and experimental preparation
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 adsorption of the test item was not determined.
Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 male and 5 female rats per dose
Control animals:
no
Details on study design:
On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information of the toxicity of the test item, a single group of animals was treated as follows:
Dose level: 2000 mg/kg.
Specific gravity: 0.887
Dose volume: 2.26 mL/kg
5 male and 5 female rats per dose
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 sin and surrounding hair wiped with cotton wool moistened with dimethyl sulphoxide followed by 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 0.5, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored.
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:
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 1 day after dosing at the test sites of one male and all females. There were no signs of dermal irritation noted at the test sites of four males.

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

1

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 2000mg/kg body weight.
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.
The test item does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals.
Executive summary:

The acute dermal toxicity of the test substance, TM 12-209, was greater than 2000mg/kg body weight according to OECD Test Guideline 402 using the fixed dose method.

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

Additional information

Acute oral toxicity

There were no deaths, no abnormalities observed at necropsy. All animals showed expected gains in body weight.

Hunched posture and ataxia were noted in the second group of animals treated at a dose level of 2000 mg/kg. There were no signs of systemic toxicity noted in animals treated at a dose level of 300 mg/kg and the initial group treated at a dose level of 2000 mg/kg.

Acute inhalation toxicity

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal from Days 3 to 6 post exposure.

All animals exhibited body weight losses or showed no body weight gains on the first day post exposure. Reasonable body weight gains were noted in all male animals during the remainder of the recovery period. In contrast, two females exhibited a slight body weight loss or no body weight gain from Days 1 to 3 post-exposure. Three females either showed a slight body weight loss or showed no body weight gain from Days 3 to 7 post-exposure, two females exhibited slight body weight losses during the final week of the recovery period.

No macroscopic abnormalities were detected amongst animals at necropsy.

Acute dermal toxicity

There were no deaths, no signs of systemic toxicity, no abnormalities observed at necropsy either. All animals showed expected gains in body weight.

Very slight erythema was noted 1 day after dosing at the test sites of one male and all females. There were not signs of dermal irritation noted at the test sites of four males.


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

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

Justification for selection of acute toxicity – dermal endpoint
The study was conducted in vivo in an appropriate test species 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 and when the acute LC50 value is ≤ 5.0 mg/L for inhalation exposure conducted with 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, TM 12-209, 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, TM 12-209, is not classified for acute dermal toxicity.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute inhalation LC50 of > 5 mg/kg and therefore the test substance, TM 12-209, is not classified for acute inhalation toxicity.