Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The acute oral toxicity of the test substance, TM 11-213 was determined according to OECD Guideline 423 using the Acute Toxic Class Method.  The acute LD50 of TM 11-213 was 500 mg/kg body weight and was therefore classified as acutely toxic via the oral route (Category IV) according to the Globally Harmonised Classification System.
The acute dermal toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 402 using a standard acute method. The acute dermal median lethal dose (LD50) of the test item was found to be > 2000 mg/kg body weight.
The acute inhalation toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 403 using a standard acute method. The LD50 of all animals was 3.49 (2.33 – 4.64) mg/L; the LD50 for males only was 4.30 (2.62 – 5.99) mg/L and for females only was 2.73 (1.23 – 4.23) mg/L.

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 31 October 2013 and 26 November 2013.
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
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Animals and Animal Husbandry

Female Wistar (RccHan™: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 were eight to twelve weeks of age. The body weight variation did not exceed ± 20 % of the body weight of the initially dosed animal.

The animals were housed in groups of up to 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 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. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. 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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
Test Item Formulation and Experimental Preparation
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 two 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:
A group of three fasted females was treated with the test item at a dose level of 300 mg/kg body weight. Based on the results from this dose level, further groups of fasted females were treated at dose levels of 300 or 2000 mg/kg body weight. Dosing was performed sequentially.

The test item was administered orally undiluted at a dose level of 2000 mg/kg and as a solution in arachis oil BP at a dose level of 300 mg/kg.

No. of animals per sex per dose:
3 female rats per dose level
Control animals:
no
Details on study design:
Procedure
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 up to fourteen days.

Individual body weights were recorded prior to dosing and seven and fourteen days after treatment or at death.

At the end of the observation period the surviving 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.


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 behavioral 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:
500 mg/kg bw
Based on:
test mat.
Mortality:
Animals treated at a dose level of 2000 mg/kg were killed for humane reasons, during the day of dosing, due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence or found dead one day after dosing. One of the second group of animals, treated at a dose level of 300 mg/kg, was found dead one day after dosing.
Clinical signs:
Signs of systemic toxicity noted at a dose level of 2000 mg/kg were ataxia, lethargy, hunched posture, prostration, labored respiration, decreased respiratory rate, occasional body tremors, hypothermia and pallor of the extremities. Hunched posture was noted in one animal treated at a dose level of 300 mg/kg.
Body weight:
Surviving animals showed expected gains in body weight over the observation period.

Gross pathology:
Abnormalities noted at necropsy of animals that were humanely killed or died during the study were pale liver, patchy pallor of the liver, pale or dark kidneys, clear liquid present in the stomach, hemorrhage and epithelial sloughing of the gastric mucosa and hemorrhage of the non-glandular epithelium of the stomach. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

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

Female

3

0

0

0

0

1

0

0

0

0

0

0

0

1/3

2000

Female

3

0

0

0

2*

1

 

 

 

 

 

 

 

3/3

*=    Animals killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

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

0

1-1

Female

0

0

0

H

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

3-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1

Female

0

0

0

0

X

 

 

 

 

 

 

 

 

 

 

 

 

 

3-2

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

H =  Hunched posture

X =  Animal dead

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

AL

HA

HA

X

 

 

 

 

 

 

 

 

 

 

 

 

 

2-1

Female

0

0

PrRl
RdTo

PrRlRd
HoEX
*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2-2

Female

0

0

PrRl
RdTo

PrRlRd
HoEX
*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0=   No signs of systemic toxicity

A =  Ataxia

L =  Lethargy

H =  Hunched posture

Pr = Prostration

Rl = Labored respiration

Rd =Decreased respiratory rate

To =Occasional body tremors

Ho =      Hypothermia

E =  Pallor of the extremities

X =  Animal dead

*=    Animals killed for humane reasons due to the occurrence of clinical signs of toxicity that exceeded the severity limit set forth in the UK Home Office Project Licence

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 (g)
at Death

Body Weight Gain (g) During Week

0

7

14

1

2

300

1-0 Female

152

174

186

 

22

12

1-1 Female

160

178

192

 

18

14

1-2 Female

156

170

187

 

14

17

3-0 Female

177

194

212

 

17

18

3-1 Female

168

-

-

158

-

-

3-2 Female

166

187

200

 

21

13

- = Animal dead

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 (g)
at Death

Body Weight Gain (g) During Week

0

7

14

1

2

2000

2-0 Female

161

-

-

154

-

-

2-1 Female

164

-

-

158

-

-

2-2 Female

158

-

-

156

-

-

-=    Animal dead

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

3-0 Female

Killed Day 14

No abnormalities detected

3-1 Female

Found dead Day 1

Liver: patchy pallor

Kidneys: dark

Gastric mucosa: epithelial sloughing

Non-glandular epithelium of the stomach: hemorrhagic

2-2 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

Found dead Day 1

Liver: patchy pallor

Kidneys: pale

Gastric mucosa:hemorrhagic

                              epithelial sloughing

2-1 Female

Humanely killed Day 0

Liver: pale

Kidneys: pale

Stomach: clear liquid present

Gastric mucosa: epithelial sloughing

2-2 Female

Humanely killed Day 0

Liver: pale

Kidneys: pale

Stomach: clear liquid present

Gastric mucosa: epithelial sloughing


 

Interpretation of results:
Toxicity Category IV
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 approximately 500 mg/kg body weight (Globally Harmonized Classification System – Category 4, >300 - 2000 mg/kg body weight and is also according to the EU C&L rules).

The test item was also classified as Category 4 according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H302: Harmful if swallowed” are therefore required.
Executive summary:

The acute oral toxicity of the test substance, TM 11-213, was assessed according to OECD TG 423 using Wistar rats. At 300 mg/kg bw one animal died at the next higher dose of 2000 mg/kg bw all animals died, within the first day. At macroscopy pale liver and kidneys were observed and effects in the gastrointestinal tract indicating severe irritation: fluid in the stomach and sloughing in the epidermis. The toxicity of this substance is somewhat unexpected. Other hydrocarbon aldehydes of similar C-atoms have toxicity > 2000 mg/kg bw. When using the scheme in the guideline for this substance an LD50 of 500 mg/kg bw is derived.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
500 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 16 February 2015 and 16 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 403 using a standard acute method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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 196g 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.
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
One day prior to the day of exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During each 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 three groups, each of ten rats (five males and five females), were subjected to a single exposure to the test item for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure.


Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator between 18 and 20 °C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The non-volatile component of the batch used during the study was found to be approximately 64.43 % (n=9). However, during preliminary generation trials it was apparent that much more test item was being lost than was expected (possibly due to the large air flows used to generate the test item causing an increase in evaporation) and as such it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations for both groups.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 to 10 liters of test atmosphere (dependent on concentration) being drawn through a glass impinger containing Acetonitrile (each made up to 80mL). The samples were then submitted for chemical analysis.

The nominal chamber concentrations were calculated by dividing the mass of test item used by the total volume of air passed through the chamber during each exposure.

The nominal concentrations were 794 %, 365 % and 524 % of the actual mean achieved atmosphere concentration for Groups 1, 2 and 3 respectively, these show that keeping the aerosol airborne was moderately difficult for Groups 1 and 3 but was relatively straightforward when generating at the lowest dose level (Group 2). This is not an unexpected occurrence as generating and maintaining test atmospheres at progressively higher concentrations becomes more difficult. For example, doubling the syringe pump rate will not necessarily result in a two fold increase of the test atmosphere concentration. For the exposures performed during this study the syringe pump rates were set as follows; 125 mL/hr, 15 mL/hr and 70 mL/hr for Groups 1, 2 and 3 respectively.


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 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 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: 4.64, 1.30, 3.99 mg/L
No. of animals per sex per dose:
Five per sex per dose
Control animals:
no
Details on study design:
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.

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 up to fourteen or twenty one 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, 14, 21* or at death.
* = Group 3 only

Necropsy
At the end of each fourteen or twenty one* day observation period, the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including those that died or were humanely killed during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
3.49 mg/L air
Based on:
test mat.
95% CL:
2.33 - 4.64
Exp. duration:
4 h
Sex:
male
Dose descriptor:
LC50
Effect level:
4.3 mg/L air
Based on:
test mat.
95% CL:
2.62 - 5.99
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
2.73 mg/L air
Based on:
test mat.
95% CL:
1.23 - 4.23
Exp. duration:
4 h
Mortality:
Group 1 - Mean achieved atmosphere concentration 4.64 mg/L
Deaths: 5/5 males, 5/5 females

Group 2 - Mean achieved atmosphere concentration 1.30 mg/L
Deaths: 0/5 males, 1/5 females

Group 3 - Mean achieved atmosphere concentration 3.99 mg/L
Deaths: 0/5 males, 2/5 females
Clinical signs:
other: See below
Body weight:
Group 1 – As none of the animals survived past the day of exposure, no significant body weight data was collected for these animals.

Group 2 – All males and three female animals exhibited body weight losses on the first day post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. Four female animals exhibited body weight gains and the remaining female showed no body weight gain from Days 1 to 3 post-exposure, however, four females exhibited a slight body weight loss or showed no body weight gains from Days 3 to 7 post-exposure. Body weight gains were noted in the four surviving female animals during the final week of the recovery period.

Group 3 – All surviving animals exhibited body weight losses on Day 1 post-exposure. One male and one surviving female animal exhibited body weight losses from Days 1 to 3 post-exposure and a further male and female exhibited body weight loss from Days 3 to 7 post-exposure. Body weight gains were then noted in all animals during the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the recovery periods.

The following macroscopic abnormalities were detected at necropsy amongst animals that were humanely killed or were found dead during the course of the study:

Lungs –abnormally dark or dark patches;
Stomach – gaseous distension;

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

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 each exposure period.

 

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

 

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

1

4.64

0.21

36.9

2

1.30

0.03

4.74

3

3.99

0.29

20.9

 

Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour during each exposure.

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:

 

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

1

4.64

2.28

75.2

2.30

2

1.30

2.23

78.1

2.14

3

3.99

2.86

66.8

2.16

[1]= Test atmospheres were generated for a total of 30, 25 and 31 minutes for Groups 1, 2 and 3 respectively prior to animal insertion to ensure test item concentrations were being achieved.

Clinical Observations

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 are considered to be associated with the restraint procedure and, in isolation, are not indicative of toxicity.

 

In addition to the observations considered to be due to the restraint procedure, the following abnormalities were detected:

 

Dose Group 1 – 4.64 mg/L

During exposure all animals exhibited decreased respiratory rate and labored respiration. On removal from the chamber all animals exhibited decreased respiratory rate and labored respiration, frequent occurrences of coma and occasional instances of prostration were also noted. By one hour post-exposure all animals were comatose. One female animal was found dead approximately 70 minutes post-exposure and another female was humanely killed at the same time as it was considered unlikely to survive. The remaining animals were all humanely killed at approximately 120 minutes post-exposure as they were considered unlikely to survive. 

 

 

Dose Group 2 – 1.30 mg/L

Isolated occurrences of increased respiratory rate were noted in two males during exposure. On removal from the chamber and one hour post-exposure all animals exhibited increased respiratory rate.

 

One day after exposure, all animals exhibited increased respiratory rate and hunched posture. Animals recovered to appear normal on Day 2 or Day 3 post-exposure. However, one female animal was found dead on Day 12 after showing no abnormal clinical signs over the previous 10 days.

 

 

Dose Group 3 – 3.99 mg/L

Decreased respiratory rate was noted in all animals during exposure. On removal from the chamber, all animals exhibited decreased respiratory rate and ataxia, one female animal also exhibited lethargy and ptosis. One hour post-exposure a deterioration in the condition of the animals was noted as they all now appeared lethargic and one female was humanely killed approximately 90 minutes post-exposure as it was considered unlikely to survive.

 

One day after exposure, one female animal was found dead. All surviving animals exhibited increased or decreased respiratory rate and hunched posture. There were frequent instances of pilo-erection and isolated occurrences of lethargy.  Slight signs of improvement were noted over the next two days, however, on Days 4 and 5 noisy respiration and sneezing became prevalent. One female animal exhibited fur loss on the right side of the head on Day 7 which persisted for eight days, however, this was considered not to be treatment related. Surviving animals recovered slowly such that they appeared normal Day 18 post-exposure.

Exposure Chamber Atmosphere Concentrations– Group 1

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

2

60

4.52

30

2

60

4.54

60

2

60

4.61

90

2

60

4.49

120

2

60

4.61

150

2

60

5.17

180

2

60

4.52

210

2

06

4.62

230

2

60

4.69

Mean achieved atmosphere concentration (mg/L) = 4.64

Standard deviation = 0.21

 

Nominal concentration:

Test item used (g)

570

Air Flow (L/min)

60

Total Generation Time (mins)

270[1]

Nominal Concentration (mg/L)

36.9

 [1]

= Test atmospheres were generated for a total of 30 minutes prior to animal insertion to ensure test item concentration was being achieved.

Exposure Chamber Atmosphere Concentrations – Group 2

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

10

10

60

1.27

30

10

60

1.32

60

10

60

1.28

90

10

60

1.29

120

10

60

1.36

151

10

60

1.35

180

10

60

1.30

210

10

60

1.27

230

10

60

1.30

Mean achieved atmosphere concentration (mg/L) = 1.30

Standard deviation = 0.03

 

Nominal concentration:

Test item used (g)

75.3

Air Flow (L/min)

60

Total Generation Time (mins)

265[1]

Nominal Concentration (mg/L)

4.74

[1]= Test atmospheres were generated for a total of 25 minutes prior to animal insertion to ensure test item concentration was being achieved.

Exposure Chamber Atmosphere Concentrations – Group 3

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

10

3

60

3.57

30

3

60

3.82

60

3

60

3.92

90

3

60

3.91

120

3

60

3.96

150

3

60

4.06

180

3

60

4.24

210

3

60

4.61

230

3

60

3.84

Mean achieved atmosphere concentration (mg/L) = 3.99

Standard deviation = 0.29

 

Nominal concentration:

Test item used (g)

340

Air Flow (L/min)

60

Total Generation Time (mins)

271[1]

Nominal Concentration (mg/L)

20.9

[1]= Test atmospheres were generated for a total of 31 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution – Group 1

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

0.04

0.16

0.07

0.09

4

7.3

0.15

0.24

0.14

0.18

5

3.6

0.36

0.52

0.41

0.43

6

1.3

0.46

0.64

0.52

0.54

7

0.94

0.65

0.83

0.67

0.72

8

0.43

0.07

0.08

0.04

0.06

Back-up Filter

<0.43

0.04

0.13

0.07

0.08

Total Mean Amount of Test Item Collected

2.10

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.01

95.7

6.72

7.3

0.863

1.83

87.1

6.13

3.6

0.556

1.40

66.7

5.43

1.3

0.114

0.86

41.0

4.77

0.94

-0.027

0.14

6.67

3.50

0.43

0.367

0.08

3.81

3.23

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) =2.28µm

Geometric Standard Deviation (GSD) =2.30

Predicted amount less than 4 µm =75.2%

Particle Size Distribution – Group 2

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

0.02

0.03

0.07

0.04

4

7.3

0.09

0.12

0.13

0.11

5

3.6

0.41

0.34

0.43

0.39

6

1.3

0.41

0.51

0.55

0.49

7

0.94

0.46

0.54

0.52

0.51

8

0.43

0.03

0.03

0.01

0.02

Back-up Filter

<0.43

0.05

0.04

0.10

0.06

Total Mean Amount of Test Item Collected

1.62

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

1.58

97.5

6.97

7.3

0.863

1.47

90.7

6.33

3.6

0.556

1.08

66.7

5.43

1.3

0.114

0.59

36.4

4.65

0.94

-0.027

0.08

4.94

3.35

0.43

0.367

0.06

3.70

3.21

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 2.23 µm

Geometric Standard Deviation (GSD) = 2.14

Predicted amount less than 4 µm = 78.1 %

Particle Size Distribution – Group 3

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

0.18

0.25

0.21

0.21

4

7.3

0.29

0.40

0.35

0.35

5

3.6

0.68

0.76

0.79

0.74

6

1.3

0.83

0.83

0.79

0.82

7

0.94

0.86

0.92

0.91

0.90

8

0.43

0.04

0.09

0.09

0.07

Back-up Filter

<0.43

0.00

0.05

0.03

0.03

Total Mean Amount of Test Item Collected

3.12

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.91

93.3

6.50

7.3

0.863

2.56

82.1

5.92

3.6

0.556

1.82

58.3

5.21

1.3

0.114

1.00

32.1

4.53

0.94

-0.027

0.10

3.21

3.15

0.43

0.367

0.03

0.962

2.66

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 2.86 µm

Geometric Standard Deviation (GSD) = 2.16

Predicted amount less than 4 µm = 66.8 %

 

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

4.64

Male

0

5[1]

-

-

-

-

-

-

-

-

10/10

Female

0

1[2]1[3]

 3*

-

-

-

-

-

-

-

-

2

1.30

Male

0

0

0

0

0

0

0

0

0

0

1/10

Female

0

0

0

0

0

0

0

0

0

1[4]

3

3.99

Male

0

0

0

0

0

0

0

0

0

0

2/10

Female

0

1[5]

1

0

0

0

0

0

0

0

[1]= Humanely killed Approximately 120 minutes post-exposure as considered unlikely to survive

[2]= Found dead Approximately 70 minutes post-exposure

[3]= Humanely killed Approximately 70 minutes post-exposure as considered unlikely to survive

[4]= Found dead on Day 12 post-exposure

[5]= Humanely killed Approximately 90 minutes post-exposure as considered unlikely to surviv

Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute inhalation median lethal concentrations (LC50) and 95% confidence limits of IFF TM 11-213 (FRET 09-0425) in the RccHanTM : WIST strain rat, were calculated to be:

All animals : 3.49 (2.33 – 4.64) mg/L
Males only: 4.30 (2.62 – 5.99) mg/L
Females only: 2.73 (1.23 – 4.23) mg/L
(Globally Harmonized Classification System – Category 4).
Executive summary:

The acute inhalation toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 403 using a standard acute method. The LD50 of all animals was 3.49 (2.33 – 4.64) mg/L; the LD50 for males only was 4.30 (2.62 – 5.99) mg/L and for females only was 2.73 (1.23 – 4.23) mg/L.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
3.49

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 25 February 2015 and 18 March 2015.
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 402 using a standard acute method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Five male and five female Wistar (RccHan: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 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 200g, and were 8 to 12 weeks of age. However, the initial female animal was slightly below the minimum weight of 200g specified in the General Study Plan but this was considered not to affect the purpose or integrity of the study. The weight variation did not exceed ±20% of the mean weight for each sex, except for one male which was slightly outside the range. This deviation was considered not to affect the purpose or integrity of the study

The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24 Hour exposure period and in groups of four, by sex, for the remainder of the study. 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 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 (06:00 to 18:00) 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 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 absorption of the test item was not determined.
Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 / sex / dose
Control animals:
no
Details on study design:
Procedure
On the day before treatment the back and flanks of each animal were clipped free of hair.

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 throughout the study. 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.

As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. The animals were caged individually for the 24 Hour exposure period. After the 24 Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item. These animals were returned to group housing for the remainder of the test period.

The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.

After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored.

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:
Very slight erythema was noted at the test sites of six animals from Day 3 up to Day 6. There were no signs of dermal irritation noted at the test sites of the remaining four animals.

No abnormalities were noted at necropsy.

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

1

1

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

3-0

Male

Erythema

0

0

1

1

1

1

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

3-1

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

3-2

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

3-3

Male

Erythema

0

0

1

1

1

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                   1 = Very slight erythema

Individual Dermal Reactions - Females

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

2-0

Female

Erythema

0

0

1

1

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

4-0

Female

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

4-1

Female

Erythema

0

0

1

1

1

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

4-2

Female

Erythema

0

0

1

1

1

1

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

4-3

Female

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                   1 = Very slight erythema

Individual Body Weights and Body Weight Changes

Dose Level mg/kg

Animal Number and Sex

Body Weight (g) at Day

Body Weight Change (g) During Week

0

7

14

1

2

2000

1-0 Male

228

234

251

6

17

3-0 Male

256

269

287

13

18

3-1 Male

280

291

306

11

15

3-2 Male

249

260

275

11

15

3-3 Male

268

281

297

13

16

2-0 Female

193

201

207

8

6

4-1 Female

208

209

214

1

5

4-2 Female

216

219

223

3

4

4-3 Female

215

217

221

2

4

4-4 Female

201

203

207

2

4

 

Individual Necropsy Findings

Dose Level

mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0

Male

Killed Day 14

No abnormalities detected

3-0

Male

Killed Day 14

No abnormalities detected

3-1

Male

Killed Day 14

No abnormalities detected

3-2

Male

Killed Day 14

No abnormalities detected

3-3

Male

Killed Day 14

No abnormalities detected

2-0

Female

Killed Day 14

No abnormalities detected

4-0

Female

Killed Day 14

No abnormalities detected

4-1

Female

Killed Day 14

No abnormalities detected

4-2

Female

Killed Day 14

No abnormalities detected

4-3

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

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

The acute dermal toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 402 using a standard acute method. The acute dermal median lethal dose (LD50) of the test item was found to be > 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 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 on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines

Justification for classification or non-classification

Acute Oral Toxicity

An in vivo study performed according to internationally recognized guidelines indicated that the test item was considered acutely toxic by the oral route. The animals treated at a dose level of 2000 mg/kg were killed for humane reasons, four hours after dosing, and an LD50 of approximately 500 mg/kg body weight was calculated. The test substance therefore meets the criteria for classification for acute oral toxicity Category 4 according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures, which is defined as an LD50 >300 - 2000 mg/kg bodyweight.

Acute Dermal Toxicity

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.

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 11-213, is not classified for acute dermal toxicity.

Acute inhalation toxicity

Substances can be allocated to one of four toxicity categories based on the acute toxicity by 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 LC50 values.

A test substance is classified according to one of these four toxicity categories 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 LC50 of 3.49 mg/kg and therefore the test substance, TM 11-213, is classified for acute oral toxicity in Category 4.