Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Oct 20, 1988 to Nov 30, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river portage,Michigen, USA
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 116-152 g
- Fasting period before study: Overnight
- Housing: Individually; metal cages with wire mesh floors
- Diet (e.g. ad libitum): Standard laboratory rodent diet(ad libitum)
- Water (e.g. ad libitum): Domestic quality portable water(ad libitum)
- Acclimation period: 14 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22⁰ C
- Humidity (%): 53%
- Air changes (per hr): Approx 15
- Photoperiod (hrs dark / hrs light): 12 h
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
VEHICLE
Test substance was administered as supplied by the sponsor

MAXIMUM DOSE VOLUME APPLIED: 4.5 mL/kg bw (specific gravity 1.1)
- Rationale for the selection of the starting dose: Preliminary study
Doses:
5000 mg/kg bw (4.54 mL/kg bw)
No. of animals per sex per dose:
5/sex/dose
Control animals:
no
Details on study design:
Five males and five females were treated at 5000 mg/kg bw using syringe and plastic catheter (8 choke). Animals were observed after 5 h on day 1 and in subsequent days the animals were observed once in the morning and again at the end of the experiment. Observation period was 14 d.

- Frequency of observations and weighing: Clinical observations done soon after dosing and remainder of Day 1. For the subsequent days observations were done in the morning and at the end of the experimental day.

- Necropsy of survivors performed: Yes along with macroscopic examinations of thoracic, abdominal and cranial cavities. Marcoscopic appearance of abnormal organs was also recorded.

- Other examinations performed: Body weights of each rat was examined at Days 1, 8 and 15 or at death.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality at 5000 mg/kg bw
Clinical signs:
Pilo-erection in all rats within 5 min after dosing which remained for 1 d followed by increased salivation during 1st 2 h after treatment. No other clinical signs were observed and recovery was complete by Day 3.
Body weight:
Slightly low body weight gains during the 1st week (for majority of female rats) and anticipated gain between Day 8 and 15 in all rats.
Other findings:
Terminal autopsy was normal.
Interpretation of results:
other: CLP criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test condition, the acute lethal oral dose of di-TMPTTA was > 5,000 mg/kg bw.
Executive summary:

A study was conducted to assess the acute oral toxicity of di-TMPTTA according to OECD Guideline 401 and EU Method B.1, in compliance with GLP. Five males and five females were administered a single oral dose of 5,000 mg/kg bw by gavage. Animals were observed for 14 d. On Day 1, pilo-erection was seen within 5 min of dosing along with increased salivation during 2 h after treatment. Low body weight gain during the first week of the study was recorded but all rats achieved anticipated weight gains between Days 8 and 15. No other clinical signs were observed and recovery, judged by appearance and behaviour, was complete by Day 3. Under the test conditions, the acute lethal oral dose of the test substance was > 5,000 mg/kg bw (Liggett 1989).

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 July, 1998 to 14 Aug, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
other: CD rats of Sprague-Dawley origin (Hsd:Sprague-Dawley(CD))
Sex:
male/female
Details on test animals and environmental conditions:
Animals chosen for this study were selected from a stock supply of healthy male and female CD rat of Sprague-Dawley origin (Hsd:Sprague-Dawley(CD)) obtained from Harlan U.K. Ltd., Bicester, Oxon, England. They were in the weight range of 125 to 152 g and approx 5-7 weeks of age prior to dosing (Day 1). All the rats were acclimatised to the experimental environment for a minimum period of 15 d prior to the start of the study. Rats were allocated without conscious bias to cages within the treatment groups. They were housed in groups of 5 rats of the same sex in metal cages (RS biotech sub-dividable rodent cages polished stainless steel (20 cm high x 39 cm wide x 39 cm long). The cages were suspended in the mobile stainless steel racks in room 6 of building R14.

A standard laboratory rodent diet (Special Diet Services RM1(E) SQC expanded pellet) and drinking water were provided ad libitum. Access to food only was prevented overnight prior to and approx 4 h after dosing. Each batch of diet used for the study was analyzed for certain nutrients, possible contaminants and micro-organisms. Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available to Huntingdon Life Sciences Ltd. at regular intervals throughout the year. Animal room environmental controls were set to maintain temperature within the range 22 ± 3°C (actual 21 to 26°C) and relative humidity 30-70% (actual 39 to 58%). These environmental parameters were recorded continously using a 7 d recorder. Lighting was controlled by means of a time switch to provide 12 h of artificial light (0700 – 1900 GMT) in each 24 h period. Each animal was identified by cage number and ear punching.
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
The test substance was administered as supplied at a volume of 4.452 mL/kg bw. The observation and characterization of the homogeneity, stability and purity of the test substance was not undertaken as part of this study and remains the responsibility of the Sponsor.
Doses:
Preliminary study: 3200 mg/kg bw
Main study: 5000 mg/kg bw
No. of animals per sex per dose:
Preliminary study: 2 rats (1 male and female)
Main study: 5/sex/dose
Control animals:
no
Details on study design:
Preliminary study: 2 rats (one male and one female) received a single oral gavage dose of the test substance at a dose level of 3200 mg/kg bw using a syringe and plastic catheter or cannula. This was conducted to help define the toxic potential of the test substance and aid in selection of a suitable dosage for the main study.
Main study:
As results at this dosage indicated the acute lethal oral dose of the test substance to be greater than 3200 mg/kg bw, in compliance with the guidelines, a further group of 10 rats (five males and five females) was similarly dosed at 5000 mg/kg bw to complete the study. The day of dosing was designated Day 1. Cages of rats were checked at least twice daily for any mortalities. For clinical signs animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity of the clinical signs and time were recorded at each observation. All animals were observed for 7 to 14 d after dosing. The bw of each rat was recorded on 1 (prior to dosing), 8 and 15 d. Individual weekly bodyweight changes and group mean bw were calculated. All animals were killed on Day 15 by carbon dioxide asphyxiation. All animals were subjected to a macroscopic examination which consisted of opening the thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths following a single oral gavage dose of the test substance in the preliminary or the main study at a dose level of either 3200 or 5000 mg/kg bw.
Clinical signs:
Preliminary study: Clinical signs of reaction to treatment comprised of abnormal piloerection, soft to liquid feces and increased salivation (in male rat only).

Main study: Clinical signs of reaction to treatment comprised of abnormal piloerection in all the animals within 7 minutes and persisted during the study. Also, soft to liquid feces and increased salivation in botht he sexes. Recovery of rats, as judged by external appearance and behaviour, was complete in males by Day 5 and in females by Day 4..
Body weight:
All animals were considered to have achieved satisfactory bw gains throughout the preliminary and the main studies.
Gross pathology:
No abnormalities were revealed at the macroscopic examination at study termination on Day 15 in the preliminary and the main studies.
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the conditions of the study, the acute oral lethal dose of the test substance to rats was found to be greater than 5,000 mg/kg bw.
Executive summary:

A study was performed to assess the acute oral toxicity of di-TMPTTA to the rat according to OECD Guideline 401 and EU Method B.1, in compliance with GLP. In a preliminary test, 2 rats (one male and one female) received a single oral gavage dose of the test substance at a dose level of 3,200 mg/kg bw. As results at this dosage indicated the acute lethal oral dose of the test substance to be greater than 3,200 mg/kg bw, a further group of 10 fasted rats (five males and five females) was similarly dosed at 5,000 mg/kg bw in the main study. No abnormalities were revealed at macroscopic examination on Day 15. Under the conditions of the study, the acute lethal oral dose to rats was > 5,000 mg/kg bw (McRae 1998).

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 April, 2012 to 07 May, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx 11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean
- Housing: Animals were group housed in labeled makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approx 15 room air changes/h, and a 12-h light/12-h dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Deviations from the minimum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 04 April - 07 May 2012
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test concentrations: 0, 0.5, 1, 2.5%
Pre-screen test was done at doses of 0.5, 1, 2.5, 5, 10, 50 and 100%. Up to doses of 5%, ear swelling and signs of erythema were too pronounced. In the OECD TG 429, it is written “excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series that does not induce systemic toxicity and/or excessive local skin irritation.“. In the case of di-TMPTTA, the threshold of 25% of ear thickness was exceeded at the doses of 5% and higher. Therefore the main study has been conducted with doses of 0.5, 1 and 2.5%.
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
Pre-screen test was done at doses of 0.5, 1, 2.5, 5, 10, 50 and 100%. Up to doses of 5%, ear swelling and signs of erythema were too pronounced. In the OECD TG 429, it is written “excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series that does not induce systemic toxicity and/or excessive local skin irritation.“. In the case of di-TMPTTA, the threshold of 25% of ear thickness was exceeded at the doses of 5% and higher. Therefore the main study has been conducted with doses of 0.5, 1 and 2.5%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on Classification, Labelling and Packaging of substances and mixtures.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 h prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 h after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.
Key result
Parameter:
SI
Value:
ca. 0.9
Variability:
five animals
Test group / Remarks:
0.5% test substance
Parameter:
other: disintegrations per minute (DPM)
Value:
ca. 448 - ca. 1 174
Variability:
Mean DPM/animal values
Test group / Remarks:
0.5, 1 and 2.5% test substance
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.5, 1 and 2.5% were 448, 613 and 1174 DPM respectively. The mean DPM/animal value for the vehicle control group was 524 DPM.
Key result
Parameter:
SI
Value:
ca. 1.2
Variability:
five animals
Test group / Remarks:
1% test substance
Key result
Parameter:
SI
Value:
ca. 2.2
Variability:
five animals
Test group / Remarks:
2.5% test substance

Results pre-screen test:

Based on the results obtained in this test, the highest test substance concentration selected for the main study was a 2.5% concentration

Other results - main study:

Skin reactions / Irritation:

The slight irritation of the ears as shown by all animals treated at 2.5% (on Days 2 and 3) was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation was shown by the other animals.

Systemic toxicity/Body weights:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area:

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:
other: CLP criteria not met
Conclusions:
In a mouse LLNA study, SI values calculated for the concentrations of 0.5, 1 and 2.5% di-TMPTTA were 0.9, 1.2 and 2.2, respectively. These results indicate that the test substance did not elicit an SI ≥ 3. The substance is therefore not considered to be sensitizing.
Executive summary:

A study was conducted to assess the skin sensitizing potential of di-TMPTTA in a mouse local lymph node assay conducted according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870.2600, in compliance with GLP. Five animals per dose were exposed to the test substance at 0.5, 1.0 or 2.5%. The slight irritation of the ears observed on Days 2 and 3 in all animals treated at 2.5% was not considered to have a toxicologically significant effect on the activity of the nodes. No irritation was shown by the other animals. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted. Mean degradations per minute (dpm)/animal at 0.5, 1 and 2.5% were 448, 613 and 1,174, respectively. The mean dpm/animal for the vehicle control group was 524. The SI values calculated for the substance concentrations of 0.5, 1.0 and 2.5% were 0.9, 1.2 and 2.2, respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 2.5%, it was established that the EC3 value (the estimated test substance concentration that will give a SI =3) exceeds 2.5%. The substance was therefore not considered to be sensitizing (Beerens 2013).

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion