Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
29 Sept 2009 to 18 Dez 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description Clear colourless liquid (determined at NOTOX)
Test substance storage At room temperature in the dark under nitrogen
Stability under storage conditions Stable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Charles River France, L¿Arbresle Cedex, France.
Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight: Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification: Tail mark with marker pen.
Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.
Reliability check: The results of a reliability test with Hexylcinnamaldehyde, performed not more than 6 months previously, are summarized in the appendix. Similar procedures were used in the reliability test and in this study.

Animal husbandry
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.1 ¿ 24.0°C), a relative humidity of 40-70% (actual range: 31 - 74%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water
Free access to tap water.
Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Study Plan (In life phase)
Start : 29 September 2009
Completion : 19 October 2009

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5 and 10%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: not indicated
- Irritation:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2; see section 6.6) at the highest concentration.
Initially, two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). The animal at 100% was treated with one concentration on two consecutive days and was found dead prior to dosing on Day 3. The animal at 50% was treated with one concentration on three consecutive days and was found dead after dosing but prior to the assessment of irritation of the ears on Day 3. No necropsy was performed
Based on the results of the initially treated animals, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage. Each of the additional animals was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was weighed in nitrogen environment in a glove box and test substance formulations (w/w) were prepared in nitrogen environment in a glove box within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
The dorsal surface of both ears was epidermally treated (25 ¿L/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
An individual Stimulation Index (SI) is calculated for each animal. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group. A mean SI is calculated for each group using the individual SI values. Standard deviations for the DPM and SI values were calculated.

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in September/October 2009, females of the CBA/J mouse strain (Charles River France, L¿Arbresle Cedex, France) were checked for sensitivity to alpha- Hexylcinnamaldehyde, technical grade. The females were approx. 11 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 ¿Skin Sensitization¿. Alpha-hexylcinnamicaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. 13102MO (Sigma- Aldrich, Steinheim, Germany) and the 3H-methyl thymidine was purchased from PerkinElmer Life Sciences, Boston, MA, USA.
HCA concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 (v/v)).

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.2 and 5.1 respectively. An EC3 value of 16.9% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI values calculated for the substance concentrations 2.5, 5 and 10% were 4.8, 13.3 and 26.2 respectively. These results show that the test substance elicits an SI > 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 2.5%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 2055, 5734 and 11294 respectively. The mean DPM/animal value for the vehicle control group was 431.

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined.

All auricular lymph nodes of control animals were considered normal in size. The majority ofauricular lymph nodes of animals treated with test substance formulations were consideredenlarged in size, except for the nodes in one animal treated at 2.5%. The largest auricular lymphnodes were found in the higher dose groups

No macroscopic abnormalities of the surrounding area were noted.

Body weights and body weight gain of experimental animals remained in the same range ascontrols over the study period.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Table: Disintegrations Per Minute (DPM) and Stimulation Index (SI).

test substance                                           mean

group                  (% w/w)                    DPM ± SEM                  SI ± SEM

2

2.5%

2055

±   302

4.8

±      1.3

3

5%

5734

±   690

13.3

±      3.3

4

10%

11294

±    2279

26.2

±      7.8

1

0% (vehicle)

431

±   94

1.0

±      0.3

            Vehicle: Acetone/Olive oil (4:1 v/v).                                                                           

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test substance TTP is sensitising in this LLNA in concentrations up to 10% (w/v) in acetone: olive oil (4+1). The EC3 value was established to be between 0 and 2.5%.
Executive summary:

Based on the results of a guideline LLNA (OECD 429, EC B.42) study :

-according to the recommendations made in the test guidelines, TTP would be regarded as skin sensitizer.

-according to the Globally Harmonized System of Classification and Labeling of Chemicals(GHS) of the United Nations (2007), TTP should be classified as skin sensitizer (Category 1).

As TTP is the main component of the submission substance, it is found to be a skin sensitiser as well.