[No public or meaningful name is available]

Endpoint summary

Description of key information

FAT  40851  was  found  to  be a skin  sensitiser

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 03 November 2009; Experiment completion date - 01 December 2009; Study completion date - 01 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.8 - 24.0 g
- Housing: Single caging
- Diet : pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): relative humidity 45-65 %
- Photoperiod (hrs dark / hrs light): 12/12
- bedding: granulated soft wood bedding

Vehicle:

methyl ethyl ketone

Concentration:

The highest test item concentration, which can be technically used was a 25 % suspension in methylethylketone.
First pre-test two mice were treated with concentrations of 10 and 25 %. Second pre-test two mice were treated with concentrations of 2.5 and 5 %. The test item in the main study was assayed at 1, 2.5, and 5 %.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 25 % suspension in methylethylketone. In the first pre-test two mice were treated with concentrations of 10 and 25 % each on three consecutive days. Within 1 hour after the first and second and 24 hours after the third application both animals showed reduced spontaneous activity and eyelid closure. In the second pre-test two mice were treated with concentrations of 2.5 and 5 % each on three consecutive days. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY

TOPICAL APPLICATION
Each of the three groups of four female mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1, 2.5, and 5% (w/v) in methylethylketone. The application volume, 25 μl, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a beta-scintillation counter.

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: once daily (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

In the study with the positive control substance alpha-hexyl cinnamic aldehyde, Stimulation Indices of 1.79, 2.09, and 6.84 were determined with test item concentrations of 5, 10, and 25 % in acetone:olive oil (4+1). The EC3 value was calculated to be 12.9 %. The test item positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitiser under the described conditions, demonstrating the validity of the study.

Parameter:

EC3

Value:

1.1

Parameter:

SI

Value:

2.83

Test group / Remarks:

1 % Test item concentration

Parameter:

SI

Value:

5.43

Test group / Remarks:

2.5 % Test item concentration

Parameter:

SI

Value:

7.16

Test group / Remarks:

5 % Test item concentration

Calculation and Results of Individual Data; Vehicle: methylethylketone

Test item concentration	Group	Measured DPM	Calculation			Result
			DPM – BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
-	BG 1	25	-	-	-	-
-	BG 2	*	-	-	-	-
-	1	1838	1813	8	226.6	1.00
1	2	5164	5139	8	642.4	2.83
2.5	3	9865	9840	8	1230.0	5.43
5	4	13009	12984	8	1623.0	7.16

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

^a)= The mean value was taken from the figures BG I and BG II

^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

* = value could not be used, due to a technical problem

 

VIABILITY / MORTALITY: No deaths occurred during the study period.

 

CLINICAL SIGNS: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

 

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The test item was found to be a skin sensitiser under the described conditions.

Executive summary:

In order to study a possible contact allergenic potential of FAT 40851/A, a local lymph node assay according to OECD guideline 429 was performed under GLP. Three groups each of four female mice were treated daily with the test item at concentrations of 1, 2.5, and 5 % (w/w) in methyl ethyl ketone by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (methyl ethyl ketone) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a scintillation counter. All treated animals survived the scheduled study period and no signs of toxicity were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 2.83, 5.43, and 7.16 were determined with the test item at concentrations of 1, 2.5, and 5 % in methyl ethyl ketone. The EC3 value calculated was 1.1 %. The test item FAT 40851/A was found to be a skin sensitiser under the described conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA study

In order to study a possible contact allergenic potential of FAT 40851/A, a local lymph node assay according to OECD guideline 429 was performed under GLP. Three groups each of four female mice were treated daily with the test item at concentrations of 1, 2.5, and 5 % (w/w) in methyl ethyl ketone by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (methyl ethyl ketone) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a scintillation counter. All treated animals survived the scheduled study period and no signs of toxicity were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 2.83, 5.43, and 7.16 were determined with the test item at concentrations of 1, 2.5, and 5 % in methyl ethyl ketone. The EC3 value calculated was 1.1 %. The test item FAT 40851/A  was found to  be a skin sensitiser under the described conditions.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

Based on the above stated assessment of the skin sensitisation potential of FAT 40851/A (EC3 = 1.1 % (w/v)) the substance needs to be classified as Category 1A (H317: May cause an allergic skin reaction) according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.

 

Respiratory sensitisation:

As no data on respiratory sensitisation is available for FAT 40851/A a classification is not possible according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.