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Description of key information

In a Local lypmph node assay (LLNA) conducted with concentrations of 100%, 50% and 25%, the test item turned out to be a skin sensitizer. The EC3 value of 26.1% indicates a weak sensitization potential

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
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
- Weight at study initiation: 18.6 - 22.9 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories GmbH, 5960 AD Horst, The Nethertlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-95 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The highest test item concentration, which can be technically used was 100 % of the undiluted test item. Test item solutions at different concentrations were formulated in acetone:olive oil (4:1).
In the pre-test, two mice were treated with test item concentrations of 50 % (w/w) and 100 %.
The test item in the main study was assayed at 25, 50 and 100 %.
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 100 % of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v) after vortexing.
In the pre-test, two mice were treated with test item concentrations of 50 (w/w) and 100 %. At the tested concentrations, the animals did not show any signs of local skin irritation or systemic toxicity.


MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50 and 100 % (w/w) in acetone/olive oil (4:1). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher 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, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA:

The proliferation 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 sensitiliser 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: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
- Clinical signs (local / systemic): 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Positive control results:
Experiment performed in May 2010 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 2.04, 3.41, and 6.14, respectively.
The EC3 value calculated was 8.5 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices of 2.78, 7.68 and 16.45 were determined with the test item at concentrations of 25, 50 and 100 % in acetone/olive (4:1 v/v), respectively. A clear dose response was observed. The EC3 value calculated was 26.1 % (w/w).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below

CALCULATION AND RESULTS OF INDIVIDUAL DATA (Vehicle: acetone/olive oil (4:1 v/v))

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

78

---

---

---

---

---

BG II

58

---

---

---

---

---

1

2249

2181

8

272.6

1.00

25

2

6131

6063

8

757.9

2.78

50

3

16820

16752

8

2094.0

7.68

100

4

35940

35872

8

4484.0

16.45

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

1  =  Control Group

2-4  = Test Groups

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

CALCULATION OF EC3 VALUE:

 

Test item concentration %

S.I.

Test Group 2

25 (a)

2.78 (b)

Test Group 3

50 (c)

7.68 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 26.1%(w/w)

EC3 = Estimated concentration for a S.I. of 3.

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

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:
sensitising
Remarks:
Migrated information
Conclusions:
The test item SAT 090073 was found to be a skin sensitiser under the described conditions.
According to the Regulation (EC) No. 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures and UN classification (Globally Harmonized System (GHS)) the test item is classified as "skin sensitiser category 1, hazard statement H317 (“May cause an allergic skin reaction”)".
According to the former European Dangerous Substance Directive (Council Directive 67/548/EEC, currently undergoing a phase-out process) the test item is classified as sensitising and assigned the symbol “Xi”, the indication of danger “Irritant” and the risk phrase R43 ("may cause sensitisation by skin contact”).
According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Bruessels), SAT 090073 would be regarded as weak sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of SAT 090073, three groups each of four female mice were treated once daily with the test item at concentrations of 25, 50, and 100 % (w/w) in acetone:olive oil (4:1) by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil (4:1)) 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 of3H-methyl thymidine measured in aß-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 results in a 3-fold or greater increase in incorporation of3HTdR 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 2.78, 7.68, and 16.45 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4:1 v/v). A clear dose response was observed. The EC3 value calculated was 26.1 % (w/w).

In view of these results, according to the Regulation (EC) No. 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures and UN classification (Globally Harmonized System (GHS)) the test item is classified as "skin sensitiser category 1, hazard statement H317 (“May cause an allergic skin reaction”)".

According to the former European Dangerous Substance Directive (Council Directive 67/548/EEC, currently undergoing a phase-out process)the test item is classified as sensitising and assigned the symbol “Xi”, the indication of danger “Irritant” and the risk phrase R43 ("may cause sensitisation by skin contact”).

According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Bruessels), SAT 090073 would be regarded as weak sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the Regulation (EC) No. 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures the test item is classified as "skin sensitiser category 1, hazard statement H317 (“May cause an allergic skin reaction”)".