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Description of key information

Skin: not sensitising, LLNA, OECD 429, Vogel 2010

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: GLP compliant, guideline study, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing:single caging
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Laboratories GmbH, 33178 Borchen), ad libidum
- Water (e.g. ad libitum): tap water, (Gemeindewerke, 64380 Rossdorf), 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 will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Vehicle:
dimethylformamide
Concentration:
10, 25, and 50% (w/v)
No. of animals per dose:
4
Details on study design:
VEHICLE AND DOSE SELECTION
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% (w/v) solution in dimethylformamide (DMF) after vortexing and warming to 37°C. The vehicle was chosen due to its solubilisation properties.
To determine the highest non-irritant test concentration that did at the same time not induce signs of systemic toxicity,a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Furthermore, prior to the first application of the test item and before sacrifice the body weight was determined. Both animals gained weight during the course of the pre-test.
In the pre-test clinical signs were recorded within 1 hour and 24±4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of local irritation or systemic toxicity.
Thus the test item in the main study was assayed at 10, 25, and 50% (w/v). The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN TEST

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 10, 25, and 50% (w/v) in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface (Æ~8 mm) of each ear once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals).

ADMINISTRATION AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were administered with 250 µl of 81.7 µCi/mL3HTdR (corresponds to 20.4 µCi3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after injection, the mice were sacrificed and the draining of auricular lymph nodes were excised and pooled per group. Single cell suspensions of lympg 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 3HTdR measured in a beta-scintilation 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; S.I.). 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 with3HTdR.
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 and 24±4 hours after the first and second application (days 1 and 2) as well as on the day of preparation (day 6). Especially the treatment sites were observed carefully.
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 performend in May 2010, 5, 10, and 25% alpha-Hexylcinnamaldehyde in acetone:olive oil (4+1) yielded a S.I. of 2.04, 3.41, and 6.14, respectively. The EC3 value calculated was 8.5%
Parameter:
SI
Value:
1.56
Test group / Remarks:
10% test item concentration (w/v)
Remarks on result:
other: see table below
Parameter:
SI
Value:
1.29
Test group / Remarks:
25% test item concentration (w/v)
Remarks on result:
other: see table below
Parameter:
SI
Value:
1.32
Test group / Remarks:
50% test item concentration (w/v)
Remarks on result:
other: see table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below

Calculation of Results

Since the lymph nodes of the animals of a dose group were pooled, the number of radioactive disintegrations per minute per lymph node (DPM/node) was determined by dividing the measured DPM-value by the number of lymph nodes pooled (8 lymph nodes). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. For the control group (vehicle group), a DPM/node value of 336.6 was determined whereas DPM/node values of 523.4, 435.8, and 444.8 were determined for the test item groups with test item concentrations of 10, 25, and 50% (w/v), respectively. The Stimulation Indices calculated for these groups were 1.56 (at a test item concentration of 10%), 1.29 (at a test item concentration of 25%) and 1.32 (at a test item concentration of 50%) (see Table 2). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

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, recordedprior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age

Summary of Results

 

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

20

---

---

---

---

---

BG II

17

---

---

---

---

---

1

2711

2693

8

336.6

1.00

10

2

4206

4188

8

523.4

1.56

25

3

3505

3487

8

435.8

1.29

50

4

3577

3559

8

444.8

1.32

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

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item was not a skin sensitiser under the described conditions.
Executive summary:

In order to study the possible contact allergenic potential of the substance, 3 groups each of 4 female mice were treated daily with the test item at concentrations of 10, 25, and 50% (w/v) in dimethylformamide by topical application to the dorsum of each ear (left and right) for 3 consecutive days. A control group of 4 mice was treated with the vehicle (dimethylformamide) 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 5 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 ab-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 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 1.56, 1.29, and 1.32 were determined with the test item at concentrations of 10, 25, and 50% (w/v) in dimethylformamide. A dose response was not observed.

 

The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two published QSAR results for salicylamide were available, both were unvalidated experimental models. Dimitrov et al (2005) predicted the substance to be sensitising, while Magee et al (2000) predicted it to be non-sensitising. Both results were discarded as being unreliable. However, both studies refer to an experimental result of non-sensitising in Cronin et al. (1994), SAR and QSAR in Environmental Research 2, 159 -179. The original Cronin paper contains no reference to salicylamide (but does refer to salicylanilide - non-sensitising). Therefore this result also had to be discarded as unreliable.

A single experimental study was available which was conducted under GLP and according to OECD guideline 429. The result as non-sensitising is considered to be relevant, reliable, and adequate for risk assessment, classification and labelling.


Justification for selection of skin sensitisation endpoint:
GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results from a mouse local lymph node assay indicate the substance is not a skin sensitiser. As a result the substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, Part 3, 3.4.2.2.