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:
03 June 2008 to 02 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed study according to existing guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD principles and German "Chemikaliengesetz"
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): o-chlorbenzaldehyd
- Stability in solvent: stable for 3-5 days when kept airtight at room temperature
- Storage condition of test material: at room temperature, under nitrogen atmosphere protected from contact with air

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: CBA/CaOlaHsd (nulliparous and non-pregnant)
- Source: Harlan Netherlands, Horst NL
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.4 to 24.2 g
- Housing: individually
- Diet: ad libitum, Harlan Winkelmann pelleted standard diet
- Water: ad libitum, tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): no data
- Photoperiod: 12 hours (6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
purity: 99.5 %
Concentration:
0 %, 25 %, 50 %, 100 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble at 50% in acetone:olive oil (4+1), dimethylformamide, methyl ethyl ketone, dimethyl sulphoxide
- Irritation: two mice were treated with concentrations of 10, 25, 50 and 100% on one ear each on three consecutive days: undiluted test item did not induce irritation or systemic toxicity, ears treated with 25 and 50% of the test item were hardened on the second day of application, the effect has gone on day 3
- Lymph node proliferation response: no data


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
a) exposure to at least one concentration of the test item resulted in an incorporation of 3H-Methyl thymidine (3HTdR) at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index
b) the data are compatibel with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression


TREATMENT PREPARATION AND ADMINISTRATION:
- the test item was kept under nitrogen atmosphere until dimethyl sulphoxide was added, preparations were made freshly before each dosing
- epidermal application of different test item concentrations of 0, 25, 50 and 100 % (w/v) in dimethyl sulphoxide to the dorsal surface of each ear lobe (left and right)
- application volume: 25 µl
- application area: entire dorsal surface (diameter about 8 mm)
- application frequency: once daily for three consecutive days
- application of 3H-Methyl thymidine (3HTdR) five days after the first topical application of test item: 250 µl of 81.8 µCi/ml 3HTdR (corresponding to 20.45 µCi 3HTdR per mouse) by intravenous injection via a tail vein
- sacrifice of all mice approximately 5 hours after 3HTdR treatment by i.p. injection of Pentobarbital-Natrium
- draining auricular lymph nodes were excised and pooled per group
- single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were washed and incubated with trichloroacetic acid over night
- level of incorporated 3HTdR was measured with a beta-scintillation counter

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated for body weight.

Results and discussion

Positive control results:
The LLNA with the positive control substance has been performed in February 2008. S.I. values of 1.78, 1.84 and 4.87 were obtained for test item concentrations of 5%, 10%, and 25% (w/v) in acetone:olive (4+1). An EC3 value of 15.7% (w/v) has been calculated.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation index for the different treatment groups: 25%: 5.21 50%: 5.62 100%: 3.31
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM were clearly increased in treated animals compared to the controls (details are presented in the table below). But there was no clear dose response. DPM were about 5-fold increased in animals treated with 25 or 50% of the test item, but only about 3-fold increased in animals treated with 100% of the test item.

Any other information on results incl. tables

Measurement of dpm:

Test item
concentration
% (w/v)

Group

Measurement
DPM

Calculation

Result

DPM-BGa)

number of
lymph nodes

DPM per
lymph node
b)

S.I.

---

BG I

42

---

---

---

---

---

BG II

35

---

---

---

---

---

1

8273

8235

8

1029.3

25

2

42933

42895

8

5361 .8

5.21

50

3

46354

46316

8

5789.4

5.62

100

4

27289

27251

8

3406.3

3.31

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

- No deaths occured during the study period.

- Body weights were not affected by treatment.

- No signs of systemic toxicity were recorded in any group during this study. Systemic toxicity were recorded in any group during this study.

- Animals treated with 50% of the test item showed hardened ears with a visible layer of test item on the second day of application. This was also observed for the low dose of 25% on the third day of application. The ears of the animals of these two groups showed a visible deformation on the day of preparation. Ears of the animals which received pure test substance were similar to the control.

- The ear weights of the animals of the 25 and 50% dose group were clearly increased in comparison to the controls. Ear weights in the highest dose group were only slightly above the vehicle control.

Ear weights:

Animal No.

Dose Group

Ear Weight
(mg) pooled per animal

Individual

Mean
± SD

1

1

26.37

28.1 ± 1.8

2

1

28.57

3

1

27.00

4

1

30.33

5

2

50.52

52.2 ± 1.8

6

2

52.85

7

2

51.14

8

2

54.44

9

3

53.63

49.9 ±4.2

10

3

53.15

11

3

48.12

12

3

44.76

13

4

37.50

35.4 ± 7.9

14

4

30.23

15

4

45.59

16

4

28.16


Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
2-Chlorobenzaldehyde was found to be an elicitor of lymph node proliferation which formally results in the conclusion that 2-chlorobenzaldehyde is a skin sensitiser under the described conditions.

Nevertheless, taking into account the missing dose-response as well as the increase in ear weight, there is some evidence, that the observed lymph node proliferation alternatively might be related to irritation effects. Clarification of the true nature of the lymphoproliferative response of o-chlorobenzaldehyde is necessary by additional testing.
Executive summary:

Allergenic potential of 2-chlorobenzaldehyde has been investigated in a local lymph node assay with female mice which were treated with 0, 25, 50, and 100% (w/v) o the test item in dimethyl sulphoxide by topical application to the dorsum of each ear lobe for three consecutive days.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortaliy were observed. On the second day of application the animals treated with 50% of the test item showed hardened ears with a visible layer of the test item. On day 3 this was also obseved for the low dose of 25%. On the day of preparation the animals of both groups showed a visible deformation of the ears. With the pure test item (100%), however, the ears looked similar to the vehicle control. Punch biopsies taken from the ear of animals of the 25 and 50% test item treated groups were also clearly increased, whereas the highest dose group had ear weights only slightly above the vehicle control.

In this study stimulation indices (S.I.) of 5.21, 5.62, and 3.31 were determined with the test item at concentrations of 25, 50, and 100% in dimethyl sulphoxide, respectively. The EC3 value could not be calculated, since all obtained S.I. values were above 3.

This well performed guideline study has been selected as key study.