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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 FEB 2010 to 10 MAR 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 429)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
o-anisidine
EC Number:
201-963-1
EC Name:
o-anisidine
Cas Number:
90-04-0
Molecular formula:
C7H9NO
IUPAC Name:
2-methoxyaniline
Details on test material:
- Name of test material (as cited in study report): o-Anisidine

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- 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.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
25, 50, and 100%
No. of animals per dose:
4
Details on study design:
In order to study a possible allergenic potential of o-Anisidine, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. 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 were excised and pooled per group. Furthermore, after the lymph nodes had been excised, both ears of the mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 square cm) and were immediately weighed pooled per animal using an analytical balance. Single cell suspensions of lymph node cells were prepared from lymph nodes pooled per group, 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.
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.

The EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
A statistical analysis was performed for the values obtained for the ear weights to assess whether the difference is statistically significant between test item groups and the negative control (vehicle) group and also to assess if there is a dose response relationship.
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performend in November 2009, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.I. of 1.78, 2.54, and 4.88, respectively. The EC3 value calculated was 12.9%

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25% test item: 2.26 50% test item: 3.43 100% test item: 1.27
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node: control: 528.5 25% test item: 1193.0 50% test item: 1813.1 100% test item: 672.2

Any other information on results incl. tables

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

19

---

---

---

---

---

BG II

19

---

---

---

---

0

1

4247

4228

8

528.5

 

25

2

9563

9544

8

1193.0

2.26

50

3

14524

14505

8

1813.1

3.43

100

4

4052

4033

6

672.2

1.27

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

 

Test item concentration %

S.I.

Test Group 2

25 (a)

2.26 (b)

Test Group 3

50 (c)

3.43 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c =40.8%(w/v)

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 / Mortalit

One animal of the group treated with the undiluted test item (100%) was found dead 24 hours after the second application.

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 with3HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Weights

The measured ear weights of all animals treated were recorded after necropsy. A statistically significant increase in ear weights was observed for the animals belonging to the group treated with a test item concentration of 50% in comparison to the control (vehicle) group (p=0.018).

Ear biopsy (punches ofØ8 mm corresponding to 0.5 cm2)

Animal No

Dose Group

Ear weights ( two ears per animal, one punch per ear in mg)

Mean

Standard deviation

1

1

19.95

22.96

2.30

2

1

23.01

3

1

23.33

4

1

25.54

5

2

23.68

25.08

1.06

6

2

24.84

7

2

25.85

8

2

25.95

9

3

32.57

29.98*

3.16

10

3

32.85

11

3

27.32

12

3

27.17

13

4

23.75

24.23

0.59

14

4

animal died 

15

4

24.89

16

4

24.04

1 = Control Group 2-4 = Test Groups * = statistically significant

Applicant's summary and conclusion

Interpretation of results:
other: equivocal
Conclusions:
Based on S.I. values one of which is slightly above 3, o-Anisidine would have to be regarded as a skin sensitiser. But due to missing dose response relationship, potentially irritating effects, and the impairment by an unexplained death of one animal this interpretation seems not to be entirely reliable. Thus the final conclusion is that the outcome of this study is considered to be equivocal. Further evidence regarding the question whether the obtained effect is truly of a sensitising nature or not can only be obtained using further appropriate studies.
Executive summary:

The present study analyses the sensitising potential of o-anisidine.

Three groups each of four female mice were treated daily with the test item at concentrations of 25% (v/v), 50% (w/v), and 100% (undiluted test item) in dimethylsulfoxide by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylsulfoxide) 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 beta-scintillation counter.

Clinical signs of systemic toxicity were not observed in the animals treated with different concentrations of the test item. However, one animal of the group treated with the undiluted test item (100%) was found dead 24 hours after the second application.

No signs of local irritation (e.g. ear swelling, reddening of the ear skin) were observed in the treated animals over the whole study period. However, a statistically significant increase in ear weights was obtained for the animals belonging to the group treated with a test item concentration of 50% in comparison to the control (vehicle) group (p=0.018).

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 2.26, 3.43, and 1.27 were determined with the test item at concentrations of 25, 50, and 100% in dimethylsulfoxide demonstrating slight excedance of threshold for one concentration but no clear dose response relationship. The S.I. for the high dose group was derived from 3 animals only due to the fact that one animal belonging to this group died. The decrease in S.I. seen at this concentration as compared to the lower doses might be related to systemic effects of the test item. The EC3 value in terms of figures was 40.8 % (w/v). However, it cannot be excluded that skin irritating properties of the test item may have contributed to the increase in lymph node cell proliferation.